Endogenous Carcinogenesis: Molecular Oncology into the Twenty-first Century-Presidential Address1
Lawrence A. LoebThe 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 thatneeds further in-depth study because it could be a major causeof human cancers. I am referring to the Damoclean possibilitythat certain normal endogenous cellular processes are inherently mutagenic and that this intrinsic mutagenesis is a significant factor in the etiology and pathogenesis of some humancancers. Moreover, I will prospectively consider the impact ofmolecular biology on cancer research. Our ability to determinethe composition of genes at the molecular and atomic levels islikely to change radically many facets of cancer research. Newtechniques in molecular biology not only have highlightedcritical deficiencies in our understanding of the differencesbetween normal and cancer cells but also have lent us confidencethat new and deeper, more fundamental insights will soon bewithin our grasp. I am pleased to report that our Associationhas taken new initiatives to incorporate recent advances inmolecular biology into the purview of cancer researchers andthereby will be a leading facilitator of the transport of molecularoncology into the 21st century.
Environmental Carcinogens
A number of environmental agents are now classified ashuman carcinogens. By far the most significant is tobaccosmoke, the cause of 30% of cancer deaths in the United States(1). Currently, the risk of the most common deadly cancer inthe United States, cancer of the lung, is 22 and 12 times greaterin male and female smokers, respectively, than in nonsmokers(2). The prevalence of cigarette smoking among males in theUnited States has decreased steadily in the last 10 years, froma high of 50% in 1965 to the current level of 31% (3). Inconjunction, after 40 years of relentless increase there has beena plateauing in the rate of lung cancer deaths in males and asignificant decrease in the 45-55-year-old age group (3). Unfortunately, smoking incidence has not decreased among American females, and there has been a substantial elevation inexport of American tobacco. In this regard, I would like tothank a person whom I have never formally met, Dr. C. EverettKoop, the Surgeon General of the United States. He hasbrought to the forefront the results of our basic investigationson the etiology of cancer and used them to motivate Americansto stop smoking. I salute him on his retirement for his visionand for his accomplishments.
Other chemicals, including industrial pollutants and naturalsubstances, have been characterized as human carcinogensmainly on epidemiológica! evidence; individuals exposed to ahigh concentration of a particular chemical have an unusuallyhigh incidence of a specific tumor. In addition, risk factors forcertain cancers have been attributed to diet and life styles.
Received 7/5/89; accepted 7/7/89.' Presented at the Eightieth Annual Meeting of the American Association for
Cancer Research, San Francisco, CA, May 25, 1989. The research work of theauthor is supported by an Outstanding Investigator Grant. CA-39903. from theNational Cancer Institute, Department of Health and Human Services.
However, despite extensive studies, many human cancers haveno apparent risk factors and do not appear to have widelyvarying incidences among individuals of diverse genetic backgrounds or among those living in different geographical areas.Human cancers with minimal variation in incidence includemany childhood malignancies, as well as carcinoma of thepancreas, 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 ofrisk factors have been established, they account for only aboutone-fourth of the incidence.
DNA Damage by Chemical Carcinogens
In the last few years we have created a veritable explosion inknowledge about how chemical carcinogens damage DNA (7,8). We have extended concepts originally formulated by theMillers (9); among chemical carcinogens are already activatedelectrophiles or those that are converted to electrophiles bycellular activating enzymes. It is the resultant activated chemicalspecies that react with many cellular macromolecules includingDNA. The interaction with DNA marks cancer as a disease ofgenes—not a classically inherited disease, but rather a diseasethat is transmitted from parent cell to daughter cell with eachdivision cycle. It is a disease that is associated invariably withaltered nucleotide sequences in DNA and/or with changes ingene transcription.
A grossly simplified model that attempts to relate damagedDNA to mutagenesis is presented in Fig. 1. Mutagenesis resultsfrom unrepaired DNA damage. During each cell division cycle,DNA polymerases copy past the damaged DNA and insertnoncomplementary nucleotides opposite the site of damage. Inthis model, mutations do not result from error-free DNA repairor from unrepaired lesions that are copied without a change insequence of the newly replicated DNA. Furthermore, mutationsdo not occur in cells that fail to undergo DNA replication. Amutational cause of malignancy presupposes that among thedispersed mutations in the genome are mutations in key genesthat alter the properties of cells, allowing them to escapehomeostatic mechanisms that regulate cell division, invade, andmetastasize. In this model, important forces in determiningwhether or not mutations are expressed are the stimuli for celldivision which include increase in autocrine and paracrinegrowth factors and regenerative stimuli brought about by thedeath of adjacent cells and tissues.
Spontaneous Mutations as a Cause of Cancer
Since mutagenesis by environmental carcinogens appears tobe a key causal event in the etiology of certain cancers, mightnot spontaneous mutagenesis also be carcinogenic? Many processes that damage DNA and could contribute to background orso-called spontaneous mutagenesis have been identified. As inthe case of DNA damage by exogenous chemical agents, spon-
Fig. 1. Schematic representation of the relationship of DNA damage tomutagenesis. This scheme emphasizes the importance of increased cell proliferation in the generation of mutations.
taneous damage would need to occur at a sufficiently highfrequency to exceed the capacity of the cell for DNA repair(Fig. 2). It should be noted that human cells possess an unusually high efficiency in repairing DNA damage (10). This repaircapacity has been postulated to be responsible for the comparatively long lifespan of our species (11). Nevertheless, not allDNA damage is repaired, as judged by the production of bothgerm line and somatic mutations. The spectrum of mutationsresulting from DNA damage by either exogenous chemicals orspontaneous lesions would be that damage that is not repaired(Fig. 2). In a similar manner, errors made by DNA polymeraseswould be those that sieve through the mechanisms of the cellfor the correction of mismatches during DNA replication (12).Spontaneous mutations could have the same potential for inducing cancer as those caused by exogenous environmentalagents.
Mutation Rate and Cancer
Clues about the relationships between mutations and cancercan be gleaned from quantitative analyses. There are "hot spots'"
for mutagenesis within genes; however, until one identifies thesites of mutations within critical genes that code for malignantchanges, the least prejudicial assumption is that DNA damageis random. Diverse studies suggest that the mutation rate insomatic cells is 10~9 to IO'12 events per nucleotide per cell
division (13). However, higher rates may occur when cells aremaintained in tissue culture. The human body contains some10' ' cells and it is estimated that within a life span our cellsundergo a total of IO16division cycles (14). If one assumes that
a single dominant mutation is oncogenic, then the spontaneousmutation rate is sufficient to produce millions of cancer cellsduring one's lifetime. If mutations at more than one site can
activate a cancer gene [consider the multiplicity of activated rasgene (15)] or if there are a large number of tumorigenic targetgenes, then an even greater number of tumor cells could beformed. However, it can be argued that many mutations withthe potential to cause cancer may not do so, since cell proliferation may be held in check by the homeostatic mechanismsthat regulate cell growth and behavior.
