MECHANISM OF
BENZO(A)PYRENE-INDUCED
ACCUMULATION OF P53
TUMOUR SUPPRESSOR
PROTEIN IN MOUSE
RAISA
SERPI
Department of Pharmacology
and Toxicology,
University of Oulu
OULU 2003
RAISA SERPI
MECHANISM OF BENZO(A)PYRENEINDUCED ACCUMULATION OF P53
TUMOUR SUPPRESSOR PROTEIN IN
MOUSE
Academic Dissertation to be presented with the assent of
the Faculty of Medicine, University of Oulu, for public
discussion in the Auditorium of the Department of
Pharmacology and Toxicology, on June 13th, 2003, at 12
noon.
O U L U N Y L I O P I S TO, O U L U 2 0 0 3
Copyright © 2003
University of Oulu, 2003
Supervised by
Professor Kirsi Vähäkangas
Reviewed by
Docent Hannu Komulainen
Professor Veli-Matti Kosma
ISBN 951-42-7039-8
(URL: http://herkules.oulu.fi/isbn9514270398/)
ALSO AVAILABLE IN PRINTED FORMAT
Acta Univ. Oul. D 731, 2003
ISBN 951-42-7038-X
ISSN 0355-3221
(URL: http://herkules.oulu.fi/issn03553221/)
OULU UNIVERSITY PRESS
OULU 2003
Serpi, Raisa, Mechanism of benzo(a)pyrene-induced accumulation of p53 tumour
suppressor protein in mouse
Department of Pharmacology and Toxicology, University of Oulu, P.O.Box 5000, FIN-90014
University of Oulu, Finland
Oulu, Finland
2003
Abstract
The tumour suppressor gene TP53 is the most commonly mutated gene in human cancers. The protein
it codes, p53, becomes activated as a response to stress signals. When activated, p53 binds to DNA
and affects the transcription of its target genes. They then cause cell cycle arrest, DNA repair and/or
induction of programmed cell death, thus preventing mutations and cancer. Specific mutations in
TP53 are associated with exposure to certain carcinogens, such as polycyclic aromatic hydrocarbons
(PAHs). These environmental chemical carcinogens are formed through incomplete combustion of
organic material. Benzo(a)pyrene (BP) is commonly used as a model compound for PAH
carcinogenesis. BP causes accumulation of p53, but the mechanism of accumulation is not known.
The aim of this study was to gain more insight into the p53 protein in the first phases of PAH
carcinogenesis in vivo in mouse, using BP as the model compound.
Mice from the inbred C57BL/6 strain were treated topically or intraperitoneally with BP or were
exposed to cigarette smoke inhalation. The amount of p53 protein was studied by immunoblotting,
immunohistochemistry and immuno electron microscopy, and the mdm2, p21 and p19ARF proteins
were studied by immunoblotting. The binding of BP to DNA was measured by synchronous
fluorescence spectrophotometry.
The p53 protein was induced in vivo in skin and lung after BP treatment and in lung after cigarette
smoke treatment. An increase in p53 was associated with an increase in the amount of BP-DNA
adducts. In skin, the induction of p53 was accompanied by induction of the p21 and mdm2 proteins,
which are transcriptional targets of p53. This indicates that the in vivo induced p53 is a wild-type
protein and functional. In lungs, the induction of p53 was accompanied by a decrease of mdm2 and
an increase of p19ARF. These results confirm that BP is metabolized and binds to DNA in mouse
tissues and indicate that BP-DNA adducts are the trigger for p53 protein induction. The in vivo
regulation of the p53 protein is different in different tissues of C57BL/6 mouse.
Keywords: benzo(a)pyrene, environmental exposure, lung, skin
Acknowledgements
This work was carried out at the Department of Pharmacology and Toxicology in the
University of Oulu during the years 1996-2003. I want to express my sincere gratitude to
my supervisor, Professor Kirsi Vähäkangas, for giving me the opportunity to prepare this
thesis as a member of her research group. Her enthusiasm and vast knowledge of science
have inspired me during these years.
I wish to thank Professor Olavi Pelkonen, head of the Department of Pharmacology
and Toxicology, for his support and for providing the most stimulating environment and
excellent conditions for scientific work. I also express my appreciation to Professors
Heikki Ruskoaho, Aarne Oikarinen and Matti Järvilehto for their valuable advice and
support during this study.
I am grateful to Docent Hannu Komulainen and Professor Veli-Matti Kosma for the
careful review of this thesis. Also, I want to thank Sirkka-Liisa Leinonen for her expert
work with the language of the manuscript.
My warmest thanks are due to the present and former members of the Department of
Pharmacology and Toxicology, Liisa Vaskivuo, Miia Turpeinen, Anna-Kaisa Lappi, Jaana
Rysä, Mika Rahkolin and Katariina Klintrup, who introduced me to the wonders of p53.
Working with you all has been a pleasure. Special thanks go to Päivi Myllynen. It has
been a rewarding experience to have you prepare your thesis simultaneously with me.
Other members of the “p53 group” in Oulu, especially Jenni Hakkarainen, and in
Kuopio deserve many thanks. Kirsi Salo, Kaisa Penttilä and Raija Hanni have been
invaluable because of their help in technical and secretarial matters and, more
importantly, because of their friendship. Tuulikki Kärnä, Esa Kerttula, Kauno Nikkilä,
Riitta Harjula and Merja Luukkonen are also acknowledged for their technical assistance,
and Ulla Hirvonen and Tuula Inkala from the Center of Experimental Animals and Marja
Räinä and Terttu Keränen from the instrument care unit deserve many thanks.
Furthermore, I wish to express my gratitude to the whole personnel of the Department of
Pharmacology and Toxicology. Liisa Kärki and Seija Leskelä from the Photographic
Laboratory of the Faculty of Medicine are warmly acknowledged for their help in
preparing posters and photographs during this study.
I am deeply grateful to my friends Riitta Pirilä and Tuomo Leino and Liisa and Tommi
Vaskivuo. Thank you for all the great moments!
Most of all, I want to thank my parents Kristiina and Jaakko for their care and
encouragement and my brother Risto for his down-to-earth attitude concerning the
wonders of the scientific world. I wish to thank my sister Sara for being there and SeijaLiisa Karvonen for her support. I am also grateful to Kaija and Yrjö Serpi for their
support.
Finally, my deepest gratitude belongs to my beloved Jussi. You have the ability to
make me remember what is really important in life. Thank you.
This work was supported financially by the Academy of Finland, the Cancer Society
of Finland, the Cancer Society of Northern Finland, and the Foundation of Oulu
University.
Oulu, April 2003
Raisa Serpi
Abbreviations
3AB
ARF
BP
BPDE
BSA
CDK
CYP
DES
DMBA
DNA-PK
DTT
EDTA
EH
ERK
FSG
GA
Hdm2
MAPK
Mdm2
NAD
PA
PAH
PARP
PBS
PKC
PMSF
PVDF
SFS
TBS
TPA
3-aminobenzamide
Alternative Reading Frame
Benzo(a)pyrene
Benzo(a)pyrene-7,8-diol-9,10-epoxide
Bovine serum albumine
Cyclin-dependent kinase
Cytochrome P450
Diethylstilbestrol
7,12-dimethylbenz[a]anthracene
DNA-dependent protein kinase
Dithiothreitol
Ethylenediaminetetraacetic acid
Epoxide hydrolase
Extracellular signal-regulated kinase
Fish skin gelatine
Glutaraldehyde
Human double minute-2
Mitogen-activated protein kinase
Murine double minute-2
Nicotinamide adenine dinucleotide
Paraformaldehyde
Polycyclic aromatic hydrocarbon
Poly(ADP-ribose)polymerase
Phosphate buffer solution
Protein kinase C
Phenylmethylsulfonyl fluoride
Polyvinylidene difluoride
Synchronous fluorescence spectrophotometry
Tris-buffered saline
12-O-tetradecanoylphorbol-13-acetate
List of original publications
This thesis is based on the following articles, which are referred to in the text by their
Roman numerals:
I
Serpi R, Piispala J, Järvilehto M & Vähäkangas K (1999) Thapsigargin has similar
effect on p53 protein response to benzo(a)pyrene-DNA adducts as TPA in mouse
skin. Carcinogenesis 20: 1755-1760.
II Turpeinen M, Serpi R, Rahkolin M & Vähäkangas K (2002) Comparison of anti-p53
antibodies in immunoblotting. Biochem Biophys Res Commun 293: 850-856.
III Serpi R & Vähäkangas K (2003) Benzo(a)pyrene-induced changes in p53 and related
proteins in mouse skin. Pharmacol Toxicol 92: 242-245.
IV Serpi R, Turpeinen M, Myllynen P, Vaskivuo L & Vähäkangas K (2003) Inhalation
exposure to cigarette smoke and intraperitoneal benzo(a)pyrene induce similar
molecular changes in mouse lung. Submitted for publication.
Contents
Abstract
Acknowledgements
Abbreviations
List of original publications
1 Introduction ...................................................................................................................13
2 Review of the literature .................................................................................................14
2.1 Chemical carcinogenesis.........................................................................................14
2.2 Polycyclic aromatic hydrocarbons in cancer ..........................................................15
2.2.1 Polycyclic aromatic hydrocarbons...................................................................15
2.2.2 Benzo(a)pyrene................................................................................................16
2.3 Mouse models for chemical carcinogenesis studies................................................16
2.3.1 Inbred strains of mice ......................................................................................16
2.3.2 C57BL/6 mice..................................................................................................17
2.4 The p53 pathway.....................................................................................................17
2.4.1 TP53 gene and p53 protein ..............................................................................17
2.4.1.1 Functions of the p53 protein .....................................................................18
2.4.1.2 Regulation of p53......................................................................................19
2.4.1.3 Aberrations of p53 function ......................................................................20
2.4.2 Mdm2 protein ..................................................................................................20
2.4.3 p19ARF protein..................................................................................................22
2.4.4 Other p53-related proteins ...............................................................................22
2.4.4.1 Poly(ADP-ribose)polymerase ...................................................................22
2.4.4.2 Oncogenic Ras ..........................................................................................23
2.4.4.3 p21WAF1/Cip1/Sdi1 ..........................................................................................24
2.4.4.4 “Family members” of p53: p63 and p73 proteins .....................................24
2.4.5 p53 in vitro and in vivo ....................................................................................24
2.5 Analysis of the p53 protein .....................................................................................25
2.6 Ethical issues of animal studies ..............................................................................26
3 Aims of the study...........................................................................................................27
4 Materials and methods...................................................................................................28
4.1 Treatment of mice (I-IV).........................................................................................28
4.1.1 Treatment of skin (I-III)...................................................................................28
4.1.2 Intraperitoneal BP treatment (IV) ....................................................................30
4.1.3 Smoke exposure (IV) .......................................................................................30
4.2 Treatment of MCF-7 cells (II) ................................................................................30
4.3 DNA isolation and BPDE-DNA adduct analysis by
synchronous fluorescence spectrophotometry (I, III, IV) .............................................31
4.4 Immunoblotting (I-IV)............................................................................................31
4.5 Immuno electron microscopy for p53 protein (I)....................................................32
4.6 Immunohistochemistry for p53 protein (I)..............................................................33
4.7 PARP enzyme activity (III).....................................................................................33
4.8 Computer analysis of protein sequences (II)...........................................................34
4.9 Statistical analysis (I, IV)........................................................................................34
5 Results ...........................................................................................................................35
5.1 p53 and related proteins in mouse skin...................................................................35
5.1.1 Expression of proteins after topical BP treatment (III) ....................................35
5.1.2 Effect of the tumour promoters TPA and thapsigargin (I)................................35
5.1.3 Effect of the PARP inhibitor 3-aminobenzamide (III) .....................................36
5.1.4 Effect of smoke exposure (IV).........................................................................37
5.2 p53 and related proteins in mouse lungs.................................................................37
5.2.1 Effect of intraperitoneal BP treatment (IV)......................................................37
5.2.2 Effect of smoke exposure (IV).........................................................................38
5.3 p53 and related proteins in mouse liver after smoke exposure
and intraperitoneal BP treatment (IV)...........................................................................38
5.4 p53 protein in MCF-7 cells (II)...............................................................................38
5.5 Comparison of anti-p53 antibodies (II)...................................................................38
5.6 Formation of BPDE-DNA adducts in mouse tissues ..............................................39
5.6.1 Adducts in mouse skin after topical BP treatment
and the effects of TPA, thapsigargin and 3AB on adducts (I, III) .............................39
5.6.2 Adducts in mouse lung and liver after intraperitoneal BP treatment (IV)........40
5.6.3 Adducts in mouse lung, liver and skin after smoke exposure (IV) ..................40
6 Discussion......................................................................................................................41
6.1 p53 induction ..........................................................................................................41
6.2 Anti-p53 antibodies.................................................................................................43
6.3 C57BL/6 mice.........................................................................................................44
6.4 Future prospects......................................................................................................45
7 Summary and conclusions .............................................................................................46
References
1 Introduction
Cancer is a disease that affects approximately every fourth person in their lifetime. The
process of cancer development includes many steps, of which most are nowadays
believed to involve genetic alterations. The tumour suppressor gene TP53 is the most
commonly mutated gene in human cancers (Hollstein et al. 1991, Greenblatt et al. 1994).
