[CANCER RESEARCH 49, 5489-5496, October 15, 1989]
Special Lecture
Endogenous Carcinogenesis: Molecular Oncology into the Twenty-first CenturyPresidential Address1
Lawrence A. Loeb
The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98I95
In this paper I will consider an aspect of carcinogenesis that
needs further in-depth study because it could be a major cause
of human cancers. I am referring to the Damoclean possibility
that certain normal endogenous cellular processes are inher
ently mutagenic and that this intrinsic mutagenesis is a signif
icant factor in the etiology and pathogenesis of some human
cancers. Moreover, I will prospectively consider the impact of
molecular biology on cancer research. Our ability to determine
the composition of genes at the molecular and atomic levels is
likely to change radically many facets of cancer research. New
techniques in molecular biology not only have highlighted
critical deficiencies in our understanding of the differences
between normal and cancer cells but also have lent us confidence
that new and deeper, more fundamental insights will soon be
within our grasp. I am pleased to report that our Association
has taken new initiatives to incorporate recent advances in
molecular biology into the purview of cancer researchers and
thereby will be a leading facilitator of the transport of molecular
oncology into the 21 st century.
Environmental Carcinogens
A number of environmental agents are now classified as
human carcinogens. By far the most significant is tobacco
smoke, the cause of 30% of cancer deaths in the United States
(1). Currently, the risk of the most common deadly cancer in
the United States, cancer of the lung, is 22 and 12 times greater
in male and female smokers, respectively, than in nonsmokers
(2). The prevalence of cigarette smoking among males in the
United States has decreased steadily in the last 10 years, from
a high of 50% in 1965 to the current level of 31% (3). In
conjunction, after 40 years of relentless increase there has been
a plateauing in the rate of lung cancer deaths in males and a
significant decrease in the 45-55-year-old age group (3). Un
fortunately, smoking incidence has not decreased among Amer
ican females, and there has been a substantial elevation in
export of American tobacco. In this regard, I would like to
thank a person whom I have never formally met, Dr. C. Everett
Koop, the Surgeon General of the United States. He has
brought to the forefront the results of our basic investigations
on the etiology of cancer and used them to motivate Americans
to stop smoking. I salute him on his retirement for his vision
and for his accomplishments.
Other chemicals, including industrial pollutants and natural
substances, have been characterized as human carcinogens
mainly on epidemiológica! evidence; individuals exposed to a
high concentration of a particular chemical have an unusually
high incidence of a specific tumor. In addition, risk factors for
certain cancers have been attributed to diet and life styles.
Received 7/5/89; accepted 7/7/89.
' Presented at the Eightieth Annual
Cancer Research, San Francisco, CA,
author is supported by an Outstanding
National Cancer Institute, Department
Meeting of the American Association for
May 25, 1989. The research work of the
Investigator Grant. CA-39903. from the
of Health and Human Services.
However, despite extensive studies, many human cancers have
no apparent risk factors and do not appear to have widely
varying incidences among individuals of diverse genetic back
grounds or among those living in different geographical areas.
Human cancers with minimal variation in incidence include
many childhood malignancies, as well as carcinoma of the
pancreas, ovary, and colon (4). Wilms' tumor has been recom
mended as an index for consistency in tumor registries (5).
Even in breast (6) and colon cancers, where a large number of
risk factors have been established, they account for only about
one-fourth of the incidence.
DNA Damage by Chemical Carcinogens
In the last few years we have created a veritable explosion in
knowledge about how chemical carcinogens damage DNA (7,
8). We have extended concepts originally formulated by the
Millers (9); among chemical carcinogens are already activated
electrophiles or those that are converted to electrophiles by
cellular activating enzymes. It is the resultant activated chemical
species that react with many cellular macromolecules including
DNA. The interaction with DNA marks cancer as a disease of
genes—not a classically inherited disease, but rather a disease
that is transmitted from parent cell to daughter cell with each
division cycle. It is a disease that is associated invariably with
altered nucleotide sequences in DNA and/or with changes in
gene transcription.
A grossly simplified model that attempts to relate damaged
DNA to mutagenesis is presented in Fig. 1. Mutagenesis results
from unrepaired DNA damage. During each cell division cycle,
DNA polymerases copy past the damaged DNA and insert
noncomplementary nucleotides opposite the site of damage. In
this model, mutations do not result from error-free DNA repair
or from unrepaired lesions that are copied without a change in
sequence of the newly replicated DNA. Furthermore, mutations
do not occur in cells that fail to undergo DNA replication. A
mutational cause of malignancy presupposes that among the
dispersed mutations in the genome are mutations in key genes
that alter the properties of cells, allowing them to escape
homeostatic mechanisms that regulate cell division, invade, and
metastasize. In this model, important forces in determining
whether or not mutations are expressed are the stimuli for cell
division which include increase in autocrine and paracrine
growth factors and regenerative stimuli brought about by the
death of adjacent cells and tissues.