Alternatively, many mutations may be required to produce aclinically detectable malignant tumor. The numerology forspontaneous mutations suggests either two dominant mutationson separate genes or two recessive mutations on the same alÃele.Assuming two dominant mutations, each in a different genewithin the same cell, then the rate of spontaneous mutagenesiswould be adequate for 1 cancer in 100 individuals. This estimateis based on 10IAcells divided by the square of the spontaneous
mutation rate. A frequency approximating one cancer per individual would be predicted on the basis of allelic recessivemutations, because of the greater number of sites that potentially could inactivate a critical gene. Recessive oncogenes as acause of human cancer have been most intensively studied inretinoblastoma (16) and have been observed in a variety ofhuman tumors (17, 18). Considering the inaccuracy of ourestimates for rates of spontaneous mutagenesis and the known"hot spots" for mutagenesis at different genetic loci in prokar-
yotes (19), either of these possibilities seems reasonable. It isof interest that the frequency of spontaneous mutations is inthe same range as the number of DNA lesions per genomeproduced upon exposure of cells in culture and animals to manychemicals at carcinogenic doses (20). Exposures of animals andcells in culture to higher doses of chemical carcinogens arefrequently lethal, and treatment at lower doses fails to yieldsignificant numbers of oncogenic transformations.
A number of observations suggest that multiple and perhapssequential events are required for carcinogenesis (21, 22). Ofimportance is age dependence in the incidence of many cancers;in different species, including humans, the incidences of cancerincrease with the fourth to sixth power of age (23). The analysisI have presented on the rate of spontaneous mutagenesis andthe number of alterations in DNA induced by a single exposureto a chemical carcinogen could set limits on the number ofmutagenic events. Either many of these age-dependent eventsare not mutagenic or one of these mutations results in theinduction of a mutator phenotype (22, 24). A mutator pheno-type could result from mutations in genes such as DNA polym-erase, with the production of an altered enzyme that is errorprone in catalyzing the synthesis of DNA (25). A mutatorphenotype would account for the chromosomal instability thatis known to characterize tumor progression (2 1). An abnormallyhigh frequency of spontaneous deletions has been observed intumor-derived cells maintained in tissue culture (17). Considering the multiplicity of proteins involved in DNA replication,DNA repair, and chromosomal segregation, the number ofpotential targets that could generate mutator phenotypes maybe larger than the number of known oncogenes (26). Nevertheless, the question of whether or not the chromosomal instabilitythat characterizes tumor progression results from increasederrors in DNA replication remains an important yet unsettledproblem for future study (27-29).
Potential Sources of Spontaneous Mutations
Until fairly recently, DNA was universally considered to bean essentially unchanged molecule within a cell. Extraordinary
EndogenousDNA Damage
Environmental preeChemical Radicals
Carcinogens Deputation
Errors^
DNA Polymerase
DNA Replication
MutationsFig. 2. Schematic representation of the production of mutations by damage
to DNA by exogenous as well as endogenous cellular processes.
stability is presumably required in germ line cells so that geneticinformation can be passed from generation to generation withfew if any alterations. We have assumed that a similar unalter-ability prevails in somatic cells. However, this assumption hasbeen repeatedly challenged by measurements on the chemicalinstability of DNA in solution and by observations of DNArearrangements within cells. I will consider three processes thatalter DNA. Each process is known to be mutagenic both invitro and in vivo and to occur at a sufficiently high frequency toaccount for spontaneous mutagenesis. In model systems, it hasbeen possible to determine the types, or spectrum, of mutationsproduced by each of these processes. A comparison of thespectra in model systems with the spectrum of spontaneousmutations occurring in somatic cells may help to quantitate thecontribution of each of these processes to spontaneous mutagenesis. As we identify mutated genes associated with cancers,it may then be possible to determine further whether spontaneous mutagenesis is responsible for mutations in those genesassociated with individual types of cancers.
The Chemical Instability of DNA
DNA is not entirely stable in aqueous solution. Of the cova-lent changes that have been documented, depurination is themost frequent (30). Depurination of DNA results from thecleavage of the W-glycosylic bond that connects the purine baseto the deoxyribose sugar. As a result, the DNA backbone is leftintact; and, during DNA replication, DNA polymerases wouldencounter a site without a base, i.e., an abasic site (31 ). Lindahland Nyberg (30) first measured the rate of release of purinebases from DNA in aqueous solution as a function of time, pH,and ionic strength. From these experiments, they calculatedthat, under physiological conditions, depurination occurs at arate of 3 x 10" events/base/s. We have confirmed their
measurements under a variety of conditions and have alsoobserved that the rate of depurination is accelerated by thepresence of several divalent metal ions (32). Since the humannuclear genome contains some 3x10' purine bases, each cell
would undergo 10,000 depurinations per day. Hydrolysis of theglycosylic bond connecting pyrimidine bases to the deoxyribosesugar occurs at a rate 100-fold slower and also results in abasicsites in DNA (33).
Because depurination is such a frequent event, it is notsurprising that cells possess multiple pathways for the repair ofapurinic sites (7). Nevertheless, it seems probable that manyabasic sites would filter through this DNA repair screen. Sequestration from DNA repair may be afforded by histones thatare tightly complexed to DNA in eukaryotic cells (34). Consequently, DNA polymerases may frequently encounter abasicsites; replication and misincorporations opposite these sitescould be a major source of spontaneous mutations. Evidencefor the mutageneicity of abasic sites is the increased misincor-poration by DNA polymerases in copying DNA templatescontaining abasic sites (35-37). Surprisingly, DNA polymerases do not randomly insert nucleotide substrates oppositeabasic sites; misincorporation is highly specific; deoxyadenosineis most frequently inserted as a single base substitution (35, 38,39). Deoxyadenosine is preferentially incorporated by a varietyof DNA polymerases using an analogue of an abasic site presentat a single position within a variety of nucleotide sequences(40). Accordingly, if depurination is a principal source of spontaneous mutations, then the spectrum of spontaneous mutations should be heavily biased toward substitutions by deoxyadenosine. However, other lesions in DNA including bulky
adducts may also direct the incorporation of deoxyadenosine(41).
