Radiation_cover def
14/10/02
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E u r o p e a n
C o m m i s s i o n
KI-NA-19-959-EN-C
Community Research
Understanding
the effects of
radiation on health
OFFICE FOR OFFICIAL PUBLICATIONS
OF THE EUROPEAN COMMUNITIES
EURATOM
L-2985 Luxembourg
EUR 19959
Editors
Dr. Ch. Desaintes, Dr. G. Neale Kelly, Ms Karin Coiffard
Contact person: Christian Desaintes, EC DG RTD J04
Address: MO75 5/1, B-1049 Brussels
E-mail: [email protected]
Legal notice
Neither the European Commission nor any person acting on behalf
of the Commission is responsible for the use which might be made
of the following information.
A great deal of additional information on the European Union is
available on the Internet. It can be accessed through the Europa server
(http://europa.eu.int).
Luxembourg: Office for Official Publications of the European Communities, 2002
ISBN 92-894-3840-1
© European Communities, 2002
Reproduction is authorised provided the source is acknowledged.
Printed in Belgium
Contents
Part 1
4
PREFACE
5
INTRODUCTION
Cellular studies:
Part 2
14
B. Michael
Experimental data for the induction of cancer by radiation of different qualities (EDICAR)
16
B. Michael
Induction, repair and biological consequences of DNA damages caused by radiations of various qualities
(RADNA)
18
A. Natarajan
Chromatin structure and DNA repair in relation to ionizing radiation-induced chromosome
aberration in mammalian cells
20
A. Natarajan
Factors modifying the yield of radiation-induced chromosome aberrations
22
L. Mullenders
Mechanisms of formation of ionizing radiation-induced chromosomal aberrations
(CHROMOSOME STRUCTURE)
24
L. Mullenders
The molecular basis of DNA damage response and radiosensitivity (DNA DAMAGE RESPONSES)
26
S. Salomaa
Genomic instability and radiation-induced cancer (RADINSTAB)
28
J. Egozcue
Role of telomere addition in the stabilisation of radiation-induced DNA breaks
30
L. Sabatier
Telomere instability and the formation and transmission of radiation induced DNA damage (TELORAD)
32
T. Jung
Evolution of genetic damage in relation to cell cycle control: a molecular analysis of mechanisms
relevant for low dose effects
34
A. van der Eb
The effects of ionising radiation on signal transduction and cell cycle control
Radiation tumorigenesis: mechanisms and pre-disposition:
36
G. Monchaux
Part 3
Risk assessment of exposure to radon decay products (RARAD)
38
R. Cox
The mechanisms and genetics of radiation tumorigenesis (MAGELLANS)
40
M. Atkinson
Genetic susceptibility to radiation carcinogenesis (GENRAD)
42
D. Llyod
Detecting differences in radiation sensitivity of people
44
A. Riches
Molecular mechanisms of radiation carcinogenesis in man
46
R. Newbold
Identification and isolation of susceptibility genes involved in radiation-induced cancer in
humans (SUS GENES IN RAD CAR)
48
D. Williams
Pathology and molecular biology of thyroid tumours in children and young adults exposed
to fallout from the Chernobyl nuclear disaster
50
G. Thomas
Chernobyl, an integrated pan-European study: morphology, oncogenes, DNA repair and outcome in
radiation carcinogenesis (CHIPS)
52
G. Thomas
The newly independent states Chernobyl tumour bank - An international scientific resource (NISCTB)
Modelling radiation cancer risk:
54
H. Paretzke
Part 4
Biophysical models for the induction of cancer by radiation (LOW DOSE RISK MODELS)
Diagnosis/treatment of cancer and radiation injury:
Part 5
56
A. Pinchera
Development of optimal protocols for the diagnosis, treatment, follow-up and preventive measures
for post-Chernobyl thyroid carcinoma (NUFISA)2
58
Ch. Reiners
Improvement of dosimetry for I-131 therapy of lung metastases with special regard to children with
thyroid cancer from Belarus following the Chernobyl accident
60
J. Bueren
Novel approaches to the treatment of radiation accident patients (NAIMORI)
Non-cancer effects:
Part 6
62
F. Stylianopoulou In utero irradiation of the foetal rodent brain: early effects
64
S. Salomaa
Minisatellite mutations and biodosimetry of population around the Semipalatinsk nuclear test site
(SEMIPALATINSK)
Annex 1: projects starting late 2002:
66
P. de Boer
Genetic factors predisposing to radiation induction of mutation during early gestation:
the role of DNA repair and cell cycle control (GEMRATE)
66
L. Mullenders
Radiation specific DNA non-double strand break lesions: repair mechanisms and biological effects
(NON-DSB-LESIONS)
66
M. Atkinson
Genetics of predisposition to radiation-induced cancer of the thyroid (GENRAD-T)
66
M. Baumann
Genetic pathways for the prediction of the effects of irradiation – European normal and tumour tissue
bank (GENEPI-ENTB)
Preface
Ionising radiation is present at low doses and low dose rates in the natural
environment, largely from radioactive atoms in minerals formed during the early
history of the planet. The technological developments of the 20th century have
resulted in the use of radioactive materials for military, industrial and medical purposes. The peaceful use of radiation includes power generation, industrial testing,
bio-medical research, disease diagnosis and cancer therapy – all an integral part
of our modern world.
These benefits to society need, however, to be weighed against the known potential of radiation to cause health effects in exposed people, principally – tissue injury,
cancer and genetic effects that are passed to offspring. Much is known on the likelihood of these health effects after exposure to radiation at high doses and high dose
rates. However, the vast majority of man-made radiation exposures to workers and
the general public occur at low doses and low dose rates where risks cannot be reliably assessed by direct observation. The majority scientific view is that risks of excess
cancer and genetic effects are likely to rise in simple proportion to radiation dose and
that even the lowest of doses carries some risk, albeit vanishingly small. However,
some suggest that there is a low dose region (the dose threshold) where there is no
excess risk of any health effect; conversely, others claim that low dose risks are
grossly underestimated. Because of scientific uncertainties, the low dose issue remains
an important source of debate in radiation protection and more widely in policy/
standard setting.
In order to weigh the socio-economic benefits of radiation against its potential effects
on health, it is essential to gain a better understanding of this low dose and low dose
rate issue. For this principal reason the European Commission has supported a
programme of epidemiological and basic research on the health effects of radiation.
This brochure provides an outline of the basic research sponsored by the Commission
in its 4th and 5th Framework Programmes. It will be of interest to a relatively broad
audience with interests in radiation protection including the safety of nuclearpower
generation, medical aspects of radiation exposure, the application of new research
technologies to environmental issues and new concepts concerning the biological
action of radiation. The brochure contains two distinct parts. The first one provides a summary of the Commission supported research in the past decade. The
main text of this part may be read at two levels with boxed notes acting to provide simplified explanations of scientifically complex issues and outcomes. The second part includes summaries of many of the projects that make up the relevant
research portfolio of the Commission. Epidemiological research is outlined in a separate publication “Epidemiology and retrospective dosimetry” (EUR 19958).
4
PART 1
Summary
T
his brochure summarises fundamental research on the health
effects of ionising radiation supported by the European
Commission in its 4th and 5th Framework Programmes. This outline is set in the context of the need for a detailed understanding of the mechanisms of radiation action on cells and tissues of
the body in order to support judgements on the health risks after
low dose and low dose-rate radiation. Also included is an indication of the main direction of research that will be pursued in
the 6th Framework Programme.
Fundamental research supported by the Commission over many
years has made a substantial contribution to the understanding
of the mechanisms that underlie radiation-induced health effects.
The overall objective is to provide a sound biological basis for
radiation protection. The research areas considered to be of particular importance are DNA damage response processes in cells,
the specific mechanisms of cancer induction, heritable susceptibility to induced cancer including individual risk, the modelling
of low dose cancer risk, heritable effects, effects on the developing brain and the diagnosis/treatment of cancer and radiation
injury (see notes 1, 2 and 3). Research in some of these areas
included consideration of the health effects of the Chernobyl
reactor accident.
The success of these research programmes has been promoted
by involving many of the key European research laboratories and
placing emphasis on interdisciplinary collaboration and communication. This success in achieving research objectives is well
illustrated by the establishment of strong biological and biophysical links between the dose-dependent appearance of DNA
damage in irradiated cells, the recognition and repair of these
initial DNA lesions and the contribution made by residual DNA
changes to cancer development in tissues. This experimentally
derived information is providing an increasingly robust biological
foundation for the development of improved judgements on
cancer risk after radiation.
However, although much has been learned of radiation mechanisms and their modelling (see notes 4, 5, 6 and 7), it is clear that
further research is needed in order to address remaining uncertainties on the risks to health after low dose and low dose rate
radiation.
1. Introduction
The damaging effects of ionising radiation on tissues in the body
have been known for about 100 years. This knowledge was followed by evidence that high doses of radiation can cause cancer.
These carcinogenic effects have been studied most closely by
following the health (epidemiological study) of radiation-exposed
survivors of the 1945 atomic (A-) bomb explosions in Japan. These
A-bomb studies provide much of the information available on the
radiation dose-response of risk of leukaemia and solid tumours
although further knowledge has come from other investigations
of people exposed to radiation accidentally, in their work or
through medical procedures. In addition, such studies have provided evidence on the more general aspects of radiation induced
tissue injury and how it may be assessed and treated. Specific forms
of non-cancer effects in tissues have also been considered, including effects on brain development following pre-natal exposures
and damage to germ cells in reproductive organs which can result
in heritable disease.
The achievement of an acceptable balance between the detrimental effects of radiation and its benefits to society requires a scientifically robust and acceptable system of radiation protection that
is applicable to public and occupational exposures. The radiation
doses that apply to these exposures are invariably below a level
where direct epidemiological study can inform on cancer risks.
At these low doses received at low dose rates the risks are very small
when compared with the 20-25% chance of a typical individual developing fatal cancer from other causes. Nevertheless, for protection
purposes, it is important to improve judgements on how the cancer risks known to apply at moderate to high doses may be projected
downwards to these low doses and dose rates. To achieve this it is
important to extend knowledge of the mechanisms through which
radiation damage to cells in tissues allows a small number to enter
a multi-stage process of mal-development which can lead to cancer (see note 3); this process can take tens of years to complete.
Stated in a simple way, with good knowledge of the biology
of radiation action within the cancer process it will be possible to construct well-founded mathematical models to describe
radiation risk at the low doses that are most relevant to
human exposures. These biologically validated models will be of
substantial value in developing firm views on whether radiation risk
increases as a simple function of dose (e.g. a linear dose-response)
or alternatively whether there is a true low dose threshold where
risk may be discounted.
An additional fundamental issue that has come to the fore in recent
years is the question of variation in inter-individual risk due to the
effects of genetic make-up on radiation response. A few rare genetic
disorders that may substantially influence radiation risk are known
but it is believed that more common genetic factors currently
escape detection because their effects are relatively weak. An understanding of such common genetic factors will aid judgements on
how radiation risks are distributed in the population and whether
certain genetic sub-groups are under-protected.
For some time the Commission has recognised the importance of
obtaining this fundamental information and to couple it with
5
Note 1 – Radiation damage to cells: tissue injury, cancer and heritable disease
When a track of ionising radiation passes through a cell in
the body it will deposit energy which can disrupt the organic
molecules and structures that make up the cell. A particularly sensitive molecule within the cell is deoxyribonucleic
acid (DNA) which is commonly termed ‘the blueprint of life’.
These sets of helical double stranded DNA molecules are
very long, they are contained in microscopic structures
called chromosomes and provide a linear series of codes
for the production of cellular proteins. The structure and
activity of these proteins allow all cellular functions to
proceed. Each protein is coded by a specific stretch of DNA
called a gene; there are around 35 000 genes in each
human cell contained within 22 chromosome pairs and two
other chromosomes that determine gender. In this way the
DNA controls all cellular activity and, in reproductive (sperm
and egg) cells, acts to pass information from one generation to the next. The total DNA constitution of a cell is termed
the genome.
There is strong scientific evidence that damage to DNA from
ionising radiation, particularly DNA breakage, underlies many
of the health effects that are recognised from clinical studies
on tissue injury in individuals, from health studies (epidemiology) of excess cancer and genetic effects in irradiated
human groups and from investigations with experimental
animals.
Cellular damage from radiation can lead to cell death. If the
radiation dose is high and enough cells are killed or functionally impaired there will be clinically observable tissue
injury, e.g. in blood forming tissues and the intestines.
Severe tissue injury can be fatal but more subtle effects may
also occur, e.g. in the developing brain.
epidemiological findings. Also, rapid advances in modern biology,
genetics and medicine are providing the background knowledge and
tools to seek answers to these outstanding questions in radiation
protection (see note 2). The research that the Commission has
identified and supported in these areas has made important progress
during the 4th and 5th Framework Programmes (FP). The research
strategies and scientific progress of these programmes are summarised in the following sections of this brochure together with a
view of how work may be extended within the 6th FP.
6
At low doses, tissue injury is not apparent but some cells
will survive radiation with changes (mutations) in their
DNA. Some of these mutations are visible at the chromosomal level and, in principle, all functional gene mutations
can alter cellular behaviour. Most importantly, certain specific mutations arising in normal cells of the body can result
in changes in cellular properties (e.g. in growth control) that
may contribute over many years to cancer development.
The full development of cancer is a multi-stage process
(see note 3) which is believed to require the acquisition of
a number of different mutations which are likely to have
different causes. Ionising radiation exposure may be most
simply viewed as one of these contributory causes of cancer.
One additional factor of growing importance is the influence of differences in genetic make-up on radiation cancer
risk – in this area our knowledge is very limited.
Radiation-induced mutations arising in reproductive (germ)
cells are not expressed as disease in the irradiated individual
but may be passed to the offspring and thereby to future
generations. Thus, radiation can contribute to the frequency
of heritable diseases of widely differing severity.
An important issue in the understanding of all the health
effects of radiation is the defence systems that cells possess
in order to protect against radiation damage. These include
cellular stress systems that act to recognise the damage that
has occurred, proteins that actively repair damage to chromosomal DNA, systems that promote the death of heavily
damaged cells and various cellular and tissue processes that
serve to minimise the probability that appropriately mutated
cells will complete the multi-stage pathway to cancer.
PART 1
2. Research within the 4th and 5th Framework
Programmes (FP4: 1994-1998; FP5: 1998-2002)
The European Commission has sponsored research in radiation
protection throughout the period since the EURATOM Treaty of 1957.
In this way the Community, through the Commission, has made a
substantial contribution to the world-wide development of knowledge on radiation effects/dosimetry and the application of that
knowledge in radiation protection.
The recognition that further information was needed on the fundamental processes underlying radiation action on cells and the subsequent development of cancer was a major factor in the design of
FP4 research in radiation protection. Previous research (note 4) had
highlighted the critical importance of the damage caused by ionising
radiation to the genetic machinery (chromosomal DNA) of cells and
how DNA damage response systems within the cell can be mobilised
to repair this damage. However, there is a certain probability that
this repair will not always be wholly correct which can result in
gene/chromosomal mutations and/or cell death. It was also becoming more clear that DNA damage response and repair/misrepair
processes in cells were critical components in cancer risk. Thus,
work on cellular responses had the potential to be coupled with
research on tumorigenic mechanisms using selected animal models
of human cancer. Cellular and animal models would also find use in
studies on the heritable factors that influence radiation cancer risk.
However of equal importance was the progressive increase in
epidemiological knowledge of radiation cancer risk from studies on
survivors of the Japanese A-bomb explosions; on groups exposed
to natural radon gas; on medically irradiated people; on nuclear
workers; and on children in the former Soviet Union (FSU) developing thyroid cancer following radioiodine exposure from the
Chernobyl accident in 1986.
In respect of these advances the Commission sought to sponsor fundamental research on the mechanisms and predisposition to radiation associated cancer. The effects of radiation were investigated
both at cellular/molecular and tissue/organism levels. The resulting gained fundamental knowledge was used to develop cancer risk
modelling. Other research that was supported included diagnosis
and treatment of post-Chernobyl thyroid cancer and the mechanisms
of tissue injury. Non-cancer research projects concerning radiation
effects on the developing brain and on biomarkers of damage to
reproductive cells were also included. Because of the long-term
nature of the research supported in this area, there has been no
major discontinuity between FP4 and FP5. Therefore, the main
findings of FP4 and FP5 research will be presented together in the
following sections. More detailed information about individual
contributions can be found in the second part of the brochure.
Note 2 – Research tools and approaches
◗ Radiation-induced human cancer: For studies on cancer
mechanisms there are few opportunities to obtain samples
of human tumours that have a high probability of being
induced by radiation; post-Chernobyl childhood thyroid
cancer and cancer resulting from radiotherapy offer the best
prospects for research.
◗ Experimental cellular models: Many mechanistic studies
of the effects of radiation utilise cells that are growing in
culture. This approach has been particularly valuable for the
identification of biologically important forms of DNA damage, cellular responses to this damage and how it is repaired.
Cells with genetically altered sensitivity to radiation may be
used to identify and characterise the genes that control
different forms of cellular response and damage repair.
Models based on cultured cells may also be used to study
certain aspects of cancer development such as cell immortalization and DNA instability.
◗ Experimental animal models: Cancer development is a
complex process and at present cellular studies cannot
adequately represent this complexity. For this reason certain problems in radiation induced cancer demand the
use of mice or rats. These animal models allow the yield and
characteristics of radiation-induced tumours to be determined; this information may be related to cellular responses.
Genetically based experiments with mouse models also
find use in investigations on the whole-body impact of
DNA repair and on how heritable factors might influence
individual cancer risk. In the past, radiation studies utilised
very large numbers of experimental animals; new animal
models coupled with cellular studies are far more efficient.
◗ Experimental methods: Research on cells and animals
benefit greatly from modern biological techniques. For example, the precise visualisation of altered chromosomes, the
high-resolution analysis of DNA damage and the rapid determination of the structure and activity of genes involved in
DNA repair, cancer development or altered brain function.
◗ Cancer risk modelling: Cancer risk modelling involves the
use of computer-based calculations using biological information on the cancer process to better describe epidemiological findings on excess cancer after radiation. A major
aim is to assess the impact of low dose radiation on excess
cancer in an irradiated human population.
7
Note 3 – Research strategies used to resolve cancer mechanisms
I. Simplified schematic model of multi-stage radiation tumorigenesis
Incorrect DNA
Initial loss of
damage response/repair
growth control
A. Cellular DNA
B. Mutant
C. Multiplication
damage from
pre-cancerous
of pre-cancerous
radiation
cell
cells
Further mutational
or non-mutational
change
E. Development of
D. Growth selection of
fully malignant
immortal/unstable
cancer
cancerous cells
Final set of cellular
changes and growth selection
II. Research strategies employed for different phases in the schematic model
Cancer mechanisms
A Cellular and biophysical studies on DNA damage induction.
A→B Cellular studies on DNA damage response/repair, bystander effects and
genomic instability.
Studies with genetically altered (knock-out) mice.
B→C→D→E Studies with normal and gene knock-out mouse models.
Studies with radiation-associated human tumours.
Cancer genetics
A→B Studies on human/rodent cells and knock-out mice with defined deficiencies in
DNA damage response or DNA instability.
B→E Studies on mutant mice fully deficient in defined tumour-associated genes.
Studies on inter-bred mouse strains having natural variation in tumour
susceptibility; identifying the responsible genes.
8
PART 1
Cellular studies on DNA damage-response
The foundations for research on DNA damage response and repair
were established in previous FPs and the new work sought to build
upon the critical mass of EU expertise and knowledge that had
been developed. At the level of initial radiation damage to DNA,
the FP4 programme greatly strengthened the view that the appearance of excess gene/chromosomal mutations in cells was principally
driven by the induction of double strand breaks in the helical DNA
molecules that make up the chromosomes. Work was undertaken
on the dependence of DNA double strand breaks induction by radiations of different qualities (different linear energy transfer – LET).
An important finding was that the complexity of this initial damage appeared to strongly influence the quality of DNA repair that
was possible. During this period of research the biologically important DNA double strand breaks were suggested to be associated with
multiply damaged sites which also contained local damage to other
constituents of the molecule, i.e. clustered damage (see note 5).
This feature seemed likely to be related to the types of gene and
chromosomal mutations that arose after radiation.
In this general area of biophysics, technical developments allowed
for the irradiation of single cells by single ionising particles of high
LET radiation. This technique allowed study of the induction of
cellular effects by the lowest possible dose of radiation and also
revealed evidence of the potential for transfer of damage signals
between cells – the bystander effect.
A number of projects addressed the specific mechanisms whereby
radiation tracks caused initial DNA damage which was then misrepaired to form chromosomal exchanges (translocations). These
studies benefited greatly from a new technique, termed FISH, that
allowed differential molecular staining of different chromosomes –
using this technique and another that prematurely condenses
chromosomes (PCC) it became possible to approach mechanistic
problems of chromosome damage that had previously been inaccessible, for example the rate of repair of initial DNA double strand
breaks in relation to chromosome damage. FISH techniques coupled
with cellular/molecular analyses were also used to explore relationships between cellular radiation response, chromosomal instability and the specialised DNA sequences termed telomeres that cap
the ends of chromosomes and occupy certain internal chromosomal sites. This and other work highlighted the potential importance
of higher order DNA structure as a factor in chromosome stability
and radiation response.
The response of cells to radiation include the imposition of checkpoints in the reproductive cycle in order to promote DNA repair –
also as part of the mechanism of programmed cell death (apoptosis) that serves to eliminate heavily damaged cells. A number of
projects succeeded in clarifying the above relationships and some
of the genes/enzymes involved in the responses.
The potential role of telomeric sequences in radiation response, highlighted in studies on chromosomal instability in tumorigenesis, was
investigated in mouse germ cells. These studies revealed upregulation of the telomere elongating enzyme telomerase in
Note 4 – Important advances in knowledge
during the period prior to FP4
◗ Knowledge on the extent to which damage to DNA and
its repair determine the sensitivity of cells to ionising
radiation.
◗ The strengthening of links between cellular radiosen-
sitivity and cancer development.
◗ The use of molecular genetic techniques for chromo-
some analysis and genetic manipulation in radiation
studies; advances in the development of animal models
for studies on cancer mechanisms.
◗ General advances in cancer epidemiology relating to
the Japanese A-bomb survivors, radon exposed miners
and post-Chernobyl childhood thyroid cancer to support the development of biologically based models of
cancer risk; also, for thyroid cancer, the availability of
tumour samples for analysis.
response to radiation and evidence that some DNA double strand
breaks can be healed by addition of telomeric sequences.
The study of mutant cells with altered radiation response had
become a most important component of radiation biology and was
used widely in many fundamental studies in FP4. Such mutants
were selected from cell cultures or derived from carriers of radiosensitive human genetic disorders e.g. ataxia-telangiectasia. A major contribution was made to the characterisation of these mutants
including the isolation of the responsible genes and others of
Note 5 - Examples of some novel features of radiation
response emerging during the period of FP4
the period of FP4
◗ That a significant fraction of DNA double strand breaks induced
by radiation are associated with a cluster of other damages
and may be difficult to repair correctly.
the period of FP
◗ That in some circumstances radiation-induced cellular damage
results in the expression of DNA instability over many cell
divisions.
◗ That in some circumstances radiation damage to a cell can result
in effects expressing in an unirradiated neighbouring cell
(bystander effects).
◗ That minisatellite DNA sequences in human germ cells show
increased mutation rates in a high dose exposed population
9
related structure/function. Other advances included the elucidation
of the relevant biochemical pathways and, latterly, the genetic
manipulation of mice to carry the mutant genes. Progress in this
whole area was most notable and the EU-supported work on DNA
repair is acknowledged world-wide. The characterisation of the
repair pathways for DNA double strand breaks repair and associated radiation responses has made a significant contribution to
our understanding of the degree of error-prone DNA repair expected
after radiation. In turn, this provides important input to the low dose
cancer risk debate – stated simply, the error-prone repair of
sometimes complex DNA double strand breaks may be used
as one element in a scientific argument against the presence
of a low dose threshold for cancer risk.
The characterisation of DNA damage response genes in FP4 has also
provided information on candidate genes determining heritable sensitivity to radiation tumorigenesis. This theme appears in a number of cellular projects including studies on the potential association
between heritable cellular radiosensitivity and breast cancer risk.
FP5 research in this area represents a more focused approach to key
issues associated with initial DNA damage and the nature, control
and consequences of the relevant DNA damage response pathways. The ongoing studies underway include detailed consideration of complex DNA lesions; post-irradiation protein-protein and
protein-DNA interactions; DNA repair fidelity; and studies with
recombinant (gene knock-out) mice to explore the consequences
of DNA repair deficiency in whole animals. The induction of
persistent genomic instability in cells and the transfer of damage
signals from irradiated to unirradiated cells (bystander effects)
are also being investigated.
Studies on the mechanisms and genetics
of radiation tumorigenesis
The principal approaches used to investigate the mechanisms and
genetics of radiation tumorigenesis were via animal models and
radiation-associated human tumours. In some projects additional
support came from cellular and chromosomal studies.
Animal models of radiation tumorigenesis were used to explore doseresponse characteristics, tumour multiplicities, early cellular events
and genetic factors in post-irradiation tumour development. The
principal tumour types investigated in mice were leukaemia, lymphoma and cancer of bone, intestine and skin. Lung cancer after
radon was investigated in rats in the broader context of a study which
included the dosimetric modelling of risk. Much of the work on early
radiation associated events in tumours centred on establishing
consistent patterns of tumour-specific gene losses. The tumour
gene categories of particular potential interest are those associated
with the control of a) the cell reproductive cycle, b) DNA stability,
c) apoptosis and d) cell growth/development. This approach was
successful particularly for myeloid leukaemia and bone cancer and
for intestinal tumorigenesis in a mouse model of a cancer-prone
human genetic disorder. In these cases it was possible to lend
support to the proposition that induced gene losses were critical
early events in multi-stage tumour development after radiation.
10
Good evidence was also obtained on heritable genes that predispose animals to cancers of bone, skin, intestine and leukaemia/lymphoma. The picture that emerged from these FP4 studies was one
of genetic complexity with different tumour types being influenced
by multiple genes, some of which interact to determine tumour yield.
Also, that the tumour-predisposing activity of strongly expressing
genes can be modified significantly in different genetic backgrounds.
Human tumours associated with radiation exposure were obtained
as sample material of second cancers after radiotherapy and of
post-Chernobyl childhood thyroid cancers. Although a relatively
small number of therapy-related cancers were available, chromosomal analyses proved possible and provided initial evidence of the
expected chromosomal loss mechanism. Molecular studies on these
tumours were initiated with emphasis on second cancers arising in
cancer-prone retinoblastoma patients.
More attention was, however, given to cytogenetic and molecular
investigations of the post-Chernobyl thyroid cancers where characteristic forms of chromosomal and RET gene-specific rearrangements were revealed. The specificity of RET rearrangement was
suggested to be associated with age-related tumorigenic processes
rather than being a particular molecular signature of radiation
action. A number of projects included consideration of thyroid
tumorigenesis in the context of cellular, histopathological and epidemiological investigation.
Overall, FP4 work on the mechanism and genetics of radiation
tumorigenesis benefited greatly from recent advances in knowledge
of mouse and human genomes. The animal studies provided valuable proof of principle evidence on tumour specific gene losses in
radiation tumorigenesis and genetic modification of radiation risk.
The human studies particularly those of thyroid tumorigenesis
showed for the first time that direct molecular investigations can
reveal much information that may be linked with parallel findings
from studies on tumour pathology and epidemiology. In this way
molecular studies, when coupled with findings from tumour
pathology, can contribute to the clinical management of radiation-associated thyroid cancer. As information on the molecular
pathology of other radiation-associated cancer types accumulate it
may be that this additional benefit from fundamental research will
become more general.
FP5 research in this area continues to be focused on the mechanisms
and genetics of the complex post-irradiation processes that contribute specifically to cancer risk. For example, selected cellular and
animal models are being utilised to explore radiation-associated
gene/chromosomal mutations that characterise different tumour
types; also the potential roles of telomerase and chromosomal
telomeres. Mechanistic study of post-Chernobyl thyroid cancer and
therapy-related cancer is also being extended; importantly, thyroid
cancer studies include the establishment of a tissue bank and data
base for collaborative follow-up. Finally, a number of projects are
seeking further information on the genetic factors that influence
radiation cancer risk.
PART 1
Studies on the modelling of radiation cancer risk
FP4 research sought to promote a closer coupling of mechanistic
studies and those centred upon the interpretation of human epidemiological data relating to radiation cancer risk. Work was initiated on the development of mathematical models of low dose cancer
risk which incorporated a current understanding of the biological
processes that underlie post-irradiation cancer development.
elegant computer-generated representations of the process at the
molecular level.
The epidemiological input to this whole area is included in the
brochure “Epidemiology and retrospective dosimetry” (EUR 19958).
Diagnosis and treatment of cancer and radiation injury
The models that were considered included mathematical approximations to describe cell mutation rates, the early post-irradiation
growth of potentially malignant cells and the time dependence of
their multistage development towards cancer; particular attention
was given to two stage models and various points of radiation
action were investigated. These cancer risk models were tested for
their validity against epidemiological data on cancer risk e.g. A-bomb
survivors, radon exposed uranium miners and in some cases animal
data. This work was conducted in parallel with the biophysical modelling of DNA damage repair and the gene/chromosomal mutation
induction noted earlier.