It is generally assumed that the protein coded by the TP53 gene, p53 protein, is nonfunctional in normal situations, but becomes activated in cells as a response to stress
signals (reviewed by Pluquet & Hainaut 2001). Activated p53 protein binds to DNA and
affects the transcription of its target genes. The target gene products of p53 then result in
cell cycle arrest, DNA repair and/or induction of programmed cell death, thus preventing
mutations and cancer.
Incorrect function of p53 is a common phenomenon in human cancers. Specific
mutations in the TP53 gene have been found to be associated with exposure to certain
carcinogens, such as polycyclic aromatic hydrocarbons (PAHs, reviewed by Hainaut &
Pfeifer 2001, Vähäkangas 2003). These important environmental chemical carcinogens
are formed when organic material is burnt incompletely, and they are present widely in
the environment in, for example, cigarette smoke, coke oven fumes and engine exhaust.
PAHs in cigarette smoke are known to bind preferentially to certain codons in the TP53
gene, where most smoking-associated mutations are also found (for a review, see Pfeifer
et al. 2002). Lung is the major target organ of PAH carcinogenicity, but the risks of skin
and bladder cancers are also increased by PAH exposure (reviewed by Boffetta et al.
1997). Tobacco smoke is known to be the main cause of lung cancer (IARC 1986).
Among PAHs, benzo(a)pyrene (BP) is the most extensively studied compound, and it
is commonly used as a model compound of PAH carcinogenesis. BP has been shown to
cause accumulation of p53 protein (Bjelogrlic et al. 1994, Rämet et al. 1995, Tapiainen et
al. 1996), but the exact mechanism of accumulation is not known. The induction of p53
protein has been studied extensively, however, mainly using in vitro models. This study
aimed to clarify the role of the p53 protein in the first phases of PAH carcinogenesis in
vivo in mouse, using BP as the model compound.
2 Review of the literature
2.1 Chemical carcinogenesis
Cigarette smoking (Lubin et al. 1984, Peto et al. 1996) and dietary factors (reviewed by
Fulgoni & Ramirez 1998) are among the most important external chemical risk factors of
cancer. Other well-known risk factors are exposure to environmental and occupational
agents. Chemical carcinogens can be classified by their function into two categories,
genotoxic and non-genotoxic carcinogens. Most genotoxic carcinogens require metabolic
activation into forms that bind to cellular macromolecules and thereafter exert their
carcinogenic effects. The formation of cancer is a very complex process, which involves
a series of changes in cancer-related genes (reviewed by Akhurst & Balmain 1999,
Vähäkangas 2003). Early studies of the carcinogenesis process in mouse skin models
gave rise to two-stage (Mottram 1944, Berenblum & Shubik 1949) and multi-stage
animal carcinogenesis models (DiGiovanni 1992). These models helped researchers to
understand the complexity of carcinogenesis and served as a basis for the classical model
of carcinogenesis, which includes the initiation, promotion and progression phases. The
initiation phase has traditionally been described to involve the induction of mutations,
and it also involves escape from DNA repair. Other steps, such as cell proliferation in the
promotion phase and additional genetic events and angiogenesis in the progression phase
(reviewed by DeFlora et al. 2001, Dempke et al. 2001), are needed for the process of
carcinogenesis to result in cancer. The carcinogenesis process can be described as a series
of consecutive genetic changes, analogous to evolution, leading to the conversion of
normal cells into cancer cells (see Hanahan & Weinberg 2000).
There are a number of chemical compounds that possess the initiating activity,
including many PAHs. There are also tumour promoters of many types, among which
phorbol esters are the most widely studied agents in skin tumour promotion. 12-Otetradecanoylphorbol-13-acetate (TPA) is the most potent of them (reviewed by
DiGiovanni 1992). The promoting mechanism of TPA is not totally understood, but is
believed to be based on the activation of protein kinase C (PKC), possibly causing a rise
in intracellular Ca2+ levels (Baranska et al. 1995). Another well-known promoter,
thapsigargin, also causes a relatively long-lasting increase in the cytoplasmic Ca2+ levels
(Thastrup et al. 1990, reviewed by Treiman et al. 1998). Its tumour-promoting function
15
probably results, at least partly, from cytotoxicity, causing a wound response in skin
(Marks et al. 1991). There are results supporting the possibility that TPA and thapsigargin
might function in the phosphorylation of the p53 protein. PKC, inducible by TPA, is
capable of phosphorylating a recombinant murine p53 protein (Delphin et al. 1997).
Milne and coworkers (1996) found that phosphorylation of murine p53 through PKC was
not stimulated by TPA. TPA did, however, cause a stimulation of p53 phosphorylation by
mitogen-activated protein kinase (MAPK). Thapsigargin has been demonstrated to
increase the extracellular signal-regulated kinase (ERK) phosphorylation of rat thyroid
epithelial cells (Jiménez et al. 2002) and to reverse the NDR1 kinase phosphorylationsuppressing activity of a Ca2+-chelating agent BAMTA-AM (Tamaskovic et al. 2003).
According to these studies, it is thus possible that both TPA and thapsigargin affect the
phosphorylation of the p53 protein.
2.2 Polycyclic aromatic hydrocarbons in cancer
2.2.1 Polycyclic aromatic hydrocarbons
Polycyclic aromatic hydrocarbons are ubiquitous environmental agents commonly
believed to significantly contribute to human cancers. PAHs are formed in the process of
incomplete combustion of organic material and are found widely in the environment, for
example, in engine exhaust, cigarette smoke, soil, water and food (for a review, see
Phillips 1999), and human exposure to PAHs is therefore unavoidable. Like many other
carcinogens, polycyclic aromatic hydrocarbons are metabolized enzymatically to various
metabolites, of which some are reactive. In the large group of enzymes involved in
carcinogenic metabolism (reviewed by Williams & Phillips 2000), cytochrome P450
enzymes CYP1A1, 1A2, 1B1, and 3A4 are the most important enzymes in the
metabolism of PAHs (Shimada et al. 1989, Shimada et al. 1996, reviewed by Pelkonen et
al. 2003). PAHs undergo metabolic activation to diol-epoxides, which bind covalently to
DNA. The DNA binding of activated PAHs is considered to be essential for the
carcinogenic effect (for reviews, see Vähäkangas & Pelkonen 1989, Guengerich 2000).
DNA adducts have been found in various human tissues (reviewed by Hemminki et al.
2000). In epidemiological studies, correlations between the level of PAH exposure and
the number of PAH-DNA adducts have been found, including that between coke oven
exposure and PAH-DNA adducts in blood cells (Harris et al. 1985, Haugen et al. 1986)
and that between cigarette smoking and PAH-DNA adducts also in blood cells (for a
review, see Poirier et al. 2000). A refined repair system has evolved to eliminate DNA
adducts from the genome. PAH adducts are repaired by nucleotide excision repair (for a
review, see Hoeijmakers 2001). If the adducts are left unrepaired, they may cause
permanent mutations (for a review, see Boysen & Hecht 2003). If these mutations are
situated at critical sites, including tumour suppressor genes or oncogenes, they may lead
to cellular transformation and the development of tumours. In some cases, specific
mutations found in the TP53 gene, the most commonly mutated gene in human cancers,
are associated with exposure to certain carcinogens (Hernandez-Boussard & Hainaut
16
1998, reviewed by Hainaut & Pfeifer 2001, Vähäkangas 2003). For example, the PAHs in
cigarette smoke bind preferentially to the sites called hotspot codons in TP53, where most
smoking-associated mutations are also found (Pfeifer et al. 2002). Such studies give
support to the link between DNA adducts and the cancer risk in humans.
2.2.2 Benzo(a)pyrene
According to the International Agency for Research on Cancer (IARC), there is sufficient
evidence to show that BP is carcinogenic in laboratory animals, and BP is probably also
carcinogenic in humans (group 2A, IARC 1983). The ability of BP to induce tumours
upon local administration is well documented (for a review, see Hecht 1999). BP is
metabolically activated, and the ultimate carcinogenic product is formed via a three-step
process. The first step includes the formation of (7R,8S)-epoxy-7,8dihydrobenzo(a)pyrene (BaP-7,8-oxide) catalyzed by cytochrome P450 enzymes (Fig. 1,
for a review, see Boysen & Hecht 2003). The second step, catalyzed by epoxide
hydrolase (EH), is the conversion to (7R,8R)-dihydroxy-7,8-dihydrobenzo(a)pyrene
(BaP-7,8-diol). Finally, cytochrome P450 enzymes catalyze the reaction, producing four
possible isomers of 7,8-diol-9,10-epoxide. Quantitatively the most important of them is
(7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE). BPDE,
which is the ultimate carcinogen (Slaga et al. 1979, reviewed e.g. by Vähäkangas &
Pelkonen 1989), binds to DNA at the guanine residues (Weinstein et al. 1976) and
produces BPDE-DNA adducts.
CYP
EH
OH
Benzo(a)pyrene
7,8 epoxide
O
HO
HO
O
Benzo(a)pyrene
CYP
Benzo(a)pyrene
7,8 diol
OH
Benzo(a)pyrene
7,8 diol-9,10
epoxide
Fig. 1. The major metabolic pathway of benzo(a)pyrene leading to the ultimate carcinogen,
benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE). CYP, cytochrome P450; EH, epoxide
hydrolase.
2.3 Mouse models for chemical carcinogenesis studies
2.3.1 Inbred strains of mice
During the past few decades, mouse has proved to be a useful model animal in
mechanistic studies of chemical carcinogenesis (D’Agostini et al. 2001, Gautam et al.
17
2002, for a review see Balmain & Harris 2000). While there are differences in the process
of carcinogenesis between mouse and human, mice do develop tumours in the same
tissues and with similar histopathology as humans. Also, the genetic events in human
cancers are extraordinarily similar to those in mice (see Balmain & Harris 2000). Mice
are small animals with a short life span and a high metabolic rate. Thus, cancer develops
more quickly, which makes the process easier to study. Different inbred mouse strains
differ widely in their susceptibility to tumour development (reviewed by Naito &
DiGiovanni 1989). This provides useful models for studying the genetic factors involved
in this process. The differences between various mouse strains in the susceptibility to
multi-stage skin carcinogenesis seem to be less closely related to the differences in
tumour initiation than to tumour promotion (for a review, see Slaga et al. 1996). Crossbreeding of different strains can be used to map the locus of a certain genetic
predisposition (for a review, see Nagase et al. 1996).