Deamination of cytidine to uridine occurs at one five-hundredth of the rate of depurination (8). Uridine base pairs withdeoxyadenosine during DNA replication and thus would produce a direct alteration in the sequence of nucleotides in thenewly replicated DNA. However, human cells have a high levelof uracil glycosylase activity (42) and thereby have the potentialto excise uridine and reduce mutagenesis. The eukaryotic genome contains 5-methylcytidine, which is believed to functionin the control of gene transcription. Deamination of 5-methyl-cytosine yields thymidine, a normal nucleoside, that presumablyif not repaired, provide a potent pathway for mutagenesis viathe formation of C:G—»T:Atransitions (43).
A number of normal, active cellular metabolites are able toform stable covalent adducts on DNA. Such covalent modifications include methylation of DNA by S-adenosylmethionine(44) and the glycosylation of DNA by reducing sugars (45). Thefrequencies of DNA alterations by these normal cellular metabolites and their mutagenic potential remain to be determinedin order to evaluate their potential contribution to spontaneousmutagenesis. Indeed, this field is likely to be a fruitful area forinvestigation, not only for carcinogenesis but also for other age-dependent disease processes.
Mutagenesis by Free Radicals of Oxygen
The rate of damage to DNA by oxygen free radicals producedin cellular metabolism (46) may be of the same order of magnitude as nucleotide alteration in DNA caused by depurination.In cells, oxygen is metabolized by a series of one electronreductions with the generation of highly active free radicalintermediates (47). Among these radicals, hydroxyl ions appearto be the most damaging. Processes that generate oxygen freeradicals include respiration, phagocytosis, and cell injury. Theresultant free radicals modify RNA, proteins, membranes, andDNA. A multiplicity of nucleotide alterations have been demonstrated by exposing DNA to systems that generate oxygenfree radicals. From measurements of 8-hydroxydeoxyguanosineand thymine glycol in urine, it can be estimated that approximately 10,000 oxygen free radical-induced alterations occur inDNA per human cell per day (48). Thus, damage to DNAoccurs at sufficiently high frequency to be a source of spontaneous mutations. In counteracting this genotoxicity, cells haveevolved a multiplicity of systems to scavenge oxygen free radicals and to excise some of the nucleotides from DNA that havebeen damaged by these radicals. Damage to DNA that escapesthese repair mechanisms would provide a mechanism for thegeneration of mutations and, in fact, the mutagenicity of oxygenfree radicals has been demonstrated in eukaryotic cells afterexposure to elevated concentrations of oxygen or upon incubation with systems that generate oxygen free radicals (49).
Even though oxygen metabolism has been well recognized asa potential source of spontaneous mutations, the contributionof oxygen-induced mutation to the spontaneous mutation ratehas been very difficult to assess. This is because multiple pathways exist in cells for the production of oxygen free radicals, avariety of reactive species are generated, a multiplicity of alterations occur in DNA, and until recently there has not beenavailable a sensitive system for evaluating the mutagenicity ofeach type of nucleotide alteration. However, new molecularsystems are sensitive enough to allow one to measure themutagenic potential of single lesions at a defined site in DNA(40, 50, 51). Thus it should be possible to test each of the
species produced by free radical damage for their potentialmutagenicity.
The mutations caused by incubating DNA with Fe2+ in air
arise via the generation of oxygen free radicals (52), providinga simple and defined chemical system to analyze the mutageniclesions in DNA induced by oxygen free radicals. In this system,DNA from bacteriophage 0X174, containing a single basechange, is incubated with Fe2+ and then transfected into Esch-
erichia coli spheroplasts. Substitutions of other nucleotides atthis single site result in the production of a wild type progenyphage that can be quantitated on appropriate indicator bacteria(53). Mutagenesis requires the presence of oxygen and can beabolished by catatase and mannitol, suggesting the involvementof 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 producedby base substitutions at the target site indicates that deoxyaden-osine is inserted preferentially opposite a deoxyadenosine inthe template strand. This preferential insertion of deoxyadenosine during replication of the damaged DNA template doesnot appear to result from the formation of an abasic site causedby oxygen free radical damage. Mutagenesis by Fe2+-treated
DNA is not abolished by incubation of the damaged DNA withan apurinic endonuclease that would cleave DNA containingabasic sites and render the DNA biologically inactive. Thechemical nature of the deoxyadenosine that is altered by exposure of DNA to the oxygen free radicals remains to be determined.
Preliminary results indicate that the generation of oxygenfree radicals by other mechanisms is also mutagenic in thissystem. 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 usinga defined system in which all components are present as chemically pure species. With Fe2+or Cul+, the extent of mutagenesis
per lethal event at the target site is greater than that observedwith any other agent thus far tested that damages DNA. Thishigh level of mutagenicity indicates that it will be feasible touse a forward mutation assay to establish the spectrum ofmutations produced by oxygen free radicals.
Mutagenesis Due to Errors in DNA Replication
In order for DNA replication not to be a key factor inspontaneous mutagenesis, DNA synthesis must be an incrediblyaccurate process. During each cellular replicative cycle, some 3x 10" nucleotides are copied. Errors in this copying (if not
corrected) are by definition mutations. The primary enzymeresponsible for the high accuracy is DNA polymerase; it catalyzes 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 templatenucleotides and the incoming deoxynucleoside triphosphatesubstrates; (b) by the capacity of the polymerase to increase thefree energy difference between the correct and incorrect base-pairings; and (c) by the ability of some, but not all, DNApolymerases to "proofread" via a 3'—>5'exonuclease that ex
cises an incorporated noncomplementary nucleotide before addition of the next complementary nucleotide. Furthermore, inbacteria, there is a postsynthetic pathway for removing incorrectly incorporated nucleotides after DNA replication (12), and
it is likely, but not proven, that a similar mechanism is presentin eukaryotic cells (55).
The primacy of DNA polymerases in guaranteeing the fidelityof DNA replication not only is apparent from their central rolein DNA replication but is also substantiated by genetic studies.Prokaryotic (56) and eukaryotic (57) mutants in DNA polymerases exhibit a mutator phenotype, i.e., one that increases inthe rate of spontaneous mutations throughout the genome.Additional evidence that errors by DNA polymerase increasemutagenesis includes experiments demonstrating enhanced mutagenesis due to alterations in the relative concentrations ofdeoxynucleoside triphosphates, the substrates of DNA polymerases (58).