In FP5, cancer risk modelling is being refined and increased attention given to age- and time-related features of the expression of
risk together with dosimetric and other uncertainties. The modelling of cancer-related chromosomal damage after radiation includes
Note 6 – Some features of biologically-based
mathematical models of radiation cancer risk
With reference to the scheme of Note 3,
these models generally consider:
◗ the different phases of multi-stage cancer development, e.g.
a simple two stage model might include a first stage (A→B)
including DNA damage to normal cells and the appearance
of mutant pre-cancerous cells plus a second stage (C→D/E)
involving a further mutation that drives a cell towards the
fully malignant state
◗ biologically realistic mathematical values to describe
the number of cells involved and rates of mutation, cell
death/development and growth selection
◗ testing and validation of the models and their chosen
mathematical values – how well does a given model fit available
information on cancer risk in irradiated humans and experimental animals; what are the implications for low dose risk;
how large are the uncertainties?
Work on the development and validation of these models also
serves to identify aspects of cancer development and radiation
risk where further information is critical. Accordingly, there is a need
for ongoing dialogue, deep integration and cross-disciplinary
research between modellers, epidemiologists and those undertaking experimental studies.
The follow-up of post Chernobyl thyroid cancer was an important
component of FP4. Work in this area centred on the development
of improved protocols for diagnosis and treatment of these cancers;
also, consideration of preventative measures based on iodine supplementation in regions of iodine deficiency. Developments in FP4
included improved diagnostic imaging techniques, the utility of
post-surgery suppressive therapy, improved dosimetry for radioiodine therapy of lung metastases and the provision of a map of
iodine deficiency in contaminated regions. An important conclusion
was that radiation induced thyroid cancer in children is in general
a well treatable disease.
One FP4 project considered new approaches to the treatment of
radiation accident patients. Developments in this area included the
provision of new data on indicators and mechanisms of damage to
critical tissues and improved preventative measure for damage to
the blood forming system. In FP5 modern techniques in cell/molecular biology are being used to investigate the role of stem cell damage,
growth factors and inflammatory responses in post-irradiation
tissue injury; also the predictive power of gene expression assays.
Studies on non-cancer effects
Although cancer risk at low doses dominates considerations in radiation protection, FP4 also gave attention to aspects of germ line
damage and to pre-natal (in utero) effects on the developing brain.
Radiation effects on in utero brain development are evident from
the follow-up of the A-bomb survivors in Japan and a relatively large
study was devoted to gaining a cellular/molecular understanding
of the processes involved; animal behaviour was also considered.
In brief, in FP4, in utero effects of radiation on the developing brain
were revealed at relatively low doses but the general pattern of such
effects were consistent with a low dose threshold of similar magnitude to that currently judged from the A-bomb data on humans.
A project sought and found evidence that hypermutable minisatellite sequences in the human germ line can act as biomarkers
of radiation damage. This study which investigated generations of
families from regions of Semipalatinsk (FSU) contaminated with
fallout from nuclear tests provides the most convincing molecular
evidence to date of such radiation-associated germ cell mutation
in humans. Selected studies on the hypermutability of certain
genomic sequences have been included in FP5 research.
11
Note 7 – Examples of findings from FP4
and FP5 research
◗ DNA double strand breaks which have a complex chemical
structure (clustered damage) probably represent the biologically critical damage to cells; there is a growing understanding of the relationships between this damage and the
physical properties of radiation tracks.
◗ The major pathways and controlling genes involved in
the response of cells to radiation induced DNA damage are
now much better understood; an intimate relationship
between DNA repair capacity, the appearance of mutations
and cancer development has been established.
◗ New information on the transfer of damage signals between
cells and the induction of persistent DNA or chromosomal
instability has been obtained; in most instances links between
these processes and the development of health effects have
yet to be established.
◗ Radiation-induced cancer in humans and mice appears to
proceed in an expected multi-stage fashion from a single
damaged cell; some specific chromosomal DNA losses have
been associated with certain radiation-induced cancers
in mice.
◗ Genetic make-up can influence individual sensitivity to
radiation-induced cancer but in most instances these genetic
factors operate in a complex fashion.
◗ Good progress has been made in the development of
computer-based biological models to describe low dose
cancer risk; these models are being tested further using
information on cancer risk in survivors of the atomic
bomb explosions in Japan and on lung cancer risk in uranium
miners.
◗ Overall, although uncertainties remain, mechanistic and modelling studies tend to support the view that there is unlikely to
be a low dose-threshold for radiation cancer risk; conversely,
other studies have provided evidence of a low dose threshold for effects on the developing brain.
◗ Understanding of the induction, diagnosis and treatment
of radiation-associated childhood thyroid cancer after
the Chernobyl accident has improved substantially; similar
improvement applies to the treatment of radiation accident
victims.
◗ A DNA marker has been used successfully to show an increased
mutation rate in the reproductive cells of humans exposed to
relatively high doses of radiation at a nuclear test site in the
former Soviet Union.
12
3. Priorities for the 6th Framework Programme
(FP6: 2002-2006)
Recent review of current knowledge and remaining uncertainty by
the Commission has served to identify research priorities in radiation protection for FP6. The main thrust of research in this area is
to resolve uncertainties in the risk from exposures to radiation at
low and protracted doses typical of those encountered in the environment and in workplaces. This remains a controversial science and
policy issue with important health and economic implications for
the use of radiation and radioactive materials in both medicine
and industry.
Quantification of these risks will be achieved through epidemiological
studies of exposed populations complemented by fundamental
research on the interaction between radiation and DNA, cells and
organs in the body.
One novel aspect of the research in FP6 is the integrated multidisciplinary approach that should gather a critical mass of scientists
to address ambitious research goals. Therefore, the studies in FP6
should be carried out within fewer projects (but with a broader perspective and possibly including more partners) involving, inter alia,
epidemiology, radiobiology, medicine and dosimetry.
Cellular and molecular biology research in FP6 may be viewed as a
more focused development of the advances made in FP4 and FP5,
particularly in key areas where coherent scientific themes on low
dose response have emerged.
All potentially important mechanisms should be addressed, from
initial damage to health effects manifested in the organism. In particular, the following aspects could be included: the biological consequences of damage to DNA and other cellular macro-molecules
and structures; interference of the various DNA repair pathways with
other cellular processes (e.g. transcription, replication, apoptosis,
chromatin remodelling); the role of intra/extra-cellular communication in tissue functions; unravelling the mechanisms of and susceptibility to radiation health effects; elucidation of the relevance
of radiation-induced cellular endpoints (e.g. genomic instability,
bystander effects, chromosomal aberrations and mutations) in the
cancer process; further development of mechanistic models describing the multi-stage process of carcinogenesis.
The research should, in the first instance, be addressed through one
or a few integrated multi-disciplinary projects involving, inter-alia,
radiobiology, genetics, molecular biology, biophysics and oncology.
The variability of radiation response within a human population and
the identification of the molecular pathways contributing to susceptibility to radiation health effects will be important outcomes
of this research.
Although it should not be expected that FP6 research would resolve
all remaining uncertainties, the Commission regards FP6 as a most
important phase of transition towards a much more complete
understanding of the potential risk to health following exposure to
low doses of ionising radiation.
13
bjectives
Estimates of the risks of exposure to
ionising radiations have to cover an
extremely wide range of radiation
types and exposure conditions.
Current estimates of risk have mainly
Experimental data for the induction
of cancer by radiation of different qualities
(EDICAR)
been derived empirically and their
refinement depends in large part on
improved mechanistic modelling
based on a more complete understanding of radiation action at the
molecular, cellular and tissue levels.
The overall aim of this project was to
provide experimental data that will
input into the development of mechanistic models of radiation carcinogenesis. The development of more
realistic and accurate models depends
on an improved knowledge of the chain
of events that leads from the earliest
physical interactions through to the
development of cancer. Such models,
based on a mechanistic understanding
of radiation effects at the cellular level,
will aid the extrapolation of human
risk data obtained following A-bomb
and other high-dose exposures to the
low levels generally occurring in the
environment and the workplace.
Challenges to be met
The research was organised into six work
packages which covered the interactions
of radiation with DNA, double-strand
break (dsb) induction and processing,
the induction of chromosome aberrations
and mutations and the effects of individual radiation tracks on cells. DNA dsb are
believed to be particularly important initial lesions in the genotoxic actions of
ionising radiations. Effects of radiation
quality (linear energy transfer (LET)) are
an important consideration in estimating risk and were an underlying theme of
the work carried out during this project.
One of the major challenges addressed
during the contract was a critical appraisal
of the methodology used to determine
the induction and processing of DNA
dsb. Another was the development of
microbeam techniques to enable investigations to be carried out under conditions that mimic low-dose exposures,
corresponding to the passage of a single
radiation track through the cell.
Achievements
The project provided data that has contributed to the development of an
improved understanding of the processes
related to radiation quality that are
involved at the molecular and cellular
levels in cancer induction by ionising
radiations. New data were obtained on
the physico-chemical processes involved
in the induction of DNA damage, especially dsb, on the yields and distributions
of dsb in cells exposed to radiations of
various types and how cells process
these damages.
The combined expertise of the partners
was able to produce a critical study of
the experimental methods used to measure the induction and cellular processing of DNA dsb following exposure to
a range of different types of radiation
and this work was published by the consortium as a review. New data were also
14
obtained on the formation of chromosomal aberrations following exposure to
radiations of different qualities and the
relation to dsb repair, as well as on the
yields and patterns of mutations induced.
Methods were developed to assess the
fidelity with which cells repair dsb. A
new aspect of mutation induction “noncontiguous deletion” was found and is
specific to radiation quality, offering the
prospect of a “signature” marking highLET exposure. New microbeam and measured-track techniques were developed
and initial data were obtained about
how cells respond to the passage of a single radiation track, which is key to understanding responses at low doses.
Overall, the data provided by the project have contributed to the development of mechanistic models for the
induction of cancer by radiation. Such
models are needed to reduce the uncertainties in extrapolating known risks
from acute high-dose exposures (mainly
from atomic bomb survivor follow-up)
down to the low doses and dose rates
that generally apply to occupational,
medical and environmental exposures.
The achievements of the project are
described in more detail in journal publications. The data obtained and the
competence developed among partners
in the various methodologies led to a further project, “RADNA”, being funded
under FP5. In this project, the new understandings gained about DNA damage
induction and processing and the induction of chromosomal aberrations and
mutations are being pursued further to
gain an improved understanding of the
mechanistic basis of radiation risk. Also,
the microbeam approaches developed
by several partners under FP4 are being
further exploited to study low-dose
mechanisms, including the involvement
of more newly recognised responses
(bystander effect, genomic instability
and adaptive response) that appear to
have significant roles in the low-dose
PART 2 : Cellular studies
EDICAR Information Column
Title:
Experimental data for the induction
of cancer by radiation of different
qualities (EDICAR)
and the development of microbeam
techniques at several centres. The development of microbeams required technology transfer between partners. These
exchanges have substantially enhanced
European competence to apply these
new techniques to research into the
effects of low-dose exposure.
Selected references
Yields of dicentric chromosomal aberrations
per cell in human lymphocytes after exposure
to different types of radiation during the G0
phase of the cell cycle. The aberrations were
visualised using the technique of premature
chromosome condensation (PCC) in which the
human cells were fused with hamster CHO K1
inducer cells. The yields of aberrations, as
revealed by PCC, are shown as functions of
the delay time tD in hours between the end of
irradiation and the start of fusion with the
inducer cells. : 150 kVp X-rays (4 Gy); :
3.45 MeV alpha-particles (2 Gy); : carbon-K
(CK) characteristic X-rays (1 Gy).
region. A new project on genomic instability funded under FP5, “RADINSTAB”,
benefits from the application of the
microbeams developed under the FP4
“EDICAR” and FP5 “RADNA” contracts.
Belli, M., Cherubini, R., Dalla Vecchia, M., Dini, V.,
Moschini, G., Signoretti, C., Simone, G.,
Tabocchini, M.A., and Tiveron, P., 2000,
DNA DSB induction and rejoining in V79 cells
irradiated with light ions: a constant field gel
electrophoresis study. International Journal of
Radiation Biology, 76, 1095-1104.
Fomina, J., Darroudi, F., Boei, J.J. and
Natarajan, A.T., 2000, Discrimination between
complete and incomplete chromosome
exchanges in X-irradiated human lymphocytes
using FISH with pan-centromeric and
chromosome specific DNA probes in
combination with telomeric PNA probe.
International Journal of Radiation Biology,
76, 807-813.
Löbrich, M., Kühne, M., Wetzel, J. and Rothkamm,
K., 2000, Joining of correct and incorrect DNA
double-strand break ends in normal human
and ataxia telangiectasia fibroblasts.
Genes, Chromosomes and Cancer, 27, 59-68.
Partnership
Under this contract, it was possible to
bring together much of Europe’s leading expertise in the field of cellular radiation biology and thereby establish a
research cooperation spanning most of
the cellular processes leading to the
induction of cancer by ionising radiations. The contract also exploited the
useful range of different radiation sources
available at the various centres. Progress
in a number of areas was made possible
by collaborations that were supported by
the project. Examples of these included
studies of chromosomal aberrations
induced by different qualities of radiation
Prise K.M., Ahnström, G., Belli, M., Carlsson, J.,
Frankenberg, D., Kiefer, J., Löbrich, M., Michael,
B. D., Nygren, J., Simone, G., and Stenerlöw, B.,
1998, A review of dsb induction data for radiations
of varying quality.
International Journal of Radiation Biology,
74, 173-184.
Stenerlöw, B., Höglund, E., Carlsson, J. and
Blomquist, E., 2000, Rejoining of DNA fragments
produced by radiations of different linear
energy transfer.
International Journal of Radiation Biology,
76, 549-557.
Co-ordinator:
Barry D. Michael
Gray Laboratory Cancer
Research Trust
Mount Vernon Hospital
PO Box 100
Northwood, Middlesex HA6 2JR
United Kingdom
Tel: +44.19238.28611
Fax: +44.19238.35210
e-mail: [email protected]
Partners:
- E.G. Sideris (NCSR-National Centre
for Scientific Research, Demokritos,
Greece)
- D. Frankenberg (Georg-AugustUniversität, Germany)
- E. Heimgartner (PSI-Institute for
Medical Radiobiology, Paul Scherrer
Institute, Switzerland)
- R. Cherubini (INFN-Istituto
Nazionale di Fisica Nazionale,
Laboratori Nazionali di Legnaro,
Italy)
- M. Belli (Istituto Superiore
di Sanità, Italy)
- F. Darroudi (Leiden University,
The Netherlands)
- J. Kiefer (Strahlenzentrum der
Justus-Liebig-Universität, Germany)
- J. Carlsson (University of Uppsala
and University of Stockholm,
Sweden)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
15
bjectives
The main objective of the RADNA
project is to make experimental
measurements of the actions of
low doses of radiation on cellular
systems. It is based on the concept
Induction, repair and biological
consequences of DNA damages caused
by radiations of various qualities (RADNA)
that the low dose risks of cancer
induction and genetic injury are
the result of damage that takes
Challenges to be met
place at the cellular level. There is
emphasis on the role of DNA damage and repair in the production of
chromosomal aberrations and
mutations. The aim is to improve
the quantification of radiation risk
at low doses. The experimental
work employs radiation types that
cover an important range of LETs in
respect of radiation quality factors.
Studies using counted-particle irradiations are designed to provide
new information about the form of
dose-effect relationships, down to
the ultimate low-dose of a single
track per cell. These measurements
relate to the development of mechanistic models that will improve
The project focuses on the role of radiation-induced DNA damage, particularly double-strand breaks (dsb), in the
production of chromosome aberrations
and mutations as critical events in the
induction of cancer. Underlying these
studies are investigations into the role
of DNA repair processes, particularly
those involving dsb, in subsequent biological effects. The project utilizes conventional irradiation methods, and
innovative microbeam technologies
coupled with single-cell assays of
response. The objective is to map out
responses at the level of individual cells.
The overall aim of the project is to provide data that will contribute to mechanistic modelling of radiation risk,
particularly in respect of radiation quality factors at low doses.
the low dose extrapolation of highdose epidemiological risk data,
Achievements
which come predominantly from
the follow-up of A-bomb survivors.
Following initial work carried out under
FP4, charged-particle microbeam facilities have been developed at several
of the participating centres; a unique
focused soft x-ray microbeam has also
been developed. Using these and other
facilities, measurements have been
made of low-dose direct and indirect
(i.e., “bystander”) responses in Chinese
hamster cells. These studies included
the induced radioresistance for a range
of radiation qualities extending from
focused soft x-rays to alpha-particles.
An assay developed under FP4 has been
used to quantify correct rejoining of
DNA double-strand breaks and compare
this with total (correct and incorrect)
rejoining. Pulsed-field gel electrophoresis (PFGE) assay for DNA dsb has been
used to measure both dsb induction
patterns and rejoining kinetics as functions of radiation quality, including studies to determine the influences of the
level of packaging of DNA and proteins
(chromatin organisation).
16
The cellular processing of DNA damage that is involved in the production
of chromosomal aberrations has been
studied using premature chromosome
condensation (PCC) and fluorescence in
situ hybridisation (FISH) techniques. The
fast and slow temporal components of
the formation of chromosomal aberrations have been studied successfully.
Radiation-sensitive cell mutants and
DNA repair inhibitors have been used to
correlate defined DNA damage processing pathways with the formation of
chromosomal aberrations. Effects of the
cell cycle status upon the formation of
chromosomal aberrations by different
radiations have been studied using
wild-type cells. Treatments that modify
chromatin structure have also been
considered.
The formation kinetics and persistence
of complex exchange aberrations have
been studied, visualising all human
chromosomes using FISH. The proportion of complex aberrations increased
with LET and might be a signature for
high-LET exposure. FISH has also been
employed to estimate low- and high-LET
induced complete and incomplete chromosome exchanges.
Analysis of mutations at the molecular
level has been made using the polymerase
chain reaction (PCR). Measurements have
been made of the cross sections for the
induction of mutations in the HPRT gene
in Chinese hamster cells for a range of
radiation qualities. Also the spectra of
damage have been observed in terms of
mutational type. A comparison of the
cross sections for mutation dsb and cell
killing as functions of radiation quality
showed differences consistent with the
functional importance of cellular processing of dsb. Measurements were also
made of preferential deletion within the
HPRT gene.
PART 2
RADNA Information Column
Title:
Induction, repair and biological
consequences of DNA damages
caused by radiations of various
qualities (RADNA)
Co-ordinator:
Barry D. Michael
Gray Laboratory Cancer Research Trust
Mount Vernon Hospital
PO Box 100
Northwood, Middlesex HA6 2JR
United Kingdom
Tel: +44.19238.28611
Fax: +44.19238.35210
e-mail: [email protected]
Microbeam apparatus used to target cells, or subcellular regions, individually with charged particles.
Each cell is imaged and located automatically and is exposed to a programmed number of traversals
by protons or by ions of helium-3 or helium-4 (α-particles). The ions are counted and “switched” so
that strictly 0, 1, 2, 3 etc. can be delivered, cell by cell. This approach enables the effects of extreme
low-dose exposures to be studied, also the effects of damage transfer between hit and non-hit cells
(“bystander effect”) can be investigated precisely.
Partnership
This consortium brings together much
of EU expertise in the field of cellular
radiation biology and exploits the useful
ranges of techniques and the different
radiation sources available at the various
centres. Examples of the collaboration
include the application of specialised
techniques such as FISH and PCC and the
development of microbeam techniques.
Selected references
Belli M, Dini V, Esposito G, Micera P, Sapora O,
Signoretti C, Simone G, Stenerlow B,
Tabocchini MA. (2001) DNA fragmentation
induced in K562 cells by nitrogen ions.
Phys Med 17 219-21
Folkard M, Schettino G, Vojnovic B, Gilchrist S,
Michette AG, Pfauntsch SJ, Prise KM,
Michael BD, (2001)
A focused ultrasoft x-ray microbeam for
targeting cells individually with submicrometer
accuracy. Radiat Res. 156 796-804
indication for a strong dependence on photon
energy of the RBE(M) for various end points.
Radiat Res 157 99-105
Kiefer J, Schmidt P, Koch S. (2001)
Mutations in mammalian cells induced by
heavy charged particles: an indicator for risk
assessment in space. Radiat Res 156 607-11
Stenerlow B, Hoglund E. (2002)
Rejoining of double-stranded DNA-fragments
studied in different size-intervals.
Int J Radiat Biol 78 1-7
Partners:
- Dieter Frankenberg (Georg-AugustUniversität, Germany)
- Roberto Cherubini (FNLN-Istituto
Nazionale di Fisica Nazionale,
Laboratori Nazionali di Legnaro,
Italy)
- Mauro Belli (ISS-Istituto Superiore
di Sanità, Italy)
- Firouz Darroudi (Leiden University,
The Netherlands)
- Eleftherios Sideris (NCSR-National
Centre for Scientific Research,
Demokritos, Greece)
- Jürgen Kiefer (Strahlenzentrum der
Justus-Liebig-Universität, Germany)
- Kai Rothkamm (Universität des
Saarlandes, Germany)
- Bo Stenerlöw (University of Uppsala,
Sweden)
- Gunnar Ahnström (University of
Stockholm, Sweden)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
Frankenberg D, Kelnhofer K, Bar K,
Frankenberg-Schwager M. (2002)
Enhanced neoplastic transformation by mammography X rays relative to 200 kVp X rays:
17
bjectives
Chromosome aberrations are
known to be associated with
human cancer as well as in spontaneous abortions and hereditary
defects in newborn children.
Chromatin structure and DNA repair in
relation to ionizing radiation-induced
chromosome aberration in mammalian cells
Ionizing radiation is very efficient
in inducing chromosomal aberrations and the main objectives of
this project were to investigate
(a) the initial induction of chromosomal damage following irradiation,
(b) heterogeneity of damage
within the genome, (c) repair at
the chromosomal level in relation
to time and status of chromatin
condensation, (d) the influence
of enzymes involved in DNA repair
in the expression of DNA damage
as chromosomal aberrations,
(e) the role of cell cycle check
points in the expression of aberra-
Challenges to be met
Though studies on radiation induced
chromosome aberrations were initiated
almost a century ago, the mechanisms
underlying the formation of these
aberrations are not fully understood.
The results of investigations from the
cooperative efforts of various groups
with expertise in different areas should
lead to a better understanding of some
of the steps involved from the initial
damage to the manifestation of radiation induced chromosomal aberrations.
Achievements
tions, (f) the influence of apoptosis
(programmed cell death) on the
yield of radiation induced aberrations, and (g) to develop methods
to identify and quantify radiation
induced aberrations employing
molecular cytogenetic techniques.
Techniques to identify and quantify
chromosomal aberrations are primary
requirements for understanding the
mechanisms involved in this process. To
achieve this, DNA libraries for human,
mouse and Chinese hamster chromosomes were generated by flow sorting of
chromosomes and micro-dissection of
specific chromosomes, chromosome arms
and chromosome regions. Fluorescence
in situ hybridization (FISH) techniques
were used to paint different chromosomes and chromosome arms and regions.
Interphase human primary fibroblasts
hybridized with arm-specific probes for
chromosome 1.
18
This allowed accurate identification of
different classes of exchange aberrations, such as, exchanges between two
different chromosomes (reciprocal
translocations), exchanges between the
two arms of a chromosome (pericentric
inversions), exchanges within an arm
of a chromosome (paracenteric inversions). These important classes of aberrations, i.e., intrachanges, were found
to be formed more frequently than
exchanges between two different chromosomes. Heterogeneity in the involvement of aberrations was found between
the chromosomes as well as between
the arms or regions of a chromosome.
An interesting factor contributing to
this heterogeneity was the presence of
intercalary telomeric sequences in Chinese
hamster chromosomes. Interstitial translocations (insertions) contributed to about
10% of total translocations induced by
high LET radiation (1 MeV neutrons)
irrespective of the dose. Since these
aberrations are stable, their frequencies
can be used as a fingerprint for past
exposures to high LET radiation.
Detailed analysis of patterns of aberrations recovered, gave some clues about
Metaphase spread of an irradiated human
lymphocyte after FISH using a combination
of arm-specific probes for chromosome 1
(p-arm is red, q-arm green) and a pancentromeric probe (yellow). A complex
exchange involving both homologues of
chromosome 1 is visualized.
PART 2
Information Column
the origin of complex exchanges (involving several chromosomes and breaks).
These analyses may help to distinguish
between two theories of exchange formation, namely, free interaction between
contemporary open ends versus buildup from multiple simple cyclical interactions, (i.e., all being in reality sequential
exchange complexes). The dose response
curve for induction of simple reciprocal
translocations was found to be linear,
whereas for complex translocations it
was quadratic. This observation was
supported by data derived from dose
fractionation experiments, in which it
was found that simple translocations
were induced in additive manner,
whereas the complexes are formed in an
interactive manner.
Frequencies of radiation induced chromosome aberrations in G2 phase of the
cell cycle have been proposed to be a
sensitive assay for determining the
radiosensitivity and cancer proneness
in human. The G2 sensitivity is very variable and the yield of aberrations depends
on several factors including contaminating irradiated S phase cells. A technique to identify S phase cells was
deployed which allowed accurate scoring of cells irradiated in G2, thus reducing inter-individual variability commonly
observed in G2 radiosensitivity assay.
Investigations of radiosensitive Chinese
hamster ovary mutant cells (xrs) which
are deficient in repair of DNA double
strand breaks, showed that these cell
lines have altered histone-DNA binding
properties which in turn influence structural properties of the chromatin and
chromosome organization. Significant
differences in the activities of DNA topoisomerase I and topoisomerase II have
also been observed between the radiosensitive Chinese hamster mutant cells and
their wild type parental cells. These
results indicated that in addition to the
primary defect in DNA repair, other
characteristics might also contribute to
the increased radiosensitivity.
fraction of the cell population leading to
increased cell survival at the expense of
genomic instability. In lymphoblastoid
cell lines derived from radiosensitive
ataxia telangiectasia (AT) patients, apoptotic response was found only after high
LET radiation and not after gamma irradiation. This indicated that apoptosis is
a complex process, since in normal
human cells, apoptosis was induced by
both high and low LET radiations.
Partnership
This project was a multinational one
involving partners who have competence
in different areas but with a common
goal. This collaboration was mutually
beneficial for the success of the project.
Selected references
Effectiveness of 0.28 keV carbon K ultrasoft
X-rays at producing simple and complex
chromosome exchanges in human fibroblasts
in vitro detected using FISH.
Griffin CS, Hill M, Papworth DG, Townsend KMS,
Savage JRK. Int. J. Radiat. Biol., 73, 591-598
(1998).
Chromosomal instability in human lymphocytes
after low dose rate gamma irradiation and
delayed mitogen simulation.
Holmberg K, Meijer A, Harms-Ringdahl M,
Lambert B. Int. J. Radiat. Biol., 73, 21-34 (1998).
Mechanisms of induction of chromosomal
aberrations and their detection by fluorescence
in situ hybridization.
Natarajan AT, Balajee AS, Boei JJ, Darroudi F,
Dominguez I, Hande MP, Meijers M, Slijepcevic
P, Vermeulen S, Xiao Y.
Mutation Res., 372, 247-258 (1996).
Title:
Chromatin structure and DNA repair
in relation to ionizing radiation
induced chromosomal aberrations
in mammalian cells
Co-ordinator:
A.T. Natarajan
LUMC-Leiden University Medical Center
Dept. Radiation Genetics and
Chemical Mutagenesis
Wassenaarseweg 72
P.O.Box 9503
NL-2300 RA LEIDEN, The Netherlands
Tel: +31.71.527.61.64
Fax: +31.71.522.16.15
e-mail: [email protected]
Partners:
- J.R.K. Savage (MRC Radiation
and Genome Instability Unit,
United Kingdom)
- P.E. Bryant (St. Andrews University,
United Kingdom)
- F. Cortes (University of Seville, Spain)
- M. Harms-Ringdahl (Stockholm
University, Sweden)
- F. Palitti (University of Tuscia, Italy)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Chromosome radiosensitivity in human G2
lymphocytes and cells progression.
Palitti F, Pichierri P, Franchitto A,
Proeitti de Santis L, Mosesso P.
Int. J. Radiat. Biol., 75, 621-627 (1999).
Human peripheral blood lymphocytes in
G0 phase of cell cycle were found to
undergo apoptosis at doses of 1 Gy and
below. Induced proliferation led to
reproductive cell death only for a
19
bjectives
Chromosomal aberrations have a
major role in the development of
neoplasia and hereditary defects in
human. European epidemiological
studies have shown that the chromo-
Factors modifying the yield
of radiation-induced chromosome
aberrations
somal aberrations in human lymphocytes to be a good bio-indicator
for future cancer risk reflecting both
Challenges to be met
exposure to genotoxic agents and
individual sensitivity. Though
radiation induced DNA double
strand breaks (DSBs) are important
lesions leading to chromosomal
aberrations, many factors influence
the ultimate yield of aberrations.