Spontaneous Mutations as a Cause of Cancer
Since mutagenesis by environmental carcinogens appears to
be a key causal event in the etiology of certain cancers, might
not spontaneous mutagenesis also be carcinogenic? Many proc
esses that damage DNA and could contribute to background or
so-called spontaneous mutagenesis have been identified. As in
the case of DNA damage by exogenous chemical agents, spon-
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CARCINOGENESIS:
Unrepaired DNA Damage —¿Â»
Mutations
DMA
mutation rate. A frequency approximating one cancer per in
dividual would be predicted on the basis of allelic recessive
mutations, because of the greater number of sites that poten
tially could inactivate a critical gene. Recessive oncogenes as a
cause of human cancer have been most intensively studied in
retinoblastoma (16) and have been observed in a variety of
human tumors (17, 18). Considering the inaccuracy of our
estimates for rates of spontaneous mutagenesis and the known
"hot spots" for mutagenesis at different genetic loci in prokar-
Quiescent _
Cells
Damage
V
Increased
Cell Prolifer •¿atlon
»No change
in sequence
Error-Prone
(SOS) "
MOLECULAR ONCOLOGY
ErroneousIncorporation
I
Mutations
Fig. 1. Schematic representation of the relationship of DNA damage to
mutagenesis. This scheme emphasizes the importance of increased cell prolifera
tion in the generation of mutations.
taneous damage would need to occur at a sufficiently high
frequency to exceed the capacity of the cell for DNA repair
(Fig. 2). It should be noted that human cells possess an unusu
ally high efficiency in repairing DNA damage (10). This repair
capacity has been postulated to be responsible for the compar
atively long lifespan of our species (11). Nevertheless, not all
DNA damage is repaired, as judged by the production of both
germ line and somatic mutations. The spectrum of mutations
resulting from DNA damage by either exogenous chemicals or
spontaneous lesions would be that damage that is not repaired
(Fig. 2). In a similar manner, errors made by DNA polymerases
would be those that sieve through the mechanisms of the cell
for the correction of mismatches during DNA replication (12).
Spontaneous mutations could have the same potential for in
ducing cancer as those caused by exogenous environmental
agents.
Mutation Rate and Cancer
Clues about the relationships between mutations and cancer
can be gleaned from quantitative analyses. There are "hot spots'"
for mutagenesis within genes; however, until one identifies the
sites of mutations within critical genes that code for malignant
changes, the least prejudicial assumption is that DNA damage
is random. Diverse studies suggest that the mutation rate in
somatic cells is 10~9 to IO'12 events per nucleotide per cell
division (13). However, higher rates may occur when cells are
maintained in tissue culture. The human body contains some
10' ' cells and it is estimated that within a life span our cells
undergo a total of IO16division cycles (14). If one assumes that
yotes (19), either of these possibilities seems reasonable. It is
of interest that the frequency of spontaneous mutations is in
the same range as the number of DNA lesions per genome
produced upon exposure of cells in culture and animals to many
chemicals at carcinogenic doses (20). Exposures of animals and
cells in culture to higher doses of chemical carcinogens are
frequently lethal, and treatment at lower doses fails to yield
significant numbers of oncogenic transformations.
A number of observations suggest that multiple and perhaps
sequential events are required for carcinogenesis (21, 22). Of
importance is age dependence in the incidence of many cancers;
in different species, including humans, the incidences of cancer
increase with the fourth to sixth power of age (23). The analysis
I have presented on the rate of spontaneous mutagenesis and
the number of alterations in DNA induced by a single exposure
to a chemical carcinogen could set limits on the number of
mutagenic events. Either many of these age-dependent events
are not mutagenic or one of these mutations results in the
induction of a mutator phenotype (22, 24). A mutator phenotype could result from mutations in genes such as DNA polymerase, with the production of an altered enzyme that is error
prone in catalyzing the synthesis of DNA (25). A mutator
phenotype would account for the chromosomal instability that
is known to characterize tumor progression (2 1). An abnormally
high frequency of spontaneous deletions has been observed in
tumor-derived cells maintained in tissue culture (17). Consid
ering the multiplicity of proteins involved in DNA replication,
DNA repair, and chromosomal segregation, the number of
potential targets that could generate mutator phenotypes may
be larger than the number of known oncogenes (26). Neverthe
less, the question of whether or not the chromosomal instability
that characterizes tumor progression results from increased
errors in DNA replication remains an important yet unsettled
problem for future study (27-29).