There are differences between different inbred mouse strains in their responsiveness to
carcinogens. A comparison of six mouse strains showed the SENCAR and C57BL/6
mouse strains to be most responsive to complete epidermal carcinogenesis when BP or
another PAH, dimethylbenzanthracene (DMBA), was used as a carcinogen, whereas the
DBA/2, CD-1, C3H and BALB/c strains are less responsive (reviewed by DiGiovanni
1992). On the other hand, with two-stage carcinogenesis (using TPA as a promoter), the
SENCAR strain is again the most responsive, but C57BL/6 mice respond rather weakly.
2.3.2 C57BL/6 mice
The C57BL/6 mouse strain was originally developed for studies of PAH carcinogenesis.
The arylhydrocarbon receptor (AhR) plays an important role in the regulation of the
cytochrome P450 enzymes CYP1A1, 1A2 and 1B1. PAHs induce these enzymes through
an AhR-dependent mechanism in inbred C57BL/6 mice (Pendurthi 1993, reviewed by
Whitlock 1999). These mice are thus classified as Ah-responsive. The Ah receptor binds
PAHs in the cytosol and starts a cascade of events leading to new synthesis of CYP1
proteins and increased metabolism of PAHs (Pendurthi 1993). The p53 protein is induced
after BPDE-DNA adduct formation in the skin of BP-treated C57BL/6 mice (Bjelogrlic et
al. 1994, Tapiainen et al. 1996).
2.4 The p53 pathway
2.4.1 TP53 gene and p53 protein
The tumour suppressor protein p53 was first described in 1979 (Lane & Crawford 1979,
DeLeo et al. 1979, Linzer & Levine 1979) and ten years later identified as a tumour
suppressor (reviewed by Levine 1990). In human, the TP53 gene that contains 11 exons is
located in chromosome 17p13.1, while the mouse gene, which also contains 11 exons, is
18
located in chromosome 11 (reviewed by Soussi & May 1996). In both human and mouse,
the coded protein is approximately 53 kDa in size, the human protein containing 393
amino acids and mouse protein 390 amino acids. The p53 protein consists of an acidic Nterminus with a transactivation domain, a hydrophobic central DNA-binding core and a
basic C-terminus with regulatory and oligomerisation domains (for a review, see e.g.
Hainaut & Vähäkangas 1997). The DNA-binding domain of the p53 protein is composed
of two beta-sheets and a zinc atom which stabilizes the structure (Cho et al. 1994).
2.4.1.1 Functions of the p53 protein
It is nowadays known that p53 is not functional or functions incorrectly in most human
cancers, and that it plays a crucial role in the prevention of tumour development
(reviewed by Vogelstein et al. 2000). The general assumption is that the p53 network in
normal, non-activated situations is non-functional, but is activated in cells as a response
to various signals that take place in the carcinogenic process (for a review, see Vogelstein
et al. 2000). Carcinogen-induced DNA damage (Fig. 2), abnormal proliferative signals,
hypoxia and loss of cell adhesion are some of the most common signals. It is thus likely
that p53 takes part in several stages of the carcinogenic process (for a review, see Woods
& Vousden 2001). The importance of a functional p53 protein is emphasized by the fact
that p53-deficient mice show a very high incidence of multiple, spontaneous tumours at
an early age (Donehower et al. 1992, Donehower et al. 1995).
Of the many functions of p53, the first ones identified were inhibition of abnormal
growth of cells (reviewed by Sionov & Haupt 1999) and triggering of programmed cell
death (Fig. 2, Heinrichs & Deppert 2003). Because these processes ensure genomic
integrity or destroy the damaged cell, p53 has been called the “guardian of the genome”
(Lane 1992). Later on, other important functions, such as DNA repair (reviewed by
Albrechtsen et al. 1999) and inhibition of angiogenesis (for a review, see Vogelstein et al.
2000), were discovered. p53 is a sequence-specific nuclear transcription factor that binds
to defined consensus sites within DNA as a tetramer and affects the transcription of its
target genes (El-Deiry 1998). p53 regulates these genes either by transcriptional
activation (Murphy et al. 1999) or by modulating other protein activities by direct
binding (Guimaraes & Hainaut 2002). The p53-induced activation of target genes may
result in the induction of growth arrest either before DNA replication in the G1 phase of
the cell cycle or before mitosis in the G2 phase. The growth arrest enables the repair of
damaged DNA. By programmed cell death, which is often referred to as apoptosis
according to its morphological appearance, the cells damaged beyond repair are
eliminated thus preventing the fixation of DNA damage as mutations. An interesting
question is what determines the choice between growth arrest and apoptosis. Extensive
studies on this topic have been made, and several factors influencing the response of the
cell have been suggested: cell type, oncogenic cell composition, intensity of stress, level
of TP53 expression, interaction of p53 with specific proteins and affinity of p53 for
promoters (reviewed by Vousden & Lu 2002). However, the details of how p53 prevents
the accumulation of mutations after DNA damage still require further studies.
19
DNA damage
Chemical carcinogens
UV, γ, and X irradiation
PARP
p53
p21
DNA repair
Cell cycle arrest
Apoptosis
Maintenance of genomic integrity
Fig. 2. The p53 pathway.
2.4.1.2 Regulation of p53
The p53 protein is effectively able to inhibit cell growth, and its activity is therefore
strictly regulated. There are several mechanisms for the regulation of p53. Although, in
some models, chemical DNA damage, by BP, for example, seems to increase TP53
transcription (Pei et al. 1999, Lu et al. 2000), it is generally believed that the principal
mechanisms governing the activity of p53 occur at the protein level. These include posttranslational modifications, regulation of the stability of p53 protein, and control of its
sub-cellular localization (for a review, see Woods & Vousden 2001). Post-translational
modifications of the protein take place in response to stress, and different agents elicit
diverse responses (reviewed by Appella & Anderson 2001). The human p53 protein has
been shown to be modified at least at 17 different sites (for a review, see Appella &
Anderson 2000). Of the post-translational modifications of p53, the most widely studied
and best-known so far is phosphorylation. After DNA damage induced by ionizing
radiation or UV light, phosphorylation takes place mostly at the N-terminal domain of
p53 (reviewed by Appella & Anderson 2001). Another important modification is
acetylation, which has been shown to occur in response to chemically induced DNA
damage and hypoxia (Ito et al. 2001). In response to DNA damage, the p53 protein is also
modified by conjugation to SUMO-1, a ubiquitin-like protein (Gostissa et al. 1999).
20
Many proteins able to interact with p53 may also play a role in p53 regulation (reviewed
by Vousden & Lu 2002).
Mdm2-mediated degradation regulates the stability of p53. The relationship between
mdm2 and p53 is discussed in more detail in chapter 2.4.2. For many of its functions, p53
needs to be localized in the nucleus. The p53 protein involves nuclear import and export
sequences, and the activity of p53 is regulated by both nuclear import and nuclear export
(Stommel et al. 1999, for a review, see Vousden & Vande Woude 2000). The p53 protein
needs an ability to interact with microtubules in order to move close to the nuclear import
machinery in the cytoplasm (Giannakakou et al. 2000). Also, any changes in the
conformation of the p53 protein and the regulation of its DNA-binding activity affect its
function (Hupp et al. 1992, Cain et al. 2000).
2.4.1.3 Aberrations of p53 function
There are many ways in which the p53 function may be altered in human cancers. p53
can be inactivated indirectly through binding to viral proteins, as a result of alterations in
the mdm2 or p19ARF genes or by localization of the p53 protein to the cytoplasm (for a
review, see Vogelstein et al. 2000). The most common aberration of p53 in human
cancers is, however, mutation of the TP53 gene. A database of TP53 mutations is
maintained by IARC (http://www.iarc.fr/p53/). Most of the mutations in the TP53 gene
occur in the exons 4-9, the coding region for the DNA-binding central domain of the
protein. A large proportion of all mutations in TP53 are single base substitutions (87 %,
Hainaut & Hollstein 2000). Of all mutations, approximately 30 % occur in six codons
(175, 245, 248, 249, 273 and 282, Greenblatt et al. 1994), which are called the hotspot
codons. These residues are located in the DNA-binding part of the protein (Cho et al.
1994), and mutations in these codons influence the protein-DNA contacts and the
conformation of the protein (reviewed by Guimaraes & Hainaut 2002). It also seems that,
in cancer cells with normal TP53 alleles, the expression or regulation of the protein is
often somehow altered. Other factors that prevent normal folding of the p53 protein, such
as cadmium, may influence its DNA-binding capacity (Méplan et al. 1999). It has
therefore been suggested that all cancer cells have some aberration of p53 (Guimaraes &
Hainaut 2002).
2.4.2 Mdm2 protein
The murine double minute 2 (mdm2, hdm2 in human) gene encodes a 90 kDa protein (97
kDa in human) that was originally identified as a dominant transforming oncogene
(Fakharzadeh et al. 1991). The mdm2 gene has been found to be amplified in human
cancers (reviewed by Momand et al. 1998). The combination of overexpressed mdm2
and p53 gives a worse prognosis than either one of them alone (Würl et al. 1998).
Deletion of the mdm2 gene in mice is embryonically lethal, probably due to increased
accumulation of p53, but this lethality can be counter-acted by deletion of the TP53 gene
21
(Jones et al. 1995, Montes de Oca Luna et al. 1995). The p53-mdm2 relationship (see
Fig. 3) is vital in the regulation of cell growth and death.
DNA damage
PARP
p53
?
Ras mRNA
mdm2
p19ARF
Ras
Fig. 3. Proteins involved in the regulation of DNA damage-induced p53 protein.
The mdm2 protein regulates the activity of the p53 protein with more than one
mechanism. It can block the transcriptional activity of the p53 protein, export p53 from
the nucleus to the cytoplasm and promote the degradation of p53 (for a review, see
Alarcon-Vargas & Ronai 2002). The mdm2 protein functions as a ubiquitin ligase and can
ubiquitinate p53 (Honda et al. 1997, Fuchs et al. 1998). Ubiquitination targets the p53
protein for degradation by the proteasome. Post-translational sumoylation of mdm2
increases this ubiquitination, whereas DNA damage reduces the sumoylation of mdm2
and thus the ubiquitination of p53. Hence, the amount of p53 increases (Buschmann et al.
2000, Buschmann et al. 2001). p53, on the other hand, affects the amount of mdm2 by
positive transcriptional activation, creating a feedback loop (for a review, see e.g. Oren et
al. 2002).
The mdm2 protein is able to shuttle between the nucleus and the cytoplasm (Roth et
al. 1998), and it is known to bind to the p53 protein in the N-terminal region (Chen et al.
1993, Picksley et al. 1994). Through binding to p53, mdm2 shuttles p53 out of the
nucleus to the cytoplasm for degradation (Freedman & Levine 1998). DNA damage
induces phosphorylation of the p53 protein at multiple sites, including those overlapping
with the mdm2-binding sites, thus preventing the association between mdm2 and p53
(Shieh et al. 1997).
22
2.4.3 p19ARF protein
The gene that encodes the p16INK4a protein also encodes p19ARF (ARF, Alternative
Reading Frame). However, the p19ARF protein is expressed by a separate promoter with a
different first exon (1β, Mao et al. 1995, Duro et al. 1995). Both p16INK4a and p19ARF
function as tumour suppressors (for a review, see Zhang & Xiong 2001). Selective
deletion of the p19ARF exon 1β in mice results in the development of spontaneous
tumours at an early age (Kamijo et al. 1997). In cells, the p19ARF protein normally
localizes to the nucleolus (Weber et al. 1999). p19ARF is able to bind the central or Cterminal portion of the mdm2 protein (Zhang et al. 1998).
The p19ARF protein is capable of interacting with mdm2 (Kamijo et al. 1998) and
interfering with the autoregulatory feedback loop between the p53 and mdm2 proteins
(Fig. 3), thus increasing the amount of p53. There are currently three competing theories
about how p19ARF inhibits mdm2-mediated p53 degradation. The first possibility is that
the p19ARF protein sequesters the mdm2 protein into the nucleolus, thus releasing p53
(Tao & Levine 1999, Weber et al. 1999). The second model suggests that nucleolar
p19ARF is relocalized by mdm2 to the nucleoplasm and forms a ternary complex with
mdm2 and p53, thus blocking the nuclear export of both mdm2 and p53 (Zhang et al.