This project was aimed at improving
our knowledge on (a) intra-genomic
heterogeneity of radiation induced
chromosomal aberrations, (b) the
initial frequencies of chromosomal
aberrations (as detected by premature chromosome condensation
technique), (c) differential repair at
the chromosomal level, (d) detection
and quantification of aberrations at
different stages of the cell cycle
following irradiation, (e) the influence of chromatin packing, namely,
heterochromatic versus euchromatic
regions, on the yield of aberrations,
(f) the role of enzymes involved in
the detoxification of free radical
damage (such as glutathione S
transferase) as modifiers of radiation
response of human cells and
(g) differential sensitivity to low
and high LET radiations.
Although DSB are primary lesions
responsible for induced aberrations, the
number of DSB induced are far more
than the number of observed aberrations, indicating the existence of several intervening processes (including
DNA repair) which operate. Utilizing
the technique of fluorescence in situ
hybridization (FISH) and premature
chromosome condensation (PCC) this
project attempted to assess low and
high LET induced chromosomal damage,
its repair and the kinetics of formation
of aberrations at different stages of
cell cycle.
Achievements
Ionizing radiation induced aberrations are supposed to occur at random
among the chromosomes and this
assumption has been used to estimate
genomic frequencies of aberrations from
the frequencies observed in two or three
FISH painted chromosomes. By PCC
technique, it is possible to visualize the
breaks and exchanges induced immediately and after different times following irradiation of human lymphocytes.
By combining PCC with FISH it was possible to study the process of exchange
aberration formation with time. Some
of the human chromosomes such as #1,
#19 are rich in actively transcribing
genes in comparison to chromosomes
#4 and #18. The initial number of breaks
induced in chromosomes #1 and #19
was more than those
induced in chromo-
somes #4 and #18, indicating that the
open structure due to transcriptional
activity leads to more initial damage.
However, these transcriptionaly active
chromosomes were also repaired very
efficiently leading to no significant differences at the metaphases.
After X-ray or neutron irradiation, the
initial frequencies of breaks, dicentrics
and translocations increased linearly
with the dose as assessed by PCC techniques. The RBE (Relative Biological
Effectiveness) for 1 MeV neutrons was
found to be in the range of 1.5-2. The
initial frequencies of translocations were
higher than dicentrics (about 2 to 3
times) both for X-rays and neutrons.
The dose response curves for induction
of exchange aberrations, generated at
different times of recovery following
irradiation, using the PCC technique
were linear-quadratic for X-rays and
linear for neutrons. For X-rays, the linear
component was formed in the early
phase (within an hour) whereas the
quadratic component developed slowly
during subsequent recovery time.
Following neutron irradiation of lymphocytes, induced breaks rejoined slowly
in comparison with X-rays. Early formed
chromosome exchanges were incomplete, namely dicentric chromosomes
were not accompanied by bicolour fragments and translocations were of one
way (incomplete) type. However, with
increasing recovery time incomplete
forms were replaced by complete forms.
There were marked differences in the
extent of induction of breaks and kinetics of their repair between low and high
LET radiations.
Metaphase spread of an irradiated human lymphocyte containing a false incomplete one-way exchange between the painted chromosome 8 (green) and an
unpainted chromosome. Both ends of the truncated painted chromosome 8 have
telomeric signals, as well as the translocated painted terminal segment (arrows).
The black and white images represent the DAPI counterstain (upper panel) and
the telomeric signals (lower panel). * Interstitial fragment (without telomeres).
20
PART 2
Information Column
In order to gain a better estimate of the
initial frequencies of induced breaks
following irradiation of human lymphocytes, an inhibitor of repair of DNA
DSB, namely ara A, was employed before
and during fusion with mitotic cells. In
the presence of ara A, the frequencies
of breaks induced by X-rays increased by
about 2 fold, whereas for neutron irradiation, ara A had no influence on the
yield of breaks, indicating the quality and
repair of breaks induced by low and
high LET radiation is different. PCC
studies on human lymphocytes X-irradiated in G2 (post DNA synthesis) showed
that chromatid exchanges formed rather
rapidly and did not increase in frequencies, whereas the frequencies of breaks
declined with time.
Whole chromosome painting was combined with FISH using pan-centromeric
and telomeric probes in order to accurately estimate the true frequencies of
incomplete (one way) and complete (two
way and complexes) exchanges. When
telomeric probing was not carried out,
the frequencies of incomplete exchanges
were 21% following 4 Gy of X-rays,
whereas with telomeric probing this
frequency was reduced to about 5%.
A similar trend was also found after irradiation with neutrons. Since, the resolution of the employed FISH technique is
limited, it may be concluded that in all
probability, all exchanges are complete.
Attempts were made to study the individual susceptibility among humans for
response to ionizing radiation. Apart
from defects in the repair of DNA damage
(such as ataxia telangiectasia patients)
which bestow increased radiosensitivity,
other factors are poorly defined. The
enzymes involved in the detoxification
of damage induced by free radicals may
play a role in determining individual
radiosensitivity. Mutations in the glutathione S transferase (GST) gene family is common in the general population.
57 individuals who have been earlier
genotyped for their status in this gene
family, namely GSTM1 and GSTT1
defective phenotypes were screened for
their response to radiation. No differences were observed between these two
groups in the background or induced
response. A Poisson model was applied
to test the contribution of the GSTM1
genotype, smoking status and age in the
yield of radiation induced chromosomal aberrations. No trend was observed
with respect to any of these parameters.
It could be concluded that GSTM1 gene
status does not affect the radiosensitivity of individuals as assessed by the
induction of chromosomal aberrations.
Title:
Factors modifying the yield
of radiation induced chromosomal
aberrations
Co-ordinator:
A.T. Natarajan
LUMC-Leiden University Medical Center
Dept. Radiation Genetics and
Chemical Mutagenesis
Wassenaarseweg 72
P.O. Box 9503
NL-2300 RA LEIDEN,
The Netherlands
Tel: +31.71.527.61.64
Fax: +31.71.522.16.15
e-mail: [email protected]
Partners:
S. Salomaa (STUK, Radiation and
Nuclear Safety Authority, Research
and Environment, Finland)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Partnership
Status: Completed
This project was bi-national and closely
linked to the project “Chromatin structure and DNA repair in relation to ionizing radiation-induced chromosome
aberration in mammalian cells”. The
collaboration was mutually beneficial
to both partners.
Selected references
Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes,
using PCC and FISH.
Darroudi F, Fomina J, Meijers M, Natarajan AT.
Mutation Res., 404: 55-65 (1998).
Premature Chromosome Condensation (PCC)
of an irradiated human lymphocyte after fusion
with a mitotic Chinese hamster cell. The p-arm
of one of the homologues of chromosome 4
(painted red) is broken. The centromeres of the
human chromosomes are painted green.
Low level of DNA repair in human
chromosome 1 heterochromatin.
Surralles J, Darroudi F, Natarajan AT.
Genes, Chromosomes & Cancer,
20, 173-184 (1997).
Kinetics of the formation of exchanges and
rejoining of breaks in human G0 and G2
lymphocytes after low-LET radiation.
Sipi P, Lindholm C, Salomaa S.
Int. J. Radiat. Biol., 76, 823-830 (2000).
Distribution of radiation-induced exchange
aberrations in human chromosomes 1, 2 and 4.
Luomohaara S, Lindholm C, Mustonen R,
Salomaa S. Int. J. Radiat. Biol.,
75, 1551-1556 (1999).
21
bjectives
DNA is the carrier for the genetic
information of almost all organisms
and hence the ‘blueprint of life ‘.
It is of vital importance to all living
organisms that the integrity of the
Mechanisms of formation of ionizing
radiation-induced chromosomal aberrations
(CHROMOSOME STRUCTURE)
DNA is well conserved and protected
against internal and external factors.
Injuries to the DNA may result from
Challenges to be met
exposure to a variety of environmental
factors including ionising radiation
(IR). IR induces various types of DNA
damage among which DNA double
strand breaks (DSB) are the most
harmful. IR induced damage can
cause genetic alterations (mutations)
that may ultimately lead to cancer
and hereditary diseases. Cellular
defence mechanisms including pathways directed towards repair of DNA
damage and cell cycle regulation,
have been evolved that can counteract the deleterious effects of IR.
The importance of these defence
mechanisms for humans is underscored by inherited disorders associated with defects in these pathways
such as ataxia telangiectasia and
Nijmegen breakage syndrome.
At the cellular level these disorders
are characterized by DNA instability
and cancer proneness.
IR is very efficient in inducing
chromosomal aberrations in cells
both in vitro and in vivo and DSB are
considered to be the most important
lesion for the induction of these
aberrations. In recent years, molecular cytogenetic techniques have
significantly increased the resolution
and accuracy of detection and
quantification of different types of
chromosomal aberrations. Along
with the availability of mutant cell
lines and knockout mouse models
sensitive to radiation, this has
offered new insights into the mechanisms of formation of chromosomal
aberrations. The broad objectives of
this project were to exploit these
developments in order to gain a better
understanding of radiation action.
22
The present proposal is a multi-disciplinary
approach aimed to unravel the mechanisms of radiation-induced chromosomal aberrations by studying the events
occurring from the initial DNA damage,
its repair or mis-repair and the biological factors influencing the ultimate yield
of chromosomal aberrations.
The major challenge in the project is to
connect the IR induced events in the
interphase nucleus (induction of DSB,
chromatin remodelling and repair) to
the formation of chromosomal aberrations as observed in cells in metaphase.
To reach this goal, different strategies
will be undertaken including the visualisation of radiation-induced DSB in
the cell nucleus, the assessment of
effects of radiation on interphase nuclear
architecture, the exploitation of mutant
cell lines known to be deficient in specific repair pathways or in genes controlling cell cycle check-points. These
studies are complemented by advanced
high resolution detection of chromosomal aberrations in the whole genome by
employing multi-colour FISH and image
analysis systems (COBRA, SKY, Q-FISH).
Achievements
The resolution of detecting different
classes of aberrations has improved by
using the technique of fluorescence in
situ hybridisation (FISH). Particularly, the
introduction of centromeric probes
along with whole chromosome painting has facilitated the discrimination
between translocations and dicentrics.
The introduction of peptide nucleic acid
(PNA) probes for telomere detection
has provided a valuable tool for detecting complete and incomplete exchange
aberrations. Employing these tools, it
appears that differences in chromosomal aberration frequencies among
chromosomes with different level of
heterochromatin or transcription are
probably small. Moreover, incomplete
aberrations (aberrations with open DNA
ends) are either very rare or do not exist
in first metaphase after IR suggesting
that there is tight control of genomic
integrity at this level.
Two factors appear to be crucial for the
formation of chromosomal aberrations:
spatial distribution of DSB and repair
efficiency. Using chromosome arm specific probes (Fig 1) it became apparent
that exchanges within a chromosome
occur with much higher frequency than
between chromosomes (Fig 2) indicating the crucial role of proximity (e.g. the
distance between DSB) in the formation
of chromosomal aberrations.
The relationship between the rate and
type of DSB repair and chromosomal
aberration formation has been studied
using either inhibitors of DSB repair or
employing cell lines deficient in the repair
of DSB. Most of the results indicated
that non-homologous end joining
(NHEJ) is the most important pathway,
though homologous recombinational
repair (HRR) at some stages of the cell
cycle can certainly not be ruled out. It
is also evident that the longer the DSB
remain unrepaired, the higher the frequencies of aberration formation suggesting that chromatin/chromosome
territories in the interphase nucleus are
dynamic and capable to interact.
Chromosome territory: localization of the two
arms of chromosome 1 in the interphase
nucleus.
PART 2
CHROMOSOME STRUCTURE Information Column
Title:
Mechanisms of formation of ionizing
radiation-induced chromosomal
aberrations: Impact of repair
pathways and nuclear architecture
(CHROMOSOME STRUCTURE)
IR induced exchanges occur
preferentielly within chromosomes. The expected value is
based on DNA content and
known frequencies for the total
genome.
Partnership
Selected references
The partners were drawn from laboratories in seven EU universities. Future
work aims at gaining much more insight
into the organisation of the interphase
nucleus by using newly developed multicolour FISH to analyse higher order chromatin structure, chromosome territories
using 4D microscopy and quantitative
image processing, before and after
irradiation of cells. Specific issues for the
future include the importance of complex DSB, DSB repair in real time, the
influence of telomere length on aberration formation and the exploitation
of techniques for genome-wide aberration analysis.
Boei JJ, Vermeulen S, Natarajan AT. (2000)
Analysis of radiation-induced chromosomal
aberrations using telomeric and centromeric
PNA probes.
Int. J. Radiat. Biol. 76, 163-7.
McIlrath J, Bouffler SD, Samper E, Cuthbert A,
Wojcik A, Szumiel I, Bryant PE, Riches AC,
Thompson A, Blasco MA, Newbold RF,
Slijepcevic P. (2001)
Telomere length abnormalities in mammalian
radiosensitive cells. Cancer Res. 61, 912-5.
Cremer M, van Hase J, Volm T, Brero A, Kreth G,
Walter J, Fischer C, Solovei I, Cremer C and
Cremer T (2001).
Non-random radial higher-order chromatin
arrangements in nuclei of diploid human cells.
Chrom.Res. 9, 541-567.
Ludwików G, Yun Xiao, Hoebe RA, Franken
NAP, Darroudi F, Stap J, van Oven CH,
van Noorden CJF and Aten JA (2002).
Induction of chromosome aberrations in
unirradiated chromatin after partial irradiation of a cell nucleus.
Int.J. Radiat.Biol. 78, 239-247.
Co-ordinator:
Leon H.F. Mullenders
Leiden University
Department of Radiation Genetics
and Chemical Mutagenesis
PO Box 9503
Wassenaarseweg 72
NL-2300 RA LEIDEN, The Netherlands
Tel: +31.71.5276126
Fax: +31.71.5221615
e-mail: [email protected]
Partners:
- Jacob A. Aten (University of
Amsterdam, The Netherlands)
- Christoph Cremer (Ruprecht-KarlsUniversität, Germany)
- Predrag Slijepcevic (Brunel
University, United Kingdom)
- Ricardo Marcos (Universitat
Autonoma de Barcelona, Spain)
- Fabrizio Palitti (Universita degli
Studi della Tuscia, Italy)
- Mats Harms-Ringdahl (Stockholm
University, Sweden)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
23
bjectives
Information on human risk from
exposure to ionising radiation (IR)
comes from epidemiological studies,
but is available largely for high doses
and high dose rates of low LET radia-
The molecular basis of DNA damage
response and radiosensitivity
(DNA DAMAGE RESPONSES)
tion. To make rational judgements in
radiation protection, it is necessary to
extrapolate to low doses and low dose
Challenges to be met
rates, and to have an appreciation of
variation in response to radiation
among the human population.
This can be done with confidence
only if we have a detailed knowledge
of the mechanisms by which radiation
induces cancer and genetic disorders.
IR induces various types of DNA dam-
The project has its focus on understanding the molecular basis of radiation
sensitivity in humans. In particular: (i) the
identification of radiation sensitive individuals, (ii) a mechanistic understanding of radiation sensitivity, and (iii) the
consequences of radiation-induced DNA
damage and repair for human health.
age among which DNA double strand
breaks (DSB) are the most harmful.
If not processed correctly, IR induced
damage can cause genetic alterations
(mutations) that may ultimately lead
to cancer and hereditary diseases.
Cellular defence mechanisms including pathways directed towards repair
of DNA damage and cell cycle regulation have been evolved that counteract the deleterious effects of IR. The
importance of these defence mechanisms for humans is underscored by
inherited disorders associated with
defects in these pathways such as
ataxia telangiectasia and Nijmegen
breakage syndrome (NBS). In this
project the major focus is on the elucidation of the molecular mechanisms
underlying the various DNA repair
pathways for IR induced DNA
damage. This is achieved by cloning
repair genes and by the characterization of the proteins encoded by these
genes. This acquired knowledge has
been used to construct mouse strains
with defined deficiencies in these
repair processes in order to study
their roles in alleviating the deleterious
effects of IR at the level of the whole
organism. Moreover, a considerable
effort has been made to identify
radiosensitive individuals within the
human population and to correlate
their radiosensitivities with possible
defects in repair pathways.
24
The various approaches include:
◗ The development of rapid methods to
identify defects in repair of DNA breaks
to be used for screening of individuals.
◗ Isolation and characterization of genes
influencing radiosensitivity including
those controlling DNA repair and the
cell cycle response to IR.
◗ Analysis of gene functions and gene
products from the molecular to the
whole organism level.
◗ Construction of transgenic mice carrying mutations corresponding to
those identified in human genetic
disorders showing IR sensitivity.
Achievements
Repair pathways: To counteract the deleterious effects of DSB, several repair pathways
have evolved. The non-homologous endjoining (NHEJ) pathway is an error prone
pathway leading to loss of genetic material during repair of DSB. In contrast, homologous recombination repair (HRR) is a error
free pathway for repairing DSB utilising an
undamaged sister chromatid or homologous chromosomes. The relative importance
of each pathway depends on the phase of
the cell cycle, on the cell type and on the
stage of development and possibly by the
complexity of the DSB. All these damage
response mechanisms are highly conserved
in evolution. A complex consisting of proteins encoded by the genes RAD50, MRE11
and NBS1 participates in NHEJ and HRR
probably as a damage sensor.
Non-homologous end joining (NHEJ). Cells
affected in this pathway are sensitive to
radiation and have reduced ability to
repair DSB. In addition they are impaired
in V(D)J recombination, demonstrating
a link between repair of IR-induced DSB
and breaks formed during the process
of immunoglobulin rearrangement in
lymphocytes. The recognition of DSB is
affected by the DNA-end binding proteins Ku80 and Ku70 together with the
large catalytic sub-unit DNA-PKCS. DNAligase IV and its partner protein XRCC4
carry out the joining step of the process.
Homologous recombination repair (HRR).
At present in yeast ten genes have been
identified to be involved in HRR and
mutations in these genes lead to sensitivity to IR and defects in mitotic and/or
meiotic recombination; human homologues of most of these genes have been
identified. To address directly the role
of HRR in mammals, mouse knockout
strains have been generated.
Cell cycle checkpoints. Apart from their
ability to repair radiation damage directly,
cells have evolved additional responses as
protection from radiation damage. Cell
cycle checkpoints are important protective mechanisms, which arrest cell cycle
progression to prevent cells containing
damaged DNA from entering critical
phases of the cell cycle, such as S phase
and mitosis. At least ten genes involved
in this process have been identified.
DNA damage response in man. The
importance of the DNA damage response
Repair proteins (rad51, green spots) accumulate at sites of X-ray induced DNA damage.
PART 2
DNA DAMAGE RESPONSES Information Column
4TH FRAMEWORK PROGRAMME
Title:
The molecular basis of radiosensitivity
Co-ordinator: Paul Lohman (retired)/
Leon H.F. Mullenders
Leiden University - Department of
Radiation Genetics and Chemical
Mutagenesis
PO Box 9503 - Wassenaarseweg 72
NL-2300 RA LEIDEN, The Netherlands
Tel: +31.71.5276126
Fax: +31.71.5221615
e-mail: [email protected]
Non-homologous end joining (NHEJ) and homologous recombination repair (HR) are the major
pathways for repair of DSB
pathways in man is shown by the
genetic disorder ataxia-telangiectasia
(A-T), in which radiation hypersensitivity is associated with defects in repair of
DSB and cell cycle checkpoint control
following radiation damage. NBS is a
related disorder with many similar features to A-T at both the clinical and cellular levels. The product of the NBS1
gene has been recently identified as a
component of a repair complex. To identify radiation sensitive individuals, cells
from cancer patients have been examined for radiation sensitivity and defects
in repair of DNA. The data suggest that
subgroups of patients are partially
defective in repair of IR induced DNA
damage. Marked overt radiosensitivity
unlinked to recognised defects therefore does exist in the human population.
Partnership
The partnership brought together research
groups with a broad range of expertise.
Future work will provide major advances
in the analysis of DNA repair pathways,
and their involvement in human radiosensitivity, by combining the study of
newly discovered human repair genes
with the use of novel analytical techniques, model organisms, and in vitro
biochemistry. Assessment of the dynamics of repair and the coordination between the different repair pathways is a
focus of future studies.
Selected references
Griffin, C.S., P.J. Simpson, C.R. Wilson and J.
Thacker (2000) Mammalian recombinationrepair genes XRCC2 and XRCC3 promote
correct chromosome segregation.
Nature Cell Biol. 2, 757-761.
Moggs J.H., Grandi P., Quivy J.P., Jönsson Z.O.,
Hübscher U., Becker P.B. & Almouzni G. (2000)
A CAF-1 / PCNA mediated chromatin assembly
pathway triggered by sensing DNA damage.
Mol. Cell. Biol. 20, 1290-1299.
Fellerhoff, B., Eckardt-Schupp, F., Friedl,
A.A. (2000) Subtelomeric repeat amplification is
associated with growth at elevated temperature
in yku70 mutants of Saccharomyces cerevisiae.
Genetics 154, 1039-1051.
Ristic, D., Wyman, C., Paulusma, C. and Kanaar,
R. (2001). The architecture of the human
Rad54-DNA complex provides evidence for
protein translocation along DNA.
Proc. Natl. Acad. Sci. USA 98, 8454-8460.
Kraakman-van der Zwet, M., van Lange, R.E.E.,
Essers, J., Wiggers, I., Swaminathan, S., van Buul,
P.P.W., Errami, A., Tan, R.T.L., Jaspers, N.G.J.,
Sharan, S.K., Kanaar, R. and Zdzienicka,
M.Z.(2002). Brca2(XRCC11) deficiency results in
radioresistant DNA synthesis and a higher
frequency of spontaneous deletions.
Mol. Cell. Biol. 22, 669-79.
Partners:
- Bridges (Medical Research Council,
United Kingdom)
- Bootsma (University of Rotterdam,
The Netherlands)
- Moustachi (Institut Curie, France)
- Thacker (Medical Research Council,
United Kingdom)
- Backendorf (University of Leiden,
The Netherlands)
- Eckhardt-Schupp (GSF-Forschungszentrum für Umwelt und Gesundheit,
Germany)
Period: Nuclear Energy 1994-1998
Status: Completed
5TH FRAMEWORK PROGRAMME
Title:
DNA damage response mechanisms in
radiation protection
(DNA DAMAGE RESPONSES)
Co-ordinator: Leon H.F. Mullenders,
Leiden University, The Netherlands
Partners:
- Alan Lehmann (Medical Research
Council, Sussex University,
United Kingdom)
- Jan Hoeijmakers (Erasmus University
Rotterdam, The Netherlands)
- Geneviève Almouzni
(Institut Curie, France)
- John Thacker (Medical Research
Council, Harwell, United Kingdom)
- Friederijke Eckardt-Schupp
(GSF - Forschungszentrum für Umwelt
und Gesundheit, Germany)
Period: Nuclear Energy 1998-2002
Status: Ongoing
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
25
bjectives
The recent discovery of radiationinduced genomic instability may
have implication in radiation
protection, especially quantifica-
Genomic instability and radiation-induced
cancer (RADINSTAB)
tion of human risk for low doses
and high LET exposure.
The role of radiation-induced
genomic instability in radiation
carcinogenesis is being studied in
animal models and mechanistic
Genomic
instability
endpoints
Mechanism
of induction and
transmission
investigations of induction and
Relevance for
cancer risk
&
radiation
protection
transmission are underway.
Genes and gene regions involved
are being studied and the basis of
individual susceptibility investigated. The relationship of genomic
Genetic
susceptibility
Dead and LET
dependance
instability endpoints to radiation
Genes and
genome regions
involved
dose, dose rate and quality is an
important issue for consideration.
A multi-disciplinary approach by radiobiological and biophysical teams is required to assess the role of
genomic instability in radiation carcinogenesis.
Challenges to be met
The main source of information on radiation-induced human cancer risk comes
from epidemiological data on exposed
populations. However, direct information is available only at relatively high
doses (above 0.1 Gy), and mostly from
low-LET radiations (X- and gamma rays).
A linear extrapolation from this data is
applied at lower doses and additional
extrapolation is applied to other radiation types. The shape of the cancer dose
response curve at the low doses is a matter of constant debate. Arguments range
from a threshold or even beneficial effect
of small radiation doses (hormesis) to
non-threshold supralinear responses
(implying that small doses are more hazardous than previously assumed).
Indirect radiation effects, such as
genomic instability (novel mutations
and cell death in the progeny of irradiated cells) and bystander effect (effects
observed in the neighbouring cells not
directly impacted by radiation), may
be important early steps in the development of radiation-induced cancer.
Gaining understanding of underlying
26
mechanisms may have consequences
for cancer risk assessment; these mechanisms could potentially be incorporated
in biological modelling of tumorigenic
responses in the future.
Achievements
It has been observed that the progeny
of irradiated cells show occurrence of
new mutations and/or new chromosomal aberrations or other genomic damage for many generations. Affected
progeny also demonstrate high levels of
lethal mutation, which may be measured as delayed reproductive cell death
and/or delayed apoptosis. Participants
in the project have developed an
approach to determine the effects of
the lowest possible dose of densely
ionising alpha particles to cells, that of
a single particle traversal. It was demonstrated that a single alpha particle is
able to induce chromosomal instability
in the progeny of cultured human cells.
The condition known as genomic instability has a strong dependence on the
type of cell, genotype and radiation quality. Genetic susceptibility to genomic
PART 2
RADINSTAB Information Column
instability induction is being studied using
animal models. Genomic instability occurs
in the progeny of irradiated cells at a frequency that is several orders of magnitude higher than would be expected for
a mutation of a specific gene. Changes
in gene expression are examined in order
to find out whether an instability phenotype is associated with sustained
alterations in the expression of genes,
rather than specific locus alterations.
Existing data of radiation-induced genomic instability suggests that transmission
involves non-traditional epigenetic
inheritance (stable non-mutational
changes). Work related to the epigenetic mechanisms of perpetuation of
instability is in progress. Increased oxidative stress seems to be a long term
characteristic of the progeny of irradiated cells. Participants in the project
have demonstrated that the increased
oxy-radical generation is actually maintained by a signal produced by the irradiated cells in the culture medium.
Partnership
The project brings together the major EU
laboratories involved in the discovery,
characterisation and mechanistic investigations of genomic instability and
bystander effect. Pooled knowledge and
resources are directed to answer key
questions concerning physics, biology
and implications for radiation protection. The multidisciplinary team has
access to a wide range of specialised
radiation sources, unique animal and
cellular models and molecular biological
techniques to address these questions.
Selected references
Kadhim, M.A., Marsden, S.J., Malcolmsson,
A.M., Folkard, M., Goodhead, D.T., Prise, K.M.,
and Michael, B.D. (2001), Long-term genomic
instability in human lymphocytes induced
by single-particle irradiation.
Radiation Research 155, 122-126.
Lyng, F. Seymour, CB Mothersill, C (2000),
Production of a signal by irradiated cells which
leads to a response in unirradiated cells
characteristic of apoptosis.
British Journal of Cancer, 83, 1223-30.
Michael, B.D., Schettino, G., Folkard, M.,
Prise, K.M., Held, K.D And Vojnovic, B., (2001).
Charged-particle and focused soft X-ray
microbeams for investigating individual and
collective radiation responses of cells.
Radiation Research, 156, 439-440.
Mothersill C, Kadhim MA, O'Reilly S,
Papworth D, Marsden SJ, Seymour CB,
Wright EG. (2000). Dose- and time-response
relationships for lethal mutations and chromosomal instability induced by ionizing radiation
in an immortalized human keratinocyte cell
line. International Journal of Radiation Biology
76, 799-806.
Ozols, A., Prise, K.M., Trott, K-R, Folkard, M.,
And Michael, B.D., (2000), The role of oxidative
stress and DNA damage in genomic instability:
targeted microbeam studies in primary human
fibroblasts. Radiation Research, 153, 229-230.
Title:
Genomic instability and radiationinduced cancer (RADINSTAB)
Co-ordinator:
Sisko Salomaa
STUK-Radiation and Nuclear Safety
Authority
Research and Environmental
Surveillance
PO Box 14
Laippatie 4
FIN-00881 HELSINKI, Finland
Tel: +358.9.75988495
Fax: +358.9.75988498
e-mail: [email protected]
Partners:
- Eric Wright (University of Dundee,
United Kingdom)
- Joerg Schmidt (GSF-National
Research Centre for Environment
and Health, Germany)
- Carmel Mothersill (Dublin Institute
of Technology, Ireland)
- Paul Schofield (University of
Cambridge, United Kingdom)
- Bo Lambert (The Karolinska
Institute, Sweden)
- Kevin Prise (Gray Laboratory Cancer
Research Trust, United Kingdom)
- Munira Kadhim (MRC-Medical
Research Council, United Kingdom)
- Laure Sabatier (CEA-Commissariat
à l'Energie Atomique, France)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
A wide range of novel molecular biological methods
is applied
to study the recently discovered new phenomena
of genomic instability and
bystander effect.