Potential Sources of Spontaneous Mutations
Until fairly recently, DNA was universally considered to be
an essentially unchanged molecule within a cell. Extraordinary
a single dominant mutation is oncogenic, then the spontaneous
mutation rate is sufficient to produce millions of cancer cells
during one's lifetime. If mutations at more than one site can
activate a cancer gene [consider the multiplicity of activated ras
gene (15)] or if there are a large number of tumorigenic target
genes, then an even greater number of tumor cells could be
formed. However, it can be argued that many mutations with
the potential to cause cancer may not do so, since cell prolif
eration may be held in check by the homeostatic mechanisms
that regulate cell growth and behavior.
Alternatively, many mutations may be required to produce a
clinically detectable malignant tumor. The numerology for
spontaneous mutations suggests either two dominant mutations
on separate genes or two recessive mutations on the same alÃ-ele.
Assuming two dominant mutations, each in a different gene
within the same cell, then the rate of spontaneous mutagenesis
would be adequate for 1 cancer in 100 individuals. This estimate
is based on 10IAcells divided by the square of the spontaneous
Endogenous
DNA Damage
Environmental
Chemical
Carcinogens Deputation
DNA
pree
Radicals
Errors
^
DNA Polymerase
Replication
Mutations
Fig. 2. Schematic representation of the production of mutations by damage
to DNA by exogenous as well as endogenous cellular processes.
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stability is presumably required in germ line cells so that genetic
information can be passed from generation to generation with
few if any alterations. We have assumed that a similar unalterability prevails in somatic cells. However, this assumption has
been repeatedly challenged by measurements on the chemical
instability of DNA in solution and by observations of DNA
rearrangements within cells. I will consider three processes that
alter DNA. Each process is known to be mutagenic both in
vitro and in vivo and to occur at a sufficiently high frequency to
account for spontaneous mutagenesis. In model systems, it has
been possible to determine the types, or spectrum, of mutations
produced by each of these processes. A comparison of the
spectra in model systems with the spectrum of spontaneous
mutations occurring in somatic cells may help to quantitate the
contribution of each of these processes to spontaneous muta
genesis. As we identify mutated genes associated with cancers,
it may then be possible to determine further whether sponta
neous mutagenesis is responsible for mutations in those genes
associated with individual types of cancers.
The Chemical Instability of DNA
DNA is not entirely stable in aqueous solution. Of the covalent changes that have been documented, depurination is the
most frequent (30). Depurination of DNA results from the
cleavage of the W-glycosylic bond that connects the purine base
to the deoxyribose sugar. As a result, the DNA backbone is left
intact; and, during DNA replication, DNA polymerases would
encounter a site without a base, i.e., an abasic site (31 ). Lindahl
and Nyberg (30) first measured the rate of release of purine
bases from DNA in aqueous solution as a function of time, pH,
and ionic strength. From these experiments, they calculated
that, under physiological conditions, depurination occurs at a
rate of 3 x 10" events/base/s. We have confirmed their
measurements under a variety of conditions and have also
observed that the rate of depurination is accelerated by the
presence of several divalent metal ions (32). Since the human
nuclear genome contains some 3x10'
purine bases, each cell
would undergo 10,000 depurinations per day. Hydrolysis of the
glycosylic bond connecting pyrimidine bases to the deoxyribose
sugar occurs at a rate 100-fold slower and also results in abasic
sites in DNA (33).
Because depurination is such a frequent event, it is not
surprising that cells possess multiple pathways for the repair of
apurinic sites (7). Nevertheless, it seems probable that many
abasic sites would filter through this DNA repair screen. Se
questration from DNA repair may be afforded by histones that
are tightly complexed to DNA in eukaryotic cells (34). Con
sequently, DNA polymerases may frequently encounter abasic
sites; replication and misincorporations opposite these sites
could be a major source of spontaneous mutations. Evidence
for the mutageneicity of abasic sites is the increased misincorporation by DNA polymerases in copying DNA templates
containing abasic sites (35-37). Surprisingly, DNA polymer
ases do not randomly insert nucleotide substrates opposite
abasic sites; misincorporation is highly specific; deoxyadenosine
is most frequently inserted as a single base substitution (35, 38,
39). Deoxyadenosine is preferentially incorporated by a variety
of DNA polymerases using an analogue of an abasic site present
at a single position within a variety of nucleotide sequences
(40). Accordingly, if depurination is a principal source of spon
taneous mutations, then the spectrum of spontaneous muta
tions should be heavily biased toward substitutions by deoxy
adenosine. However, other lesions in DNA including bulky
MOLECULAR ONCOLOGY
adducts may also direct the incorporation of deoxyadenosine
(41).