In order to measure frequencies and types of misincorpora-tions by DNA polymerases or by DNA replicating complexes,we designed a genetic assay (53). A single-stranded circular0X174 DNA template containing an amber mutation is primedwith a complementary oligonucleotide and copied in vitro witha purified DNA polymerase. Incorporation of any noncomplementary nucleotide at position 587 opposite the adenosine inthe amber codon results in reversion to the wild type phenotype.After copying proceeds past the amber site, the partially double-stranded DNA product is transfected into E. coli spheroplastswhere synthesis is completed. The error rate of the DNApolymerase is determined from the reversion rate of the progenyphage after plating on bacteria that are permissive or nonpermissive for the amber colon. This assay is able to detect mis-incorporations at a frequency of 10~6 when the four deoxynu
cleoside triphosphate substrates are at equal concentrations inthe reaction mixture and even at a lower frequency of ~10~8
when one noncomplementary nucleotide substrate is present ata very high concentration (59).
The frequencies of misincorporation by various DNA polymerases have been analyzed using the t¡>\fidelity assay (60)and assays to measure forward mutations (61). The combineddata indicate that DNA polymerases are highly error prone incomparison with the low frequency of spontaneous mutationsexhibited by human cells. DNA polymerase a, the major replicating DNA polymerase found in eukaryotic cells, has an errorrate of approximately 1/30,000 when purified as a monomericspecies (60) and 1/200,000 when purified by antibody affinitychromatography as a 4-subunit complex containing DNA primase (62). The most frequent errors are single-base substitu
tions and, of these, transitions are more frequent than transversions. DNA polymerase ß,the enzyme presumed to functionprimarily in DNA repair, has an in vitro error rate of about1/5000 (61). Its most frequent errors are single-base substitutions, particularly involving T:G and C:A mispairs, followed bysingle-base deletions opposite stretches of identical templatenucleotides. DNA polymerase 6, an enzyme also postulated tobe involved in DNA replication, shares many properties withDNA polymerase a but contains a 3'—»5'proofreading exonu
clease (25, 63). As a result, this DNA polymerase is moreaccurate; the error frequency has been estimated at 1/500,000(64). One current model for eukaryotic DNA replication involves coordinate synthesis by DNA polymerases a and 5, ofthe lagging and leading strand, respectively (65), and it hasbeen proposed that the exonuclease activity associated withpolymerase 6 is able also to excise errors produced by DNApolymerase a on the opposite, newly replicated strand.1
Studies on the fidelity of DNA synthesis by eukaryotic DNApolymerases may not be adequate. Many of the purified en-
*S. Klebanoff and L. A. Loeb, unpublished results. 3 F. W. Ferrino and L. A. Loeb, unpublished observations.
zymes could be unphysiological in that they lack a proofreadingexonuclease present in cells but lost during purification. Alsoin bacteria, there is a potent system for the postsyntheticcorrection of errors during DNA replication, and there is someevidence for such a system in eukaryotes (55). Nevertheless, thecombined studies on the fidelity of DNA synthesis in vitroindicate that misincorporations by eukaryotic DNA polymer-ases are very frequent events; they occur at a much higherfrequency than spontaneous mutations. The most frequent errors are single-base substitutions and "minus one" base frame-
shifts (66). These results are in accord with studies on spontaneous DNA replication mutations in bacteria. Schaaper andDunn (67) determined the mutation spectrum of errors in DNAreplication by using bacteria lacking the genes for postreplica-tive mismatch repair. Of all mutants, 25% were single-basedeletions and 75% were single-base substitutions. Among thesingle-base substitutions, 96% were transitions (67).
The Spectrum of Spontaneous Mutations in Eukaryotic Cells
Detailed comparisons of the frequencies and types of mutations produced by singular pathways in vitro with actual spontaneous mutations in eukaryotic cells may yield important cluesconcerning the contribution of a specific pathway to spontaneous mutagenesis. In principle, the insertion of recombinantshuttle vectors into eukaryotic cells should allow one to samplethe eukaryotic environment for the production of spontaneousmutations. However, these studies have been complicated by ahigh frequency of mutagenesis, presumably due to DNA damage produced during the DNA transfection process (68, 69). Incontrast, recent studies in which chromosomal genes frommutant cells are cloned or amplified using the polymerase chainreaction are beginning to yield information on the nucleotidesequence alterations that characterize spontaneous mutagenesis. Studies with cells containing a single copy of the nonessen-tial adenine phosphoribosyltransferase locus (APRT) have beenparticularly informative. Results from two laboratories (58, 70)indicate that the majority of spontaneous mutations are single-base substitutions; in one study, 22 of 27 mutants were G:C toA:T transitions (70). These limited studies are in accord withthe hypothesis that infidelity of DNA synthesis is a major factorin spontaneous mutagenesis. There is, however, a caveat; ifgenes adjacent to the hemizygous-4/>/?7'gene are essential, then
many deletion and frameshift mutants in APRT may go undetected, since these alterations might inactivate the essentialneighboring genes.
Mutations within the three ras genes have been observed ina significant percentage of all human tumors (15, 71-74). Eventhough the role of these mutations in carcinogenesis remainsto be established, these genes provide an interesting probe forsampling the spectrum of spontaneous mutations associatedwith tumorigenesis (75). However, the interpretation of resultsobtained with mutant ras genes should be restricted, since onlya subset of all possible activating ras mutations are found intumors; thus, those detected may be mutational "hot spots"
and not representative of random mutagenesis. As many as65% of human colon cancers contain mutant ras genes (71). Inone study, 71% of mutations in codon 12 in the K-ras genewere transitions (74), while in another 25 of the 26 mutationswere G to A transitions (71, 76). The high frequency of transitions favor errors by DNA replication as the source of spontaneous mutations within the K-ras gene. On the other hand,studies of K-ras gene mutations in human pancreatic adenocar-cinoma yielded a different result; the predominant changes were
A—*Ttransversions (73), suggesting depurination as a possiblecause for these mutations. However, in another study, the mostfrequent mutation was a G—»Atransition (72). Even thoughthese early studies are only beginning to yield data about theorigin of spontaneous mutations, they indicate that recent molecular techniques are applicable, and they offer evidence thatthe source(s) of spontaneous mutations will soon be identified.