27
bjectives
Telomeres are specialized structures at
the end of chromosomes composed
of short tandem DNA repeat
sequences and proteins. The primary
role of the telomeres is to protect
chromosome ends from recombination. Telomeres, by means of TRF2
binding protein, allow the cell to
distinguish the natural chromosome
ends from DNA double strand breaks
(DSB). These lesions are created by
exogenous agents such as ionising
radiations. Chromosomal DSB are particularly dangerous lesions for cells.
DSB are usually stabilised through the
acquisition of a pre-existing telomere
by recombination, but it has been
suggested that in some cells they can
also be stabilized by the direct addition of simple telomeric repeats to
the free ends of chromosomes by
telomerase. Persistent DSB can lead to
carcinogenesis through activation of
oncogenes, loss of tumor-supressor
genes or loss of heterozygosity.
If a DSB is stabilised in germ cells,
they may lead to terminal deletions
which are known to be associated
with abortions and a wide variety of
birth defects in humans. The overall
objective of the project was to determine the extent to which telomere
sequence processing influences radiation response.
a
c
b
d
Metaphase I spermatocyte. A small fragment
is present (purple arrow) with telomere signals
at both ends (b,d). A candidate broken bivalent is indicated (orange arrow). Also present
are a bivalent with a split centromeric region
(blue arrow) and a multivalent (pink arrow).
a. DAPI filter. b. FITC filter. c.TRITC filter.
d. Triple bandpass filter.
28
Role of telomere addition
in the stabilisation of radiation-induced
DNA breaks
Challenges to be met
Insight into the mechanisms of DSB
repair and stabilisation in mammals is
crucial in order to understand the biological consequences of exposure to ionising radiations. The challenge was to
gain understanding of the role of telomere acquisition processes in the capping
of radiation-induced DSB and how this
molecular mechanism can modulate the
formation and stabilisation of chromosome aberrations. To this end mouse
germ and embryo cells have been examined. The partners investigated the
expression of telomerase in these cells
and their upregulation following in vivo
exposure to X-rays, as well as the radiation-induced chromosomal rearrangements and their telomere status.
Achievements
Cells have been assayed for telomerase
activity using a PCR based Telomerase
Repeat Amplification Protocol (TRAP).
High levels of telomerase activity were
detected in mature female germ cells
(oocytes) and in zygotes. Very low levels of telomerase activity were detected
in mature spermatozoa. Telomerase
activity was also measured in male germ
cell precursors. Cell extracts from adult
mice testes containing mixed spermatogenic cell populations showed a
relatively high activity. The cells responsible for the telomerase expression
observed in these extracts were probably premeiotic spermatogonia.
A dose-dependent increase in the telomerase activity up to 3 Gy X-ray exposure
of female mice was observed in mature
oocytes. Increased levels of telomerase
were also found in zygotes after in vivo
irradiation of male mice. Again, these
upregulations were found to be dosedependent from 0 to 3 Gy, and were less
marked at 4 Gy. The results of our experiments suggest a role of telomerase in
DNA repair mechanisms. Chromosomal
damage and telomere capping of radiation-induced chromosomal breaks in
spermatogenic cells was assayed in spermatocytes at metaphase I. Adult male
mice were given 3Gy of X-rays to the
caudal third of the body and were sacrified at different intervals post-irradiation
to sample spermatocytes irradiated at
different spermatogenic stages. Exposure
of pre-meiotic cells to ionizing radiation
gave a considerably lower proportion of
cells with aberrations (figure 1) as compared to the exposure of meiotic stages.
Inter-stage variations in chromatin condensation and chromosome behaviour
may contribute to differences in the
response to irradiation.
When analysing the telomere status of
radiation-induced chromosomal breaks,
we observed that the majority of centric fragments had only the proximal
telomere pair present. However, in two
out of 378 spermatocytes analysed with
a complete set of chromosomes and
telomeres an extra pair of telomeres was
observed at the distal end of the centric fragments (figure 1). We conclude
that although formation of new telomeres on radiation-induced chromosome
fragments does not occur regularly in
mouse spermatocytes, in a small proportion of cells there is evidence of the
generation of new telomeres.
Chromosomal damage and telomere
capping of radiation-induced chromosomal breaks were also analysed in early
embryos after in vivo irradiation of male
germ cells at the stages of epididymal
sperm, spermatocyte and spermatogonia. A total of 1282 zygote and 486 twocell embryo chromosome complements
were analysed. The results showed a peak
in the frequency of aberrant chromosome complements in both stages when
matings were carried out after spermatid
irradiation. The analysis of the telomere
status of embryo metaphases demonstrated that the acentric fragments
associated with dicentric chromosomes
PART 2
Information Column
always contained telomeres at each end.
They were probably compound acentric fragments with pre-existing telomeres resulting from the rejoining of the
free ends of the broken chromosomes by
illegitimate recombination (non homologous end joining, NHEJ).
Excess acentric fragments were also frequently found in zygotes and embryos
(figure 2). They originated from terminal deletions, incomplete exchanges or
interstitial deletions. After irradiation of
spermatozoa at the epididymal stage,
about 40-50% of excess acentric fragments had telomeres at both ends. This
percentage increased to about 100% in
embryos obtained after fertilisation with
spermatozoa irradiated at the spermatocyte and spermatogonial cell stages.
Assuming similar probabilities of centric and acentric fragments to rejoin,
the capped excess acentric fragments
observed in our study cannot be explained by NHEJ. Therefore, they represent the traces of telomere acquisition
events other than simple end-joining.
Overall, capping of fragments in spermatocyte I metaphases was very low
when prophase of the same cell stage
was irradiated. The proportion of capped
fragments was increased in embryo
metaphases when spermatozoa and
spermatids were irradiated and was relatively high when spermatogonial stages
were exposed to X-rays. This suggest
that the telomere acquisition events may
be dependent on DNA synthesis. In addition, our results show that telomerase is
active in oocytes, zygotes and male germ
cell precursors. The upregulation of
telomerase in the same cells after in vivo
irradiation points to the participation of
telomerase dependent mechanisms in
the capping process.
In summary, the origin of capped fragments remains uncertain but, for some
of these, there are strong arguments
that favour real capping events rather
than simple NHEJ. According to our
results, both chromosome healing by
telomerase and telomere capture by a
replication based mechanism can
explain the capping observed in spermatogenic and embryonic cells. It is
also possible that these two mechanisms
could be operating together following
DNA damage. Whatever the mechanism, chromosome capping in cells may
influence the induction of terminal deletions, and gene amplifications and may
reduce chromosome stability.
The ongoing FP5 project, TELORAD, is
expected to allow the determination of
the mechanisms underlying the stabilization of radiation induced DNA breaks
as well as the mechanistic links between
telomerase, DNA repair genes and
telomerase binding proteins.
Title:
Role of telomere addition in the
stabilization of radiation-induced
DNA breaks
Co-ordinator:
Josep Egozcue
Universitat Autònoma de Barcelona
E-08193 BELLATERRA, Spain
Tel: +34.93.581.1025
Fax: +34.93.581.10.25
e-mail: [email protected]
Partners:
- A. Genescà (UAB-Universitat
Autònoma de Barcelona, Spain)
- M. A. Hultén (BHH- Birmingham
Heartlands Hospital, United
Kingdom)
- R. Rosell (University Hospital
Germans Trias I Pujol, Spain)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Partnership
The partners in this contract were EU
investigators with an appropriate range
of expertise in telomere structure and
function.
Selected references
A
B
(A) Partial metaphase of a zygote with telomeric and centromeric hybridization showing a
deletion (del), an acentric fragment with
telomeres at only one end (ace t1) and an
acentric fragment with telomeres at both ends
(ace t2). (B) The same metaphase hybridized
with the painting of the Y chromosome (Y).
Barlow AL, Benson FE, West SC,
Hultén MA (1997).
Distribution of the Rad51 recombinase in
human and mouse spermatocytes.
EMBO J. 16: 5207-5215.
Lerner B, Clocksin WF, Dhanjal S,
Hultén MA Bishop CM (2001).
Automatic signal classification in fluorescence
in situ hybridization images.
Cytometry 43: 87-93.
Scherthan H, Jerratsch M, Li B, Smith S,
Hultén MA, Lock T, de Lange T (2000).
Mammalian telomeres: protein composition
and redistribution in relation to nuclear pores.
Mol Bio. Cell 11:4189-4203.
Tussell L, Latre L, Miro R, Egozcue J, Genesca A.
Radiation induced chromosome breaks in mouse
early embryonic cells: telomere capping.
Int. J. Radiat. Biol. (submitted).
Ponsa I, Barquinero JF, Miró R, Egozcue J,
Genescà A (2001). Non-disjunction and
chromosome loss in gamma irradiated cells.
Radiat. Res. 155: 424-431.
29
bjectives
Ionising radiation damages DNA
and induces mutations and chromosomal anomalies. Broken chromosomes are highly unstable
(fusions, deletions) and are quickly
Telomere instability and the formation
and transmission of radiation induced DNA
damage (TELORAD)
repaired. Telomeres, the very ends
of the chromosomes act as natural
caps and prevent chromosomes
from fusion with one another. The
proteins involved in telomere maintenance play a role in the distinction between radiation-induced
DNA breaks which are subject to
repair and telomeres. TELORAD
aims to determine the mechanistic
link between telomeres, telomerase, telomere binding proteins
and DNA repair genes and their
respective roles in cellular radiosensitivity. Mechanisms of stabilisation
of broken chromosome ends, a
starting point in the transmission
of radiation induced damage, will
be studied. Short telomeric arrays
are also present at intrachromosomal locations, probably reflecting
species evolution. The stability of
these Interstitial Telomeric
Sequences (ITS) will be studied.
Long term consequences, such as
gene amplification will be investigated to identify the involvement
of telomere maintenance in
radiosensitivity of individuals.
Challenges to be met
The aim of this proposal is to study
telomere instability and the formation
and transmission of radiation induced
DNA damage. The work is divided into
four workpackages (WP):
The work of WP1 aims to determine the
mechanistic link between telomerase,
telomeric proteins and DNA repair genes
in radiosensitivity. WP2 is focused on
the determination of the propensity of
interstitial telomeric sequence (ITS) to
breakage and on the determination of
the role of telomerase and DNA repair
genes in the sentivity to breakage and in
the efficiency of repair of ITS. The main
challenge of WP2 is to answer the question : Are ITS hot spots for radiation breakage? WP3 deals with the characterisation
of capping of radiation-induced DNA
breaks. Two mechanisms of stabilization
of radiation-induced breaks, recombination mediated telomere capture, and
chromosome healing by telomerase, will
be particularly studied. In WP4, the role
of telomerase, telomeric proteins and
DNA repair genes in the transmission of
DNA damage in the progeny of irradiated
cells will be questioned.
Achievements
Work on radiosensitivity of telomerase-,
telomeric proteins- and DNA repair deficient mice and cells has provided different
biological endpoints indicating that
telomere length is one of the biological
determinants of radiation sensitivity in
mammals.
Mice with short telomeres are highly sensitive to split dose gamma radiation.
Components of the non-homologous
end joining pathway DNA repair process, Ku86 and DNA-PKcs, have an
important role in protecting telomeres
from fusions. Telomere length is a marker
for chromosomal radiosensitivity. Tumorderived cell lines with shorter telomeres
are usually more radiosensitive.
30
Telomere localisation on a metaphase
spread of human male lymphocyte.
Metaphase was hybridized with telomeric
repeat sequence-specific PNA probe (yellow).
Chromosomes were counter-stained with
DAPI. Bottom left shows Interstitial telomeric
sequences located on chromosome 2 (inverse
DAPI and FISH signals).
Double mutant mice for telomerase and
DNA repair and also telomeric proteins
have been generated and the study of
their radiosensitivity is ongoing. The
effect of expression of telomeric protein is being analysed. Telomere dynamics were studied as a function of the
targeting of telomere binding proteins
to a specific telomere. The influence of
cell irradiation on telomere binding by
proteins will be analysed.
The radiosensitivity of interstitial telomeric
sites seems different in hamster and
human cells.
Interstitial telomeric repeat sequences
are detected as breakage hotspots in
hamster cells. Up to now such results
have failed to be observed in human
cells. Further studies are ongoing to
address the proneness of natural ITS to
breakage. To identify why hamster ITSs
are sensitive to ionizing radiation, it has
been proposed that radiation-induced
DNA DSBs located within ITSs may be
preferentially targeted by telomerase
PART 2
TELORAD Information Column
Studies on transmission of DNA damage has
shown that DNA repair gene defects increase
the probability of gene amplification:
Interest is focused on gene amplification
and chromosome imbalances as detected
in human radiation-induced tumours.
Variation in these processes may rely on
the selection of specific chromosome
imbalances in tumours due to abnormal
telomere maintenance.
Partnership
Human cell line containing a selectable
marker gene located adjacent to a telomere
(short arm of chromosome 16) to isolate
cells undergoing Breakage/Fusion/Breakage
cycles and to analyze the types of events
involved in telomere acquisition.
Induction of gene amplification.
The same metaphase spread was sequentially
hybridized with a telomeric repeat sequencespecific PNA probe (red)(largest picture) and
with (from top right to bottom for the normal
chr16 and from the bottom left to right for
the marker chromosome) 1) the cosmid
(green) specific for the region adjacent to the
plasmid integration site on 16p, 2) the 16qspecific subtelomeric BAC clone GS-240G10(red) and the 16p-specific subtelomeric
BAC clone GS-121-I4 (green), 3) a chromosome 16-specific painting probe (green).
Chromosomes were counter-stained with
either propidium iodide (red) or DAPI (blue).
The location of the translocation on marker
chromosome is shown (arrow).
The nonreciprocal translocation occurred after
several rounds of BFB and induced amplification of the DNA located near the broken end.
because of their complementarity with the
telomerase RNA template. The consequence of this may be chromosome
breakage within ITSs as a result of telomerase-mediated healing. Alternatively,
preferential breakage may be the result of
the hyper-recombination activity of ITSs.
The analysis of the mechanisms of capping of radiation-induced DNA breaks
are crucial for understanding the biological consequences of exposure to ionizing
radiation.
Most of the preliminary studies, optimisation of the techniques, development
of cell lines have been performed; work
is in progress in mice and human cells.
The consortium is composed of seven
partners, two of which are assistant contractors, and two subcontractors. TELORAD brings together partners from
national research laboratories from cancer institutes and from university laboratories. This team has expertise in
molecular biology, cell biology, genetics, radiation biology and animal sciences. Unique cellular and animal
models are available in the team.
Selected references
Ancelin K., Bruneli M., Bauwens S., Brun C.,
Ricoul M., Pommier J.P. Sabatier L. , Gilson E.
(2002) Targeting assay to study the cis functions
of human telomeric proteins: evidence for
inhibition of telomerase by TRF1 and for
activation of telomere degradation by TRF2.
Mol Cell Biol. 22: 3474-87.
Fernández J.L., Vázquez-Gundín F., Rivero M.T.,
Genescá A., Gosálvez J., Goyanes V. (2001)
DBD-FISH on neutral comets: simultaneous
analysis of DNA single- and double-strand
breaks in individual cells. Experimental Cell
Research 270: 102-109.
Goytisolo FA, Samper E, Martin-Caballero J,
Finnon P, Herrera E, Flores JM, Bouffler SD,
Blasco MA (2000) Short telomeres result in
organismal hypersensitivity to ionizing radiation
in mammals. J. Exp. Med. 192:1625-1636.
Title:
Telomere instability and the formation
and transmission of radiation induced
DNA damage (TELORAD)
Co-ordinator:
Laure Sabatier
Commissariat à l'Energie Atomique
BP 6
F-92265 FONTENAY-AUX-ROSES,
France
Tel: +33.1.46.54.83.51
Fax: +33.1.46.54.87.58
e-mail: [email protected]
Partners:
- Maria Blasco (Consejo Superior de
Investigaciones Cientificas, Spain)
- Anna Genesca (Autonomous
University of Barcelona,
Barcelona, Spain)
- Predrag Slijepcevic (Brunel
University, United Kingdom)
- José-Luis Fernandez Garcia
(Laboratorio de Genetica Molecular
y Radiobiologica, Spain)
- Elena Giulotto (Università degli
Studi di Pavia, Italy)
- Chiara Mondello (Istituto de
Genetica Biochimica ed
Evoluzionistica, Italy)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
McIlrath J., Bouffler S., Samper E., Cuthbert A.,
Wojcik A., Szumiel I., Bryant P.E., Riches A.C.,
Thompson A., Blasco M.A., Newbold R.F.,
Slijepcevic P. (2001) Telomere length
anormalities in mammalian radiosensitive cells.
Cancer Res. 61: 912-915.
Mondello C., Faravelli M., Pipitone L., Rollier A.,
Di Leonardo A., Giulotto E. (2001) Gene amplification in fibroblasts from ataxia telangiectasia
patients and in X-ray hypersensitive Chinese
hamster mutants. Carcinogenesis 22: 141-145.
31
bjectives
The project aims to understand the
mechanisms by which radiationinduced effects on cellular control
mechanisms will affect the evolution of permanent genetic damage
in cells. Evolution of permanent
Evolution of genetic damage in relation
to cell cycle control:
a molecular analysis of mechanisms
relevant for low dose effects
damage means the transformation
of initial radiation-induced DNA
damage into permanent cellular
modifications. This may be
expressed as reproductive cell
death, chromosomal aberrations,
mutations, malignant transformation, and disturbances in embryonic development in utero or cell
differentiation in adults. The damage evolution process is dependent
on cellular mechanisms such as
DNA repair, cell cycle control,
induction of programmed cell
death and other adaptive cell
mechanisms to cellular stress.
The principal objective of this
Challenges to be met
The project focuses on the analyses of
cell cycle regulating enzyme activities at
the G2-phase/mitosis transition in relation to DNA damage, DNA repair and
the development of chromosomal damage. The working hypothesis is that the
higher the endogenous cdk1/cyclin B
activity is in a cell, the higher will be the
non-repaired damage in DNA at the
time when the cell enters mitosis. To test
this hypothesis different human cell lines
and embryonic cells from different
mouse strains with known differences in
radiation sensitivity were analysed for
cell cycle activity, chromosomal damage,
and DNA repair capacity.
project is to gain a better understanding of these adaptive cell
mechanisms by analysing cell cycle
regulating enzyme activity levels at
cell cycle transition control points
in relation to radiation induced
DNA damage, DNA repair and the
development of chromosomal
damage; emphasis is placed on
the G2-phase/mitosis (G2/M)
transition.
The project was separated in two major
parts, one on adult somatic cells and a
second on embryonic cells. The two
major parts were further subdivided in
4 work packages each. The work package aims were, (I) to analyse changes in
cdk/cyclin enzyme complexes in different human cell lines in response to irradiation, (II) to study the evolution of
chromosomal damage after premature
chromosome condensation in these cell
lines, (III) to measure the repair capacity in cells by using the comet assay,
and (IV) to analyse intracellular signal
transduction pathways in irradiated cells.
Achievements
Irradiation induces changes in phosphorylation of the central cell cycle driving
enzyme complex at the G2/M transition
point (cdk1/cyclin B protein kinase). In
the cell lines analysed there was an about
50% decrease in the kinase activity in
irradiated cells. At least up to an irradiation of 2 Gy, there was no difference in
the amount of initial radiation-induced
DNA damage for each particular dose in
exponentially growing mammalian cells
in vitro. Repair was comparable in all four
cell lines.
The conversion of primary radiationinduced DNA damage into visible chromosomal damage under the influence
of different levels of cdk1/cyclin B activity was analysed by premature chromosome condensation (PCC). The PCC
breaks increased linearly with dose and
the yield was dependend on the type of
the mitotic cells used as PCC inducers.
From these experiments it is concluded,
that the differences in the conversion of
radiation-induced initial DNA lesions for
each particular dose into PCC breaks
reflect differences in the cdk1/cyclin B
activity levels during chromosome condensation in early mitosis.
Using the G2 PCC radiosensitivity assay,
cancer patients (n=185, with different
types of cancer) on the average showed
an increased in vitro radiosensitivity of
their peripheral lymphocytes when compared to controls (n=25). Because of
our observation of a strong correlation
between G2 chromosomal radiosensitivity and cdk1/cyclin B activity during
the G2/M transition and of our failure
Evolution of genetic damage in relation to cell cycle control:
A molecular analysis of mechanisms relevant for low dose effects
Cdk1/cyclin B kinase activity in four different cell lines (MCF7, HeLa, SCL2, TK6) after irradiation (1)
and in non-irradiated controls (0).
32
PART 2
Information Column
to detect this strong correlation between
sensitivity and DNA repair, we conclude
that cell cycle regulation is a key player
in determining human radiosensitivity.
To study cell cycle regulation in early
mouse embryos and effects of irradiation on this regulation, the activity of the
cdk1/cyclin B protein kinase was determined in single oocytes or embryos. After
fertilisation, cdk1/cyclin B protein kinase
activity in mouse embryos is low during
interphase of the first cell cycle, increases
during the first mitosis, and decreases
again during the next interphase.
Embryos irradiated in the first cell cycle
were arrested during G2. However, the
dynamic of this radiation-induced G2block in the first embryonic cell cycle is
totally different between the mouse lines
tested. This differential dynamic in the
G2-block in one-cell embryos of the different strains is strictly paralleled by a
similar dynamic in the cdk1/cyclin B
activity.
Using the comet assay the amount of
initial radiation induced DNA damage
in embryos for a particular dose was
comparable to the amount in the cell
lines mentioned above. The repair in
embryos of the mouse strains tested is
extremely fast and almost complete after
30 minutes. This is significantly faster
than in all cell lines and primary cells
tested so far.
The results of these studies will allow a
better understanding and assessment
of risks from those effects which are
characteristic consequences of exposure to small radiation doses, i.e. cancer, genetic damage and disturbance
of development in utero. All results
obtained so far are consistent with the
proposed hypothesis that following
exposure to ionising radiation, the onset
and the efficiency of chromatin condensation-decondensation which is
dependent on cdk1/cyclin B activity levels is the important determinant of the
process that converts initial radiationinduced DNA damage into chromosomal breaks. This may, therefore, explain
the differential radiosensitivity observed
at the various stages of the cell cycle as
well as among mutant cells and cells of
different origin. More important we
were able to show differences in in vitro
chromosomal radiosensitivity of peripheral lymphocytes of cancer patients
compared to lymphocytes from healthy
control individuals.
Partnership
The project integrated the expertise of
five laboratories all of which have made
important contributions to progress of
the project and the development of new
methods needed. Their co-operation
allowed the concerted approach to correlate key mechanisms in cell cycle control with damage to the DNA molecules
and chromosomes at different stages of
the cell cycle.
Selected references
Terzoudi GI, Jung T, Hain J, Vrouvas J,
Margaritis K, Donta-Bakoyianni C,
Makropoulos V, Angelakis P, Pantelias GE.
Increased G2 chromosomal radiosensitivity in
cancer patients: the role of cdk1/cyclin-B
activity level in the mechanisms involved.
Int J Radiat Biol. 2000 May; 76(5):607-15.
Baatout S, Jacquet P, Michaux A, Buset J, Desaintes C.
Histone H1 kinase activity in ovulated oocytes.
Anticancer Res. 1999 Nov-Dec; 19 (6B):
5117-8.
Baatout S, Jacquet P, Jung T, Hain J, Michaux A,
Buset J, Vandecasteele C, De Saint-Georges L,
Baugnet-Mahieu L.
Histone H1 kinase activity in one-cell mouse
embryos blocked in the G2 phase by X-irradiation.
Anticancer Res. 1999 Mar-Apr; 19 (2A):
1093-100.
Title:
Evolution of genetic damage in
relation to cell cycle control:
a molecular analysis of mechanisms
relevant for low dose effects
Co-ordinator:
Thomas Jung
BFS-Bundesamt für Strahlenschutz
Institut für Strahlenhygiene
Postfach 10 01 49
D-38201 SALZGITTER, Germany
Tel: +49.89.31603.142
Fax: +49.89.31603.111
e-mail: [email protected]
Partners:
- Paul Jacquet (CEN/SCK Centre
d’Etude de l’Energie
Nucléaire/Studiecentrum voor
Kernenergie, Belgium)
- Rolf Jaussi (Paul Scherrer Institut,
Switzerland)
- Gabriel Pantelias (National Centre
of Scientific Research ‘Demokritos’,
NCSR, Greece)
- Christian Streffer (Universitätsklinikum
Essen, Germany)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
33
bjectives
The project has been divided in
four work-packages, which address
the radiation response at various
levels of the signalling cascades.
The effects of ionising radiation on signal
transduction and cell cycle control
Challenges to be met
Ionising radiation can activate a large
number of responses in mammalian
cells. The most important of these are
the activation of a number of signal
transduction pathways which in turn
activate specific transcription factors,
transient inhibition of cell division (cell
cycle arrest), and induction of programmed cell death (apoptosis). The
function of these reactions is to protect
the organism as a whole against the
consequences of radiation-induced damage. Elucidation of the mechanism by
which these responses are activated by
ionising radiation is a main objective of
this study.
mean numbers (S.E.) of skin carcinomas
Micrograph of Scotin, a novel p53-inducible
protein, which is localised in the endoplasmic
reticulum and the nuclear membrane.
H1299 cells (human lung cancer cells, devoid
of p53) were transfected with Scotin expression vector. Cells were fixed 24h after
transfection and indirectly stained for Scotin
protein using FITC- labeled antibody. Scotin
expression is revealed as green fluorescence.
time (weeks)
DFMO treatment suppresses the development
of skin carcinomas during UV-carcinogenesis in
XPA-knockout mice. At each point, the average
numbers of skin carcinomas per mouse is
depicted. Closed circles are data from animals
that have only received normal drinking water,
closed squares from animals that received 1%
DFMO until week 14, and closed triangles
from animals that received 1% DFMO starting
at week 14. The daily UV irradiation (16 J/m2)
was discontinued at week 14.
34
Two important transcription factors that
are activated by radiation are NFkB and
cJun-ATF2. Two other consequences of
exposure of cells to ionising radiation are
a transient inhibition of cell division (cellcycle arrest) and programmed cell death
(apoptosis). The function of cell-cycle
arrest is probably to provide sufficient
time for the cells to repair the DNA damage in the absence of DNA replication
(in this way preventing mutations).
Programmed cell death is a suicide
mechanism that is activated if the radiation damage is too extensive to be
repaired. Both phenomena require the
transcription factor and tumour-suppressor protein p53. The p53 protein is
the product of a tumour suppressor
gene, which is involved in the control
of cell division. Its role in protecting
genomic integrity is suggested by the
finding that the protein accumulates
upon irradiation and that cells lacking
p53 do not show the radiation-induced
cell-cycle arrest. The fact that apoptosis is also a function of p53 has important consequences for cancer therapy,
since tumour cells containing mutant
p53 are much less responsive to
chemotherapy than tumours containing
functional p53 (chemotherapeutic
agents normally kill tumour cells by
apoptosis). One of the genes that is
activated by radiation-induced signalling
is the gene coding for ornithine decarboxylase (ODC). Among others, ODC
plays a role in the response of cells to
radiation damage. Interestingly, it was
found that certain human cells fail to
activate ODC after radiation damage,
and that these cells are derived from
individuals who appear to be resistant
to UV-induced cancer. This suggests that
the activation of certain genes may
increase the risk for oncogenic transformation, while at the same time contributing to cell survival.
Achievements
Radiation-activated signalling cascades
In this workpackage the activation by
ionising radiation of two important transcription factors in the cellular stress
response, NFkB and cJun/ATF2, was
studied. Activation of NFkB depends on
degradation of its inhibitor IkB. This is
caused by phosphorylation of IkB by a
mechanism different from that induced
by ultraviolet (UV) light. Similarly, while
UV light causes phosphorylation of
both cJun and ATF2, ionising radiation
activates only ATF2. Thus, ionising radiation activates two important transcription factors via mechanisms distinct
from those used by UV light. Finally,
evidence was found that not only
nuclear DNA damage but also processes
in the cytoplasm (plasma membrane)
involving inactivation of tyrosine phosphatases play a role in activation of the
signalling pathways by radiation.
Activation of p53 by ionising radiation
The tumour suppressor protein p53
plays a central role in the response to
ionising radiation and for a large part
determines the fate of the irradiated
cell. It was shown that the stabilization
(and thus increase) of p53, which is the
key event in its activation, is probably
independent of phosphorylation of the
p53 protein, as was generally believed.
PART 2
Information Column
Rather, it seems likely that modulation
of Mdm2, the degradation-inducing
enzyme which binds to p53, is the
important factor.
and how it differs from other stresses.
This knowledge may contribute to a
better understanding of the harmful
effects of ionising radiation.
Constituents of protective responses
Ornithine decarboxylase (ODC) is the
key enzyme in polyamine biosynthesis
and is strongly activated by ionising
radiation. Intracellular levels of polyamines are important factors in protecting cells from radiation damage. It was
shown that in cells derived from certain “cancer-resistant” individuals, ODC
was not activated after exposure to ionising radiation or UV light. Thus, the
absence of ODC induction can possibly
protect against carcinogenesis. This idea
was tested in radiation-sensitive mice
which were irradiated with UV-B and
simultaneously treated with DFMO, a
specific inhibitor of ODC. Indeed, it was
found that application of inhibitor
strongly diminished the frequency of
skin tumours.