Deamination of cytidine to uridine occurs at one five-hun
dredth of the rate of depurination (8). Uridine base pairs with
deoxyadenosine during DNA replication and thus would pro
duce a direct alteration in the sequence of nucleotides in the
newly replicated DNA. However, human cells have a high level
of uracil glycosylase activity (42) and thereby have the potential
to excise uridine and reduce mutagenesis. The eukaryotic ge
nome contains 5-methylcytidine, which is believed to function
in the control of gene transcription. Deamination of 5-methylcytosine yields thymidine, a normal nucleoside, that presumably
if not repaired, provide a potent pathway for mutagenesis via
the formation of C:G—»T:A
transitions (43).
A number of normal, active cellular metabolites are able to
form stable covalent adducts on DNA. Such covalent modifi
cations include methylation of DNA by S-adenosylmethionine
(44) and the glycosylation of DNA by reducing sugars (45). The
frequencies of DNA alterations by these normal cellular metab
olites and their mutagenic potential remain to be determined
in order to evaluate their potential contribution to spontaneous
mutagenesis. Indeed, this field is likely to be a fruitful area for
investigation, not only for carcinogenesis but also for other agedependent disease processes.
Mutagenesis by Free Radicals of Oxygen
The rate of damage to DNA by oxygen free radicals produced
in cellular metabolism (46) may be of the same order of mag
nitude as nucleotide alteration in DNA caused by depurination.
In cells, oxygen is metabolized by a series of one electron
reductions with the generation of highly active free radical
intermediates (47). Among these radicals, hydroxyl ions appear
to be the most damaging. Processes that generate oxygen free
radicals include respiration, phagocytosis, and cell injury. The
resultant free radicals modify RNA, proteins, membranes, and
DNA. A multiplicity of nucleotide alterations have been dem
onstrated by exposing DNA to systems that generate oxygen
free radicals. From measurements of 8-hydroxydeoxyguanosine
and thymine glycol in urine, it can be estimated that approxi
mately 10,000 oxygen free radical-induced alterations occur in
DNA per human cell per day (48). Thus, damage to DNA
occurs at sufficiently high frequency to be a source of sponta
neous mutations. In counteracting this genotoxicity, cells have
evolved a multiplicity of systems to scavenge oxygen free radi
cals and to excise some of the nucleotides from DNA that have
been damaged by these radicals. Damage to DNA that escapes
these repair mechanisms would provide a mechanism for the
generation of mutations and, in fact, the mutagenicity of oxygen
free radicals has been demonstrated in eukaryotic cells after
exposure to elevated concentrations of oxygen or upon incu
bation with systems that generate oxygen free radicals (49).
Even though oxygen metabolism has been well recognized as
a potential source of spontaneous mutations, the contribution
of oxygen-induced mutation to the spontaneous mutation rate
has been very difficult to assess. This is because multiple path
ways exist in cells for the production of oxygen free radicals, a
variety of reactive species are generated, a multiplicity of alter
ations occur in DNA, and until recently there has not been
available a sensitive system for evaluating the mutagenicity of
each type of nucleotide alteration. However, new molecular
systems are sensitive enough to allow one to measure the
mutagenic potential of single lesions at a defined site in DNA
(40, 50, 51). Thus it should be possible to test each of the
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CARCINOGENESIS:
species produced by free radical damage for their potential
mutagenicity.
The mutations caused by incubating DNA with Fe2+ in air
arise via the generation of oxygen free radicals (52), providing
a simple and defined chemical system to analyze the mutagenic
lesions in DNA induced by oxygen free radicals. In this system,
DNA from bacteriophage 0X174, containing a single base
change, is incubated with Fe2+ and then transfected into Escherichia coli spheroplasts. Substitutions of other nucleotides at
this single site result in the production of a wild type progeny
phage that can be quantitated on appropriate indicator bacteria
(53). Mutagenesis requires the presence of oxygen and can be
abolished by catatase and mannitol, suggesting the involvement
of H2O2 and hydroxyl radicals, respectively (52). In E. coli,
mutagenesis is dependent on the induction of the "error-prone"
SOS pathway (54). The DNA sequence of mutants produced
by base substitutions at the target site indicates that deoxyadenosine is inserted preferentially opposite a deoxyadenosine in
the template strand. This preferential insertion of deoxyaden
osine during replication of the damaged DNA template does
not appear to result from the formation of an abasic site caused
by oxygen free radical damage. Mutagenesis by Fe2+-treated
DNA is not abolished by incubation of the damaged DNA with
an apurinic endonuclease that would cleave DNA containing
abasic sites and render the DNA biologically inactive. The
chemical nature of the deoxyadenosine that is altered by expo
sure of DNA to the oxygen free radicals remains to be deter
mined.