I have considered the possibility that spontaneous mutationscontribute causally to human cancer. This contribution is likelyto be most significant for cancers with no known risk factorsand/or for cancers the incidence of which is age dependent andsimilar, with respect to genetic background or geography. Molecular methods to identify mutations in genes that determinethe malignant phenotype should allow us to evaluate the contribution of spontaneous mutagenesis to carcinogenesis in different human cancers. Thus, for each individual it could be onlya matter of time before a random mutation occurs in a keygene, the thread that holds the sword of Damocles breaks, andthe carcinogenic process starts. A superficial analysis wouldsuggest that many cancers are inevitable. A deeper inspectionwould highlight our lack of understanding of the moleculardifferences between normal and cancer cells and the mechanisms of the cell for accurate replication of DNA; this knowledge is required before we can even begin to consider how tomanipulate these factors. Thus, molecular biology offers boththe power and the promise to unravel the mysteries of humancancer.
Molecular Biology within the AACR
I have used studies on the origin of spontaneous mutationsand their possible relationships to human cancer to illustratethe potential of molecular biology to define differences betweennormal and malignant cells. I believe we are currently witnessing a quantum leap in our understanding of how cells work.This revolution is spearheaded by molecular biology. It seemsto me that many facets of cancer research will be redirected. Asa practitioner of molecular biology, I find it difficult to beobjective. Yet, despite my zeal, let me consider, "How should
the American Association for Cancer Research respond to thisnew discipline? What have we so far accomplished?"
The history of cancer research is a record of considerableaccomplishments. Most childhood malignancies are now potentially curable; but as recently as 20 years ago, they were invariably fatal. The years of productive lives saved by just this oneof many successes have more than justified the cost of cancerresearch on economic grounds, to say nothing about humanitarian grounds. Members of our Association have been majorcontributors to this success. In this regard the Associationsalutes the awarding of the Nobel Prize in Medicine this yearto Gertrude Elion and George H. Hitchings, two very activemembers of our Association. (Dr. Elion served as President ofthe American Association for Cancer Research in 1983, andDr. Hitchings was elected an Honorary Member in 1981.)
Even though advances in research have furthered our understanding of the malignant process and have resulted in improvements in the treatment of specific adult cancers, all too frequently new directions in cancer research have not met ourexpectations. Is the new emphasis on molecular biology simplyanother chapter in this uneven ascent? I submit not. Previousnew pathways in cancer research were limited at the start bymethodology; and perhaps the absence of this limitation is thepromise of molecular biology.
Molecular biology has been classified as a discipline with its5493
own curriculum, rules, and practitioners. But this definitionmay be short-sighted. Instead, I submit that molecular biologyconstitutes a series of new experimental approaches of unprecedented power—approaches that will increasingly changemost aspects of cancer research. Therefore, it is imperative thatthis new technology become part of the armamentarium ofcurrent and future cancer investigators. To take full advantageof this technology, our society has initiated a series of newprograms. These new directions are not limited to molecularbiology. They are designed to provide new avenues to enable alarge society such as ours to stay at the forefront of science andyet also to maintain a forum for interdisciplinary discussion.We must maintain this interdisciplinary approach to cancerresearch, since this disease has so many ramifications.
This year, the American Association for Cancer Research hasinaugurated a series of satellite meetings focused on molecularoncology. The first of these meetings, "Oncogenes and Transcription," was organized by Phillip Sharp. At this meeting was
presented for the first time a series of reports documenting thattwo oncogenes, fos and jun, are transcription factors. Thesediscoveries foretell a new direction in cancer research, i.e., theassociation of oncogenes with the control of transcription. Thesecond meeting, "Human DNA Tumor Viruses," was organizedby Harald zur Hausen. The thematic question was, "To what
extent are common pathogenic human DNA viruses responsiblefor human tumors?" This year's national meeting was preceded
by two short satellite meetings. One, organized by B. Singerand D. Patel, addressed the structure of DNA adducts and theirmutagenic potential. Another, organized by Inder Verma, considered the relationship of oncogenes to growth factors. Ourannual meeting was followed by three workshops in molecularbiology and a joint U.S./Japanese Meeting on "GrowthControl and Cancer." I should emphasize that even though
many of these meetings have been focused on molecular approaches to human cancer, other meetings that will focus on adiversity of topics are planned. We will choose the topics offuture meetings based in part on new exciting findings and thepotential impact of these fields on future cancer research.
In January 1990, the American Association for Cancer Research will publish a new journal, "Cell Growth and Differentiation." The Journal will be edited by Dr. George Vande
Woude and a cadre of distinguished scientists. Our goal is toexpeditiously publish exciting papers that merge the fields ofmolecular and cellular biology with cancer research.
Perhaps the three workshops following the most recent Annual Meeting best convey the power of this new moleculartechnology. The workshop on cloning of genes consideredcurrent methods for identifying, recovering, and expressinggenes in both prokaryotic and eukaryotic cells. We now havethe ability to isolate a single cellular gene, insert it into arecombinant DNA vector, and amplify it into a million functional copies. Thus, we can approach the question of how genesin cancer cells are altered during carcinogenesis. The workshopon the polymerase chain reaction (77) presented a new technology allowing one to choose a region of a gene in a singlecell, hybridize onto it oligonucleotide primers and then, withDNA polymerase, synthesize a million copies of that region invitro in a single day. Moreover, one can use archival materialas a source of DNA and thus reconstruct the genetic history ofa tumor (71, 78). I predict that this in vitro technique is likelyto replace DNA cloning. The third workshop focused on techniques for the production of transgenic mice. It is now possibleto insert genes or artificial constructs into embryos and producemice containing these transgenes in many tissues. By the use of
a specific promotor, one can study the expression of oncogenesin selective tissues and do so at different times during development (79). Thus, one has the potential to determine howmutations in genes and inappropriate gene expression lead tomalignant changes in vivo.
The molecular biology approaches already at hand will haveprofound effects as molecular oncology enters the 21st century.I predict that not only will this new discipline alter fundamentalcancer research, but its tentacles will also extend into mostrelated disciplines. Let us consider some fields and examples.
Cancer Epidemiology. Since cancer frequently arises 20 yearsafter exposure to a chemical carcinogen, retrospective epidemiológica! studies are difficult. It is frequently impossible toquantitate exposure to environmental agents or to trace thechronology of tumor progression. Sensitive methods are nowavailable to measure DNA adducts when present at a concentration of 1 adduct in 10'°nucleotides (80). Further, with other
new techniques, it is becoming feasible to detect mutations atthe DNA sequence level occurring during tumor progression.
Cancer Genetics. It has long been recognized that somecancers occur more frequently in individuals with deficits inDNA repair (7). Our ability to transplant genes from one humancell to another, or from a human cell to an animal cell deficientin DNA repair, should allow us to carry out biochemical studieson human DNA repair enzymes. The identification of thealtered genes and their functions will give us clues into themolecular basis for carcinogenesis.