1998). p19ARF has also been shown to bind the p53 protein directly, indicating that it can,
in addition to mdm2, recruit p53 into ternary complexes (Kamijo et al. 1998). The third
model proposes that, because the p19ARF protein is able to bind to the mdm2 protein and
inhibit its ubiquitin ligase activity, p19ARF might prevent p53 nuclear export by blocking
the ubiquitination of p53 (Honda & Yasuda 1999). It was shown by Weber and coworkers
(2000) that triple knock-out mice lacking functional p53, mdm2 and p19ARF proteins
develop tumours at a greater frequency than mice lacking p53 and mdm2 or p53 alone.
This suggests that p19ARF is a tumour suppressor independent of mdm2 and p53. The
p19ARF protein itself is regulated primarily at the transcriptional level. Both Myc and E1A
oncoproteins have been shown to induce the synthesis of p19ARF (Zindy et al. 1998, De
Stanchina et al. 1998, respectively). In summary, these three proteins form a system that
regulates their localization, amount and function (Fig. 3).
2.4.4 Other p53-related proteins
2.4.4.1 Poly(ADP-ribose)polymerase
Poly(ADP-ribose)polymerase (PARP) has long been known to play a role in the
recognition of DNA damage and in DNA repair (for a review, see Tong et al. 2001). One
of the first events to take place after DNA strand breakage, caused either directly by
genotoxic agents or indirectly by enzymatic incision of a DNA-base lesion, is the
increased synthesis of poly(ADP-ribose) by PARP (Fig. 3). This is followed by
poly(ADP-ribosyl)ation of proteins localized near the DNA strand breaks. The PARP
protein detects DNA strand breaks and catalyzes the attachment of ADP-ribose units from
23
NAD to itself and to other proteins (reviewed by Lindahl et al. 1995). The substrates of
PARP can then influenze the architecture of chromatin or act in DNA metabolism.
PARP is known to be involved in the regulation of p53, although the relationship is not
clear. There are studies showing that PARP upregulates p53. Cell lines derived from
Chinese hamster V79 cells that are defective in poly(ADP-ribosyl)ation have lower basal
p53 levels and fail to activate p53 in response to etoposide (Whitacre et al. 1995). Also,
in lymphoblastoid cells with normal PARP activity, the PARP inhibitor 3aminobenzamide (3AB, Purnell & Whish 1980) suppresses the accumulation of p53 after
BPDE (Venkatachalam et al. 1997). On the other hand, contradictory results have also
been published. X-ray-induced p53 accumulation was prolonged in mouse prostate cells
treated with a PARP inhibitor 3AB (Lu & Lane 1993). Also, alkylating N-methyl-Nnitrosourea (MNU) caused increased accumulation of p53 in PARP-deficient splenocytes
(Ménissier de Murcia et al. 1997). It has been suggested that PARP plays a positive role
in the activation and upregulation of p53 (Malanga et al. 1998). Smulson and coworkers
(2000) have shown that, in human osteosarcoma cells, p53 is poly(ADP-ribosyl)ated by
PARP. PARP has also been shown to activate DNA-dependent protein kinase (DNA-PK)
activity in vitro and thus to regulate the activity of p53 by phosphorylation (Ruscetti et al.
1998). The PARP inhibitor 3AB also acts as an inhibitor of tumour promotion in mouse
skin (Ludwig et al. 1990). On the other hand, it can act as a cocarcinogen for UV-induced
carcinogenesis (Epstein & Cleaver 1992). Altogether, the relationship between PARP and
p53 seems to differ in different models and still needs further studies to be thoroughly
understood.
2.4.4.2 Oncogenic Ras
Mammalian ras genes are considered crucial in the regulation of cell proliferation
(Johnson et al. 1997, reviewed by Bos 1989). In mammals, the ras family consists of
three genes located in different chromosomes, encoding the homologous 21 kDa proteins
H-Ras, N-Ras and K-Ras. It has been estimated that 30 % of all human cancers express
mutated forms of ras (for a review, see McMahon & Woods 2001). The signal of Ras can
have either negative or positive effects on cell growth, differentiation and death
(reviewed by Frame & Balmain 2000). The signal is subsequently transmitted by a
cascade of kinases, which results in the activation of MAPK. The Ras-MAPK pathway is
apparently involved in the regulation of basal and induced levels of p53 (Fukasawa &
Vande Woude 1997, Serrano et al. 1997). In vascular smooth muscle cells, BP treatment
has been shown to cause an increase in Ras mRNA levels (Kerzee & Ramos 2000). Ras,
in turn, induces p19ARF in murine fibroblasts (Ferbeyre et al. 2002, Groth et al. 2000).
There are also data that support a linear model from Ras through the induction of p19ARF
to p53. Palmero and coworkers (1998) showed that an oncogenic form of Ras protein
increases significantly p19ARF mRNA. Also, in ARF-/- mouse embryonic fibroblasts
(MEF), the p53 level is not affected by oncogenic Ras. In an earlier work on wild-type
MEFs, the p53 level increased after oncogenic Ras (Serrano et al. 1997). It can thus be
concluded that p19ARF is required for oncogenic Ras-induced accumulation of p53 (Fig.
3).
24
2.4.4.3 p21WAF1/Cip1/Sdi1
The p21 protein was the first cyclin-dependent kinase (CDK) identified (El-Deiry et al.
1993, Harper et al. 1993, Noda et al. 1994). The p21 protein has multiple functions. ElDeiry and coworkers (1993) named this gene WAF1 and found it to code a protein that
mediates p53-induced growth arrest of the cell cycle. Almost simultaneously, another
group showed it to be a regulator of CDK activity (Harper et al. 1993). Yet another group
demonstrated its gene expression to be induced in relation to cellular senescence (Noda et
al. 1994). p21 can inhibit CDK-cyclin activity (reviewed by Boulaire et al. 2000) and
directly inhibit DNA replication (Li et al. 1994, Shivji et al. 1994, Chen et al. 1995). The
gene is transcriptionally upregulated by wild-type p53 (El-Deiry et al. 1993). The
activation of p53 causes induction of p21, which in turn inhibits CDK-cyclin activity and
arrests the cell cycle at the G1 (reviewed by Gartel et al. 1996, Colman et al. 2000) or G2
(reviewed by Taylor & Stark 2001, Winters 2002) cell cycle checkpoint. This gives time
for DNA repair before replication or mitosis and thus links p21 directly to the tumour
suppressor function of p53.
2.4.4.4 “Family members” of p53: p63 and p73 proteins
Recently, two new genes notably similar to the TP53 gene have been found. One of these
genes is called p63, p51 or KET, (Yang et al. 1998a, Osada et al. 1998, Schmale &
Bamberger 1997, respectively) and the other p73 (Kaghad et al. 1997). They encode
proteins that share high sequence similarity and conserved functional domains with p53
and can exert p53-like functions, such as transactivation of p53 target genes and
induction of apoptosis (reviewed by Yang et al. 2002). Both give rise to differentially
spliced mRNAs and, respectively, to several different proteins homologous to p53
(reviewed by Levrero et al. 2000). There are at least three different forms of the p63
protein differing at the C-terminal end (α, β and γ) that may also differ at the
transactivation domain (p63TA and p63∆N) and six different variants of the p73 protein,
p73α-ζ. The p73 protein, like p53, accumulates in response to DNA damage, and it is
noteworthy that different types of inducers of DNA damage seem to affect p73 in
different ways (reviewed by Levrero et al. 2000). Both p63 and p73 take part in the
regulation of normal cell development and apoptosis (reviewed by Lohrum & Vousden
2000). Different forms of p63 protein can act in a dominant-negative manner towards p53
(Yang et al. 1998a), but whether p63 dysregulation has a role in tumorigenesis remains to
be seen. p73, on the other hand, has been suggested to be a tumour suppressor protein
(see Levrero et al. 2000), although opposite opinions have also been presented (Irwin &
Kaelin 2001). The function of p63 or p73 as a tumour suppressor still remains unclear
(reviewed by Michael & Oren 2002).
2.4.5 p53 in vitro and in vivo
Because of its importance in human cancers, p53 is nowadays one of the most intensively
studied proteins. Significant progress has been made in the recent years, including the
25
identification of the types of cellular stress that induce p53, identification of the posttranslational modifications that regulate p53 and characterization of the proteins that
interact with p53 and regulate its function (see Fig. 3). All of these important findings
have been made in vitro. The question that still remains to be answered concerns the
relevance of these results in vivo (reviewed by Sansom & Clarke 2000). The significance
of individual aspects concerning the p53 protein, including its function, modification and
regulation, needs to be studied in vivo. The progress in understanding the p53 pathway in
vitro and the introduction of transgenic mice models (reviewed by Herzig & Christofori
2002) will facilitate this work.
2.5 Analysis of the p53 protein
The identification and characterization of the p53 protein has relied extensively on
immunological methods, including immunohistochemistry (Bártek et al. 1990, Davidoff
et al. 1991, Moll et al. 1992, Isola et al. 1992) and immunoblotting (Banks et al. 1986,
Hebel et al. 1986). p53 immunohistochemistry has been used to study the p53 protein in
urothelial carcinomas and dysplasias (Soini et al. 1993), in lymphatic malignancies (Soini
et al. 1992a) and in prostatic, lung and breast carcinomas (Soini et al. 1992b, Bartley &
Ross 2002). p53 is constitutively repressed in most tissues (for a review, see Guimaraes
& Hainaut 2002) and is thus almost undetectable by immunological methods in normal
cells. It can, however, be detected in a variety of tumours and transformed cells because
of the extended half-life of the wild-type or mutated-type protein. Immunoblotting is
widely used for studies on p53 and has been called “the gold standard for demonstration
of p53 expression” (Nickels et al. 1997a). Despite its usefulness in studying the p53
protein, immunoblotting has its limitations. There have been problems with anti-p53
antibodies, especially PAb421, in the detection of the protein (Bonsing et al. 1997,
Nickels et al. 1997b, Danks et al. 1998).
A variety of antibodies have been developed for immunological detection of p53. The
mouse p53 protein can be detected with monoclonal PAb240 and PAb421 antibodies and
with CM5 serum, among others (Table 1). The monoclonal PAb240 antibody (Gannon et
al. 1990) detects both mutated and wild-type proteins under denaturing conditions, e.g. in
immunoblotting. The p53 proteins of both mouse and human origin are detectable with
PAb240, reflecting the high level of sequence homology of the p53 protein between these
species. The epitope for the monoclonal PAb421 antibody (Harlow et al. 1981) is also
highly conserved between human and mouse (Lane et al. 1996). The CM5 polyclonal
serum from rabbits immunised with mouse wild-type p53 was originally developed for an
immunohistochemical analysis of mouse p53 expression (Midgley et al. 1995). It has
high affinity for mouse p53 and low affinity for human p53 and is also very useful in
immunoblotting. CM5 recognizes several antibody epitopes of the mouse p53 protein,
including the epitope for PAb240 (Lane et al. 1996).
26
Table 1. Most frequently used antibodies for mouse p53 protein
Antibody/Clone
Epitope in mouse p53 protein
Reference
PAb122
363-372
PAb240
203-212
Gannon et al. 1990
PAb246
88-93
Yewdell et al. 1986
Yewdell et al. 1986
PAb248
43-52
PAb421
363-372
PAb1620
conformational epitope
CM5
multiple epitopes
Gurney et al. 1980
Harlow et al. 1981
Ball et al. 1984
Midgley et al. 1995
2.6 Ethical issues of animal studies
The use of animals in scientific experiments raises questions that need to be considered.