Partnership
Novel ionising radiation-activated genes
Attempts were made to isolate genes
that are specifically induced by ionising
radiation in intact mice and in human
cells. In the mouse studies a gene has
been identified, named Scotin, which
seems involved in the apoptotic response to ionising radiation. Induction of
the gene is dependent on p53 since it
is not activated in p53-null mice.
Surprisingly, in the experiments involving human cells no genes were isolated
that were specifically induced by ionising radiation. In the same study, however, several genes were identified that
were induced by the alkylating chemical agent methyl-methanesulfonate
(MMS) (but not by radiation). One of
these genes is a novel member of the
signalling cascade from the endoplasmic
reticulum to the nucleus, and is activated by the presence of unfolded proteins, the so called unfolded protein
response (“protein stress”).
This work was dependent upon the complementary expertise of three EU laboratories with long standing interests in
cellular stress responses.
Title:
The effects of ionising radiation on
signal transduction and cell cycle
control
Co-ordinator:
Alex J. van der Eb
Leiden University
Faculty of Medicine
Wassenaarseweg 72
NL-2333 AL LEIDEN, The Netherlands
Tel: +31.71.5276109 or .5276000
Fax: + 31.71.5276173
e-mail: [email protected]
Selected references
Bender K, Goettlicher M, Whiteside S,
Rahmsdorf HJ and Herrlich P.
Sequential DNA damage-independent and –
dependent activation of NF-kappaB by UV.
EMBO J 17: 5170-5181, 1998.
van Dam H, Huguier S, Kooistra K, Baguet J,
Vial E, van der Eb AJ, Herrlich P, Angel P and
Castellazzi M. Autocrine growth and anchorage
independence: two complementing
Jun-controlled genetic programs of cellular
transformation. Genes Dev. 12:1227-39, 1998.
Blattner C, Tobiasch E, Litfin M, Rahmsdorf HJ
and Herrlich P.
DNA damage induced p53 stabilization:
no indication for an involvement of p53
phosphorylation.
Oncogene 18: 1723-1732, 1999.
Partners:
- P. Herrlich (Forschungszentrum
Karlsruhe, Germany)
- D.P. Lane (University of Dundee,
United Kingdom)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Bottger V, Bottger A, Garcia-Echeverria C,
Ramos YF, van der Eb AJ, Jochemsen AG
and Lane DP.
Comparative study of the p53-Mdm2 and
p53-MDMX interfaces.
Oncogene 18: 189-199, 1999.
Rebel H, Van Steeg H, Beems R.B., Schouten R.,
De Gruijl F.R. and Terleth C.
Suppression of UV carcinogenesis by difluoromethylornithine in nucleotide excision repairdeficient Xpa knockout mice.
Cancer Research 62: 1338-1342, 2002.
Overall these studies have shown that
ionising radiation activates different
mechanisms than other forms of stresses
such as UV light. Unravelling these mechanisms will further help understanding
what ionising radiation does to the cell
35
bjectives
Risk assessment of exposure
to radon decay products
(RARAD)
The aim of this contract was to
assess the risk due to inhalation of
radon and its decay products that
escape from certain forms of rock.
The central objective was the
assessment of human risk that
requires combination of several
topics and a multidisciplinary
approach. Five main topics were
addressed: radioactive aerosol
studies, modelling, human studies,
animal studies and retrospective
assessment of radon exposure studies.
These tasks were distributed
between five working groups.
Challenges to be met
Aerosol Studies Group sought to determine the properties and behaviour of
the radon decay products in order to
provide improved characterisation of the
indoor atmosphere. Studies were focused
on the size distribution of the unattached
and aerosol-associated radon decay products and the amount of the unattached
activity. These are the most important
parameters in models used for calculating
lung doses.
Deposition
ICRP Model:
Monte Carlo Model:
Bronchial
Bronchial
Pulmonary
Pulmonary
Diameter (µm)
Comparison of bronchial and pulmonary deposition as functions
of particle size for sedentary breathing conditions using the
Human Respiratory Tract Model (ICRP Publication 66) and
a stochastic model.
Bq/m3 OBJ2
Bq/m3 OBJ1
Calculated average 222Rn concentration, for more than
20 years in the past, from retrospective measurements on
225 pairs of personal objects.
36
Modelling Group was acting to
develop two complementary
approaches. One used the
model based on the new ICRP
dosimetric model for the respiratory tract (ICRP Publication 66, 1994), and the
other used a stochastic model
that included the clearance
process and radiation interaction at the cellular level.
Human Studies Group aimed
to conduct inhalation studies on human volunteers in
order to provide a better definition of radon progeny specific parameters, including
the deposition pattern of the
unattached fraction and the
dependence of deposition on
age and gender.
Animal Studies Group sought to
assess the effect of exposurerate at low cumulative radon
exposure for the induction
of lung cancer in rats. The
extent to which early biological dosimetric markers,
such as nuclear aberrations
and cell proliferation might
be used to predict late effect
from exposure to radon and
its progeny was also to be
investigated.
RARE (Retrospective Assessment of Radon
Exposure) Group was acting to provide
data for the retrospective reconstruction
of indoor radon exposure. The studies
were focused on techniques allowing
measurement of long-lived radon decay
products either by measuring 210Po in
indoor glass surfaces (surface traps) or in
porous materials (volume traps).
Achievements
Progress was made in improvement,
calibration and automation of experimental techniques for continuous and integrated measurements of the unattached
fraction fp- and equilibrium factor F- values.
Measurements were performed to determine the variation of size distributions of
unattached and aerosol-associated radon
decay products under typical living conditions and were considered for updated
dose calculations. Controlled chamber
studies to understand the basic behaviour of airborne activity concentrations
were undertaken. Measurements were
performed to determine neutralisation
rates of 218Po, to understand the cluster
growth with residence time and to
understand the hygroscopic growth of
aerosol particles.
The programme RADEP has been developed to calculate the weighted committed equivalent lung dose per unit
exposure of radon progeny (Hw/Pp)
which implements the ICRP Publication
66 Human Respiratory Tract Model
(HRMT). The stochastic deposition model
(IDEAL) was compared with the deposition model used by the HRTM; the agreement was good. A deterministic radon
progeny dosimetry model (RADOS) has
also been developed. Initial calculations
with RADOS show that the basal and
secretory cell doses are slightly smaller
compared with that of the HRTM.
A sensitivity analysis has been performed
that has identified those HRTM model
parameters that most affect the Hw/Pp.
PART 3 : Radiation tumorigenesis: mechanisms and pre-disposition
RARAD Information Column
A stochastic rat deposition model (RALMO)
and a clearance model for the rat based
on the HRTM have been developed to
calculate equivalent dose.
Selected references
Human studies were focused on the
deposition patterns of the unattached
fraction and the dependence of deposition on age and gender, the transfer
of inhaled radon progeny to blood and
the comparison of total deposition of
radon progeny for adults and children
in the domestic environment.
Marsh JW, Birchall A : Determination of lung-toblood absorption rates for lead and bismuth
that are appropriate for radon progeny.
Radiat Protect Dosim 1999;89:331-337.
The results of experiments on the influence of exposure-rate on lung cancer
induction results indicate that at relatively
low cumulative exposures comparable to
lifetime exposures in high-radon houses or
current underground mining exposures,
the risk of lung cancer in rats decreases
with decreasing exposure-rates. These
data suggest that in terms of risk of induction of lung cancer, there is a complex
interplay between cumulative exposure
and exposure rate. A positive dose rate
response was seen for proliferating epithelial cells at relatively high exposure levels.
The response of proliferating epithelial
cells was found to depend on dose rate
at higher doses, whereas at lower cumulative doses no significant elevations in
proliferation were observed.
Protocols were also developed for selection of the most suitable surface and
volume traps to be used for radon measurements. In addition a questionnaire
was developed to obtain estimates of
room parameters that control the deposition and build up of 210Po on surfaces.
Computer models have been produced
which make it possible to make a retrospective estimation of radon exposure in
the past using surface trap measurements and questionnaire information.
Hofmann W, Bergmann R : Predictions of particle
deposition patterns in human and rat airways .
Inhal Toxicol 1998;10:557-583.
Mc Laughlin JP : The application of techniques
to restrospectively assess radon and radon
exposure.
Radiat Protect Dosim 1998;78:1-6.
Monchaux G, Morlier JP, Altmeyer S,
Debroche M, Morin M : Influence of exposure
rate on lung cancer induction in rats exposed to
radon progeny.
Radiat Res 1999;152:S137-S140.
Vargas A, Ortega X, Porta M :
Dose conversion factor for radon concentration
in indoor environments using a new equation for
the F-fp correlation.
Health Phys 2000;78:80-85.
Title:
Risk Assessment of Exposure to Radon
Decay Products (RARAD)
Co-ordinator:
Georges Monchaux
IRSN
BP 17
F-92262 FONTENAY-AUX-ROSES
CEDEX, France
Tel: +33.1.58.35.70.48
Fax: +33.1.58.35.79.71
e-mail: [email protected]
Partners:
- Porstendörfer (University of
Göttingen, Germany)
- Tymen (University of Brest, France)
- Ortega (Univ. Catalunya-Politécnica,
Spain)
- Samuelsson (University
of Lund, Sweden)
- Birchall (NRPB, Chilton,
United Kingdom)
- Hofmann
(University of Salzburg, Austria)
- Falk (SSI, Stockholm, Sweden)
- Collier (AEA Technology,
United Kingdom)
- McLaughlin (University College
Dublin, Ireland)
- Vanmarcke (CEN/SCK,
Mol, Belgium)
- Poffijn (University of Gent, Belgium)
- Schuler (Paul Scherrer Institute,
Switzerland)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Partnership
Status: Completed
This contract has pooled the expertise
of EU laboratories working on different
fields of radiation protection. Collaborations were developed with other
groups of the Nuclear Fission Safety
Programme involved in research on
alpha particle carcinogenesis, dosimetry of alpha-emitters and with the different epidemiology programmes.
37
bjectives
Radioactive materials play an
important socio-economic role in
most developed countries. In addition to their industrial uses, especially
in energy generation, there is increa-
The mechanisms and genetics
of radiation tumorigenesis
(MAGELLANS)
sing utilization of ionising radiation
in the therapeutic and diagnostic
procedures of modern medicine.
Ionising radiation is also present
in the natural environment where
it may also have a socio-economic
impact. Since ionising radiation is
a proven human carcinogen it is
clearly important to improve our
knowledge of the risks involved in
its use – this applies particularly to
health effects that result from low
dose exposure of the public and
workers. Since epidemiological
studies have insufficient power to
directly measure excess cancer
after these low doses it is necessary
to project risks from high dose
studies. Establishing the biological
validity of these projections is an
increasingly important issue in
radiological protection research.
Accordingly, the experimental
studies outlined here utilized a
combination of experimental models
of cancer induction and molecular
biology techniques to investigate
the mechanisms and genetics of
Challenges to be met
The specific challenges to be met in the
project are best illustrated by the following questions. First, radiation is a
proven carcinogen but where does it act
in the complex multi-stage process of
cancer development operating over
many years and what are the implications for low dose risk? Second, we know
from radiotherapy observations that rare
human genetic conditions pre-dispose
to radiation-induced cancer but should
we expect more common genetic variation that might distort populationbased estimates of low dose risk? At this
stage in knowledge it is necessary to
seek proof-of-principle evidence on
these questions using relevant experimental models of cancer-induction. The
project was designed ahead of FP4 to
make full use of information expected
from human and mouse genome
research. In the project the partners are
seeking detailed knowledge of radiation-induced myeloid leukaemia, lymphoma, skin tumours and breast cancer
in selected mouse models.
Achievements
radiation carcinogenesis. The work
was initiated in FP4 and is currently
being extended in FP5; there is
close collaboration with the
GENRAD consortium.
38
Induction of myeloid leukaemia and lymphoma: Changes in the normal structure
of chromosomes are frequent in myeloid
leukaemia and lymphoma arising in
humans and mice; both these tumour
types are thought to originate in bone
marrow cells. Chromosomal studies
relating to radiation-induced myeloid
leukaemia in the mouse have shown
that the crucial radiation-induced damage during multi-stage tumour development occurs in primitive, normal
bone marrow cells. This damage is consistently expressed in bone marrow cells
and tumours as loss of a small segment
of chromosomal DNA and the segment
has been isolated in fragments and
reconstructed. Using genome information and technology the likely target
genes have been positioned in this
chromosomal region and analysed
further to determine their importance
for tumour initiation. Similar progress is
being made for lymphoma development but, here, the critical DNA loss
occurs on a different chromosome.
The picture emerging from this subproject, and indeed from others in the
same EU programme, is that radiation
is principally acting very early in multistage carcinogenesis as a DNA-deleting
agent for tumour-specific genes in single cells. There is already good knowledge of this mechanism of DNA damage
and, overall, the research is adding
important support to the view that cancer-induction by radiation does not
occur through an unusual mechanism.
On this basis, cancer risk will tend to rise
with dose without a low dose-threshold
where risk can be discounted.
Genetic susceptibility to myeloid leukaemia,
skin tumours and breast cancer: Previous
studies with mouse models had provided some evidence of common,
genetic variation in radiation cancer risk
but little specific information. As given
below, work on this sub-project has
added substantially to knowledge. In
the case of myeloid leukaemia, a single
heritable gene distributed amongst
mouse strains appears to be the major
determinant of risk. The evidence suggests that the variant gene acts to reduce
the ability of bone-marrow cells to
respond adequately to DNA damage in
certain chromosomal regions; the gene
appears to be tissue-specific in its action.
The chromosomal location of the gene
has been estimated and work on its
identification is underway. The genetics
of skin-tumour susceptibility is more
complex involving the interaction of a
number of common variant genes, some
of which have been located. Importantly,
there appears to be shared genetic
mechanisms for susceptibility to radiation and chemical carcinogens. One
susceptibility gene which produces an
PART 3
ACRONYM
???
Information
Column
MAGELLANS
Information
Column
4TH FRAMEWORK PROGRAMME
A
Title:
The Mechanisms and Genetics
of Radiation Oncogenesis
B
Partnership
The partnership brought
together EU expertise
Chromosomes from a radiation-induced mouse myeloid leukaemia
in animal radiobiology,
cell highlighted with molecular stains. In panel A the critical loss
tumour biology, chrofrom chromosome number 2 is seen in the reduction in size of one
mosome analysis, molecof the two copies (2 versus del 2). Panel B shows a cell from the
ular biology and genetics.
same leukaemia stained for a very small part of the chromosome.
Without this combinaThe paired stained spots on the upper copy (2) are lost from the
lower copy (del 2) that carries the critically deleted segment.
tion it would not have
been possible to fully
exploit the scientific
altered hormone-related protein has potential of the mouse models; in this
been isolated and its mechanism of area collaborative studies are essential.
action is under investigation; studies on The progress made in MAGELLANS and
a new mouse genetic model are also GENRAD illustrates the growing power
underway. Finally, a third sub-project on of these models, coupled with genome
genetic variation in the genes that con- research, to comment upon complex
trol response to radiation damage in biological problems associated with low
DNA led to an external collaboration on dose risk. The work is also providing
breast cancer risk. This work which guidance on the future development of
involved gene location, isolation and new approaches based upon human celcharacterisation showed that partial lular material; this applies particularly to
deficiency in a single key gene was asso- the issue of cancer-susceptibility.
ciated with tissue radiosensitivity, chromosomal instability and breast cancer Selected references
risk. Further work on the origins and
impact of this variant gene, which is not Manenti G, Peissel B, Gariboldi M, Falvella FS,
common, is being shared with GENRAD. Zaffaroni D, Allaria B, Pazzaglia S, Rebessi S,
The principal outcome from work in this
area is evidence that variant genes influencing radiation cancer risk can be, but
are not always, common. They tend to be
tissue-specific in their action and frequently work in concert; cross-sensitivity
to other carcinogens will probably apply
to some variant genes. Overall, the work
adds support to the view that, for
genetic reasons, tissue-specific cancer
risk after radiation will not be uniformly
distributed in the human population.
Also, that we should expect much of the
common genetic variation in low dose
cancer risk to be inherited in a complex
fashion – predictions on the response of
most individuals are likely to remain
most difficult.
Covelli V, Saran A and Dragani TA.
A cancer modifier role for parathyroid
hormone-related protein.
Oncogene 19: 5324-5328 (2000).
Peissel B, Zaffaroni D, Pazzaglia S, Manenti G,
Zanesi N, Zedda I, Rebessi S, Covelli V,
Dragani TA and Saran A.
Use of intercross outbred mice and single
nucleotide polymorphisms to map skin cancer
modifier loci.
Mamm. Genome 12: 291-294 (2001).
Meijne E, Huiskamp R, Haines J, Moody J,
Finnon R, Wilding J, Spanjers S, Bouffler S,
Edwards A, Cox R and Silver A.
Analysis of loss of heterozygosity in lymphoma
and leukaemia arising in F1 hybrid mice locates a
common region of chromosome 4 loss.
Genes Chrom. Cancer 31: 373-381 (2001).
Yu Y, Okayasu R, Weil M, Silver A, McCarthy M,
Zabriskie R, Long S, Cox R and Ullrich R.
Elevated breast cancer risk in irradiated BALB/c
mice associates with unique functional
polymorphism of the Prkdc (DNA-PKcs) gene.
Cancer Res. 61: 1820-1824 (2001).
Co-ordinator:
Roger Cox
NRPB-National Radiological
Protection Board
Radiation Effects Department
Chilton, Didcot
UK-OX11 0RQ OXFORDSHIRE,
United Kingdom
Tel: +44.1235.831648
Fax: +44.1235.833891
e-mail: [email protected].
Partners:
- R. Huiskamp (ECN, The Netherlands)
- V. Covelli (ENEA, Italy)
- B. Young ( ICRF, United Kingdom)
- B. Dutrillaux (CEA, France)
- T. Dragani (INT, Italy)
- A. Van der Eb (Leiden University,
The Netherlands)
- R. Newbold (Brunel University,
United Kingdom)
- F-S. Pedersen (University Aarhus,
Denmark)
Period: Nuclear Energy 1994-1998
Status: Completed
5TH FRAMEWORK PROGRAMME
Title:
The mechanisms and genetics of
radiation tumorigenesis
(MAGELLANS)
Co-ordinator:
Roger Cox, NRPB, United Kingdom
Partners:
- Emmy Meijne (NRG-Nuclear Research
and Consultancy Group,
The Netherlands)
- Anna Saran (ENEA-Ente par le
Nuove Tecnologie, l’Energia e
l’Ambiente, Italy)
- Tommaso A. Dragani (INTIstitutonazionale per lo Studio
e la Cura dei Tumori, Italy)
Period: Nuclear Energy 1998-2002
Status: Ongoing
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
39
bjectives
One limitation to the recommendations for dose limits in radiation
protection is the tacit assumption
that all individuals receiving the
Genetic susceptibility to radiation
carcinogenesis (GENRAD)
same dose of ionising radiation are
equally at risk of developing cancer.
It is now apparent that the human
genome is highly variable, and that
inherited genetic information influences individual sensitivity to many
environmental carcinogens. At the
moment radiosensitive individuals
are recognised post-hoc, i.e. after
tumours appear. The GENRAD
consortium, in partnership with the
MAGELLANS group, is striving to
identify the genes that are responsible for determining individual
cancer risk after irradiation.
This knowledge will ultimately be
used to develop a genetic screening platform for use in determining
individual cancer risk following or
even prior to, an exposure to
ionising radiation.
Challenges to be met
The risk of developing cancer after exposure to ionising radiation is currently
extrapolated from epidemiological data
derived from exposed human populations. Although these cohorts are well
defined, exposure is primarily high-dose
radiation applied at a high dose rate, and
no allowance is made for inter-individual
variability. At the low doses and low
dose rates more conventionally encountered the contribution of genetic variability may be more significant.
The most readily identified source of
genetic variability is the rarely encountered group of genes responsible for
familial cancer syndromes or acute radiation sensitivity. An inherited mutated
form of one of these genes greatly
increases the risk of developing cancer
after radiation exposure. A less dramatic,
but equally important, source of genetic
variation is gene polymorphisms arising
through the natural variability of the
human genome (current estimates suggest there may be over 4 million sequence
Incidence of osteosarcoma
Inheritance of a set of 5 susceptibility genes (red curve)
increases radiation-induced tumour formation and
shortens letancy
BALB/c x CBA F2 hybrid with
5 susceptibility loci
BALB/c strain
BALB/c x CBA F2 hybrid with
0 susceptibility loci
CBA strain
Latency (days after injection)
40
differences between normal individuals). These polymorphisms are responsible for only small differences in gene
function and individually make a minor
contribution to overall risk. However, in
individuals who by chance inherit several such polymorphisms the net effect
on risk may be appreciable.
The ability to more accurately predict
the outcome of radiation exposure will
greatly change modern radiation protection. Not only can individuals with
workplace exposure be more accurately
evaluated prior to exposure, but also
adverse effects of radiation therapy can
be minimized. Following accidental
exposure those individuals at greater
risk can be identified and offered more
comprehensive care when resources are
limited or enrolled in more frequent follow-ups. An additional product of this
research will be a firmer understanding
of the mechanisms of radiation carcinogenesis, which will allow a more
direct approach in the evaluation of the
risk, and hopefully the development of
targeted radioprotective therapies.
Before we can use genetic profiling to
determine individual risk we must first
establish which genes influence risk.
There is very little chance of using
human epidemiological data to retrospectively identify genetic risk factors.
However, as an alternative, recent
advances in molecular genetics make it
possible to identify susceptibility genes
present in the mouse and rat genomes.
During FP4 the consortium has developed animal models of human radiation
carcinogenesis, selected to reflect the
most clinically relevant target tissues.
These models were validated to establish proof of principle for the use of
genetic screens designed to identify the
relevant genes.
In FP5 we are deploying genetic screens
covering the entire mouse and rat
genomes aimed at identifying the genes
PART 3
GENRAD Information Column
responsible for determining radiation
cancer risk. Two different screening
strategies are used. In the first, a genome-wide scan for loci exhibiting allelic
imbalance (AI-screen) is used to map
and ultimately identify regions of the
genome harbouring genes with the
potential to act through the germ-line
to modify risk of radiation carcinogenesis. This screen is based on the premise
that such genes manifest themselves
through a change in the number of
gene copies in the tumour tissue. Such
a change is detectable through an assay
of allelic status.
In the second screen, linkage analysis is
used to identify those genes that exert
a more modest influence on individual
susceptibility. In a cohort of genetically
heterogeneous animals those that
inherit predisposing genes will be more
likely to develop cancer. Consequently
these genes will be found to be inherited more frequently by tumour-bearing
animals (such a gene is said to be linked
to the risk of developing cancer) (see
figure).
Achievements
Five animal models of radiation carcinogenesis, breast, intestine, lung, lymphoid tissue, and bone, have been
established in genetically defined systems
amenable to analysis. In all models,
genome-wide screens for allelic imbalance have already been conducted.
A number of known and novel gene loci
have been identified. For example in
bone tumours (alpha-particle-induced
osteosarcoma) a set of 10 loci, each
believed to harbour a gene capable of
increasing tumour risk when present
as a germ-line mutation, have been
mapped in the mouse genome.
Partnership
Modern genetic analysis requires largescale coordinated efforts from a multidisciplinary team, with contributions
required in the fields of animal sciences,
radiation biology, tumour pathology,
molecular genetics, and statistics as well
as a mastery of highly complex molecular biology technologies. Radiation protection research is encountering increased
public awareness, but no single European
laboratory can hope to maintain expertise in all of these fields. The specialties
required for this research project are
provided by the partners in the GENRAD
consortium.
Selected references
Dano L., Guilly M.N., Dutrillaux B.,
and Chevillard S., and Laurent D.
Clonal evolution of a radon-induced rat
lung tumor.
Cancer Genet Cytogenet 125, 52-58, 2001.
Title:
Genetic susceptibility to radiation
carcinogenesis (GENRAD)
Co-ordinator:
Michael J. Atkinson
GSF Institut für Pathologie
Ingolstädter Landstrasse 1
D-85764 NEUHERBERG, Germany
Tel: +49.89.3187.2983
Fax: +49.89.3187.3360
e-mail: [email protected]
Partners:
- Michael Rosemann (Technische
Universitaet Muenchen, Germany)
- John Harrison
(NRPB, United Kingdom)
- Sylvie Chevillard (CEA, France)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
Ellender M, Barr M, Houghton O, Harrison JD
and Carr KE.
Gastrointestinal tumour development in irradiated
neonatal and adult ApcMin/+ CHB6 F1 mice.
J Anat. Soc 199 217-227, 2001.
Haines, J., Dunford, R., Moody, J., Ellender, M.,
Cox, R. and Silver, A.
Loss of heterozygosity in spontaneous and
X-ray-induced intestinal tumours arising in F1
hybrid min mice: evidence for sequential loss of
Apc(+) and Dpc4 in tumour development.
Genes Chromosomes Cancer 28 387-394, 2000.
Heidenreich W.F., Atkinson M.J., Paretzke H.G.
Radiation induced cell inactivation can increase
the cancer risk.
Radiat. Res. 155, 868-870, 2001.
Rosemann M., Lintrop M., Favor J., Atkinson M.J.
Mapping of Loci on Mouse Chromosome 7 and
14 that Confer Susceptibility to Osteosarcoma.
Radiat. Res. 157:426-434, 2002.
In the search for genetic polymorphisms
influencing cancer risk the linkage analysis studies have been performed, and
statistical evaluations are underway. In
one tissue this has already led to the
identification of a polymorphism in a
gene controlling the rate of cell proliferation. A highly significant linkage
between inheritance of this polymorphism and the development of cancer
after irradiation has been demonstrated.
41
bjectives
There is strong evidence that some
people have a high sensitivity to
radiation. People with rare cancerprone genetic disorders such as
Detecting differences in radiation
sensitivity of people
ataxia-telangiectasia (AT), if
treated by radiotherapy suffer
severe side effects such as burned
skin. However there are also real
differences in sensitivity between
seemingly healthy people in the
general population. There is an
incentive to developing a reliable
test that could identify radiosensitive individuals. It could, for example be used to avoid unacceptable
normal tissue damage if such a
person developed a cancer and
received radiotherapy. In addition,
there is the socio-legal question of
whether identified sensitive subjects should be employed to work
with radiation. The question of
whether such people are more
liable to develop radiation induced
cancer is still open. The broad
objective of the project was to
develop and validate cellular assays
for human radiosensitivity.
Challenges to be met
A technically demanding assay was
developed, using skin fibroblasts which
were cultured, irradiated in the G2 stage
of their cell cycle and analysed in the
metaphase stage to measure the frequency of radiation induced chromosomal aberrations. Cells from sensitive
subjects showed a higher level of damage. A slightly simpler test was then
developed using blood lymphocytes
also irradiated in the G2 stage. A more
straightforward test was developed, also
using lymphocytes, but irradiated in the
G0 stage and scored for the frequency
of micronuclei seen shortly after the
following metaphase. All three tests have
been applied to cells taken from known
radiosensitive persons such as AT cases
and their relatives and they scored sensitive compared with apparently healthy
control people. There was also evidence
that some cancer patients had a greater
likelihood of being radiosensitive than
healthy controls.
The three tests measure different types
of chromosomal damage all of which
could potentially cause gene rearrangements or losses that are important steps
in cancer formation. Chromosomal damage is broadly classified into two types;
stable and unstable. The former, as its
name suggests, is able to pass through
cell divisions into daughter cells. This
type is therefore more likely to be associated with early steps in the cancer
process. Analysing for randomly distributed stable aberrations – translocations – in large numbers of cells is now
possible by using the fluorescence in
situ hybridisation method (FISH).
A FISH image of a human lymphocyte
metaphase with chromosome pairs 2,3 and 5
highlighted in green, the remainder in blue and
centromeres in red. This cell contains radiation
induced unstable aberrations involving some of
the green chromosomes; a dicentric, a ring and
their associated fragments.
42
The study had two distinct aims. First,
to extend the database of subjects tested
by the G2 and G0 lymphocyte assays to
determine the extent to which they are
comparable in distinguishing radiosensitivity. This work concentrated on sampling newly diagnosed breast cancer
patients and healthy controls. Second to
select four small groups from the data
base, patients and controls who scored
at the high or low extremities of the sensitivity scale by one of the tests; the G0
micronucleus test. Lymphocytes from
these subjects were test irradiated in G0
with gamma rays and examined by FISH
to determine whether any systematic
trend could be found in induced translocation frequencies that correlated with
their sensitivity status.
Achievements
Data were collected for the G2 test on 105
healthy persons and 135 patients and
for the G0 test 68 and 130 respectively.
Eleven controls and 80 patients were
tested by both methods. In each test a
significantly higher proportion, about
40%, of the breast cancer patients showed higher aberration levels compared
with about 10% of the healthy subjects.
Within each test group variability from
repeatedly sampling the same people
was quite small; variability between persons was more marked.