Cancer Diagnoses. In most cases the diagnosis of tumors andthe origin of metastasis is based on tissue histology and cellmorphology. However, 5 to 10% of tumors are too inadequatelydifferentiated to establish the tissue of origin. For pathologists,new molecular probes are becoming available to unequivocallyestablish the tissue of origin, a diagnosis increasingly relevantto the choice of appropriate chemotherapy.
Cancer Prognoses. Current cancer therapy can be debilitating.Thus, a major dilemma for the clinician is to choose a therapybased on prognosis. If the multiplicity of mutations in certainhuman cancers determines the tumor phenotype, methods areat hand to predict the ability of individual tumors to spread andmetastasize. Also, the early recurrence of tumors can be established by using the polymerase chain reaction to detect raretumor cells with known changes in DNA sequence among alarge population of normal cells.
Cancer Therapy. Most, but not all, effective chemotherapeuticagents are directed against DNA replication. They bind to theDNA template, inhibit DNA replication, or interfere with chromosome segregation. The identification of genes involved incancers should yield a new approach to cancer therapy, i.e.,gene therapy. This approach might include the use of antisenseDNA or RNA to interfere with gene expression in cells likelyto become malignant and even methods to inactivate selectivelycells harboring specific mutations.
Acknowledgments
I would like to thank Drs. Sam Sorof, Ann Blank, and Keith Chengfor generous advice in reviewing and Mary Whiting for preparing thismanuscript.
Now that I have completed my term as President of the AmericanAssociation for Cancer Research, I can state unequivocally that this isan exciting position, a conclusion shared by the 79 presidents whopreceded me. I can highly recommend this office to Drs. Busch andWeinstein, who will succeed me, as well as to future presidents. I havehad the support and tolerance of two families: my wife, Phyllis, and mychildren who endured the many trips to Philadelphia, as well as a
second family of students, postdoctoral fellows, and colleagues whohave provided ongoing counsel. I wish I could thank them all by name;in particular, let me again thank Sam Sorof, as well as RichmondPrehn, Michael Sirover, Bea Singer, and Brad Preston. Every recentpresident has thanked Marge Foti and her staff; these tributes aregenuine and are not phatic statements. We are indeed very fortunate tohave our organization staffed by such competent and dedicated people.
References
1. Loeb, L. A., Ernster, V. L., Warner, K. E., Abbotts, J., and Laszlo, J.Smoking and lung cancer: an overview. Cancer Res., 44: 5940-5958, 1984.
2. United States Public Health Service. The Health Consequences of Smoking—Cancer: A Report of the Surgeon General. Rockville, MD: UnitedStates Department of Health and Human Services, Office on Smoking andHealth, 1989.
3. Marcus, A. C, Shopland, D. R., Crane, L. A., and Lynn, W. R. Prevalenceof cigarette smoking in the United States: estimates from the 1985 currentpopulation survey. J. Nati. Cancer Inst., SI: 409-414, 1989.
4. Muir, C. S., and Nectoux, J. International patterns of cancer. In: D. Shotten-feld and J. F. Fraumeni, (eds.), Cancer Epidemiology and Prevention, pp.119-135. Philadelphia: W. B. Saunders Co., 1982.
5. Miller, R. W. Geographical and ethnic differences in the occurrence ofchildhood cancer. In: D. M. Prkin, C. A. Stiller, G. J. Draper, C. A. Bieber,B. Terracini, and J. L. Young (eds.), International Incidence of ChildhoodCancer, pp. 3-7. Lyon, France: International Agency for Research on Cancer,1988.
6. Kelsey, J. L., and Berkowitz, G. S. Breast cancer epidemiology. Cancer Res.,¥«.•5615-5623,1988.
7. Friedberg, E. C. DNA Repair. San Francisco: W. H. Freeman and Co., 1985.8. Singer, B., and Grunberger, D. Molecular Biology of Mutagens and Carcin
ogens, New York: Plenum Publishing Corp., 1983.9. Miller, E. C., and Miller, J. A. Biochemical mechanisms of chemical card
nogenesis. In: H. Busch (ed.), The Molecular Biology of Cancer, pp. 377-402. New York: Academic Press, 1974.
10. Bohr, V. A., Okumoto, D. S., and Hanawalt, P. C. Survival of UV-irradiatedmammalian cells correlates with efficient DNA repair in an essential gene.Proc. Nati. Acad. Sci. USA, 83: 3830-3833, 1986.
11. Hart, R. W., and Setlow, R. B. Correlation between deoxyribonucleic acidexcision—repair and life-span in a number of mammalian species. Proc.Nati. Acad. Sci. USA, 71: 2169-2173, 1974.
12. Su, S-S., Lahue, R. S., Au, K. G., and Modrich, P. Mispair specificity ofmethyl-directed DNA mismatch correction in vitro. J. Biol. Chem., 263:6829-6835, 1988.
13. Drake, J. W., Allen, E. F., Forsberg, S. A., Preparata, R. M., and Greenberg,E. O. Spontaneous mutation, Nature (Lond.), 221: 1128-1132, 1969.
14. Duesberg, P. H. Cancer genes: rare recombinants instead of activated onco-genes. Proc. Nati. Acad. Sci. USA, 84: 2117-2124, 1987.
15. Almoguera, C., Shibata, D., Forrester, K., Martin, J., Arnheim, N., andPerucho, M. Most human carcinomas of the exocrine pancreas containmutant c-K-ras genes. Cell, 53: 549-554, 1988.
16. KHudson, A. G., Jr. Genetics and etiology of human cancer. Hum. Genet.,8:1-66, 1977.
17. Kaden, D. A., Bardwell, L., Newmark, P., Anisowicz, A., Skopek, T. R., andSager, R. High frequency of large spontaneous deletions of DNA in tumor-derived CHEF cells. Proc. Nati. Acad. Sci. USA, 86: 2306-2310, 1989.
18. Lee, E. Y-H. P., To, H., Shew, J-Y., Bookstein, R., Scully, P., and Lee, W-H. Inactivation of the retinoblastoma susceptibility gene in human breastcancers. Science (Wash. DC), 241: 218-221, 1988.
19. Miller, J. H., and Schmeissner, U. Genetic studies of the lac represser X.Analysis of missense mutations in the lad gene. Mol. Biol., 131: 223-248,1979.
20. Mita, S., Monnat, R. J., Jr., and Loeb, L. A. Resistance of HeLa cellmitochondria! DNA to mutagenesis by chemical carcinogens. Cancer Res.,48: 4578-4583, 1988.