The first question is the one that attracts most attention: is it justified to use animals for
scientific purposes. There has been a steady increase (approximately 6 % per year in
Finland) in the number of animals used in scientific research, but this increase has now
levelled off (Ministry of Agriculture and Forestry in Finland 2001). Especially the
number of mice used as experimental animals has increased greatly. This is largely due to
the implementation of transgenic techniques and the interesting new opportunities opened
up by them. In addition to these new techniques, the more traditional models are still
widely used. Compensatory methods have been developed in an attempt to reduce the
number of animals used in these studies (for reviews, see Watts 1998, Worth & Balls
2001). The environment in which laboratory animals are maintained is mainly poor in
stimuli compared to the natural environment of the species. Some of the experiments also
cause discomfort or even pain to the animals. It is therefore extremely important to weigh
carefully the rationale of each experiment and the number of animals needed in it. In that
respect, the animal care and use committees of universities are essential.
A frequently asked question is whether animal studies are irreplaceable or whether
some other models could be used instead. In vitro systems compared to in vivo systems
have some important advantages: apart from the fact that they do not involve animals,
they are often technically easier (Peterson 1998). In in vitro models, the arrangement is
simplified and interfering factors are minimised. On the other hand, in vivo studies take
into account the homeostatic system of a living animal and allow more comprehensive
understanding of the system as a whole. Especially in studies of carcinogenesis, which is
a highly complex process and involves multiple steps, the entire process can only be seen
using a living animal.
The credibility and usefulness of animal studies in research on human diseases is also
sometimes questioned. There are differences between humans and the animal species
used in biomedical studies. However, the basic cellular mechanisms are strikingly similar
(reviewed by Balmain & Harris 2000), and therefore animal studies, even though they do
not give direct answers, are at least indicative of human disease.
3 Aims of the study
The complex mechanism by which the p53 protein is induced after environmental
carcinogenic exposure has mainly been studied after exposure causing DNA breaks. The
mechanism of p53 induction after exposure to chemicals causing bulky DNA adducts is
less well known and has mostly been studied in vitro. Therefore, the aim of this study was
to gain deeper insight into the mechanism of the PAH-induced p53 response in vivo in
mouse.
The specific aims of this study were:
1. To confirm the induction of the p53 protein after BP treatment in mouse skin and to
find out if the tumour promoters TPA and thapsigargin affect this induction.
2. To evaluate the different anti-p53 antibodies in immunoblotting and to estimate
whether p53 homologues can cross-react with anti-p53 antibodies.
3. To determine the correlation between the p53, p21 and mdm2 proteins after BP
exposure in mouse skin and to find out whether poly(ADP-ribose)polymerase has a
role in p53 induction.
4. To find out if environmental cigarette smoke exposure causes BPDE-DNA adducts
and how it affects the p53, mdm2, p19ARF and H-Ras proteins in mouse lung, skin,
and liver and to compare these effects to those caused by BP treatment.
4 Materials and methods
4.1 Treatment of mice (I-IV)
The permission for studies using mice was given by the Animal Care and Use Committee
of the University of Oulu. Male C57BL/6 mice that were nine to twenty weeks of age at
the beginning of the studies were maintained in plastic cages at a constant 25°C
temperature with a 12 h light-dark cycle. The mice had free access to food (standard
rodent pellets, Special Diets Services, England) and water. After different treatments
(which are described in detail in the following chapters), the samples from the treated
tissues (skin, lung and/or liver) were divided for immunoblotting, benzo(a)pyrene
diolepoxide (BPDE)-DNA adduct measurements, immunohistochemistry, immuno
electron microscopy and poly(ADP-ribose)polymerase (PARP) enzyme activity assay, as
appropriate.
4.1.1 Treatment of skin (I-III)
Altogether 83 mice were used to study the effect of benzo(a)pyrene (BP) on the p53
protein in skin (for the number of mice in the different treatment groups and in the
original papers, see Table 2). The backs of the mice were shaved three days prior to the
treatments, and only mice with fur in a non-growing phase were used in the studies. The
mice were treated with 500 µg of BP (Sigma, USA), which was applied topically in 100
µl of acetone by dropping slowly on the shaved backs. After 24 hours, the mice were
killed by cercival dislocation and samples were collected. The time for sampling was
chosen based on the knolwedge that the p53 response is strongest 24 hours after the BP
treatment (Bjelogrlic et al. 1994).
29
Table 2. Number of mice in the different treatment groups
Group
Number of Treatment
publications
BP
I, II, III
Solvent control
I, II, III
BP and thapsigargin
I
Thapsigargin control
I
BP and TPA
I
TPA control
I
X-ray and thapsigargin
Studied tissue
Painting of skin
Skin
Painting of skin
Skin
27
7
8
5
Painting of skin
Skin
I
Irradiation and
Skin
X-ray control
I
painting of skin
BP and 3AB
III
Painting of skin
Skin
3AB control
III
BP
IV
Intraperitoneal
Lungs and liver
Solvent control
IV
injection
Smoke exposure
IV
Inhalation exposure
Sham-treated control
IV
Altogether
Number of mice in
the group
10
5
1
1
19
2
9
5
Lungs, liver and skin
6
6
111
For tumour promoter studies, the mice were treated twice, on two consecutive days, with
a tumour promoter, i.e. either thapsigargin (Sigma, USA) or 12-O-tetradecanoylphorbol13-acetate (TPA, Sigma, USA), in 100 µl of acetone. The control animals were treated on
the first day with 100 µl of acetone and those used for the promoter studies with the
promoters as described. Twenty-four hours after the last applications, the mice were
killed by cervical dislocation, and skin samples from the exposed area, which was about 2
cm × 2 cm, were collected.
For comparison, one mouse was treated by X-ray irradiation (50 cGy, 120 kV, 10 mA,
38 s) and another by X-rays and with two doses of thapsigargin (first treatment 17 hours
before and the second 3 hours after the X-ray treatment). The mice were killed by
cervical dislocation, and their skins were collected 24 hours after the first thapsigargin
treatment.
To study the effect of the PARP enzyme on the p53 protein, the backs of male
C57BL/6 mice, which in this particular study (III) were 12-15 weeks of age, were shaved
three days prior to the treatments. The skins of mice with fur in a non-growing phase
were treated topically with either 500 µg of BP alone or with both BP and the PARP
inhibitor 3-aminobenzamide (3AB, Sigma, USA), both in acetone. The control animals
were treated with acetone. 3AB was added at different time points in relation to BP (5 mg
of 3AB, except when stated otherwise): 6 or 2 hours before the BP treatment, at the same
time with BP or 2, 6, 9, 12 (1, 5, or 25 mg), 15, 22 or 24 and 48 hours after the BP
treatment (for the detailed setup of the BP and 3AB -treatments, see Table 3). The skin of
the mouse treated with 3AB twice (24 and 48 hours after the BP treatment) was collected
72 hours after the BP treatment. Otherwise, the skin was collected 24 hours after the BP
treatment.
30
Table 3. Topical treatments of the back skin of C57BL/6 mice for the PARP inhibition
study (III)
Time
a
N
9
1
1
1
1
1
2
2
5
2
1
1
1
a
-6 h
-2 h
Treatment
3ABd
3AB
0h
BPb
BP
BP
BP
3AB
BP
BP
BP
BP
BP
BP
BP
BP
BP
2h
6h
9h
12 h
15 h
22 h
24 h
48 h
72 h
3AB
S
Sc
S
S
S
3AB
3AB
3AB
3ABe
3ABd
3ABf
3AB
3AB
S
S
S
S
S
S
S
S
3AB
Number of mice in the group, b 500 µg BP, c Sampling, d 5 mg 3AB, e 1mg 3AB, f 25 mg 3AB
4.1.2 Intraperitoneal BP treatment (IV)
To study the effect of BP on the lungs and livers of mice, two mice were treated
intraperitoneally with 2.4 mg and seven mice with 3 mg of BP diluted in DMSO and corn
oil (1:1, total volume 150 µl). The lung and liver samples were collected 24 hours after
the treatment from the mice sacrificed with cervical dislocation.
4.1.3 Smoke exposure (IV)
Six mice were treated with smoke from commercial filter cigarettes containing 12 mg tar
and 0.9 mg nicotine per cigarette, as stated by the manufacturer. The mice were placed in
a smoking chamber (Vähäkangas et al. 1982) once a day and five times a week for 31
days. Smoke from two cigarettes per day was forced into the chamber by positive air
pressure. The control mice were placed in a similar chamber without cigarette smoke for
an equal time. The skin, lung and liver samples from mice were taken after cervical
dislocation on the 32nd day.
4.2 Treatment of MCF-7 cells (II)
MCF-7 breast adenocarcinoma cells expressing wild-type p53 protein (Rämet et al. 1995)
were cultured in RPMI 1640 medium containing non-essential amino acids, 10 % fetal
31
bovine serum, 1 mM streptomycin, 1 mM penicillin, 2 mM glutamine (all from Gibco,
UK), 1 µg/ml estradiol (Sigma, USA) and 1 µg/ml insulin (Novo Nordisk, Denmark) at
37°C in an atmosphere of 95 % humified air and 5 % CO2. To study p53 protein, subconfluent cell cultures were treated with either 1 µM or 5 µM BP (Sigma, USA) in
acetone (50 µl of the dilution was added to the media) for 48 hours. Control cells were
treated with acetone.
4.3 DNA isolation and BPDE-DNA adduct analysis by synchronous
fluorescence spectrophotometry (I, III, IV)
For the measurement of adducts, a sensitive fluorescence assay, which detects
specifically BPDE-DNA adducts, was used (Vähäkangas et al. 1985). The principle of
synchronous fluorescence spectrophotometry (SFS) was originally presented by Lloyd
(1971) for analytical chemistry and applied by Vähäkangas and coworkers (1985) to
detect BPDE-DNA adducts. For SFS, mouse skin was incubated in 50°C water for 30
seconds, and the epidermis was then scraped off with a surgical blade into SDS-EDTA
buffer and stored at –20°C (Bjelogrlic et al. 1994). The lungs and livers were
homogenized in SDS-EDTA buffer and stored at –20°C. DNA from the tissues was
isolated using a phenol extraction/ethanol precipitation method (Vähäkangas et al. 1985).
One hundred µg of DNA was hydrolysed in 0.1 M HCl at 90°C for three hours. BPDEDNA adducts were then measured by SFS using a Perkin-Elmer 650-40 fluorescence
spectrophotometer at room temperature. The solvent for fluorescence measurements was
Tris-EDTA. In SFS, when the excitation wavelength is 345 nm, the formed peak
correlates linearly with the level of adducts when scanned synchronously with a
wavelength difference of 34 nm (Vähäkangas et al. 1985). One fluorescence unit equals
approximately 1.1 fmol BPDE/µg DNA. The sensitivity of SFS is about 1 adduct/107
nucleotides.
4.4 Immunoblotting (I-IV)
Immunoblotting was used to study p53 and related proteins in different tissues after
various treatments. For immunoblotting analysis, the treated skin was removed and
subcutaneous fat was removed on ice. The skin and other tissue samples were
homogenized on ice in nuclear lysis buffer (20 % glycerol, 20 mM HEPES, 500 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol (DTT), 0.1 % NP40, 100
µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, 1 µg/ml pepstatin A and
1 µg/ml antipain). MCF-7 cells were washed with ice-cold phosphate-buffered solution
(PBS) and lysed in nuclear lysis buffer. The samples were then incubated for 20 minutes
on ice and centrifuged for 15 minutes at 12 000 rpm at +4°C. The supernatant, a wholecell extract, was collected and samples were maintained at –70°C. A subset of skin
samples was divided into nuclear and cytoplasmic fractions. First, the sample was
homogenized in cytoplasmic lysis buffer (similar to the nuclear lysis buffer with the
32
exception of 10 mM NaCl) and centrifuged for 4 minutes at 2 000 rpm, after which the
supernatant (cytoplasmic extract) was collected. Then nuclear lysis buffer was added to
the pellet, the sample was incubated for 30 minutes on ice and centrifuged for 15 minutes
at 12 000 rpm at +4°C, and the supernatant (nuclear extract) was collected.