The question was asked whether the two
tests correlate, i.e., does the same person
score high or low in both. Of the 80
patients only 3 scored doubly high; 44
were sensitive in just one whilst 33 were
doubly negative. This poor correlation
indicates that different mechanisms are
responsible for the types of damage measured by the two tests. The well documented radiosensitive syndromes, like AT,
that exhibit sensitivity in both G2 and G0
tests are conditions that involve multisystem disorders. They are characteristic of inherited mutations in regulatory
genes that influence the activity of several other genes. By contrast in breast
cancer patients the latter genes themselves may be where the mutations arise,
with typically only one mutation event
per patient. These would include genes
involved in different DNA damage repair
pathways. A mutation of this type would
PART 3
Information Column
Title:
Investigate mechanisms underlying
inter-individual differences in
sensitivity to ionising radiation
Co-ordinator:
David Lloyd
NRPB-National Radiological
Protection Board
Chilton Didcot
UK-OX11 0RQ OXFORDSHIRE,
United Kingdom
Tel: +44.1235.831.600
Fax: ++44.1235.833.891
e-mail: [email protected]
0
0
Radiation induced G2 aberrations in normal
healthy donors and breast cancer patients.
The vertical line (90th percentile of normals) is
the suggested cut-off point between sensitive/
non-sensitive individuals. About 40% of patients
are sensitive compared with 10% of normals.
Radiation induced micronucleus yields in normal healthy donors and breast cancer patients.
The vertical line (90th percentile of normals) is
the suggested cut-off point between
sensitive/non-sensitive individuals.
About 30% of patients are sensitive compared
with 10% of normals.
therefore tend to confer either G2 or G0
sensitivity but rarely both.
sensitive end of the range. The presumption can be made that this would
also apply to some other cancers. More
research is required on characterising
cellular radiosensitivity and it is highly
likely that several distinct molecular processes operate. Whatever these processes
might be, it appears that they can not be
distinguished by measuring stable chromosomal translocations which are final
products of DNA misrepair.
The FISH study concentrated on 16 subjects; 8 patients and 8 controls with 4 of
each having tested high or low in the G0
sensitivity method. Various combinations
of 11 chromosomes, together comprising
62% of the human genome, were highlighted and their involvements in translocations measured. Following a 2 Gy test
dose given in G0 the induced translocations yields in all 16 study subjects were
very similar and they fell generally within
15% of expected values based on previous dose response studies using a small
pool of healthy donors. There was no significant variability between subjects and
especially no trend could be discerned
that correlated with the radiosensitivity
scores. The relative involvements of the
11 chromosomes tested in different
classes of aberrations was also examined
and again no trend relative to sensitivity
could be found.
The conclusion of this study is that it is
possible to distinguish a spectrum of
radiosensitivity among apparently normal healthy persons and that breast
cancer patients tend to fall within the
Partners:
- D. Scott (PICR-Paterson Institute
for Cancer Research, Manchester,
United Kingdom)
- J.L. Fernandez (COE-Centro
Oncológico de Galicia, Spain)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Partnership
Selected references
This EC funded study enabled two radiobiological disciplines to collaborate. One
partner had a long term research objective to develop sensitivity tests that could
assist with radiotherapy treatment planning. The other partners had developed
FISH analysis for use in biological dosimetry but also had the brief to exploit it as
a tool for more fundamental research.
Much was learned about the sensitivity
tests and the likelihood is that, with further research, they will prove of practical value.
Mitchell EL and Scott D, G2 chromosomal
radiosensitivity of fibroblasts of ataxia-telangiectasia heterozygotes and a Li-Fraumeni syndrome
patient with radioresistant cells.
International Journal of Radiation Biology,
72, 435-438, 1997.
Scott D, Barber JBP, Spreadborough AR,
Burrill W and Roberts SA, Increased chromosomal
radiosensitivity in breast cancer patients:
a comparison of two assays.
International Journal of Radiation Biology,
75, 1-10, 1999.
43
bjectives
The project aimed at investigating
radiation-induced cancer in man
focusing on the molecular and
chromosomal changes that are
Molecular mechanisms of radiation
carcinogenesis in man
induced so that a better understanding of these mechanisms
could be obtained. The approaches
used compared these molecular
Challenges to be met
and chromosomal changes in:
a) The cancers that developed in
children in Belarus induced by
irradiation of their thyroid glands
by radioactive iodine released
following the Chernobyl reactor
accident.
b) Experimentally induced human
tumours produced following irradiation of human thyroid epithelial
cells in the laboratory in cell
culture.
Using this integrated approach
with the expertise of a number of
laboratories across Europe, the aim
was to identify key molecular and
cytogenetic events, which were
important in the development of
radiation-induced cancers in man.
Radiation-induced tumorigenesis is
potentially an emotive issue particularly
when cancers develop in children. They
are caused by exposure of individuals to
environmental sources of radioactivity,
to material released at accidents or following military use to exposure in the
work place and to medical irradiation.
It would be useful to determine whether
signatures of radiation-induced cancers
could be identified. An improved understanding of the molecular mechanisms
and chromosomal changes induced by
radiation would lead to a better understanding of the process of cancer formation. It is also important to establish
whether particular sub-groups of the
population are at increased risk of developing cancer.
Achievements
Radiation-induced childhood cancers
from Belarus.
The consortium have painstakingly
developed a tumour tissue bank from
childhood cancers of the thyroid induced
by radioactive iodine. Using this resource,
it was possible to grow cells in tissue
culture and thus study the chromosomes in the tumours. Specific hot spots
in specific chromosomes where breakpoints occurred have been identified.
Using molecular techniques, the DNA
from specific regions can be amplified and
checked for differences between normal DNA and tumour DNA. Characteristic
rearrangements of the DNA were identified in the childhood tumours where
the chromosomes are broken then
repaired inappropriately leading to
defects in the cells.
Radiation-induced human tumours
induced in cell culture.
An alternative approach is to develop a
laboratory model where normal human
cells can be exposed to radiation and the
stepwise process of cancer development
44
followed. Using this in vitro approach
tumours have been produced following exposure of cell cultures to gamma
irradiation and alpha particle irradiation. Studies of the resulting tumour
cells were able to establish characteristic chromosome changes and changes
in gene expression.
Structural organisation of chromosomal
regions in radiation-induced human
cancers.
A genetic map of chromosome 10 has
been constructed around the region
where breakpoints occur in thyroid
tumours. This was undertaken to try
and explain why breaks occur at specific
regions on the chromosomes and link
this with DNA structure.
The programme has formed a useful
transition into FP5 where new and established collaborations have developed.
Using improved models of human
epithelial cell cultures we have further
established molecular and chromosomal changes in human cancers and will
be applying this model to human breast
cancer.
Partnership
The laboratories taking part in this programme have been collaborating for a
long period of time and the EC funding
has been essential in allowing good links
to be maintained. The range of expertise being applied to this important issue
of cancer induction in man could only
be achieved by collaboration between
laboratories experienced in this field.
Future research will build upon the
advances made in this project.
PART 3
Information Column
Title:
Human cell systems for investigating
the molecular mechanisms of
radiation carcinogenesis in man
Selected references
Gamble S., Cook M., Arrand J., Herceg Z.,
Bryant P. and Riches A.
p53 mutations in tumours derived from irradiated human thyroid epithelial cells.
Mutat. Res. 425: 231-238 (1999)
Riches A., Herceg, Z., Bryant P., Stevens, D
and Goodhead D.
Radiation-induced transformation of SV40immortalised human thyroid epithelial cells
by single exposure to plutonium alpha
particles in vitro.
Int. J. Radiat. Biol. 72: 515-521 (1997)
Smida J., Salassidis K., Hieber L., Zitzelsberger
H., Kellerer A., Demidchik E., Spelsberg F.,
Negele T., Lengfelder E., Werner M. and
Bauchinger M.
Distinct frequency of ret rearrangements
in papillary thyroid carcinomas of children
and adults from Belarus.
Int. J. Cancer 80: 32-38 (1999)
Zitzelsberger H., Lehmann L., Hieber L.,
Weier H-UG., Janish C., Fung J., Negele T.,
Spelsberg F., Lengfelder E., Demidchik E,
Salassidis K., Kellerer A., Werner M. and
Bauchinger M.
Cytogenetic changes in radiation-induced
tumours of the thyroid.
Cancer Res. 59: 135-140 (1999)
Zitzelsberger H., Bruch J., Smida J., Hieber L.,
Peddie C., Bryant P., Riches A., Fung J.,
Weier H-UG and Bauchinger M.
Clonal chromosomal aberrations in simian virus40 transfected human thyroid cells and in derived tumours developed after in vitro irradiation.
Int. J. Cancer 96: 166-177 (2001)
Co-ordinator:
A.C. Riches
University of St. Andrews,
Scotland
Bute Medical Building
ST ANDREWS, Fife KY16 9TS
United Kingdom
Tel: +44.1334.463603
Fax: +44.1334.463600
e-mail: [email protected]
Partners:
- J. Arrand (Brunel University,
United Kingdom)
- A.M. Kellerer (University of Munich,
Germany)
- H. Zitzelsberger/L. Hieber
(GSF, Germany)
- E.M. Geigl (Institut Jacques Monod,
University of Paris, France)
- R. Mustonen/K. Servomaa/T.
Rytomaa (STUK, Finland)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Complex chromosomal changes detected in a
human tumour induced by irradiating human
epithelial cells in culture.
45
bjectives
Ionizing radiation (IR) is an environmental carcinogen to which humans
can be exposed. The precise molecular mechanisms by which IR induces
cancer are poorly understood,
despite advances in knowledge
about the molecular mechanisms of
cancer in general. To be able properly to assess the risks and provide
updated advice both to the public,
as well as to nuclear workers, a
detailed understanding of radiobiological mechanisms is essential. In
particular, more reliable scientific
information is needed about the
nature of the molecular genetic
alterations involved in radiation carcinogenesis in humans, and also of
inter-individual genetic variations in
susceptibility to radiation effects.
Identification of key target genes will
furnish a basis for molecular epidemiological studies aimed at determining the precise role of IR in the
development of human cancers, and
may also lead
to rapid screening procedures for
predicting individual risk.
The project has been designed to
provide an improved understanding
of the genetics of radiation-induced
cancer. This core objective will be
realized by work on human cancers
for which there exists a strong link
with radiation exposure, and work
with cell culture models for human
cancer development – ultimately
based on clinically relevant human
cell types.
Identification and isolation of susceptibility
genes involved in radiation-induced
cancer in humans
(SUS GENES IN RAD CAR)
Challenges to be met
Sources of tissue from human cancers
that can be reliably associated with
radiation exposure are rare. However,
members of the project consortium have
collected panels of such materials from
two sources: thyroid tumour samples
from children from Belarus (associated
with radioiodine contamination from the
Chernobyl accident) and tumour samples
from secondary cancers induced (in the
irradiated field) following radiotherapy for
an unrelated primary tumour. A major
challenge in the project is to employ
state-of-the-art molecular cytogenetic
and genomics techniques to characterize the genetic changes that drive the
process of IR-induced human cancer,
including the identification of individual
cancer susceptibility genes.
It has recently been discovered that activation of the enzyme telomerase which
generates protective caps for chromosomes leads to the immortalization of
normal human cells. This has provided
a means for developing cell culture models of human cancer that permit early
critical steps in the process to be induced
and characterized, in the absence of confounding genetic instability typically
found in many overt cancers. A second
main challenge is to develop such systems and to compare key genetic
changes involved in IR-induced malignant transformation in culture, with
those found in radiation-induced tumour
material. The means by which telomerase is induced during radiation carcinogenesis needs to be established, as
well as the role of telomeres in radiationinduced genetic instability.
Achievements
Analysis of human radiation-induced cancers has revealed common chromosomal and genetic alterations. Progress
towards cloning of translocation breakpoints present in cells from childhood
46
a
b
Example of the use of the monochromosome
transfer technique to screen the human
genome for novel tumour suppressor genes.
(a) shows whole copies of chromosome-1 and
fragments (yellow fluorescence) in a human
breast cancer cell. In (b) an additional normal
copy of chromosome-1 has been transferred
to the cancer cells, which completely represses
their malignancy. (Courtesy Prof. Rob
Newbold, Brunel University. UK)
thyroid tumours has exceeded expectations. Molecular cytogenetic analysis
has identified a number of interesting
translocations including seven involving
a known oncogene (RET). Cytogenetic
and molecular analysis of secondary
IR-induced cancers (following radiotherapy) is also yielding novel results. Rearranged chromosomes have been observed
in these cancers with several common
rearrangements detected. Molecular
studies on 7 tumours have provided evidence for the involvement of the known
tumour suppressor genes p53 and RB1 in
the cancer process. An abrupt change in
chromatin structure in the chromosomal
region carrying RET has also been identified.
Major advances have been made in
establishing cell culture models for radiation carcinogenesis based on human
cells. Repeated exposure of teomerase
immortalized retinal epithelial cells to Xrays converted them into tumour-forming
populations. A large panel of radiationinduced malignant cell clones has been
assembled for molecular and cytogenetic analyses. Three out of seven such
lines were found to have suffered characteristic chromosomal losses. Human
ductal breast epithelial cells have been
PART 3
SUS GENES IN RAD CAR Information Column
Title:
Identification and isolation of
susceptibility genes involved in
radiation-induced cancer in humans
(SUS GENES IN RAD CAR)
immortalized by the same procedures,
and induced to undergo malignant
transformation following IR exposure. A
panel of around 50 transformants are
being isolated for study.
Substantial progress has been made in
the fine mapping by chromosome fragment transfer of a putative telomerase
‘master regulator’ gene on the short arm
of chromosome 3. Several candidate
genes from the region are being studied
for the presence of mutations in human
cancers. In addition, transgenic mouse
model systems have established conclusively that lack of telomerase confers resistance to carcinogenesis and point
towards an important role for telomere
length in radiation sensitivity. Conversely,
experiments in which telomerase has
been constitutively expressed in specific
tissues clearly show that the enzyme substantially enhances susceptibility to cancer induction, and consequently that
telomerase activation is likely to be a key
event in radiation-induced cancer.
Partnership
The partnership brings together a group
of 8 European laboratories that have
considerable expertise directly related to
the scientific goals of the project. The
technical and scientific expertise of the
partners provides a suitable balance
without significant overlap. The collaborations established will be important for
achieving the goals of the project.
Selected references
Riches, A., Peddie, C.M., Rendell, S., Bryant, P.,
Zitzelsberger, H., Bruch, J., Smida, J.,
Bauchinger, M.:
Human tumour formation following ionising
radiation exposure of human epithelial cells.
Radiat. Research, 155: 222-229, 2001.
Goytisolo, F.A., Samper, E., Edmonson, S.,
Taccioli, G.E., and Blasco, M.A. (2001).
Absence of DNA-PKcs in mice results in
anaphase bridges and in increased telomeric
fusions with normal telomere length and
G-strand overhang.
Mol. Cell Biol. 21: 3642-3651.
Lefevre, S. H., Vogt, N., Dutrillaux, A. M.,
Chauveinc, L., Stoppa-Lyonnet, D., Doz, F.,
Desjardins, L., Dutrillaux, B., Chevillard, S.,
and Malfoy, B.
Genome instability in secondary solid tumors
developing after radiotherapy of bilateral
retinoblastoma,
Oncogene. 20: 8092-9, 2001.
Pommier, J. P. and Sabatier, L.
Telomere length distribution. Digital image
processing and statistical analysis,
Methods Mol Biol. 191: 33-63, 2002.
Ducrest, A.L., Amacker, M., Matthieu, Y.,
Reichenbach, P., Cuthbert, A.P., Trott, D.A.,
Newbold, R.F., Nabholz, M. and Lingner, J.
Telomerase reverse transcriptase (TERT) levels are
regulated in the nuc leus: repression by
chromosome 3 does not involve c-Myc.
Cancer Research 61: 7594-7602, 2001.
Co-ordinator:
Robert F. Newbold
Brunel University
Dept. of Biological Sciences
Kingston Lane
UK-UB8 3PH UXBRIDGE, Middlesex,
United Kingdom
Tel: +44.1895.203090
Fax: +44.1895.274348
e-mail: [email protected]
Partners:
- Andrew Riches (University of
St. Andrews, United Kingdom)
- Bauchinger Manfred (GSF,
Forschungszentrum für Umwelt
und Gesundheit, Germany)
- Bernard Dutrillaux (Institut Curie,
France)
- Eva-Maria Geigl (Institut Jacques
Monod, France)
- Laure Sabatier (Commissariat à
l’Energie Atomique, France)
- Maria Antonia Blasco (Consejo
Superior de Investigaciones, Spain)
- Carrol Terleth (Leiden University
Medical Centre, The Netherlands)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
47
bjectives
Thyroid cancer in children and
young people is usually extremely
rare (of the order of 1-1.5 per million per year for those under
15 years of age at operation).
However, in the areas of Belarus,
Pathology and molecular biology of thyroid
tumours in children and young adults
exposed to fallout from the Chernobyl
nuclear disaster
Ukraine and Russia that were
contaminated by fallout from the
nuclear disaster at the Chernobyl
Power Plant in 1986, there has
been a very large increase in
thyroid cancer in those who were
exposed to fallout as children,
highest in those areas which
received the highest levels of
Challenges to be met
70
To develop a network which enabled the
application of a number of highly specialised assay techniques to the material in order to maximise the information
available. To correlate molecular biological marker with clinicopathological
information.
radioiodine contamination.
The largest incidence has been in
80
60
50
40
30
20
10
0
SF
Achievements
CP
DSV
UKRAINE & BELARUS
SF
CP
DSV
ENGLAND & WALES
Gomel oblast in Southern Belarus
(90 per million per year for those
under 15 at operation). To date
there have been approximately
1800 cases of thyroid cancer in
children and young adults in
Belarus, Northern Ukraine and in
those areas of Russia contaminated
by fallout. So far, the increase in
thyroid cancer is the only unequivocal carcinogenic consequence of
exposure to radioactive fallout
from the accident. The objectives
of this project were therefore to
continue to monitor the outbreak
of thyroid cancer and to compare
the molecular biology of these
tumours of radiation aetiology with
those not known to be radiation
induced.
We studied the pathology of over 800
thyroid tumours from patients aged up
to 20 years at operation who were
exposed to fallout from the Chernobyl
accident. The majority (96%) were a
type of thyroid cancer called papillary
carcinoma. In those aged under 14 at
operation, nearly 75% were of a particular subtype of papillary carcinoma
(called solid/follicular SF). In adolescents only just over half of papillary
carcinomas were of this morphology.
This subtype is also found in young
children from a non-radiation exposed
area (England and Wales), but the
frequency was much higher in the
Chernobyl – related cases.
Two other types of thyroid cancer (follicular and medullary carcinoma) were
much less frequent in the study population. More detailed study combined
with previous data suggests that the
latency for the solid/follicular subtype
may be shorter than that for other
thyroid tumour types. If this is true,
adjustments to the prediction of risk of
development of thyroid cancer following exposure of young children may
require revision.
We have also studied the involvement
of certain genes causing thyroid cancer
in 180 of these tumours. These genes
have been shown to be broadly involved
in non-radiation associated thyroid
carcinogenesis; some of these genes are
known to be involved in carcinogenesis in other tissues e.g. the ras and p53
48
Proportion of different subtypes of papillary
carcinoma in children from areas exposed to
fallout from Chernobyl and from England and
Wales.
genes, others appear to be specific to
carcinogenesis in the thyroid, e.g. alterations in the TSH receptor and ret genes.
The ret oncogene codes for a cell surface receptor that is not normally
expressed in thyroid follicular cells. It is
activated when a breakage of chromosome 10 occurs in such a position that
splits the DNA coding for the ret gene
product in two. When the break is
repaired, in some cases the DNA coding for the intracellular part of the ret
gene becomes fused to part of another
gene which is normally expressed in
thyroid follicular cells i.e. the gene is
rearranged. This leads to inappropriate
activation of a signalling pathway inside
the cell. This has been shown to occur
with ret being fused to a variety of
70
PTC 1
60
50
PTC 3
40
Other
30
20
10
0
CP +DSV
SF
Subtype of Papillary Carcinoma
Correlation of type of rearrangement of the
ret oncogene with morphology.
PART 3
Information Column
different genes; however, the most common rearrangement appears to involve
inversion of part of chromosome 10 to
generate two different rearrangements
called PTC1 and PTC3.
Rearrangements of the ret oncogene
have been shown to be associated with
papillary carcinoma. The results from
this study show that only papillary carcinomas show ret gene rearrangement,
follicular tumours from those exposed to
fallout from Chernobyl do not show this
alteration. In addition, we have shown
that a particular type of rearrangement,
PTC3, is associated with the solid/follicular type of papillary carcinoma (see
figure 2). However, the frequency of
involvement of the ret oncogene in post
Chernobyl thyroid tumours does not differ from that of a non-exposed age
matched population, indicating that ret
rearrangement cannot be used as the
sole marker for papillary carcinoma as a
result of exposure to radiation.
We have shown that mutation in ras,
TSH receptor and p53 is not found in
papillary carcinomas, and that a similar
frequency of ras mutation is observed in
thyroid follicular tumours post Chernobyl
to that observed in those from populations not exposed to radioactive fallout.
There is also no evidence from our studies
on loss of heterozygosity for involvement of any of the known tumour suppressor genes in post Chernobyl thyroid
tumourigenesis or for genetic instability in these tumours. In summary, the
molecular biology of post Chernobyl
thyroid tumours does not seem to be
markedly different from that observed
in non radiation associated thyroid
tumours, but there are differences in
the frequency of tumour types. Because
of the rarity of thyroid cancer in children
in a non-exposed population it is difficult to be certain that no differences
exist with respect to the frequency of the
type of ret rearrangement found.
Partnership
cate that further studies will be required
to fully understand the consequences of
the Chernobyl accident, and that we
do not yet have sufficient data to make
accurate risk assessments because of the
changing frequencies in the morphological type of thyroid tumour.
Selected references
Williams GH, Rooney S, Thomas GA,
Cummins G, Williams ED (1996).
Ret activation in adult and childhood papillary
thyroid carcinoma using a RT-nPCR approach
on archival material.
British Journal of Cancer 74: 585-589.
Abrosimov A Yu, Lushnikov EF, Tsyb AF,
Thomas GA, Williams ED (1999)
Thyroid carcinoma post Chernobyl in the Russian
Federation, a pathological study. In: Proceedings
of the First International Symposium on Radiation
and Thyroid Cancer., Thomas GA, Karaoglou A,
Williams ED (eds) pp145-150.
Thomas GA, Bunnell H, Cook HA, Williams ED,
Nerovnya A, Cherstvoy ED, Tronko MD,
Bogdanova TI, Chiapetta G, Viglietto G,
Pentimalli F, Salvatore G, Fusco A, Santoro M,
Vecchio G (1999).
High prevalence of RET/PTC rearrangements
in Ukrainian and Belarussian post Chernobyl
thyroid papillary carcinomas: a strong
correlation between RET/PTC3 and the
solid/follicular variant.
JCEM 84: 4232-4238.
Santoro M, Thomas GA, Vecchio G, Williams GH,
Fusco A, Chiappetta G, Pozcharskaya V,
Bogdanova TI, Cherstvoy ED, Voscoboinik L,
Tronko MD, Carss A, Bunnell H, Tonnachera M,
Parma J, Dumont JE, Keller G, Höfler H,
Williams ED (2000).
Gene rearrangement and Chernobyl related
thyroid cancers. Br J Cancer 82: 315-322.
Title:
Pathology and molecular biology
of thyroid tumours in children and
young adults exposed to fallout from
the Chernobyl nuclear disaster
Co-ordinator:
Dillwyn Williams
Thyroid Carcinogenesis
Research Group
University of Cambridge
Strangeways Research Laboratory
Wort's Causeway
UK-CB1 8RN CAMBRIDGE,
United Kingdom
Tel: +44.1223.243.231
Fax: +44.1223.411.609
e-mail: [email protected]
Gerry A. Thomas
South West Wales Cancer Institute
Singleton Hospital
Sketty
UK- SA2 8QA SWANSEA,
United Kingdom
Tel: +44.1792.285407
Fax: +44.1792.285201
e-mail: [email protected]
Partners:
- J. E. Dumont (IRIBHN, Université
Libre de Bruxelles, Belgium)
- G. Vecchio (Department of Cell
Biology, Cellular Pathology and
Molecular Biology, Università
degli Studi di Napoli, Italy)
- H. Höfler (Institute for Pathology,
Techische Universität München,
Germany)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
The partnership involved European scientists with expertise in pathology and
molecular biology plus collaboration
with colleagues in the former Soviet
Union. The data from this project indi49
bjectives
The key aims of this project are to
(a) provide a better assessment of
thyroid cancer risk posed by
radioactive fallout from the
Chernobyl reactor accident. This
would help to decide appropriate
Chernobyl, an integrated pan-European
study: morphology, oncogenes, DNA repair
and outcome in radiation carcinogenesis
(CHIPS)
population screening procedures
and therefore improve healthcare
economic planning in the event of
a future accident, as well as informing on prophylactic measures,
prognostic markers and health care
surveillance; (b) indicate new therapeutic avenues, which may be of
use in other human cancers; (c)
lead to a better understanding of
the relationship between radiation
exposure, gene defects and DNA
repair in carcinogenesis.
Challenges to be met
The consequences of the Chernobyl
accident have provided a unique possibility to advance our understanding of
thyroid carcinogenesis in general, and
in particular the factors involved in the
genesis of a tumour where the aetiological agent is known – exposure to ionising radiation in childhood. It is clear
from our earlier results that alterations
other than those in the ret oncogene
must be involved in thyroid carcinogenesis, but that these factors are not
those which have already been shown
to be involved in non-radiation related
thyroid carcinogenesis. This project will
continue study of the involvement of the
ret oncogene to see if the pattern of
involvement changes with time; it also
seeks to identify novel markers involved
in thyroid carcinogenesis. It is essential
that an integrated approach is applied,
as it is increasingly likely that several different oncogenes will participate in the
development of a tumour.
Achievements
FISH on a follicular carcinoma from Ukraine.
The FISH technique uses three overlapping
probes to the ret locus on 10q11.2. Probe
55A10 binds to 3’ end (red), probe 214H10
to mid region (green) and probe 313F4 to 5’
(green). The majority of the nuclei show
overlapping signals. One nucleus (arrowed)
shows a clearly split signal, indicating a rearrangement of that region of chromosome 10.
It has been suggested recently that rearrangement of the ret oncogenes may occur in lymphoid cells and it has been shown to occur in
fibroblasts in vitro. Thyroid cancers are composed of a number of cell types; the finding of
a single cell that contains a split signal could
be the result of a rearrangement occurring in
a non-epithelial cell.
50
Work package (WP 1) will obtain RNA
and paraffin sections from post Chernobyl
thyroid tumours from the NISCTB (EC
project no. FIS5 2001 008). Thus far
the majority of thyroid cancers post
Chernobyl have been of the papillary
type, but follicular tumours are becoming more frequent. We will document
any change in the pathology of post
Chernobyl tumours with time by comparing the results from these studies
with those of our previous project.
We will also use a variety of different
techniques to study the frequency of
ret rearrangement (WP3 and 4), and to
look for rearrangement involving other
chromosomes using fluorescence in situ
(FISH) analysis (an example is provided
in figure 1). It is clear from our previous
studies that ret is not the only gene that
is involved in thyroid carcinogenesis.
We have already shown that other genes
known to be involved in other types of
thyroid cancer with different aetiologies are not involved in the post
Chernobyl thyroid cancers. WP2 will use
the new technology of cDNA array to
identify other genes which may be
altered in the carcinogenic pathway.
WP4 will develop epithelial cell cultures
from both normal thyroid and from thyroid tumours and will investigate
whether culturing the cells alters the ret
rearrangement status or alters any other
tumour related rearrangement identified
using FISH. This could indicate that a
rearrangement of the ret oncogene was
present in a cell type other than the
epithelial cells which compose the
majority of the tumour. FISH is a very
powerful technique which allows identification of chromosomal abnormalities in single cells and will be very useful
in approaching problems of this type.
Control of cell division involves a complicated cascade of signals initially
received at the cell surface (through
proteins such as ret) which can trigger
replication of the DNA. DNA replication is strictly controlled in normal cells;
one hallmark of cancers is that DNA
replication is dysregulated. There are
many factors which control DNA replication; unwinding of the DNA to allow
replication is one of these. There are a
number of proteins which have recently
been identified, called minichromosome
maintenance (mcm) proteins, which
form the origin recognition complex
where DNA synthesis starts. The consortium involved in this project has
developed an assay which allows assessment of the presence of these proteins
in isolated nuclear and cytoplasmic
cytosol.