21. Foulds, L. The experimental study of tumor progression: a review. CancerRes., 14: 317-339, 1954.
22. Nowell, P. C. The clonal evolution of tumor cell populations. Science (Wash.DC), 194: 23-28, 1976.
23. Ames, B. N., Saul, R. L., Schwiers, E., Adelman, R., and Cathcart, R.Oxidative DNA damage as related to cancer and aging: The assay of thymineglycol, thymidine glycol, and hydroxymethyluracil in human and rat urine.In: R. S. Sohal, L. S. Birnbaum, and R. G. Cutler (eds.), Molecular Biologyof Aging: Gene Stability and Gene Expression. New York: Raven Press,1985.
24. Loeb, L. A., Springgate, C. F., and Battuta, N. Errors in DNA replication asa basis of malignant change. 34: 2311-2321, 1974.
25. Fry, M., and Loeb, L. A. Animal Cell DNA Polymerases. Boca Raton, FL:CRC Press, 1986.
26. Giroux, C. N., Mis, J. R. A., Pierce, M. K., Kohalmi, S. E., and Kunz, B. A.DNA sequence analysis of spontaneous mutations in the SUP4-O gene ofSaccharomyces cerevisiae. Mol. Cell. Biol., 8:978-981, 1988.
27. Seshadri, R., Kutlaca, R. J., Trainor, K., Matthews, C., and Morely, A. A.Mutation rate of normal and malignant human lymphocytes. Cancer Res.,47:407-409,1987.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
Elmore, E., Kakunaga, T., and Barrett, J. C. Comparison of spontaneousmutation rates of normal and chemically transformed human skin fibroblasts.Cancer Res., 43: 1650-1655, 1983.Cifone, M. A., and Fidler, 1. J. Increasing metastatic potential is associatedwith increasing genetic instability of clones isolated from murine neoplasms.Proc. Nati. Acad. Sci. USA, 78:6949-6952, 1981.Lindahl, T., and Nyberg, B. Rate of depurination of native deoxyribonucleicacid. Biochemistry, 11: 3610-3618, 1972.Loeb, L. A., and Preston, B. D. Mutagenesis by apurinic/apyrimidinic sites.Annu. Rev. Genet., 20: 201-230, 1986.Schaaper, R. M., Koplitz, R. M., Tkeshelashvili, L. K., and Loeb, L. A.Metal-induced lethality and mutagenesis: possible role of apurinic intermediates. Mutât.Res., 777: 179-188, 1987.Lindahl, T., and Nyberg, B. Heat-induced deamination of cytosine residuesin deoxyribonucleic acid. Biochemistry, 13: 3405-3410, 1974.Loeb, L. A. Apurinic sites as mutagenic intermediates. Cell, 40: 483-484,1985.Boiteux, S., and Laval, J. Coding properties of poly(deoxycytidylic acid)templates containing uracil or apyrimidinic sites: in vitro modulation ofmutagenesis by DNA repair enzymes. Biochemistry, 21:6746-6751, 1982.Shearman, C. W., and Loeb, L. A. The effects of depurination on the fidelityof DNA synthesis. J. Mol. Biol., 72«:197-218, 1979.Kunkel, T. A., Schaaper, R. M., and Loeb, L. A. Depurination-inducedinfidelity of DNA synthesis with purified DNA replication proteins in vitro.Biochemistry, 22: 2378-2384, 1983.Sagher, D., and Strauss, B. Insertion of nucleotides opposite apurinic/apyrimidinic sites in deoxyribonucleic acid during in vitro synthesis: uniqueness of adenine nucleotides. Biochemistry, 22: 4518-4526, 1983.Schaaper, R. M., Kunkel, T. A., and Loeb, L. A. Infidelity of DNA synthesisassociated with bypass of apurinic sites. Proc. Nati. Acad. Sci. USA, 80:487-491, 1983.Takeshita, M., Chang, C-N., Johnson, F., Will, S., and Grollman, A. Oli-godeoxynucleotides containing synthetic abasic sites. J. Biol. Chem., 262:10171-10179, 1987.Moore, P., and Strauss, B. S. Sites of inhibition of in vitro DNA synthesis incarcinogen- and UV-treated 0X174 DNA. Nature (Lond.), 278: 664-666,1979.Seal, G., Arenaz, P., and Sirover, M. A. Purification and properties of thehuman placenta! uracil DNA glycosylase. Biochim. Biophys. Acta, 925:226-233, 1987.Erlich, M., and Wang, R. Y-H. 5-Methylcytosine in eukaryotic DNA. Science(Wash. DC), 212: 1350-1357, 1981.Barrows, L. R., and Magee, P. N. Nonenzymatic methylation of DNA by S-adenosyl-methionine in vitro. Carcinogenesis (Lond.), 3: 349-351, 1983.Lee, A., and Cerami, A. Elevated glucose 6-phosphate levels are associatedwith plasmid mutations in vivo. Proc. Nati. Acad. Sci. USA, 84: 8311-8314,1987.Cathcart, R., Schwiers, E., Saul, R. L., and Ames, B. N. Thymine glycol andthymidine glycol in human and rat urine: a possible assay for oxidative DNAdamage. Proc. Nati. Acad. Sci. USA, 81:5633-5637, 1984.Klebanoff, S. J. Phagocytic cells: products of oxygen metabolism. In: J. I.Gallin, I. M. Goldstein, and R. Snyderman (eds.), Inflammation: BasicPrinciples and Clinical Correlates, pp. 391-444. New York: Raven Press,1988.Richter, C., Park, J-W., and Ames, B. N. Normal oxidative damage tomitochondria! and nuclear DNA is extensive. Proc. Nat!. Acad. Sci. USA,«5:6465-6467, 1988.Hsie, A. W., Recio, L., Katz, D. S., Lee, C. Q., Wagner, M., and Schenley,R. L. Evidence for reactive oxygen species inducing mutations in mammaliancells. Proc. Nat!. Acad. Sci. USA, «5:9616-9620, 1986.Preston, B. D., and Loeb, L. A. Enzymatic synthesis of site-specificallymodified DNA. Mutât.Res., 200: 21-35, 1988.Basu, A. K., and Essigmann, J. M. Site-specifically modified oligodeoxynu-cleotides as probes for the structural and biological effects of DNA-damagingagents. Chem. Res. Toxicol., /: 1-18, 1988.Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J.Mutagenesis by the autoxidation of iron using isolated DNA. Proc. Nati.Acad. Sci. USA, «5:3918-3922, 1988.Weymouth, L. A., and Loeb, L. A. Mutagenesis during in vitro DNAsynthesis. Proc. Nati. Acad. Sci. USA, 75: 1924-1928, 1978.Walker, G. C. Mutagenesis and inducible responses to deoxyribonucleic aciddamage in Escherichia coli. Microbio!. Rev., 48: 60-93, 1984.Brown, T. C., and Jiricny, J. Different base/base mispairs are corrected withdifferent efficiencies and specificities in monkey kidney cells. Cell, 54: 705-711, 1988.Speyer, J. F. Mutagenic DNA polymerase. Biochem. Biophys. Res. Commun., 21:6-8, 1965.Liu, P. K., Chang, C-C, Trosko, J. E., Dube, D. K., Martin, G. M., andLoeb, L. A. Mammalian mutator mutant with an aphidicolin-resistant DNApolymerase-a. Proc. Nati. Acad. Sci. USA, «0:797-801, 1983.Phear, G., Nalbantoglu, J., and Meuth, M. Next-nucleotide effects in mutations driven by DNA precursor pool imbalances at the APR T locus of Chinesehamster ovary cells. Proc. Nati. Acad. Sci. USA, 84:4450-4454, 1987.Kunkel, T. A., and Loeb, L. A. On the fidelity of DNA replication X: effectof divalent metal ion activators and deoxyribonucleoside triphosphate poolson i/i vitro mutagenesis. J. Biol. Chem., 254: 5718-5725, 1979.Kunkel, T. A., and Loeb, L. A. Fidelity of mammalian DNA polymerase.Science (Wash. DC), 213: 765-767, 1981.