Preliminary results indicate that the generation of oxygen
free radicals by other mechanisms is also mutagenic in this
system. Mutagenesis has been observed with myeloperoxidase,
cytochrome oxidase, activated polymorphonuclear leukocytes,
as well as Cu'V The advantage of this molecular approach is
that DNA damage and mutations can be produced in vitro using
a defined system in which all components are present as chem
ically pure species. With Fe2+or Cul+, the extent of mutagenesis
per lethal event at the target site is greater than that observed
with any other agent thus far tested that damages DNA. This
high level of mutagenicity indicates that it will be feasible to
use a forward mutation assay to establish the spectrum of
mutations produced by oxygen free radicals.
Mutagenesis Due to Errors in DNA Replication
In order for DNA replication not to be a key factor in
spontaneous mutagenesis, DNA synthesis must be an incredibly
accurate process. During each cellular replicative cycle, some 3
x 10" nucleotides are copied. Errors in this copying (if not
corrected) are by definition mutations. The primary enzyme
responsible for the high accuracy is DNA polymerase; it cata
lyzes the sequential addition of deoxynucleoside triphosphates.
The fidelity of this process is governed by a multistep process:
(a) by the exactness of base pairings between the template
nucleotides and the incoming deoxynucleoside triphosphate
substrates; (b) by the capacity of the polymerase to increase the
free energy difference between the correct and incorrect basepairings; and (c) by the ability of some, but not all, DNA
polymerases to "proofread" via a 3'—>5'exonuclease that ex
cises an incorporated noncomplementary nucleotide before ad
dition of the next complementary nucleotide. Furthermore, in
bacteria, there is a postsynthetic pathway for removing incor
rectly incorporated nucleotides after DNA replication (12), and
MOLECULAR ONCOLOGY
it is likely, but not proven, that a similar mechanism is present
in eukaryotic cells (55).
The primacy of DNA polymerases in guaranteeing the fidelity
of DNA replication not only is apparent from their central role
in DNA replication but is also substantiated by genetic studies.
Prokaryotic (56) and eukaryotic (57) mutants in DNA poly
merases exhibit a mutator phenotype, i.e., one that increases in
the rate of spontaneous mutations throughout the genome.
Additional evidence that errors by DNA polymerase increase
mutagenesis includes experiments demonstrating enhanced mu
tagenesis due to alterations in the relative concentrations of
deoxynucleoside triphosphates, the substrates of DNA poly
merases (58).
In order to measure frequencies and types of misincorporations by DNA polymerases or by DNA replicating complexes,
we designed a genetic assay (53). A single-stranded circular
0X174 DNA template containing an amber mutation is primed
with a complementary oligonucleotide and copied in vitro with
a purified DNA polymerase. Incorporation of any noncomple
mentary nucleotide at position 587 opposite the adenosine in
the amber codon results in reversion to the wild type phenotype.
After copying proceeds past the amber site, the partially doublestranded DNA product is transfected into E. coli spheroplasts
where synthesis is completed. The error rate of the DNA
polymerase is determined from the reversion rate of the progeny
phage after plating on bacteria that are permissive or nonper
missive for the amber colon. This assay is able to detect misincorporations at a frequency of 10~6 when the four deoxynu
cleoside triphosphate substrates are at equal concentrations in
the reaction mixture and even at a lower frequency of ~10~8
when one noncomplementary nucleotide substrate is present at
a very high concentration (59).
The frequencies of misincorporation by various DNA poly
merases have been analyzed using the t¡>\fidelity assay (60)
and assays to measure forward mutations (61). The combined
data indicate that DNA polymerases are highly error prone in
comparison with the low frequency of spontaneous mutations
exhibited by human cells. DNA polymerase a, the major repli
cating DNA polymerase found in eukaryotic cells, has an error
rate of approximately 1/30,000 when purified as a monomeric
species (60) and 1/200,000 when purified by antibody affinity
chromatography as a 4-subunit complex containing DNA pri
mase (62). The most frequent errors are single-base substitu
tions and, of these, transitions are more frequent than transver
sions. DNA polymerase ß,the enzyme presumed to function
primarily in DNA repair, has an in vitro error rate of about
1/5000 (61). Its most frequent errors are single-base substitu
tions, particularly involving T:G and C:A mispairs, followed by
single-base deletions opposite stretches of identical template
nucleotides. DNA polymerase 6, an enzyme also postulated to
be involved in DNA replication, shares many properties with
DNA polymerase a but contains a 3'—»5'
proofreading exonu
clease (25, 63). As a result, this DNA polymerase is more
accurate; the error frequency has been estimated at 1/500,000
(64). One current model for eukaryotic DNA replication in
volves coordinate synthesis by DNA polymerases a and 5, of
the lagging and leading strand, respectively (65), and it has
been proposed that the exonuclease activity associated with
polymerase 6 is able also to excise errors produced by DNA
polymerase a on the opposite, newly replicated strand.1
Studies on the fidelity of DNA synthesis by eukaryotic DNA
polymerases may not be adequate. Many of the purified en3 F. W. Ferrino and L. A. Loeb, unpublished observations.