MCM protiens can also be demonstrated in intact sections of tissue. Nuclei
in cells which are primed to undergo
DNA replication stain red in this system;
PART 3
CHIPS Information Column
those that are at rest stain blue. In Selected references
figure 2, the upper panel shows normal
adult mouse thyroid which has a very Salassidis K, Bruch J, Zitzelsberger H,
low growth rate, and therefore few cells Lengfelder E, Kellerer AM, Bauchinger M. (2000).
are positive (arrow). The lower panel Translocation t(10;14)(q11.2:q22.1)
shows actively growing mouse thyroid, fusing the kinetin to the RET gene creates a
where clearly many cells are in a state novel rearranged form (PTC8) of the RET
of readiness to undergo DNA replication. proto-oncogene in radiation-induced childhood
WP5 will compare the results obtained papillary thyroid carcinoma.
using mcm assay with those using Cancer Res. 60: 2786-2789.
conventional markers of
Monaco C, Visconti R,
dividing cells. We will relate
Barone MV, Pierantoni GM,
cellular growth rate to the
Berlingieri MT, De Lorenzo C,
presence and type of ret
Mineo A, Vecchio G, Fusco A,
rearrangement, clinical
Santoro M. (2001).
parameters which measure
The RFG oligomerization
the invasiveness and the
domain mediates kinase
ability of the tumour to
activation and re-localization
metastasise. A key aim is to
of the RET/PTC3 oncoprotein
investigate in vitro the
to the plasma membrane.
factors which are involved
Oncogene 20:599-608.
in the regulation of DNA
Stoeber K, Tlsty TD,
replication and compare
Happerfield L, Thomas GA,
these with results obtained
Romanov S, Bobrow L,
on paraffin embedded
Williams ED. Williams GH
Mcm2 protein in mouse
material from the primary
(2001).
thyroid
visualised
using
tumour. These studies will
DNA replication licensing and
immunocytochemistry.
help us to assess whether
human cell proliferation.
in vitro cell cultures of thyJ Cell Science 114: 2027-2041.
roid tumours could be
used as a good guide to the anomalies Basolo F, Giannini R, Monaco C, Melillo RM,
that are present in the primary tumour. Carlomagno F, Pancrazi M, Salvatore G,
The comparison of all these scientific Chiappetta G, Pacini F, Elisei R, Miccoli P,
data with information on the clinical Pinchera A, Fusco A and Santoro M (2002).
aspects of the disease in the patient The potent mitogenicity of the RET/PTC3
from which the tumour was removed oncogene correlates with its prevalence in
will help us to assess which of the mark- agrressive variants of papillary thyroid
ers we have identified provide informa- carcinomas.
tion on the prognosis for the patient. Am J Pathol. 160: 247-254.
The identification of key factors involved
in the generation of the tumour will
also open new avenues for therapy.
Title:
Chernobyl, an integrated pan-European
study: morphology, oncogenes,
DNA repair and outcome in radiation
carcinogenesis (CHIPS)
Co-ordinator:
Gerry A. Thomas
South West Wales Cancer Institute
Singleton Hospital
Sketty
UK- SA2 8QA SWANSEA,
United Kingdom
Tel: +44.1792.285407
Fax: +44.1792.285201
e-mail: [email protected]
Partners:
- Mykola Tronko/T.I. Bogdanova
(Institute of Endoctrinology
and Metabolism, Ukraine)
- Massimo Santoro (Università
di Napoli "Federico II", Italy)
- Jacques E. Dumont (IRIBHN,
Université Libre de Bruxelles,
Belgium)
- Manfred Bauchinger
(GSF - Forschungszentrum für
Umwelt und Gesundheit, Germany)
EC Scientific Officer:
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
Partnership
This project is a collaboration involving
5 main European centres. It is a development of a longstanding partnership
between these centres to investigate
the consequences of the Chernobyl accident with particular reference to thyroid
cancer; it will extend information provided by our earlier project “Pathology
and molecular biology of thyroid tumours
in children and young adults exposed
to fallout from the Chernobyl nuclear
disaster”.
51
bjectives
This project will form an internationally supported collaborative
research resource for many groups
working on the health effects of
the Chernobyl accident. The basis
The newly independent states Chernobyl
tumour bank - an international scientific
resource (NISCTB)
of the project is a collaborative
approach to the collection and documentation of specimens of thyroid tumour from patients who
were under 19 years at exposure to
fallout from Chernobyl. It will provide an internationally agreed
pathological diagnosis for each
specimen and make nucleic acid
available to researchers worldwide. The project will provide a
valuable research tool for present
and future researchers interested in
the mechanism of radiation tumorigenesis as well as providing data
for assessment of radiological risk.
Challenges to be met
This project aims to promote collaboration and avoid competition for a very
valuable but limited resource; to properly document in a uniform manner
pathological specimens and to collect
blood samples from those exposed as
children to radioactive fallout from the
Chernobyl accident, who subsequently
develop thyroid tumours; to provide
aliquots of nucleic acid extracted from
thyroid tumour, normal thyroid and
blood from these cases to the worldwide
scientific community for research; to
collate and record data on a case by
case basis from scientific projects that
have access to material and to provide
a database which permits later multifactorial analysis for each case.
the tissue was removed. e) International
agencies should participate in such a
project, providing financial support,
training, equipment and expert advice.
f) The relevant countries of the former
Soviet Union (FSU) will be fully represented in all committees related to
the implementation of the project.
There should be full agreement within
the committees of the thyroid tissue
and data bank and the scientists intimately involved in the project regarding the protocols used for the
collection of material and to maintain
the archive.
The project is a cooperation between the
governments of Belarus, Russia and
Ukraine and the European Commission,
the National Cancer Institute of the
US, the Sasakawa Memorial
Health Foundation and the
World Health Organisation.
This project is a unique
response to a unique situation. The occurrence of
such a large number of
human tumours of a single tissue, the majority of
which are of a single histological type, due to a
known cause on a known
date provides an opportunity to fully investigate the
link between exposure to
Geographical location of the Chernobyl Nuclear Power Plant
radiation and thyroid carcinogenesis. The study cohort
Achievements
is composed of all cases of thyroid carcinoma and cellular follicular adenoma
The project is governed by six important from patients who were born after 26th
principles:
April 1967 (i.e. under 19 years at the
time of the Chernobyl accident) and
a) The interests of the patient must who are or were at the time of the
come first. b) Spare tissue of potential accident, resident in Belarus, Northern
value should not be wasted, c) Blood oblasts of Ukraine or 4 contaminated
and tissue samples should only be used oblasts of Russia. Thyroid cancer relaif appropriate consent is given by the tively rare in this age group – therefore
patient, and the laws of the country the majority of cases are due to expoconcerned are observed. d) A tissue sure to radioiodine. Informed consent
bank and a nucleic acid bank should is obtained from the patient or his/her
be established in the country in which parent or guardian, a sample of blood
52
PART 3
NISTCB Information Column
is taken pre-operatively for serum separation and extraction of DNA from
blood. The patient is then operated on
and the operative specimen is seen by
the pathologist. The pathologist then
records the location of the tumour or
tumours on a simple diagram which is
later scanned into a database, noting the
positions from which he or she has taken
blocks of tissue that will be processed to
paraffin for diagnosis. When the material that is necessary for accurate diagnosis has been taken, small blocks of
tissue from the remainder of the tumour
and normal thyroid are snap frozen. The
majority of these tumours are small, but
where possible 3 blocks of tissue from
tumour and three from normal thyroid
are taken. The position of these blocks
relative to those taken for paraffin are
noted on the form.
It is very important to ensure that all the
information relating to these specimens
is stored in a way that can be accessed
easily. Each of the three Institutes involved
in the project has an identical Access
database and a back-up integrated database is kept at the Coordinating Centre.
All patient data is anonymised and is
totally confidential. The identical format enables regular updates to be made
to the Coordinating Centre and facilitates exchange of data between centres
if so wished.
began in 2000. The extraction of RNA
and DNA from tissue commenced in
March 2000. The current status of the
bank is as follows: full pathology documentation on 1380 cases, (720 from
Belarus, 447 from Ukraine and 213 from
Russia); of these 913 are papillary carcinoma. A significant number of benign
follicular tumours are also included in the
NISCTB. Frozen material is available from
1139 cases (614 from Belarus, 361 from
Ukraine and 164 from Russia). The majority of cases have samples of both normal
tissue and tumour available. Aliquots of
DNA and RNA are available from 421
cases with more than 4000 individual
aliquots of RNA and DNA from tissue and
tumour. In over half of these cases there
are more than 5 standard aliquots for
both tissue and tumour available. In
addition serum and samples of blood
from which DNA will be extracted are
available from 171 of these cases.
Applications for access to materials and
information are now invited. All applications are reviewed by an External
Panel of experts nominated by the
governments and the sponsors of the
project. Further details of the project,
together with information on how
to make an application for access
can be found on the project website
(http://nisctb.swan.ac.uk).
Partnership
One of the major aims of the project is
the release of nucleic acid (DNA and
RNA) to researchers. Provision of pieces
of tissue to individual researchers would
result in wastage of a lot of nucleic acid.
DNA and RNA are therefore extracted
from the same piece of tumour or normal thyroid, and standard-sized aliquots
are provided to researchers. Quality control is carried out on each extract by RTPCR (reverse transcriptase polymerase
chain reaction) for RNA and PCR for
DNA. The quality control information is
recorded on the database. DNA will also
be extracted from blood and this will be
available in the near future.
This is an experienced consortium with
skills in clinical medicine, pathology,
molecular biology and data maintenance; the success of the project is
dependent upon ongoing collaboration.
Selected references
Thomas GA, Williams ED, et al., (2000).
Thyroid Tumour Banks.
Science 289: 2283.
Title:
The newly independent states
Chernobyl tumour bank an international scientific resource
(NISCTB)
Co-ordinator:
Gerry A. Thomas
South West Wales Cancer Institute
Singleton Hospital
Sketty
UK- SA2 8QA SWANSEA,
United Kingdom
Tel: +44.1792.285407
Fax: +44.1792.285201
e-mail: [email protected]
Partners:
- Vladislav Ostapenko
(Research and Clinical Institute for
Radiation Medicine and
Endocrionology, Belarus)
- Mykola Tronko
(Institute of Endocrinology and
Metabolism of the Academy of
Sciences of Ukraine, Ukraine)
- Anatoly Tsyb (Medical Radiological
Research Centre of the Russian
Academy of Sciences, Russian
Federation)
- Aldo Pinchera
(Universita di Pisa, Italy)
EC Scientific Officer:
Neale Kelly
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1998-2002
Status: Ongoing
Williams ED, Abrosimov A, Bogdanova TI,
Ito M, Rosai J, Sidorov Yu, Thomas GA (2000).
Two proposals regarding the terminology of
thyroid tumours.
Int J Surgical Pathology 8 181-183.
The documentation of the pathology of
thyroid tumours and collection of frozen
tissue from tumour and normal thyroid
(where available) began on 1st October
1998, and collection of blood specimens
53
bjectives
The aim of this project was to
improve the radiation protection of
humans and their environment by
a better understanding of the
Biophysical models for the induction
of cancer by radiation
mechanisms of radiation action at low
doses and dose rates for different
radiation qualities. The group is
confident that it contributed
substantially with this first internationally coordinated project on this
topic to a more solid basis for such
urgently needed, risk estimation
models.
In particular the project aimed to
integrate a maximum of present
knowledge in radiation research
into comprehensive mechanistic
models for the induction by
radiation of somatic late effects.
These models included the formation of chromosome aberrations
and of mutations in humans and
the many physical, chemical and
biological processes that determine
these consequences. These models
Challenges to be met
An important problem is the quantification of radiation cancer risks to
humans at low doses and low dose rates
of ionising radiation of various qualities.
Since this problem cannot be solved by
biological investigations nor by epidemiological studies alone, the scientific
knowledge from both areas have to
be combined to develop and test quantitative mechanistic models for the
dose-time-effect surfaces of radiation
carcinogenesis. Existing data and theories have to be evaluated and used for
the modelling of radiation transport and
interaction, for the modelling of subsequent damage and repair of DNA,
genes, cells and organs taking normal
oxidative damage into account, and for
ultimately serve as a better basis
for extrapolation of epidemiological
data for human radiation risks to
low doses and dose rates, other
types of ionizing radiation and
different exposed populations,
and individual radio-sensitivities.
Simulation of the track of a 1 MeV proton traversing a piece of the 30 nm chromatin fiber
with a stochastic crossed-linker structure (red:
ionisations and excitations by the proton and
secondary electrons, blue: phosphate, white:
sugar, gold: bases of the DNA, turquoise:
histones).
the multi-step modelling of carcinogenesis. Conclusions have to be drawn
for the general radiation protection of
the worker and the public.
Achievements
The project contains six work packages
(WP):
WP 1 “Mechanistic Models for Radiation
Oncogenesis”: The two-stage clonal
expansion model of Moolgavkar, Venzon
and Knudson was selected as the basic
model. The model can help in extrapolation to low dose rates: when applied to
data on lung tumours in radon-exposed
miners and rats, radiation was found to
act on both initiation and promotion;
the model predicts the possibility of a
dose-rate effect at low dose rates, and an
inverse dose-rate effect at high dose rates.
WP 2 “Mechanistic Models
for Chromosome Aberrations”:
A biophysical model for chromosome
aberration induction by ionizing radiation was developed. The code allows
the simulation of dose response curves
for different kinds of aberrations, under
the main assumption that a subclass of
DNA double strand breaks (dsb), namely
complex lesions, are responsible for
chromosome aberrations, less severe
damages being repaired systematically.
WP 3 “Mechanistic Models
for Mutagenesis”:
Mutation at the hprt gene locus has
been selected as a paradigm for modelling radiation mutagenesis because
this locus has been most broadly studied
experimentally. For this purpose an
extensive database of about 400 reference records has been compiled of hprt
radiation mutagenesis. Measured patterns of exon-deletions in hprt-mutants
after X-ray irradiation could be reproduced by simulations with mutants
arising from pairs of dsb due to the nonrandom DNA fragmentation below 10
base pairs.
54
PART 4 : Modelling radiation cancer risk
ACRONYM
ColumnColumn
LOW DOSE ???
RISKInformation
MODELS Information
4TH FRAMEWORK PROGRAMME
WP 4 “Mechanistic Models for DNA
Damage and Repair”:
Computations of clustered and simple
DNA damage have shown a relevant
role of clustered DNA damage determining cell killing. Inclusion of chemical
pathways for indirect effects in the models show that >50% of low LET induced
dsb are complex. For high LET radiations, the spectrum of complex lesions
shifts to more complexity and some of
the very complex lesions may be unique
to high LET radiation. Protons produce
a more complex spectrum of dsb than
alpha-particles for the some LET.
WP 5 “Chemical Pathways Involving
Track Species in Cells”.
Comparisons of various Monte Carlo
track structure codes show similarities in
the distribution of damage at the chromatin level whereas differences are
noticed at the level of naked DNA.
WP 6 “Production of Initial Track
Species in Mammalian Cells”:
An atomic model of DNA able to simulate higher order DNA structures has
been developed and coupled with the
track structure modules in the PARTRAC
code. A new set of cross section for
electron transport in liquid water has
been derived and implemented.
WP 7 “Transport of Radiation to Cells
of Interest”:
Data from related GSF- and PSI- work
provided the necessary start spectra.
The FP5 project Low Dose Risk Models
It is the main goal of this project to
improve our knowledge on the risk estimates for somatic health effects in various groups of humans after exposures
to low doses of various types of ionising
radiation at low dose rates.
The project is organized in a "bottom up
design" manner into five work packages:
1) Critical processes and data
evaluation,
2) Primary damage models,
3) Repair consequences models,
4) Mechanistic cancer model
development,
5) Conclusions for radiation protection.
The modelling work of the present project can consider results of experimental work done in other consortia and
draw conclusions on the consequences
for risk to humans exposed to radiation.
The research concentrates on radiation
induced cancer, but the mechanisms
are likely to be to some extent universal for cancer induced by other environmental or nutritional agents. So the
project may also help to estimate small
additional cancer risks due to other reasons than radiation.
Partnership
The success of these projects has depended upon the combined multidisciplinary expertise of a total of eight EU
laboratories.
Selected references
M. Dingfelder, M. Inokuti, H.G. Paretzke:
Inelastic-collision cross section of liquid water for
interactions of energetic protons,
Radiat. Phys. Chem. 59, 255-275 (2000).
F. Ballarini, M. Biaggi, M. Merzagora,
A. Ottolenghi, M. Dingfelder, W. Friedland,
P. Jacob, H.G. Paretzke: Stochastic aspects and
uncertainties in the prechemical and chemical
stages of electron tracks in liquid water:
a quantitative analysis based on Monte Carlo
simulations.
Radiat. Environ. Biophys. 39, 179-188 (2000).
F.A. Cucinotta, H. Nikjoo, P. O'Neill,
D.T. Goodhead: Kinetics of DSB rejoining and
formation of simple chromosome aberrations.
Int. J. Rad. Biol. 76, 1463-1474 (2000).
W.F. Heidenreich, M.J.P. Brugmans, M.P. Little,
H.P. Leenhouts, H.G. Paretzke, M. Morin,
J. Lafuma: Analysis of lung tumour risk in
radon-exposed rats: an intercomparison of
multi-step modelling.
Radiat. Environ. Biophys. 39, 235-263 (2000).
D.T. Goodhead, J. Hendry: Radiological risks of
depleted Uranium. In: The Health Hazards of
Depleted Uranium Munitions, Part 1, The Royal
Society, London, p. 59-73 (2001).
Title:
Biophysical models for the induction
of cancer by radiation
Co-ordinator:
Herwig G. Paretzke
GSF-Forschungszentrum für Umwelt
und Gesundheit GmbH
Institut für Strahlenschutz
Ingolstädter Landstrasse 1
D-85764 NEUHERBERG, Germany
Tel: +49.89.3187.006
Fax: +49.89.3187.3323
e-mail: [email protected]
Partners:
- D. Goodhead/P.O'Neill (Medical
Research Council, United Kingdom)
- H.P. Leenhouts (Rijksinstituut voor
Volksgezondheit en Milieuhygiene,
The Netherlands)
- M. Terrissol (Association pour le
Développement de la Physique
Atomique de Toulouse, France)
- C. von Sonntag (Max Planck Institut
für Strahlenchemie, Germany)
- A. Ottolenghi (Università degli Studi
di Milano, Italy)
- A. Edwards/M. Little
(NRPB, United Kingdom)
- J. Stepanek (Paul Scherrer Institut,
Switerland)
Period: Nuclear Energy 1994-1998
Status: Completed
5TH FRAMEWORK PROGRAMME
Title:
Improved cancer risk quantification
for environmental, medical and
occupational exposures to low dose
of ionising radiation by mechanistic
models (LOW DOSE RISK MODELS)
Co-ordinator:
Herwig G. Paretzke, GSF, Germany
Partners:
- Dudley Goodhead (Medical Research
Council, United Kingdom)
- Andrea Ottolenghi (Università
degli Studi Milano, Italy)
- Henk Leenhouts (RIVM, Bilthoven,
The Netherlands)
- Alan Edwards (NRPB, United Kingdom)
- Michel Terrissol (Université Paul
Sabatier, France)
- Werner Hofmann (University of
Salzburg, Austria)
- Annette Kopp-Schneider (DKFZ,
Germany)
Period: Nuclear Energy 1998-2002
Status: Ongoing
EC Scientific Officer:
Diederik Teunen - Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
55
bjectives
This post Chernobyl project had
the following objectives:
a) development and implementation of protocols for the correct use
of l-thyroxine suppressive therapy
in childhood thyroid cancer.
Development of optimal protocols
for the diagnosis, treatment, follow-up
and preventive measures for post-Chernobyl
thyroid carcinoma (NUFISA)
b) development of new diagnostic
imaging techniques for the postsurgical monitoring of thyroid
carcinoma, including the possible
use of recombinant human thyroid
stimulating hormone (TSH).
c) study of autoimmune phenomena in radiation-induced childhood
thyroid cancer.
d) evaluation of the biological and
clinical behavior of radiationinduced thyroid carcinoma as
compared with sporadic thyroid
carcinoma.
e) assessment of iodine deficiency
and the need for iodine supplementation and thyroid suppressive
therapy in the prevention of
radiation-induced thyroid cancer.
Number of cases
Challenges to be met
The Chernobyl nuclear reactor accident
took place in April 26, 1986. The accident was unprecedented in scale and in
size of the population exposed. Very
large amounts of radioactivity, including
isotopes of iodine were released from
the reactor. The most apparent health
consequence of the accident was the
increase in the incidence of thyroid
cancer in the following years. Thyroid
carcinoma occurred mostly in children
of southern Belarus, northern Ukraine,
and, to a lesser extent in Russia. Western
experts have independently confirmed
the pathological diagnosis of the great
majority of cases. Tumors are virtually all
papillary carcinomas, relatively aggressive, with the majority showing invasion of extra-thyroid tissues, and lymph
node metastases. Children have been
shown to be more susceptible than
adults to the carcinogenic affect of radiation to the thyroid. Radiation dose to
the thyroid from isotopes of iodine was
probably higher in areas of iodine deficiency, such as the regions surrounding
Chernobyl. Thus, the most likely cause
of the post-Chernobyl increase in childhood thyroid cancer found in Belarus
and Ukraine is the release of radioactive
isotopes of iodine
from the reactor.
Children
Adolescents
Year of diagnosis
Number of children and adolescent patients affected by postChernobyl thyroid cancer and distribution according to the year of
diagnosis.
56
In addition to the
basic question of the
causal relationship
between the postChernobyl outbreak of
thyroid cancer and
exposure to the radioactive isotopes of
iodine released by the
reactor, several questions have been raised
by the "epidemic" of
post-Chernobyl thyroid carcinomas:
1) Does thyroid cancer in young children
differ in its clinical behavior from that in
older individuals? 2) Is there evidence
suggesting that thyroid cancer caused
by radiation behaves differently from
sporadic thyroid cancer? 3) What should
be the guidelines in managing children
with thyroid cancer, especially in regard
to surgical treatment and post-operative medical treatment and follow-up
procedures. 4) Is there a possible role
for the prophylactic use of iodine and/or
thyroxine to suppress pituitary TSH in
the very large population exposed to
the Chernobyl accident?
The present project was aimed to answer
these questions, having in mind the
concept that the outcome of differentiated thyroid carcinoma in children is
largely dependent on the adequacy of
the management, at the diagnostic,
therapeutic and follow-up levels.
Achievements
The present project was aimed to improve
the health status of the population
exposed to the Chernobyl accident and
to develop effective means for managing the radiobiological consequences of
the radioactive contamination, with
particular regard to the post-Chernobyl
thyroid carcinoma. The project was
designed as a natural continuation in
the first collaborative project (JSP4)
between CIS countries and European
Union.
According to the project objectives, the
definition of guidelines for diagnosis,
treatment and follow-up of thyroid
cancer, including the correct use of
l-thyroxine suppressive therapy after
surgery have been developed and validated in the affected territories. This
resulted in a dramatic amelioration of
the metabolic controls of hypothyroidism in thyroid cancer children and
in the standardization of laboratory procedures to be used for the correct follow-up of thyroid carcinoma.
PART 5 : Diagnosis/treatment of cancer and radiation injury
NUFISA Information Column
Title:
Development of optimal protocols for
the diagnosis, treatment, follow-up
and preventive measures for postChernobyl thyroid carcinoma
(NUFISA)
Development of new diagnostic imaging techniques and standardization of
traditional techniques for the post-surgical monitoring of thyroid carcinoma
have been developed and made available to the CIS colleagues.
New insights in the non-tumoral thyroid
consequences of the Chernobyl accident
have also been studied The main achievement was the discovery of autoimmune
phenomena in children and adolescents
exposed to the radioactive fallout. This
finding opens the important question of
following exposed subjects in the future
not only for the early diagnosis of thyroid nodules and cancer but also for the
development of autoimmune thyroiditis with or without hypothyroidism.
The evaluation of the biological and
clinical behavior of radiation-induced
thyroid carcinoma, including the response to treatment, compared to that
of naturally-occurring differentiated thyroid cancer in western European children
and adolescents has been accomplished
through analysis of data on more than
500 children treated in western Europe
in the past 20 years. The results of this
study have shown that thyroid cancer
in children is a very well treatable disease in general, and that the radiationinduced variant of papillary thyroid
cancer has the same response to modern treatment as naturally occurring
tumors. The assessment of iodine deficiency and the employment of iodine
prophylaxis, has been the most difficult
task to perform, due both to logistic
difficulties and to lack of well documented historical information. However,
the project developed a detailed map of
iodine deficiency in contaminated territories that will constitute the basis
for future development of programs
devoted to the correction of iodine deficiency disorders. Furthermore, a simple
method for measurement of urinary
iodine excretion has been developed and
validated in a large number of patients.
All the information collected during the
three years period of the project has,
whenever possible, been described in
written documentation in the form of
books, flow-charts, guidelines and recommendations translated into Russian
and distributed to a large number of
specialists.
Partnership
The project brought together four
European partners with complementary
expertise in clinical medicine and
tumour diagnosis and therapy.
Selected references
F Pacini, T Vorontsova, E Molinaro, E Shavrova,
L Agate, E Kuchinskaya, R Elisei, EP Demidchik,
A. Pinchera. Thyroid consequences of the
Chernobyl nuclear accident.
Acta Paediatr (Suppl) 433:23-7, 1999.
Co-ordinator:
Aldo Pinchera
University of Pisa
Via Paradisa
I-56124 PISA, Italy
Tel: +39.050.544723
Fax: +39.050.578772
e-mail: [email protected]
Partners:
- F. Pacini (University of Pisa, Italy)
- F. Delange (Brussels, Belgium)
- C. Reiners (Wurzburg, Germany)
- M. Schlumberger (Paris, France)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Elisei R, Romei C, Vorontsova T, Cosci B,
Veremeychik V, Kuchinskaya E, Basolo F,
Demidchik EP, Miccoli P, Pinchera A, Pacini F.
Ret/ptc rearrangements in thyroid nodules:
studies in irradiated and not irradiated,
malignant and benign thyroid lesions in
children and adults.
J Clin Endocrinol Metab 86:3211-6, 2001.
Pacini F, Molinaro E, Lippi F, Castagna MG, Agate
L, Ceccarelli C, Taddei D, Elisei R, Capezzone M,
Pinchera A. Prediction of disease status by
recombinant human TSH-stimulated serum
Tg in the postsurgical follow-up of differentiated
thyroid carcinoma.
J Clin Endocrinol Metab 86:5686-90, 2001.
Lippi F, Capezzone M, Angelini F, Taddei D,
Molinaro E, Pinchera A, Pacini F. Radioiodine
treatment of metastatic differentiated thyroid
cancer in patients on L-thyroxine, using
recombinant human TSH.
Eur J Endocrinol 144:5-11, 2001.
Oliynyk V, Epshtein O, Sovenko T, Tronko M,
Elisei R, Pacini F, Pinchera A. Post-surgical
ablation of thyroid residues with radioiodine in
Ukrainian children and adolescents affected by
post-Chernobyl differentiated thyroid cancer.
J Endocrinol Invest 24:445-7, 2001.
57
bjectives
The frequency of thyroid cancer in
children from Belarus, the Ukraine and
the Western part of Russia is increasing
since 1990. Totally, in the 3 republics
afflicted by radioactive fallout from
the Chernobyl accident more than
Improvement of dosimetry for I-131 therapy
of lung metastases with special regard to
children with thyroid cancer from Belarus
following the Chernobyl accident
1000 cases of thyroid cancer in children
and adolescents have been diagnosed
between 1990 and 1996 as compared
to approximately 100 cases diagnosed
between 1986 and 1989. Standard
treatment is surgery followed by
radioiodine therapy. About 15% of the
cases present with distant metastases,
mainly pulmonary. The main problem
in treating disseminated pulmonary
metastases in children with papillary
thyroid cancer is to find a balance
between an insufficient dose for the
ablation of metastatic tissue and
unnecessary high radiation exposure
to the lungs and the bone marrow.
Visualisation of the spatial dose distribution for 131I incorporation in a focal metastasis in the left lung
(right side) and in disseminated metastases in the left lung (left side) in a phantom. Different colours
mean different percentages of the maximum dose (green: 0-20%, cyan: 20-40%, blue: 40-60%,
magenta: 60-80%, red: 80-100%; within each colour code, darker colours stand for a lower
percentage, brighter colours for a higher percentage).
The major goal of this project was to
improve the concept for treating
patients with lung metastases induced
Challenges to be met
by thyroid carcinoma. Almost all of the
patients with lung metastases are
treated by more than one therapy
course. After each course the knowledge of the doses to the tumor tissue,
the lung, and the bone marrow is of
crucial importance for a well funded
decision about further treatment.
In the cases of either the tumor doses
being inefficient for ablation or substantial impairment of residuing pulmonary metastases or the cumulated
doses to lung and bone marrow
exceeds empirically defined “critical”
limits, the fractionated radioiodine
treatment has to be stopped to avoid
side effects such as lung fibrosis or
leukemia in patients with papillary
thyroid cancer. The decision which has
to be taken must consider that prognosis
with respect to mortality and quality
of life even in the case of persisting
lung metastases may be better than
after the induction of progressive
pulmonary fibrosis. The broad
objective was to seek the knowledge
necessary for these clinical decisions.
58
The principle challenges were:
To improve the measuring techniques
for the determination of iodine kinetics
and mass of thyroid carcinoma metastases particularly for lung tissue. To
improve dosimetric methods and models. To determine cross sections for the
interaction of electrons and photons
with lung tissue for the purpose of
Monte-Carlo simulations. To coordinate
European efforts to treat patients with
pulmonary metastases of thyroid cancer;
to propose, test and apply an optimal
protocol for the treatment of those
patients. To follow-up the treated patients
in order to observe late effects of radioiodine therapy.