61. Kunkel. T.A. The mutational specificity of DNA polymerase-fi during in vitroDNA synthesis: production of frameshift, base substitution and deletionmutations. J. Biol. Chem.. 260: 5787-5796, 1985.
62. Reyland. M., and Loeb, L. A. On the fidelity of DNA replication XX:Isolation of high fidelity DNA polymerasc-primase complexes by immunoaf-finity chromatography. J. Biol. Chem.. 262: 10824-10830. 1987.
63. Kunkel, T. A. Exonucleolytic proofreading. Cell, 53: 837-840. 1988.64. Kunkel. T. A., Sabatino, R. D., and Bambara, R. A. Exonucleolytic proof
reading by calf thymus DNA polymerase-Ã. Proc. Nati. Acad. Sci. USA, 84:4865-4869, 1987.
65. Ottiger. H. P., and Hubscher, U. Mammalian DNA polymerase-a holoen-zymes with possible functions at the leading and lagging strand of thereplication fork. Proc. Nati. Acad. Sci. USA, 81: 3993-3997, 1984.
66. Kunkel. T. A., and Bcbenek, K. Recent studies of the fidelity of DNAsynthesis. Biochim. Biophys. Acta, 951: 1-15, 1987.
67. Schaaper, R. M., and Dunn. R. L. Spectra of spontaneous mutations inEscherichia coli strains defective in mismatch correction: the nature of invivo DNA replication errors. Proc. Nati. Acad. Sci. USA, 84: 6220-6224,1987.
68. Calos, M. P., Lebkowski, J. S., and Botchan, M. R. High mutation frequencyin DNA transfected into mammalian cells. Proc. Nati. Acad. Sci. USA, 80:3015-3019, 1983.
69. Razzaque, A., Mizusawa, II.. and Seidman, M. M. Rearrangement andmutagenesis of a shuttle vector plasmid after passage in mammalian cells.Proc. Nati. Acad. Sci. USA, 80: 3010-3014, 1983.
70. De Jong, P. J., Gorsovsky, A. J., and Glickman, B. W. Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysisat the DNA sequence level. Proc. Nati. Acad. Sci. USA, 85: 3499-3503.1988.
71. Burmer, G. C., and Loeb, L. A. Mutations in the KRAS2 oncogene during
progressive stages of human colon carcinoma. Proc. Nati. Acad. Sci. USA,«6:2403-2407, 1989.
72. Mariyama, M., Kishi. K., Nakamura. K.. Obata, H.. and Nishimura, S.Frequency and types of point mutation at the 12th codon of the c-Ki-ros genefound in pancreatic cancers from Japanese patients. J. Cancer Res., in press,1989.
73. Smitt, V. T. H. B. M., Boot, A. J. M., Smits, A. M. M., Fleuren, G. J.,Cornelisse, C. J., and Bos. J. L. KRAS codon 12 mutations occur very'frequently in pancreatic adenocarcinomas. Nucleic Acids Res., 16: 7773-7782, 1988.
74. Vogelstein, B., Fearon. E. R., Hamilton. S. R., Kern. S. E.. Preisinger, A.C., Leppert, M., Nakamura, Y., White, R., Alida, M. M. S., and Bos, J. L.Genetic alterations during colorectal-tumor development. N. Engl. J. Med.,39: 525-532, 1988.
75. Balmain. A., and Brown, K. Oncogene activation in chemical carcinogenesis.Adv. Cancer Res., 51: 147-182, 1988.
76. Burmer, G. C., Rabinovitch, P. S., and Loeb, L. A. Analysis of c-Ki-rosmutations in human colon carcinoma by cell sorting, polymerase chainreaction and DNA sequencing. Cancer Res., 49: 2141-2146, 1989.
77. Saiki, R. K., Bugawan, T. L., Horn, G. T., Mullis, K. B.. and Erlich, H. A.Analysis of enzymatically amplified (j-globin and HLA-DQ n DNA withallele-specific oligonucleotide probes. Nature (Lond.). 324: 163-166, 1986.
78. Goelz, S. E., Hamilton. S. R.. and Vogelstein, B. Purification of DNA fromformaldehyde fixed and paraffin embedded human tissue. Biochem. Biophys.Res. Commun.. 130: 118-125, 1985.
79. Ornitz. D. M.. Hammer, R. E., Messing, A., Palmiter. R. D., and Brinster,R. L. Pancreatic neoplasia induced by SV40 T-antigen expression in acinarcells of transgenic mice. Science (Wash. DC), 238: 188-193. 1987.
80. Randerath. K.. Reddy, M. V., and Gupta. R. C. "P-labeling test for DNAdamage. Proc. Nati. Acad. Sci. USA. 78: 6126-6129. 1981.
81. Ames, B. N. Mutagenesis and carcinogenesis: endogenous and exogenousfactors. Environ. Mutagen., in press, 1989.
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