*S. Klebanoff and L. A. Loeb, unpublished results.
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CARCINOGENESIS:
zymes could be unphysiological in that they lack a proofreading
exonuclease present in cells but lost during purification. Also
in bacteria, there is a potent system for the postsynthetic
correction of errors during DNA replication, and there is some
evidence for such a system in eukaryotes (55). Nevertheless, the
combined studies on the fidelity of DNA synthesis in vitro
indicate that misincorporations by eukaryotic DNA polymerases are very frequent events; they occur at a much higher
frequency than spontaneous mutations. The most frequent er
rors are single-base substitutions and "minus one" base frameshifts (66). These results are in accord with studies on sponta
neous DNA replication mutations in bacteria. Schaaper and
Dunn (67) determined the mutation spectrum of errors in DNA
replication by using bacteria lacking the genes for postreplicative mismatch repair. Of all mutants, 25% were single-base
deletions and 75% were single-base substitutions. Among the
single-base substitutions, 96% were transitions (67).
The Spectrum of Spontaneous Mutations in Eukaryotic Cells
Detailed comparisons of the frequencies and types of muta
tions produced by singular pathways in vitro with actual spon
taneous mutations in eukaryotic cells may yield important clues
concerning the contribution of a specific pathway to sponta
neous mutagenesis. In principle, the insertion of recombinant
shuttle vectors into eukaryotic cells should allow one to sample
the eukaryotic environment for the production of spontaneous
mutations. However, these studies have been complicated by a
high frequency of mutagenesis, presumably due to DNA dam
age produced during the DNA transfection process (68, 69). In
contrast, recent studies in which chromosomal genes from
mutant cells are cloned or amplified using the polymerase chain
reaction are beginning to yield information on the nucleotide
sequence alterations that characterize spontaneous mutagene
sis. Studies with cells containing a single copy of the nonessential adenine phosphoribosyltransferase locus (APRT) have been
particularly informative. Results from two laboratories (58, 70)
indicate that the majority of spontaneous mutations are singlebase substitutions; in one study, 22 of 27 mutants were G:C to
A:T transitions (70). These limited studies are in accord with
the hypothesis that infidelity of DNA synthesis is a major factor
in spontaneous mutagenesis. There is, however, a caveat; if
genes adjacent to the hemizygous-4/>/?7'gene are essential, then
many deletion and frameshift mutants in APRT may go unde
tected, since these alterations might inactivate the essential
neighboring genes.
Mutations within the three ras genes have been observed in
a significant percentage of all human tumors (15, 71-74). Even
though the role of these mutations in carcinogenesis remains
to be established, these genes provide an interesting probe for
sampling the spectrum of spontaneous mutations associated
with tumorigenesis (75). However, the interpretation of results
obtained with mutant ras genes should be restricted, since only
a subset of all possible activating ras mutations are found in
tumors; thus, those detected may be mutational "hot spots"
and not representative of random mutagenesis. As many as
65% of human colon cancers contain mutant ras genes (71). In
one study, 71% of mutations in codon 12 in the K-ras gene
were transitions (74), while in another 25 of the 26 mutations
were G to A transitions (71, 76). The high frequency of transi
tions favor errors by DNA replication as the source of sponta
neous mutations within the K-ras gene. On the other hand,
studies of K-ras gene mutations in human pancreatic adenocarcinoma yielded a different result; the predominant changes were
MOLECULAR ONCOLOGY
A—*Ttransversions (73), suggesting depurination as a possible
cause for these mutations. However, in another study, the most
frequent mutation was a G—»A
transition (72). Even though
these early studies are only beginning to yield data about the
origin of spontaneous mutations, they indicate that recent mo
lecular techniques are applicable, and they offer evidence that
the source(s) of spontaneous mutations will soon be identified.