The protein samples were heated for 4 min at 96°C in Laemmli buffer and separated
electrophoretically at 200 V (Bio-Rad Power Pac 200, Bio Rad, Hercules, USA) in a 1014 % polyacrylamide gel containing SDS. The proteins were transferred to
polyvinylidene difluoride (PVDF) filter (Immobilon P, 0.45 µm pore size, Millipore,
USA) by electroblotting for 1 h at 100 V. To avoid nonspecific binding, the filters were
blocked overnight at +4ºC in Tris-buffered saline (TBS) containing 5 % non-fat cow’s
milk powder. The filters were then incubated with primary antibody at room temperature
or at +4ºC for 1 h (for the antibodies used in immunoblotting, see Table 4).
Table 4. Antibodies used in immunoblotting
Antigen
Primary
antibody
Source
Secondary
antibody
Source
anti-rabbit IgG
Calbiochem-Novabiochema
Prof. D. Lane, Novocastrab
anti-rabbit IgG
Calbiochem-Novabiochem
PAb240
Oncogenec, Santa Cruzd
anti-mouse IgG
Amershame
PAb421
Oncogene
anti-mouse IgG
Amersham
PAb 1801
Oncogene, Santa Cruz
anti-mouse IgG
Amersham
DO7
Novocastra, Serotecf
anti-mouse IgG
Amersham
H-221
Santa Cruz
anti-rabbit IgG
Calbiochem-Novabiochem
SMP14
Santa Cruz
anti-mouse IgG
Amersham
p19ARF
G-19
Santa Cruz
anti-goat IgG
Santa Cruz
PC435
Oncogene
anti-rabbit IgG
Calbiochem-Novabiochem
p21
(C-19)-G
Santa Cruz
anti-goat IgG
Santa Cruz
H-Ras
C-20
Santa Cruz
anti-rabbit IgG
Calbiochem-Novabiochem
β-catenin
H-102
Santa Cruz
anti-rabbit IgG
Calbiochem-Novabiochem
p53
CM5
Prof. D. Lane (Dundee,
CM1
Scotland)
mdm2
a
Calbiochem-Novabiochem Corporation (USA), bNovocastra Laboratories (UK), cOncogene Research Products
(USA), dSanta Cruz Biotechnologies (USA), eAmersham Life Science (UK), fSerotec (UK)
After treatment with a primary antibody, the filters were washed five times with TBS
containing 0.05 % Tween-20, incubated at room temperature for 1 h with secondary
antibody and washed again with TBS-Tween. As a loading control, β-catenin was
analyzed from some of the same filters. An enhanced chemiluminescence system (ECL+,
Amersham Life Science, UK) was used. The size of the bands was defined by Rainbow
Coloured Protein Molecular Weight Marker (Amersham Life Science, UK).
4.5 Immuno electron microscopy for p53 protein (I)
Immuno electron microscopy was used to study the accumulation and localization of p53.
For immuno electron microscopy, samples were collected from the treated skin with a
stance two millimeters in diameter. The samples were first fixed at room temperature for
33
two hours in 4 % paraformaldehyde (PA) or 2 % PA and 0.2 % glutaraldehyde (GA) in
0.1 M PBS, pH 7.4. They were then dehydrated through a graded ethanol series and
propylenoxide twice for 15 minutes and embedded in araldite (Araldite 502 Kit, Electron
Microscopy Sciences, USA) overnight at 60ºC. Sections of 60-90 nm were cut and
collected in copper and nickel grids. The sections were etched with 10 % H2O2 for 10
minutes and treated with glycine (4 % PA as fixative) or 0.1 % borohydride (2 % PA and
0.2 % GA as fixatives) for 10 minutes and with 8 % BSA (bovine serum albumine) in
PBS, pH 7.4, for 15 minutes. Then the sections were labelled with primary antibody CM5
(dilutions 1:2 000 and 1:10 000) in 1.5 % BSA/0.8 % fish skin gelatine (FSG)/0.05 %
Tween 20 in PBS for two hours at room temperature. The formed p53-protein-antibody
complexes were coupled with 10 nm gold particles (Prot-A-Gold, Sigma, USA) diluted in
1.5 % BSA/0.15 % FSG/0.1 % Tween 20 in PBS for 45 minutes at room temperature. The
sections were double-stained with uranylacetate and Pb citrate. Finally, the sections were
screened and photographed in a transmission electron microscope (Jeol JEM-100 CX II,
Japan).
4.6 Immunohistochemistry for p53 protein (I)
Immunohistochemistry was used to study the p53 protein and its sub-cellular localization.
The skin sections were fixed in neutral 10 % formalin, embedded in paraffin and cut into
5 µm sections. The sections were mounted on slides coated with poly-L-lysin,
deparaffinized in xylene and dehydrated in graded ethanol. The sections were then
incubated in 0.1 % hydrogen peroxide in absolute methanol to block endogenous
peroxidases and in 20 % foetal calf serum in phosphate-buffered saline to block nonspecific binding. The avidin-biotin complex method was used. The sections were
incubated with polyclonal antibody CM5 for 12 hours. Sections treated in a similar
manner without CM5 were used as negative controls. The sections were then incubated
with secondary anti-rabbit immunoglobulin and the avidin-biotin complex (Dako A/S,
Denmark). The brown colour for positive staining was developed with diaminobenzidine,
and the samples were lightly counterstained with haematoxylin. The nucleus thus stains
brown in a positive and blue in a negative case. The percentages of immunopositive
keratinocytes were counted in five high-power (40 × objective) fields including at least
500 keratinocytes each.
4.7 PARP enzyme activity (III)
To measure the activity of PARP enzymes in mouse skin, a poly(ADP-ribose)polymerase
assay kit (Trevigen Inc., USA) was used. The kit determines the incorporation of
radiolabelled nicotinamide adenine dinucleotide (NAD). Samples for the measurement of
PARP activity were prepared according to the instructions of the kit’s manufacturer. The
treated skin was detached from the mice and subcutaneous fat was removed on ice. The
sample was then homogenized for five minutes in sonication buffer (50 mM Tris-Cl, pH
34
8.0, 25 mM MgCl2 and 0.1 mM PMSF). The sample was sonicated for 10 × 10 seconds
on ice and centrifuged for 5 minutes at 3 000 × g. The supernatant was collected and used
for enzyme activity measurement.
4.8 Computer analysis of protein sequences (II)
To judge the likelihood that p53 homologues would interfere with the immunoblotting of
p53, the epitopes of anti-p53 antibodies were compared to the sequences of different
forms of p63 and p73 proteins. Computer analysis of the protein sequences was done by
using a SWISS-PROT/TrEMBL database (http://www.expasy.ch/cgi-bin/sprot-search-de).
Sequences of p53 homologues were compared against a theoretical epitope area of each
anti-p53 antibody using a SIM-Alignment analysis tool (http://www.expasy.ch/tools/simprot.html) with default parameters. The comparison of the protein and the epitope
sequences is presented as a SIM score, which represents the SIM -program’s best score
for the alignment of two sequences.
4.9 Statistical analysis (I, IV)
Statistical analysis of the results of SFS measurements and immunohictochemistry was
done by Student’s t-test. One-tailed t-test was used for the results from SFS and twotailed t-test for results from immunohistochemistry. P-values<0.05 were considered
statistically significant.
5 Results
5.1 p53 and related proteins in mouse skin
5.1.1 Expression of proteins after topical BP treatment (III)
Treatment of mouse skin with BP for 24 hours increased the p53 protein in the skin of all
of the seven studied mice as assessed by immunoblotting (Table 5). Also, the amount of
p21 protein was clearly higher in six out of seven BP-treated mice. The amount of mdm2
protein (studied with two different antibodies SMP14 and H-221) was slightly higher in
the skin in six (the same six with both antibodies) out of seven BP-treated mice compared
to the six acetone-treated controls. The amount of p19ARF protein was not affected in
three BP-treated mice compared to the controls.
Table 5. Effect of benzo(a)pyrene on p53 and related proteins in mouse skin (topical
treatment), lung and liver (intraperitoneal treatment)
Tissue
Skin
Lung
Liver
p53
mdm2
p19ARF
p21
induction
induction
no effect
induction
n.a.*
induction
reduction
induction
n.a.
induction
no effect
n.a.
n.a.
n.a.
n.a.
H-Ras
* Not analyzed
5.1.2 Effect of the tumour promoters TPA and thapsigargin (I)
Both TPA and thapsigargin decreased the number of BP-induced p53-positive nuclei as
assessed by immunohistochemistry (Fig. 4). The effect of TPA was more marked than
that of thapsigargin.
% of p53 positive cells (mean ± s.d.)
36
16
14
12
10
8
6
4
2
0
*
1. BP and acetone
(n=6)
*
2. BP and thapsigargin 3. BP and TPA (n=3)
(n=5)
Fig. 4. Effect of thapsigargin and TPA on benzo(a)pyrene (BP)-incuded p53 protein in mouse
skin in immunohistochemistry. * P<0.05 (compared to group 1).
The decrease of p53 protein after TPA and thapsigargin was confirmed by
immunoblotting. Both TPA and thapsigargin decreased the amount of p53 protein induced
by BPDE-DNA adducts. Based on immunoblotting, too, the effect of TPA was more
pronounced than that of thapsigargin. Thapsigargin also decreased the amount of p53
induction after X-ray irradiation.
Immuno electron microscopy, which was used to study the amount and localization of
p53 protein, confirmed the effect of TPA and thapsigargin on BP-induced p53. The
density of p53-antibody-gold particles was greatest in the sample treated with BP only. A
slightly lower density was seen in the BP- and acetone-treated sample and even lower in
the sample treated with BP and thapsigargin. The lowest density was found in the sample
treated with BP and TPA. The number of particles indicated that slightly more p53
protein is localized in the nuclei of cells than in the cytoplasm. The p53 protein seemed to
be present in all cell organelles except in the mitochondria.
5.1.3 Effect of the PARP inhibitor 3-aminobenzamide (III)
The treatment of mouse skin with 3AB at different time points and with different doses
(see Table 3) had no effect on BP-induced p53 protein in immunoblotting. 3AB did not
affect p21 and mdm2 after BP treatment, either.
Because the PARP inhibitor 3AB did not affect the BP-induced p53 protein, it was
studied whether 3AB really inhibits PARP activity in vivo in our mouse skin model.
When 3AB was painted on skin 12 hours after topical BP treatment, the PARP activity
was inhibited to 62 % compared to the sample treated with BP only (Table 6). Acetone
alone did not cause any inhibition of PARP activity: the activity was 98 % of the activity
in the sample treated with BP.
37
Table 6. Effect of benzo(a)pyrene (BP) and 3-aminobenzamide (3AB) on PARP activity in
mouse skin
Treatment
Acetone
BP for 24 hours
BP and 3AB 12 hours later
BP and 3AB 22 hours later
PARP activity*
98 %
100 %
62 %
90 %
* Relative to mouse treated with BP for 24 hours
5.1.4 Effect of smoke exposure (IV)
Because the skin of the mice was also in contact with the smoke in the smoking chamber,
the p53, mdm2 and p19ARF proteins from the skin were studied. However,
immunoblotting revealed no changes in the skin of the smoke-treated mice compared to
the control mice kept in a similar chamber without cigarette smoke (Table 7).
Table 7. Effect of cigarette smoke treatment on p53 and related proteins in mouse skin,
lung and liver
Tissue
Skin
Lung
Liver
p19ARF
p53
mdm2
p21
H-Ras
no effect
no effect
no effect
n.a.*
n.a.
induction
reduction
induction
n.a.
no effect
no effect
no effect
no effect
n.a.
n.a.
* Not analyzed
5.2 p53 and related proteins in mouse lungs
5.2.1 Effect of intraperitoneal BP treatment (IV)
Intraperitoneal BP treatment changed the levels of the p53, mdm2, p19ARF and H-Ras
proteins in lungs as assessed by immunoblotting (Table 5). Both p53 and p19ARF were
slightly induced in BP-treated lungs (two out of five and four out of five mice,
respectively). The amount of mdm2 decreased after BP treatment in two out of five of the
studied mice. The H-Ras protein also increased after BP treatment in lungs compared to
the controls in four out of five studied mice.