Achievements
In the course of this project it could be
shown that it is possible to measure the
biokinetics of I-131 under therapeutic
conditions, particularly during the first
24 hours after the application of radioiodine. Monte-Carlo calculations for the
decay of I-131 in small spheres representing small lung metastases were performed. In addition, as some new data
point out, the decay of I-125 in small
spheres was simulated as this radionuclide might be superior for the treatment of small metastases. Further
calculations were carried out simulating the two extreme cases, diffuse
metastases and focal metases, in the
lungs for a phantom (see figure). The
results show that in the case of a focal
metastasis the dose to the lung is more
than ten-fold lower than in the case of
diffuse metastases.
Applying all these data to the treatment
of children it was shown that the achievable dose to the lung and/or tumor with
one radioiodine therapy is limited to a
few Gy. It may not be possible to reach
the tumoricidal dose of 80 Gy with just
one therapy as exceptionally high activities of I-131 would have to be administered. This might cause severe side
effects as the blood dose may well
exceed 2 Gy. Several courses of radioiodine treatment will probably reduce the
dose delivered to the metastases per
therapy.
An ongoing treatment project in
Germany with children from Belarus
shows a comparatively high rate of
successful treatments without severe
side effects. With comparatively low
PART 5
Information Column
Title:
Improvement of dosimetry for I-131
therapy of lung metastases with
special regard to children with
thyroid cancer from Belarus following
the Chernobyl accident
cumulative mean doses to the lung of
approximately 7 Gy (range: 0.1 Gy - 25
Gy) the secondary tumors could be eliminated in 52 out of 80 cases. In only
one child the development of lung fibrosis was related to I-131 treatment.
As a result of this study the following
recommendations can be given for
the treatment of lung metastases:
a) Radioiodine therapy is a safe and efficient therapy in the case of lung metastases from thyroid carcinoma; a
comparatively high rate of successful
treatments without directly related
severe side effects can be achieved.
b) The per therapy dose to the lung
and/or tumor achievable with one
radioiodine therapy is limited to a few
Gy. Administering higher activities of I131 to the patients in order to achieve
higher doses might cause severe side
effects. c) Care has to be taken in the
case of exceptionally high lung uptake
as the lung doses can become excessive
(more than 10 Gy). d) Several courses
of radioiodine treatment will probably
reduce the dose delivered to the metastases per therapy. e) The local dose to
small foci is much higher than the lung
dose and might be sufficiently high for
ablation. f) If the metatases are smaller
than a few millimeter the dose to the surrounding tissue cannot be neglected.
g) Using other radioisotopes such as
I-125, perhaps in combination with
I-131 may enhance the efficacy of the
treatment of lung metastases. h) More
research is necessary on the histology,
size and shape of micrometastases from
thyroid cancer for a better understanding of the therapeutic efficacy of
radioiodine. i) A long-term follow-up of
the children with thyroid carcinoma
from Belarus, the Ukraine and the
Western part of Russia is mandatory.
Partnership
The success of the project was dependent upon the bringing together of
European expertise in clinical and
nuclear medicine and medical physics
plus collaboration with colleagues in
Belarus.
Selected references
Lassmann M, Schelper L, Hanscheid H, Biko, J,
Reiners CHR, Paretzke H, Hoefnagels C, Clarke,
S, Allen S. (1999).
Determination of Iodine Kinetics in Lung
Metastases During Radioiodine Therapy
of Belarussian Children with Thyroid
Carcinoma. In G.Thomas, A. Karaoglou,
E.D.Williams Eds.), Radiation and Thyroid
Cancer (pp.443-448), World Scientific
Publishing Co. Pte. Ltd, Singapore 1999.
Li W, Friedland W, Pomplun E, Jacob P,
Paretzke H, Lassmann M, Reiners CHR (2001).
Particle track structure and dose distributions
from decaysof 131I and 125I in and around
micrometastases of thyroid cancer.
Radiat Res., 156, 419-429.
Co-ordinator:
Christian Reiners/M. Lassmann
Clinic for Nuclear Medicine
University of Würzburg
Josef-Schneider-Str. 2
D-97080 WÜRZBURG, Germany
Tel: +49.931.201.35868
Fax: +49.931.201.35079
e-mail: [email protected]
Partners:
- H. Paretzke (GSF-Institute for
Radiation Protection, Research
Center for the Environment and
Health, Germany)
- C. Hoefnagel (NCI-The Netherlands
Cancer Institute, The Netherlands)
- S. Clarke (GHL-Guy´s Hospital,
United Kingdom)
- E.P. Demidchik (RCTT-Republican
Center for Thyroid Tumors, Belarus)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
59
bjectives
This project aims to develop a
novel, multidisciplinary and complementary approach for improving the management of the
radiation injury of victims exposed
Novel approaches to the treatment
of radiation accident patients
(NAIMORI)
to high doses of radiation. With the
purpose of addressing this objective three main tissues, which
Challenges to be met
determine the fate of irradiated
victims, have been chosen: the
hematopoietic, the epidermal and
the vascular tissues. In addition,
studies in endothelial cells, which
play a critical role in the response
of these two tissues to irradiation,
have been considered. In particular, our study proposes the development of new indicators capable
of predicting the radiation injury
produced in both hematopoietic
and the epithelial tissues. Our project also aims to improve the
understanding of the damage
within the most critical cells in
both tissues and, finally, to offer
new alternatives for ameliorating
the hematopoietic cellular damage,
for preventing the inflammatory
reaction and for the therapy of the
cutaneous syndrome produced
after irradiation.
Development of new indicators
of radiation injury:
Early intervention is of pivotal importance to limit the consequences of
accidental radiation exposure. Unfortunately, quantitative indicators of radiation injury are largely lacking at present,
which also makes it difficult to establish the optimal treatment at an early
stage. The project seeks to establish
such indicators for damage to blood
cell production and to the skin. Procedures based on the quantification of
hematopoietic stem cells (HSCs) mobilised to peripheral blood will be used to
estimate the HSC reserve of accidentally exposed subjects. Using cDNA
microarrays and proteomic analysis,
genes that are modulated by radiation
will be investigated in human skin, and
more specifically in keratinocytes. It is
expected to identify clusters of gene
expression/suppression, which may elucidate the basis of the molecular events
leading to the injury of the tissue.
Better understanding of the radiationinduced damage in critical tissues:
Optimal medical management of radiation injury requires profound understanding of the pathophysiology in critical
tissues. Using in vivo repopulation assays,
the response of the different subpopulations of human HSCs to radiation will
be investigated. The
pathophysiological
Peripheral blood cell counts after 5 Gy TBI
mechanisms of radiaand growth factor treatment
tion-induced eosinophilic inflammation
will be investigated by
thrombocytes (x10 /L) reticulocytes (x10 /L)
neutrophils (x10 /L)
characterising the role
104
100
102
of the CC-chemokine
10
3
-2
10
10
receptor CCR3 and
100
102
10-3
its ligand eotaxin.
10-1
10
10-4
10-2
Since TGF-β1 repretime (day)
sents a key molecule
and a master switch
huTPO
G-CSF
for the general fibrohuTPO + G-CSF
placebo
tic programme, the
9
60
12
role of TGF-β1 at the level of signal transduction will be further investigated.
Better treatment of the radiation
damage:
Although much progress has been made
in the treatment of the hematopoietic
syndrome, current insights highlight the
possibility of more efficacious treatment,
while treatment of skin damage is still
largely conservative and eventually
based on surgery. Although it has been
recognised that the acute inflammatory
reaction to radiation is a key element in
the generation of damage and eventually fibrosis, this knowledge has not yet
resulted in clinically effective interventions. The direct effect of cytokines, such
as the blood platelet stimulator thrombopoietin, on the survival of hematopoietic stem cells exposed to radiation will
be investigated. The capability of synergistic combinations of growth factors
to promote amplification of surviving
progenitors and stem cells will be also
determined. Novel therapeutic approaches will target endothelial cells (ECs)
with cytokines and antithrombotic
agents. The activation state of endothelial cells following radiation will be
assessed. For the chronic stage of the
radiation syndrome, fibrosis and keratosis will be studied in radiation accident and radiotherapy patients, as well
as in a pig experimental model. A
detailed investigation describing the
antifibrotic action of superoxide dismutase (SOD) on the metabolism of
myofibroblasts will be conducted to
contribute to the understanding of SOD
as an anti-fibrotic agent.
9
Achievements
The demonstration that thrombopoietin – if immediately administered after
irradiation – potentiates the hematopoietic response to other growth factors
and markedly prevents the leukopenia,
thrombocytopenia and anemia associated to the HSC damage, makes this
PART 5
ACRONYM
??? Information
NAIMORI Information
Column Column
4TH FRAMEWORK PROGRAMME
agent a key in the future treatment of
radiation accident patients. Hence, it is
recommended to provide such patients
immediately with thrombopoietin and
G-CSF (fig.1).
Improved harvesting of HSCs from
peripheral blood and the isolation of
these cells will greatly promote the
chances of survival in radiation exposure
dose ranges refractory to growth factor
treatment. This may therefore replace
the bone marrow transplantation
approach and may serve as a back-up
treatment for those patients which otherwise cannot be saved. In this context,
the use of peripheral blood HSCs will
enable transplantation of large numbers of these cells if sufficient additional
immunosuppression is given to the
recipients. Experimental evidence was
obtained that the use of very high numbers of HSCs results in the further abrogation of recipient resistance to donor
HSC engraftment, even at sublethal
radiation doses in combination with
immunosuppressive antibodies.
Understanding of the intercellular communication between cutaneous cells,
blood vessels and peripheral blood cells
after treatment with ionising radiation
gives the opportunity for new therapeutic targets and treatment approaches
of radiation accident patients. Concerning late radiation damage, the postulate of irreversibility of fibrosis has been
challenged. The most impressive results
were obtained with SOD, which could
rapidly reduce fibrosis in patients. In
combination with the antioxidant
alpha-tocopherol, pentoxifylline treatment resulted in a striking regression of
fibrosis as well. Understanding of the
underlying mechanisms may open
several effective treatment options.
Partnership
The consortium is based upon European
scientists working on three critical tissues
i.e. hematopoietic, epithelial and endothelial tissues. In addition, the activities
of the consortium are multidisciplinary
in nature and include molecular biology,
cell biology and animal models of radiation response.
Selected references
Neelis KJ, Visser TP, Dimjati W, Thomas GR,
Fielder PJ, Bloedow D, Eaton DL, Wagemaker G.
A single dose of thrombopoietin early after
myelosuppressive total body irradiation prevents
pancytopenia by promoting short-term multilineage spleen repopulating cells at the transient
expense of bone marrow repopulating cells.
Blood 92: 1586-1597, 1998.
B. Albella, J.C. Segovia, G.Güenechea and
J.A. Bueren. Preserved long-term repopulation and
differentiation properties of hematopoietic grafts
subjected to ex vivo expansion with stem cell factor
and interleukin 11.
Transplantation 67: 1348-57, 1999.
Griffiths NM, Linard C, Dublineau I, François A,
Esposito, Neelis KJ, Niemer-Tucker MMB, van der
Hage M, Broerse JJ, Wagemaker G. Long term
effects of x-irradiation on gastrointestinal function and regulatory peptides in rhesus monkeys.
Int J Radiat Biol 75: 183-91, 1999.
M. Ramírez, J.C. Segovia, I. Benet, C. Arbona, G.
Guenechea, C. Blaya, J. Gracía-Conde, J. A. Bueren
and F. Prosper. Ex vivo expansion of umbilical cord
blood (UCB) CD34+ cells alters the expression and
function of α4β1 and α5β1 integrins.
Br. J. Hematol. 115: 213-221, 2001.
Gottlober P, Steinert M, Weiss M, Bebeshko V,
Belyi D, Nadejina N, Stefani FH, Wagemaker G,
Fliedner TM, Peter RU. The outcome of local
radiation injuries: 14 years of follow-up after the
Chernobyl accident.
Radiat Res. Mar 155: 409-16, 2001.
Title:
New approaches to diagnosis and
treatment of individuals exposed
accidentally to ionizing radiation
Co-ordinator:
Gerard Wagemaker
Erasmus University
Dr Molewaterplein 50
NL - 3015 GE, Rotterdam
The Netherlands
Tel: +31.10.408.77.66
Fax: +31.10.436.23.15
e-mail: [email protected]
Partners:
- J.W. Hopewell (The Research
Institute, United Kingdom)
- T.M. Fliedner (Universität Ulm, Germany)
- B.L. Ziegler (Universität Tübingen,
Germany)
- A. Fischer (INSERM, Paris, France)
- M. Martin (CEA, France)
- T. Egeland (ITT-Institute of Transplantation Immunology, Norway)
Period: Nuclear Energy 1994-1998
Status: Completed
5TH FRAMEWORK PROGRAMME
Title:
Novel approaches to the treatment
of radiation accident patients
(NAIMORI)
Co-ordinator:
Juan A. Bueren
CIEMAT-Centro de Investigaciones
Energeticas, Institute of Environment
Avenida Complutense 22
E-28040 MADRID, Spain
Tel: +34.91.3466518
Fax: +34.91.3466393
e-mail: [email protected]
Partners:
- Gerard Wagemaker (Erasmus
University, The Netherlands)
- Ralf U. Peter (Universität Ulm, Germany)
- Michèle Martin (CEA, France)
- Anne Van der Meeren (IRSN, France)
Period: Nuclear Energy 1998-2002
Status: Ongoing
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
61
bjectives
The developing human brain is
very sensitive to ionising radiation,
as shown by the increased number
In utero irradiation of the foetal
rodent brain: early effects
of cases of mental retardation
among survivors of the atomic
bombing in Japan, who were
exposed in utero during the sensitive period. However, the minimal
Challenges to be met
dose level, which induces such
pathology, remains to be determined. The objectives of this project were to provide an answer to
the question as to whether a real,
functional threshold exists and to
investigate the effects of low doses.
Statistical analysis of the epidemiological data on the in utero irradiated Abomb survivors revealed a dose dependent
increase in the frequency of mental retardation. On the basis of some analyses it
could be argued that there is no dose
threshold and that prenatal irradiation
of the brain produces an all or none
impairment. On the other hand, the
great complexity of the target tissue
which has great compensatory abilities
as well as the criteria used to measure
multifunctional IQ make it more likely
that deterministic effects with thresholds
represent the underlying mechanisms
for effects on the developing brain. If a
threshold is to be determined, studies
must move to lower doses. In animals,
it is clear from previous brain studies,
also supported by the EC, that dose levels as low as 10 mGy can cause a small
(2%) but statistically significant atrophy
of the rat brain. However, given the
plasticity of the brain and its great capacity to compensate for loss of function,
the question is, whether such small
changes would impair brain physiology
or function.
The specific objectives of the project
included the determination of the effects
of irradiation on the developing brain
with respect to: apoptosis and its underlying molecular mechanisms; growth
factor gene expression, cell formation
and migration; gross anatomical parameters; cortical and commissural diameters, synaptogenesis and synaptic
remodelling, as well as behaviour. The
effects of different doses, acute or
protracted irradiation, as well as the
developmental stage at which the brain
was irradiated were evaluated.
Achievements
It was shown both in vivo and in vitro that
irradiation-induced cell death follows
the apoptotic pathway. The transcription
62
factor c-Jun, which binds to regulatory
regions of DNA, and controls gene
expression, was shown to be involved
in irradiation-induced apoptosis. NMDA
type glutamate receptors are known to
be involved in neurodegeneration, by
allowing increased calcium influx.
Calcium, in turn, activates processes
leading to cell death. Our results showed
that a calcium-dependent protease, calpain, plays a role in irradiation-induced
apoptosis. p53 protein which is known
as the guardian of the genome, inducing either repair or apoptosis when DNA
is damaged by irradiation, was increased
in apoptotic cells. Both classical neurotrophins such as NGF, BDNF, NT-3
and other growth factors such as IGF-II
were increased following irradiation,
suggesting that the damaged tissue
reacts in a compensatory way, producing substances that ameliorate the injury.
Indeed it was shown that some growth
factors had ameliorative effects on irradiation-induced damage in the in vitro
systems. Prenatal irradiation also produced alteration in the patterns of cell
proliferation and migration in the neuroepithelium, leading to long-term morphological changes such as reduction of
brain weight, atrophy of the cingulum,
and reduction of the hippocampal area.
Even 60 mGy was shown to be effective
in producing diminution of the diameter of the parietal cortex. The effect of
prenatal irradiation on the animal’s adult
behaviour, the final output of brain
function was also determined. Spatial
memory appeared to be the most vulnerable to prenatal irradiation effects.
These results provide an integrated view
of the cellular processes that might
underlie radiation-induced mental
deficits: Irradiation, even at doses of
100-500 mGy, leads to DNA damage.
If this damage is not repaired, cells enter
the apoptotic pathway and are eventually eliminated. This leads to lower cell
numbers in the respective brain areas
and consequent smaller structures. If
PART 6 : Non-cancer effects
Information Column
Title:
In utero irradiation of the foetal
rodent brain: early effects
Selected references
Nieder C, Price R.E., Rivera B, et al.
"Experimental data for insulin-like growth factor
I (IGF-I) and basic fibroblast gowth factor
(bFGF) in prevention of radiation myelopathy".
Strahlenther Onkol 178 (3): 147-152,
March 2002.
New, P. "Radiation injury to the nervous system".
Current Opinion in Neurology 14 (6):725-734,
Dec. 2001.
Li Y.Q., Wong C.S. "Radiation-induced apoptosis
in the neonatal and adult rat spinal cord".
Radiation Res. 154 (3): 268-276, Sept. 2000.
the affected brain structures are involved
in the regulation of a specific behaviour
(e.g. the hippocampus in spatial learning), then the ability to express that
particular behaviour is impaired.
Partnership
Verheij M., Bartelink H. "Radiation-induced
apoptosis".
Cell Tissue Research 301 (1): 133-142, July 2000.
Reyners H, Gianfelici de Reyners E, Yan J,
de Saint-Georges L, Desaintes C. Delayed
effects of prenatal low-dose irradiation in the
white matter of the rat brain.
Int. J. Radiat. Biol. 75: 1327-1334, 1999.
Co-ordinator:
Hubert Reyners (retired - CEN/SCK,
Belgium)
Fotini Stylianopoulou
University of Athens
PO Box 14224
GR-11510 ATHENS, Greece
Tel: +30.1.746.14.67
Fax: +30.1.778.18.29
e-mail: [email protected]
Partners:
- L. de Saint-George
(CEN-SCK, Belgium)
- H. Korr
(University of Aachen, Germany)
- I. Ferrer
(University of Barcelona, Spain)
- Z. Sienkiewicz
(NRPB, United Kingdom)
- G. Konermann
(University of Freiburg, Germany)
- Y. Dimberg
(Swedish University of Agricultural
Sciences, Uppsala, Sweden)
- H. Coffigny (CEA-FAR, Paris, France)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
The participation of different laboratories, allowed the use of a variety of experimental techniques and in vivo/in vitro
systems. This multidisciplinary approach
resulted in the view that doses as low as
100-250 mGy can induce a variety of
effects on the developing central nervous system. The coincidence of the
threshold-levels for experimental and
A-bomb epidemiological studies suggests that similar types of cellular damage might underlie the induction of
structural cognitive and mental deficits.
Status: Completed
63
bjectives
The Semipalatinsk nuclear polygon
in Kazakhstan has been the site for
470 nuclear tests performed by
the Soviet Union during the
period 1949-89. Tests carried out
Minisatellite mutations and biodosimetry
of population around the Semipalatinsk
nuclear test site (SEMIPALATINSK)
in 1949-63 were atmospheric and
surface explosions and, in 1963-89,
underground explosions.
Challenges to be met
More than 1.5 million people living
in the region were exposed to
ionising radiation, partly from the
radioactive cloud and partly from
the environmental fallout.
Very high radiation doses – up to
several Gy – have been previously
reported for the inhabitants of the
most exposed villages as a result of
the tests.
The objectives of the SEMIPALATINSK
project were 1) To establish a bank
of blood samples of three generations of families living close to the
Semipalatinsk nuclear test site and
control families from clean areas,
2) To reconstruct radiation doses
by using biomarkers of radiation
exposure, and 3) To determine the
germline minisatellite mutation rates
of exposed people and the control
families of the same ethnic origin
living in non-contaminated areas.
The overall objective was to provide information relevant to the
genetic risk assessment of exposure
to ionising radiation in chronically
exposed human populations and to
provide advice for local authorities
in order to help them mitigate
the effects of ionising radiation on
the population around the
Semipalatinsk nuclear test site.
64
Information on the exposure of the local
population has been available to the
international scientific community only
since the 1990's. Many of the people
that received the highest doses after the
surface tests performed in 1949-1956
are now old and soon no longer available for examinations. To learn more
on radiation effects in the future using
technologies not yet available, biological samples need to be stored.
Reconstruction of radiation doses is an
essential step in order to obtain quantitative information on the health risk of
radiation. The participants in the project
have used biomarkers of radiation exposure to assess the exposure of people
that have been living in the most contaminated villages since the first Soviet
nuclear test in August 1949. Stable
chromosomal translocations in blood
lymphocytes were analysed for dose
reconstruction. In addition, glycophorin
A (GPA) mutations in erythrocytes were
analysed by a US laboratory (University
of Pittsburgh).
Genetic risk estimates for man from
exposure to ionising radiation have been
based mainly on the information from
animal experiments and general knowledge of radiation biology. No significant elevation of heritable mutation rate
has been observed in the children of
A-bomb survivors in Hiroshima and
Nagasaki (Japan) by using standard
genetic methods. Tandem repeat minisatellite loci monitoring of germline
mutation in man is a new and sensitive
approach.
The first Soviet nuclear weapon test in 1949
was a surface explosion.
Achievements
A biological sample database with an
accompanying registry of background
information on the examined subjects
has been established for long-term storage of frozen lymphocytes, blood cell
DNA and EDTA blood. Biosamples from
361 individuals living near the Semipalatinsk test site and 251 controls from
Taldy Kurgan area are available for future
studies addressing genetic effects of
ionising radiation.
Chromosomal translocation frequencies
in the Semipalatinsk and the control
groups were similar. This suggests that
on average, the magnitude of exposure to the examined group in the
Semipalatinsk area has been considerably smaller than that reported in the
local registries. Previously reported doses
of the order of 1-4.5 Gy (2.9 Gy on average among the grandparent generation that lived in the villages at the time
of testing) cannot be confirmed by the
PART 6
SEMIPALATINSK Information Column
Title:
Minisatellite mutations and
biodosimetry of population around
the Semipalatinsk nuclear test site
(SEMIPALATINSK)
present data. A more likely dose estimate is below 0.5 Gy. A similar conclusion was drawn on the basis of the
GPA mutation data.
Differences in hereditary minisatellite
mutation rates between the two rural
populations from the Semipalatinsk and
Taldy Kurgan Districts of Kazakhstan
were small in general. However, the
minisatellite mutation rate in the P0 generation (grandparents) directly exposed
to radioactive fallout from the surface
and atmospheric nuclear tests was 1.7fold higher than in the control nonexposed population from the Taldy
Kurgan District. Moreover, the minisatellite mutation rate in the F1 generation
(parents) from the affected area showed
a significant negative correlation with
the year of birth. The mutation rate
among those that were born in the
1950's (during atmospheric testing) was
significantly elevated, whereas no
increase in the germline minisatellite
mutation rate was observed amongst
those that were born in the 60's and 70's
and thus were exposed to considerably
smaller radiation doses.
Selected references
Dubrova, Yu. E., Bersimbaev, R. I.,
Djansugurova, L. B., Tankimanova, M. K.,
Mamybaeva, Z. Zh., Mustonen, R.,
Lindholm, C., Hulten, M., Salomaa, S.
Nuclear weapons tests and human germline
mutation rate.
Science 295:1307; 2002.
Salomaa, S.; Lindholm, C.; Tankimanova, M.K.;
Mamyrbaeva, Z.Zh.; Koivistoinen, A.;
Hulten, M.; Mustonen, R.; Dubrova, Y.E.;
Bersimbaev, R.I. Stable chromosome aberrations
in the lymphocytes of a population living in the
vicinity of the Semipalatinsk nuclear test site.
Submitted to Radiat. Res. 2002.
Co-ordinator:
Sisko Salomaa
STUK-Radiation and Nuclear Safety
Authority
Research and Environmental
Surveillance
PO Box 14
Laippatie 4
FIN-00881 HELSINKI, Finland
Tel: +358.9.75988495
Fax: +358.9.75988498
e-mail: [email protected]
Partners:
- Rakhmetkaji Bersimbaev (Institute
of General Genetics and Cytology,
Kazakstan)
- Peter Townsend (University of
Leicester, United Kingdom)
- Roger Whittenbury (University
of Warwick, United Kingdom)
EC Scientific Officer:
Diederik Teunen
Christian Desaintes
Tel: +32.2.295.82.73
Fax: +32.2.295.49.91
e-mail: [email protected]
Period: Nuclear Energy 1994-1998
Status: Completed
Partnership
The project brought together five competent Kazakh, European and US laboratories having knowledge on radiation
dose reconstruction, genetic effects of
ionising radiation, epidemiology, molecular biology and radiation protection.
Three Kazakh post doctoral scientists
were trained in European laboratories
on molecular cytogenetic and mutation analysis techniques.
65
Annex 1: projects starting late 2002:
Genetic factors predisposing to radiation induction of
mutation during early gestation: the role of DNA repair
and cell cycle control
(GEMRATE)
Genetics of predisposition to radiation-induced cancer
of the thyroid
(GENRAD-T)
Recent reports in the mouse indicate considerable radiation sensitivity of particularly the early zygote and the
postimplantation gastrula stages. Data on the chromatin
stability of human sperm and chromosome stability of
preimplantation embryos point at the genetic vulnerability of these stages in human. We propose to assess, for
the mouse, the roles of single and double strand DNA
break repair at the zygote and gastrula stages in response
to radiation-caused DNA damage of male postmeiotic
haplophase stages, the zygote stage and the gastrula
stage. Various genetic, and some cell cycle control endpoints will be utilised to understand the importance of
DNA break repair and cell cycle control for the transmission of genetic damage of sperm, zygote and gastrula
stages, such that risk assessment and mechanistic insight
are coupled.
Carcinoma of the thyroid is a serious public health problem in populations at risk of exposure to radionuclides
accumulating in thyroid tissue. However, due to genetically determined differences in individual susceptibility
to ionising radiation, simple dose-response relationships
do not exist. Identification of those subjects who are
predisposed to developing thyroid tumours would be of
value for setting individual exposure limits and for tailoring
post-exposure monitoring. The GENRAD-T consortium
aims to identify the gene loci that are responsible for determining individual susceptibility to thyroid cancer. To do
this we will use a mouse model of radioiodine-induced
thyroid cancer combined with molecular genetic screening methods (QTL-mapping, microsatellite allelotyping
and CGH). The applicability of the gene loci thus identified for use in human populations will be confirmed by
analysing human thyroid tumours.
Co-ordinator: Peter de Boer
e-mail: [email protected]
Co-ordinator: Michael Atkinson
e-mail: [email protected]
Radiation specific DNA non-double strand break lesions:
repair mechanisms and biological effects
(NON-DSB LESIONS)
Genetic pathways for the prediction of the effects of
irradiation - European normal and tumour tissue bank
(GENEPI-ENTB)
Ionising radiation induces a plethora of DNA non-double strand break lesions such as oxidised bases, abasic sites
and single strand breaks. Strong evidence exists that ionising radiation induces, besides isolated DNA lesions, an
unique form of DNA damage termed clustered damage
consisting of closely spaced lesions on opposite DNA
strands. There is also evidence that repair of lesions within
clustered damage sites is compromised compared with
the effective repair of isolated lesions. We will investigate
the following aspects of radiation-induced single and
clustered DNA damage: (i) frequency and nature of the
damage, (ii) mechanisms of repair, (iii) interference with
transcription, replication and chromatin dynamics and
(iv) biological consequences (stress responses, mutations, lethality). To achieve these goals we will exploit a
variety of new technologies and utilise different organisms with defined mutations in damage response genes.
There is strong evidence for a genetic basis for reaction
to radiation in normal tissues as well as in tumours. The
aim of GENEPI is to create a tissue bank linked to a
detailed outcome-database of a large cohort of patients
receiving radiotherapy as an essential infrastructure for
present and future genetic research in the field of radiation protection and health. A central database will be
established that provides a link to exististing decentral databases and tissue banks, to foster optimal utilisation and
access to data and material. Dummy runs with modern
statistical tools will be applied to monitor the quality of
this novel research infrastructure. Protocols for outcome
assessment, tissue handling, and use and access of the
infrastructure will be developed and ethical and legal
issues addressed. The tissue bank will remain open for at
least 20 years. In parallel, a cohort of large kindred french
families will be recruited, and peripheral blood lymphocytes will be stored. These samples will be made available
to study the genetics of individual radiosensitivity.
Co-ordinator: Leon F.H. Mullenders
e-mail: L.Mullenders@ lumc.nl
Co-ordinator: Michael Baumann
e-mail: [email protected]
66
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