I have considered the possibility that spontaneous mutations
contribute causally to human cancer. This contribution is likely
to be most significant for cancers with no known risk factors
and/or for cancers the incidence of which is age dependent and
similar, with respect to genetic background or geography. Mo
lecular methods to identify mutations in genes that determine
the malignant phenotype should allow us to evaluate the con
tribution of spontaneous mutagenesis to carcinogenesis in dif
ferent human cancers. Thus, for each individual it could be only
a matter of time before a random mutation occurs in a key
gene, the thread that holds the sword of Damocles breaks, and
the carcinogenic process starts. A superficial analysis would
suggest that many cancers are inevitable. A deeper inspection
would highlight our lack of understanding of the molecular
differences between normal and cancer cells and the mecha
nisms of the cell for accurate replication of DNA; this knowl
edge is required before we can even begin to consider how to
manipulate these factors. Thus, molecular biology offers both
the power and the promise to unravel the mysteries of human
cancer.
Molecular Biology within the AACR
I have used studies on the origin of spontaneous mutations
and their possible relationships to human cancer to illustrate
the potential of molecular biology to define differences between
normal and malignant cells. I believe we are currently witness
ing a quantum leap in our understanding of how cells work.
This revolution is spearheaded by molecular biology. It seems
to me that many facets of cancer research will be redirected. As
a practitioner of molecular biology, I find it difficult to be
objective. Yet, despite my zeal, let me consider, "How should
the American Association for Cancer Research respond to this
new discipline? What have we so far accomplished?"
The history of cancer research is a record of considerable
accomplishments. Most childhood malignancies are now poten
tially curable; but as recently as 20 years ago, they were invari
ably fatal. The years of productive lives saved by just this one
of many successes have more than justified the cost of cancer
research on economic grounds, to say nothing about humani
tarian grounds. Members of our Association have been major
contributors to this success. In this regard the Association
salutes the awarding of the Nobel Prize in Medicine this year
to Gertrude Elion and George H. Hitchings, two very active
members of our Association. (Dr. Elion served as President of
the American Association for Cancer Research in 1983, and
Dr. Hitchings was elected an Honorary Member in 1981.)
Even though advances in research have furthered our under
standing of the malignant process and have resulted in improve
ments in the treatment of specific adult cancers, all too fre
quently new directions in cancer research have not met our
expectations. Is the new emphasis on molecular biology simply
another chapter in this uneven ascent? I submit not. Previous
new pathways in cancer research were limited at the start by
methodology; and perhaps the absence of this limitation is the
promise of molecular biology.
Molecular biology has been classified as a discipline with its
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CARCINOGENESIS:
own curriculum, rules, and practitioners. But this definition
may be short-sighted. Instead, I submit that molecular biology
constitutes a series of new experimental approaches of unpre
cedented power—approaches that will increasingly change
most aspects of cancer research. Therefore, it is imperative that
this new technology become part of the armamentarium of
current and future cancer investigators. To take full advantage
of this technology, our society has initiated a series of new
programs. These new directions are not limited to molecular
biology. They are designed to provide new avenues to enable a
large society such as ours to stay at the forefront of science and
yet also to maintain a forum for interdisciplinary discussion.
We must maintain this interdisciplinary approach to cancer
research, since this disease has so many ramifications.
This year, the American Association for Cancer Research has
inaugurated a series of satellite meetings focused on molecular
oncology. The first of these meetings, "Oncogenes and Tran
scription," was organized by Phillip Sharp. At this meeting was
presented for the first time a series of reports documenting that
two oncogenes, fos and jun, are transcription factors. These
discoveries foretell a new direction in cancer research, i.e., the
association of oncogenes with the control of transcription. The
second meeting, "Human DNA Tumor Viruses," was organized
by Harald zur Hausen. The thematic question was, "To what
extent are common pathogenic human DNA viruses responsible
for human tumors?" This year's national meeting was preceded
by two short satellite meetings. One, organized by B. Singer
and D. Patel, addressed the structure of DNA adducts and their
mutagenic potential. Another, organized by Inder Verma, con
sidered the relationship of oncogenes to growth factors. Our
annual meeting was followed by three workshops in molecular
biology and a joint U.S./Japanese
Meeting on "Growth
Control and Cancer." I should emphasize that even though
many of these meetings have been focused on molecular ap
proaches to human cancer, other meetings that will focus on a
diversity of topics are planned. We will choose the topics of
future meetings based in part on new exciting findings and the
potential impact of these fields on future cancer research.
In January 1990, the American Association for Cancer Re
search will publish a new journal, "Cell Growth and Differen
tiation." The Journal will be edited by Dr. George Vande
Woude and a cadre of distinguished scientists. Our goal is to
expeditiously publish exciting papers that merge the fields of
molecular and cellular biology with cancer research.