38
5.2.2 Effect of smoke exposure (IV)
Changes in the p53, mdm2 and p19ARF proteins were seen in the lungs of the smoketreated mice compared to the control mice (Table 7). These changes were mostly similar
in nature to those caused by intraperitoneal BP treatment. In two out of six studied mice,
the smoke exposure caused an induction of the p53 protein as assessed by
immunoblotting. Interestingly, the effect of cigarette smoke on the mdm2 protein was
more pronounced: the amount of mdm2 protein decreased clearly in the lungs of all of the
six tobacco smoke-treated mice. The p19ARF protein was also induced by smoke exposure
in all of the six studied mice. The amount of p19ARF was higher in one of the control mice
than in the other controls. Thus, although the effect of cigarette smoke on p53 in lungs
was very similar compared to intraperitoneal BP treatment, the effects on lung mdm2 and
p19ARF were stronger. Smoke exposure seemed to cause no consistent changes in the HRas protein in the lungs.
5.3 p53 and related proteins in mouse liver after smoke exposure and
intraperitoneal BP treatment (IV)
No changes were seen in the p53, mdm2 and p19ARF proteins in the livers of the smoketreated mice compared to the control mice in immunoblotting (Table 7). Furthermore, no
changes were seen in the p53 protein in the livers of the BP-treated mice, either (Table 5).
5.4 p53 protein in MCF-7 cells (II)
The p53 protein was detectable in MCF-7 cells with polyclonal CM1 antibodies and with
monoclonal PAb1801 and DO7 antibodies. The amount of p53 protein increased after BP
treatment, and even more clearly when the cells were treated with 5 µM BP than when
treated with 1 µM BP.
5.5 Comparison of anti-p53 antibodies (II)
In mouse skin, all the antibodies used (CM5, PAb421 and PAb240 from two commercial
sources, see Table 4) detected the p53 protein and also the increase of p53 due to BP
treatment. The intensity of the p53 band and the degree of induction differed, however,
depending on the antibody. The CM5 antibody detected no 53 kDa band in untreated
skin, but showed a clear p53 band after BP treatment. Also, PAb240 from SantaCruz
Biotechnologies showed a clear p53 response. With PAb240 from Oncogene Research
Products, the p53 induction seemed slightly weaker than with PAb240 from SantaCruz
Biotechnologies. With PAb421, the induction was barely detectable. The detected band
was slightly larger in size with the PAb240 and PAb421 antibodies than the band detected
39
by CM5. All the four antibodies showed a strong additional band approximately 66 kDa
in size. The BP treatment did not affect the intensity of this band. The CM5 antibody also
showed a smaller band of 46 kDa, and both the PAb240 antibodies and the PAb421
antibody showed a weak band 60 kDa in size. BP treatment did not affect the intensity of
these bands compared to the controls, either.
With MCF-7 cells, both CM1 antibodies (see Table 4) detected bands about 35, 48, 53,
63 and 66 kDa in size. Both DO7 antibodies detected one clear band at 53 kDa, while
both PAb1801 antibodies showed bands at 53 and 46 kDa. PAb1801 from Oncogene
Research Products also detected a band at approximately 80 kDa. The dose-dependent
accumulation of p53 protein after BP treatment was most obvious with DO7 antibodies.
In MCF-7 extracts, the monoclonal antibodies PAb421 or PAb240 did not produce a
visible band at 53 kDa. Instead, both PAb240 antibodies detected a band at 63 kDa, and
PAb240 from Oncogene Research Products also detected a strong band at 73 kDa.
It was unsure whether these additional bands were caused by the use of whole-cell
lysates. Therefore, a comparison of immunoblots made from whole cell lysates against
samples where nuclear and cytoplasmic fractions of mouse skin had been separated was
made. The results from whole cell lysates and from nuclear fractions in immunoblotting
were similar. A comparison of the PAb240 sequence with equivalent epitopes in p53 and
its homologues showed high similarity only with p53, making it unlikely that the
homologues would interfere in immunoblotting. With PAb421, the result was the same.
5.6 Formation of BPDE-DNA adducts in mouse tissues
5.6.1 Adducts in mouse skin after topical BP treatment and the effects of
TPA, thapsigargin and 3AB on adducts (I, III)
Topical BP treatment clearly increased the amount of BPDE-DNA adducts in mouse skin
compared to acetone treatment (P<0.001, Table 8). Compared to BP-treated skin, the
tumour promoters TPA and thapsigargin caused no statistically significant differences in
fluorescence as measured by SFS, indicating that they do not affect the level of BPDEDNA adducts.
In a small subset of samples, where the PARP inhibitor 3AB was additionally applied
to skin 9, 12 or 15 hours after the BP treatment, the amount of BPDE-DNA adducts was
slightly decreased as measured by SFS. The amount of adducts was 75, 80 or 74 %,
respectively, of the amount in the sample treated with BP alone.
Table 8. Effect of benzo(a)pyrene (BP) or cigarette smoke treatment on BPDE-DNA
adducts measured by synchronous fluorescence spectrophotometry in mouse skin, lung
and liver
Control
Skin
0±0b,c
0.95±0.42
(9)d
(6)
0.03±0.04
0.43±0.10
(4)
(6)
0.01±0.01
0.39±0.41
(4)
(4)
Lung
Liver
a
BPa
Tissue
P-value
P<0.001***
P<0.001***
P=0.054
Control
Cigarette
smoke
P-value
P=0.310
0.06±0.02
0.07±0.06
(5)
(5)
0.03±0.02
0.07±0.04
(6)
(6)
0.03±0.02
0.03±0.02
(6)
(6)
P<0.05*
P=0.381
Topical treatment of skin and intraperitoneal treatment of lung and liver
b
Acetone- and thapsigargin-treated controls
c
Mean fluorescence units ± s.d.
d
Number of samples in parenthesis
5.6.2 Adducts in mouse lung and liver after intraperitoneal BP
treatment (IV)
The amount of BPDE-DNA adducts in the lungs of the C57BL/6 mice treated
intraperitoneally with BP was statistically significantly higher (P<0.001, Table 8) than
that in the lungs of the control mice. In the livers of the mice treated with intraperitoneal
BP, the mean adduct level was also higher than in the control mice, but the difference was
not statistically significant.
5.6.3 Adducts in mouse lung, liver and skin after smoke exposure (IV)
Exposure of the C57BL/6 mice to cigarette smoke in vivo for 31 consecutive days
induced the formation of BPDE-DNA adducts in lungs, as measured by SFS, statistically
significantly (P<0.05, Table 8).
In skin and liver, the cigarette smoke treatment caused no difference in the mean
fluorescence level compared to the controls.
6 Discussion
6.1 p53 induction
Although the induction of the p53 protein has been studied extensively, a vast majority of
the studies have been done using in vitro models. Induction of p53 after BP exposure has
been seen both in vitro (Rämet et al. 1995, Venkatachalam et al. 1997, Pei et al. 1999,
Binkova et al. 2000) and in vivo in mouse (Bjelogrlic et al. 1994, Tapiainen et al. 1996).
The advantage of in vivo studies is that they take into account the regulatory systems of a
living animal and give a more extensive picture, whereas in cell cultures it is easier to
concentrate on an individual phenomenon. In this work, the mechanism of BP-induced
p53 accumulation was studied in vivo in different mouse models.
The p53 protein is known to be induced by various DNA-damaging agents, including
PAHs, but the mechanisms of induction seem to differ, depending on the type of the agent
causing the damage (Gangopadhyay et al. 2002, for a review see Pluquet & Hainaut
2001). BP is a widely used model PAH compound, and it was also used in this study. The
induction of p53 after BP treatment seen in skin (I-III) supports the previous results in
C57BL/6 mice (Bjelogrlic et al. 1994, Tapiainen et al. 1996). This work, where the
increase of p53 was detected by immunoblotting and immunohistochemistry, extends the
earlier work, where only immunohistochemistry was used. Because the induction of the
p53 protein by BP was seen by both of these methods, the induction can be regarded as
proven. The utilization of four different antibodies, CM5, PAb240 (from two suppliers)
and PAb421, which all detected the accumulation of p53 (II), increases the reliability of
this result.
In line with the earlier skin studies (Bjelogrlic et al. 1994, Tapiainen et al. 1996), the
p53 accumulation was associated with an increase in the amount of BPDE-DNA adducts
in skin (I) and in lungs (IV) here, too. BP administered to mice (in this study as a pure
compound or in cigarette smoke) is metabolized to BPDE, which then binds to DNA,
forming bulky DNA adducts (Weinstein et al. 1976, reviewed by Gelboin 1980,
Vähäkangas & Pelkonen 1989). The removal of bulky DNA adducts causes DNA strand
breaks. Ching and coworkers (2001) showed, in a model developed for monitoring
genotoxicants in marine waters, that BP markedly increased DNA strand breaks in
mussels. Strand breaks were also formed in plasmid DNA by Escherichia coli uvrABC
42
endonuclease after modification with BPDE (Seeberg et al. 1983). It has been proposed
that strand break formation may be a common denominator in the effect of most p53activating agents (Nelson & Kastan 1994). Therefore, this could be at least one of the
pathways from BP exposure to p53 activation.
The induction of p53 in skin after BP treatment was earlier shown to be decreased by
the tumour promoter TPA (Tapiainen et al. 1996). In this study, the result obtained with
TPA was confirmed, and another promoter, thapsigargin, was found to decrease p53
induction (I). However, because TPA has been found to be involved in the
phosphorylation of p53 (Milne et al. 1996), it could thereby stabilise the p53 protein by
preventing the binding of mdm2 to p53 (reviewed e.g. by Alarcon-Vargas & Ronai 2002).
Thus, the effect of TPA and thapsigargin on p53 is not yet clear and needs to be studied
further.
The induction of p53 in the skin of C57BL/6 mice was accompanied by induction of
the p21 and mdm2 proteins (III). Because p53 is a transcription factor for the genes of
these proteins, the result indicates that the in vivo induced p53 protein is functionally
active and wild-type. Also, the period between the BP treatment and the collection of
samples was so short that it did not allow time for clonal expansion of possibly mutated
cells. Because, in mouse skin, the mdm2 protein was induced together with p53 after the
BP treatment, the BP-induced p53 accumulation cannot be explained by the lack of
mdm2 in the skin. According to the literature, the PARP protein is involved in the
regulation of the p53 protein (reviewed by Chiarugi 2002) at least in primary mouse
embryonic fibroblasts (Valenzuela et al. 2002), in rat cells overexpressing p53
(Węsierska-Gądek & Schmid 2000) and in high-grade lymphomas (Menegazzi et al.
1999). A binding site for PARP has been identified in the p53 protein (Pleschke et al.
2000), and the phosphorylation of p53 by DNA-PK is stimulated by PARP (Ruscetti et al.
1998). Nonetheless, BP-induced p53 accumulation in mouse skin was not affected when
PARP was inhibited by 3AB (III). If PARP were necessary for the p53 response caused
by BP, 3AB should have inhibited the p53 response. The conclusion is that, in mouse
skin, the induction of p53 by BP is probably independent of PARP. The inhibition of
PARP by 3AB did not affect the BP-induced p21 or mdm2 proteins, either, suggesting
that the p21 and mdm2 proteins in mouse skin are also independent of PARP activity in
vivo. Other proposed factors causing the BP-induced accumulation of p53 are changes in
the sub-cellular localization of the protein (reviewed by Liang & Clarke 2001) or changes
in the phosphorylation status of p53 (Meek 2002, Furihata et al. 2002). These
possibilities remain to be studied.
In the lungs of mice treated with BP or cigarette smoke, the relationship between p53
and mdm2 was, however, different compared to skin: the induction of p53, again
associated with an increase in the amount of BPDE-DNA adducts, was accompanied by a
clear decrease in mdm2 and with an increase in p19ARF (IV). However, the effects of BP
and cigarette smoke on the different proteins were not clearly concentrated in the same
animals, and the phenomenon thus requires further studies. These results implicate that
there are differences in the regulatory systems of p53 induction between different tissues.
This is supported by the finding of organ-specific induction of the p53 protein after lowdose X-ray irradiation (Wang et al. 1996). It is thus possible that BP-induced p53
accumulation in the lungs of C57BL/6 mice is due to induction of the p19ARF protein,
which then prevents mdm2 from functioning. The fact that, in lungs, both cigarette smoke
43
and intraperitoneal BP treatment caused similar results concerning these proteins, gives
support to the hypothesis. Also, the connection between p53, mdm2 and p19ARF has been
demonstrated in many studies in cultured cells (Pomerantz et al. 1998, Zhang et al. 1998,
reviewed by Zhang & Xiong 2001).
The connection between p19ARF and BP could be established by the Ras protein: BP
treatment was shown to cause an increase in the Ras mRNA levels in vascular smooth
muscle cells (Kerzee & Ramos 2000), and Ras was demonstrated to induce the p19ARF
protein in mouse embryo fibroblasts (Groth et al. 2000, Ferbeyre et al. 2002). Besides,
the induction of Ras in the primary skin keratinocytes of mice was found to be directly
accompanied by an increase in the p19ARF and p53 proteins and the activation of the cellcycle arrest program (Lin & Lowe 2001). Moreover, the Ras protein was induced in the
lungs of the C57BL/6 mice by intraperitoneal BP treatment (IV). It is therefore possible
that Ras is the mediating factor between BP and p19ARF, leading to p53 accumulation in
vivo in mouse lungs. On the other hand, the cigarette smoke treatment did not cause an
induction of Ras protein (IV). This could be because of the longer duration of the smoke
treatment compared to the intraperitoneal BP treatment. It was shown by Kerzee &
Ramos (2000) that Ras mRNA increased within a few hours after DNA damage by BP in
vascular smooth muscle cells. Also, different ways of administration of BP (through the
alveolar space in cigarette smoke or through the peritoneal cavity after an intraperitoneal
BP injection) could explain the difference in Ras induction. As a conclusion, the main
finding of this thesis is that, in C57BL/6 mice, BP, after activation and binding to DNA as
BPDE-DNA adducts, induces the p53 protein and the p19ARF, mdm2 and Ras proteins are
associated with the induction in lung. However, p19ARF and mdm2 are not associated with
p53 induction in skin. The role of Ras in skin remains unknown.
6.2 Anti-p53 antibodies
The role of anti-p53 antibodies has been pivotal in the studies of p53 protein.
Immunological detection of proteins is, at its best, easy, quick and sensitive. The number
of commercially available antibodies is large, but as shown in paper II of this thesis and
in the literature, such problems as unidentified additional bands and weak detection have
arisen (Bonsing et al. 1997, Nickels et al. 1997b, Danks et al. 1998).
In mouse tissues, however, the four anti-p53 antibodies compared here showed similar
p53 responses to BP (II). The most widely used anti-p53 antibody in this work was
polyclonal serum CM5, originally developed for immunohistochemical analysis of mouse
p53 (Midgley et al. 1995). CM5 had a high affinity for the mouse p53 protein, as already
shown earlier (Bjelogrlic et al. 1994, Midgley et al. 1995, Tapiainen et al. 1996).
According to the manufacturer, a widely used commercial anti-p53 antibody PAb421
(Harlow et al. 1981) is assumed to detect both human and mouse p53 proteins. In
immunoblotting it did, indeed, detect the BP-induced murine p53 protein in this study
(II). However, problems have been reported with PAb421. It did not detect p53 in human
MCF-7 cells in this study (II) or in the human-derived cell lines Rh28 and Dayo in
immunoblotting (Danks et al. 1998). Well in line with these results, an old paper by
Banks and coworkers (1986) describes that PAb421 has better reactivity against murine
44
than human p53 protein. On the other hand, the problems are not universal: PAb421
antibody has been successfully used even with the human p53 protein in immunoblotting
(Mogaki et al. 1993, Sadji-Ouatas et al. 2002) and in binding assays (Kaku et al. 2001,
Melle & Nasheuer 2002). There seems to be no clear connection between the cases where
the PAb421 antibody has been used successfully or where it has been problematic and the
cause of the discrepancy thus remains unclear. As seen in paper II of this study, dilution
of the antibody is important. Unfortunately, this is not always mentioned in papers, and
its relevance to the problems is therefore difficult to estimate. Cross-reactivity is a
common problem in immunoblotting (Fouraux et al. 2002, Le Bras et al. 2002), and great
care should be practised when drawing conclusions. In this study, the use of more than
one antibody increased the reliability of the results and conclusions.
Another important method for utilizing anti-p53 antibodies is immunohistochemistry
(Bártek et al. 1990, Davidoff et al. 1991, Moll et al. 1992, Isola et al. 1992). The
advantage of immunohistochemistry compared to immunoblotting is that it gives more
information about the sub-cellular localization of proteins. By immunohistochemistry, the
BP-induced accumulation of p53 was seen predominantly in the nuclei of cells (I). The
relationship between the p53, mdm2 and p19ARF proteins is, as described earlier, strongly
influenced by the distribution in cell compartments. The results obtained by
immunohistochemistry in this work concerned only the p53 protein and are limited in that
respect.
6.3 C57BL/6 mice
The C57BL/6 mouse strain has proved useful in studies of the mechanistic aspects of
chemical carcinogenesis with BP (e.g. Bjelogrlic et al. 1994, Tapiainen et al. 1996, Kim
& Lee 1996, Shimada et al. 2002) and with other agents (Seril et al. 2002, Shirai et al.
2002), and it was also used in all of the studies of this thesis. The C57BL/6 strain is
relatively resistant to two-stage skin carcinogenesis but is, however, quite sensitive to
complete carcinogenesis with BP (DiGiovanni 1992). Former studies with this mouse
strain showed the formation of BPDE-DNA adducts in lungs and livers (Bjelogrlic et al.
1989, Bjelogrlic & Vähäkangas 1991) and the induction of the p53 protein in skin
(Bjelogrlic et al. 1994, Tapiainen et al. 1996). It seemed logical to continue with the same
mouse strain to study the effect of cigarette smoke on p53 and related proteins.
Neoplastic processes in mouse and human tissues are genetically and biologically
similar (reviewed by Balmain & Harris 2000). The studies of this thesis did not concern
the inter-species differences or similarities. Nonetheless, some important findings from
the literature should be mentioned. Wadhwa and coworkers (2002) found that there is a
major functional difference between the mouse and human ARF proteins (p19ARF in
mouse and p14ARF in human): p19ARF interacts with the cytosolic Pex19p protein, which
leads to nuclear exclusion and repression of its p53 activation function. Human p14ARF
was not subject to this functional inactivation by Pex19p. The authors therefore suggested
that this could contribute to stricter tumour suppression in human compared to mouse.
The induction of the p19ARF protein after cigarette smoke or BP treatment in the mouse
lung (IV) but not after BP treatment in the skin (III) indicates that there are also
45
differences in the function of the p19ARF protein between different tissues. Also, the
mdm2 protein may act differently in different tissues of C57BL/6 mice according to this
study. In skin, after BP treatment, it is accompanied by the induction of p53 (III), but in
lungs, after intraperitoneal BP or cigarette smoke, it decreases when p53 is induced (IV).
Correspondingly, Salleh and coworkers (2003) recently showed that, in the liver of
C57BL/6J mice, carcinogenic diethylstilbestrol (DES) induces the expression of the TP53
gene. This induction by DES was accompanied by down-regulation of the expression of
the mdm2 gene. However, there are still too few studies to warrant extensive conclusions
concerning the relationship between p53 and mdm2 in vivo.
6.4 Future prospects
Studies have shown abnormal sequestration of wild-type p53 protein in the cytoplasm of
human breast cancer cells, neuroblastoma cells and colon cancer cells (Moll et al. 1992,
Moll et al. 1995, Bosari et al. 1995, respectively). This suggests that a defect in the
regulation of the nuclear import of p53 could result in tumorigenesis (reviewed by Liang
& Clarke 2001). The BP-induced p53 protein in this study (I) was seen more in the nuclei
than in the cytoplasm of the cells. It remains unclear which signals regulate the subcellular localization of p53 in vivo. The effect of the mdm2 and p19ARF proteins on the
localization as well as stabilization of p53 protein in vivo could explain the accumulation
of p53 after PAH exposure and should therefore be studied further. The difference in the
Ras protein in lungs after intraperitoneal and cigarette smoke treatment of C57BL/6 mice
(IV) shows that Ras may also have an important role in the regulation of p53 protein.
An important issue is the difference in the accumulation of p53 between tissues in
vivo. This difference was seen in this study after BP treatment (III-IV) and has also been
seen after whole body γ-ray irradiation (Midgley et al. 1995) and low-dose X-ray
irradiation (Wang et al. 1996) of mice. The variation in p53 accumulation between tissues
seems important. However, only a few studies concerning the tissue variations of p53
have been published, and the causative background of such differences is not clear.
Phosphorylation of proteins, such as SV40 large T antigen, cyclin B1 and nucleolin,
can affect their nuclear import or export (Jans et al. 1991, Yang et al. 1998b, Schwab &
Dreyer 1997, respectively). The tumour promoter TPA may cause phosphorylation of the
p53 protein (Milne et al. 1996), and the other promoter used in this study, thapsigargin, is
also involved in the phosphorylation of other proteins and could putatively be involved in
the phosphorylation of p53 (Jiménez et al. 2002, Tamaskovic et al. 2003). In this study
(I), both TPA and thapsigargin reduced the BP-induced accumulation of p53 in skin. It
would therefore be interesting to study whether this is a common phenomenon with
tumour promoters and with PAHs, and what is the impact of the phosphorylation of p53
on its stability in vivo.
7 Summary and conclusions
1. p53 protein was induced after BP treatment in skin and lungs and after cigarette
smoke treatment in lungs in C57BL/6 mice in vivo. Accumulation of the p53 protein
was associated with an increase in the amount of BPDE-DNA adducts. This
confirmed that BP as a pure compound or in cigarette smoke is metabolized and then
binds to DNA in mouse tissues and indicated that BPDE-DNA adducts are the trigger
for the induction of p53. The p53 induction was decreased by the tumour promoters
TPA and thapsigargin, suggesting that phosphorylation of p53 could affect its
stability.
2. Four different anti-p53 antibodies (CM5, PAb421 and PAb240 from two different
suppliers) detected the induction of p53 by BP in mouse skin in immunoblotting. No
shared sequences were found with the anti-p53 antibodies studied and the p53
homologues p63 and p73, indicating that the additional bands seen in
immunoblotting are not caused by cross-reactivity with them.
3. The induction of the p53 protein in mouse skin by BP was accompanied by an
induction of the p21 and mdm2 proteins, whereas the p19ARF protein was not
affected. Because p53 is a transcription factor for the genes expressing the p21 and
mdm2 proteins, it appears that the in vivo induced p53 protein is wild-type and
functionally active.
4. Treatment of mouse skin with a PARP inhibitor 3AB did not affect the p53, p21 or
mdm2 proteins induced by BP. Although the inhibition of PARP by 3AB was not
very marked, it indicates that, in mouse skin, the induction of the p53, p21 and
mdm2 proteins by BP is independent of PARP.
5. In the lungs of mice treated with BP or cigarette smoke, the induction of the p53
protein was accompanied by a clear decrease of the mdm2 protein and an increase of
the p19ARF protein, suggesting that BP in cigarette smoke, after metabolic activation
to BPDE and binding to DNA, induces p19ARF, which then prevents mdm2 from
functioning and thus allows p53 to accumulate.
6. BP treatment induced the accumulation of the p53 protein in MCF-7 breast
adenocarcinoma cells.
7. The regulation of p53 differs between different tissues in C57BL/6 mouse in vivo.
8. The C57BL/6 mouse strain was reactive to BP and cigarette smoke treatments. To
find out whether this is a more common phenomenon, it would be necessary to
47
utilize other in vivo models to confirm the observations and to help the extrapolation
of the results to man.
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