Perhaps the three workshops following the most recent An
nual Meeting best convey the power of this new molecular
technology. The workshop on cloning of genes considered
current methods for identifying, recovering, and expressing
genes in both prokaryotic and eukaryotic cells. We now have
the ability to isolate a single cellular gene, insert it into a
recombinant DNA vector, and amplify it into a million func
tional copies. Thus, we can approach the question of how genes
in cancer cells are altered during carcinogenesis. The workshop
on the polymerase chain reaction (77) presented a new tech
nology allowing one to choose a region of a gene in a single
cell, hybridize onto it oligonucleotide primers and then, with
DNA polymerase, synthesize a million copies of that region in
vitro in a single day. Moreover, one can use archival material
as a source of DNA and thus reconstruct the genetic history of
a tumor (71, 78). I predict that this in vitro technique is likely
to replace DNA cloning. The third workshop focused on tech
niques for the production of transgenic mice. It is now possible
to insert genes or artificial constructs into embryos and produce
mice containing these transgenes in many tissues. By the use of
MOLECULAR ONCOLOGY
a specific promotor, one can study the expression of oncogenes
in selective tissues and do so at different times during devel
opment (79). Thus, one has the potential to determine how
mutations in genes and inappropriate gene expression lead to
malignant changes in vivo.
The molecular biology approaches already at hand will have
profound effects as molecular oncology enters the 21st century.
I predict that not only will this new discipline alter fundamental
cancer research, but its tentacles will also extend into most
related disciplines. Let us consider some fields and examples.
Cancer Epidemiology. Since cancer frequently arises 20 years
after exposure to a chemical carcinogen, retrospective epide
miológica! studies are difficult. It is frequently impossible to
quantitate exposure to environmental agents or to trace the
chronology of tumor progression. Sensitive methods are now
available to measure DNA adducts when present at a concen
tration of 1 adduct in 10'°nucleotides (80). Further, with other
new techniques, it is becoming feasible to detect mutations at
the DNA sequence level occurring during tumor progression.
Cancer Genetics. It has long been recognized that some
cancers occur more frequently in individuals with deficits in
DNA repair (7). Our ability to transplant genes from one human
cell to another, or from a human cell to an animal cell deficient
in DNA repair, should allow us to carry out biochemical studies
on human DNA repair enzymes. The identification of the
altered genes and their functions will give us clues into the
molecular basis for carcinogenesis.
Cancer Diagnoses. In most cases the diagnosis of tumors and
the origin of metastasis is based on tissue histology and cell
morphology. However, 5 to 10% of tumors are too inadequately
differentiated to establish the tissue of origin. For pathologists,
new molecular probes are becoming available to unequivocally
establish the tissue of origin, a diagnosis increasingly relevant
to the choice of appropriate chemotherapy.
Cancer Prognoses. Current cancer therapy can be debilitating.
Thus, a major dilemma for the clinician is to choose a therapy
based on prognosis. If the multiplicity of mutations in certain
human cancers determines the tumor phenotype, methods are
at hand to predict the ability of individual tumors to spread and
metastasize. Also, the early recurrence of tumors can be estab
lished by using the polymerase chain reaction to detect rare
tumor cells with known changes in DNA sequence among a
large population of normal cells.
Cancer Therapy. Most, but not all, effective chemotherapeutic
agents are directed against DNA replication. They bind to the
DNA template, inhibit DNA replication, or interfere with chro
mosome segregation. The identification of genes involved in
cancers should yield a new approach to cancer therapy, i.e.,
gene therapy. This approach might include the use of antisense
DNA or RNA to interfere with gene expression in cells likely
to become malignant and even methods to inactivate selectively
cells harboring specific mutations.
Acknowledgments
I would like to thank Drs. Sam Sorof, Ann Blank, and Keith Cheng
for generous advice in reviewing and Mary Whiting for preparing this
manuscript.
Now that I have completed my term as President of the American
Association for Cancer Research, I can state unequivocally that this is
an exciting position, a conclusion shared by the 79 presidents who
preceded me. I can highly recommend this office to Drs. Busch and
Weinstein, who will succeed me, as well as to future presidents. I have
had the support and tolerance of two families: my wife, Phyllis, and my
children who endured the many trips to Philadelphia, as well as a
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ENDOGENOUS
CARCINOGENESIS:
second family of students, postdoctoral fellows, and colleagues who
have provided ongoing counsel. I wish I could thank them all by name;
in particular, let me again thank Sam Sorof, as well as Richmond
Prehn, Michael Sirover, Bea Singer, and Brad Preston. Every recent
president has thanked Marge Foti and her staff; these tributes are
genuine and are not phatic statements. We are indeed very fortunate to
have our organization staffed by such competent and dedicated people.
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Endogenous Carcinogenesis: Molecular Oncology into the
Twenty-first Century−−Presidential Address
Lawrence A. Loeb
Cancer Res 1989;49:5489-5496.
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