Search

We have proposed that an early step in tumor progression is the expression of a mutator phenotype resulting from mutations in genes that normally function in the maintenance of genetic stability. There is new and strong experimental evidence that supports the concept of a mutator phenotype in cancer. As technologies for chromosomal visualization and DNA advance, there are increasing data that human cancer cells contain large numbers of mutations. First, I will review the concept of a mutator phenotype. Second, I will present the recent evidence that individual cancer cells contain thousands of mutations. Third, I will explore potential target genes that are required for maintenance of genetic stability in normal cells and ask if they are mutated in cancer cells. Fourth, I will address the timing of a mutator phenotype; is it an early event during tumor progression? Do tumors already contain cells that harbor mutations rendering them resistant to most chemotherapeutic agents? Lastly, I will speculate on the theoretical and practical implication of a mutator phenotype in cancer and consider the possibility of cancer prevention by delay, i.e., a reduction in mutation rates early during carcinogenesis might slow the progression of tumors.

Thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluoro-2'-deoxyuridine 5'-monophosphate, a tight binding covalent inhibitor of thymidylate synthase. Recent studies have identified 5-fluoro-2'-deoxyuridine (5-FdUR) and antifolate-resistant mutants of human thymidylate synthase (TS) that contain single residue substitutions within the highly conserved Arg50-loop, which binds the pyrimidine substrate (Y. Tong et al., J. Biol. Chem. 273: 11611-11618, 1998). We have used random sequence mutagenesis to gain structure-function information about the TS and to create novel drug-resistant mutants for gene therapy. A library of 1.5 million mutants of the Arg50-loop and the nearby residue Tyr 33 was selected to identify mutants of the human enzyme with the ability to complement a thymidylate synthase-deficient Escherichia coli strain and form colonies in the presence of 5-FdUR. E. coli-harboring plasmids that were encoding TS with single, double, and triple amino acid substitutions were identified that survive at dosages of 5-FdUR clearly lethal to E. coli harboring either wild-type thymidylate synthase or constructs encoding previously characterized drug resistant mutants. Four 5-FdUR-resistant mutants were purified to apparent homogeneity. Kinetic studies indicate that these enzymes are highly efficient. Inhibition constants (Ki) for the double mutant K47Q;D48E and the triple mutant D48E;T51S;G52C in the presence of 5-fluoro-2'-deoxyuridine 5'-monophosphate were determined to be 75 to 100 times higher, respectively, than that of the wild-type enzyme. These mutant TSs, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of 5-FU chemotherapy.

O6-Alkylguanine-DNA alkyltransferase (AGT) is a suicide enzyme that repairs alkylation damage at the O6 position of guanine in DNA. The essentiality of a limited number of amino acid residues at the active site has been determined by site-directed mutagenesis. We used random mutagenesis techniques to create a plasmid library of > 10(6) human AGT mutants with substitutions at residues 150-172 and selected for clones with mutations rendering Escherichia coli resistant to both the alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and the AGT inhibitor, O6-benzylguanine (BG). On sequencing surviving clones resistant to MNNG in the presence and absence of BG, we found that a majority of the clones contained multiple substitutions at mostly nonconserved positions. We selected nine mutants resistant to a combination of MNNG and BG, and the survival of these mutants was as much as 341-fold higher than that of cells harboring wild-type AGT under the same conditions. Each of the mutants contained at least three amino acid substitutions and as many as eight, suggesting that maximum resistance to MNNG in the presence of BG requires even more substitutions than resistance to MNNG alone. BG is being tested clinically as a way to sensitize tumors to chemotherapeutic alkylating agents. Therefore, our BG-resistant mutants hold strong potential as gene therapy candidates for protecting normal tissue in patients receiving BG in combination with alkylating agents for the treatment of cancer.

O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents. Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression. One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment. We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene. A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli. Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG. One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E. coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.

In this review we will summarize recent data on reactive oxygen species-induced mutagenesis and consider its relationship to tumorigenesis in humans. With the use of a single-stranded DNA template it has been possible to correlate oxygen radical-induced chemical alterations at specific nucleotides with the types of mutations that occur when these altered bases are copied by DNA polymerases. This has allowed us to identify the types of mutations that occur secondary to a variety of oxidative stresses and study several of the mechanisms by which they arise. The most frequent mutations that result from reactive oxygen species-induced damage to DNA in bacteria are C to T transitions. These mutations, however, are not pathoneumonic for mutagenesis by oxygen-free radicals since they result from DNA damage caused by other genotoxic agents as well as by DNA polymerase errors. One type of mutation, a tandem CC to TT double substitution, has been shown to be induced by reactive oxygen species generated by a variety of systems and may be diagnostic for such damage. In studies with mammalian DNA polymerases, DNA damaged by reactive oxygen species yields mutations different from those observed in Escherichia coli. This diversity of mutagenic changes in these in vitro studies highlights the role of DNA replicating enzymes in specifying the types of mutations produced by reactive oxygen species. In conclusion, we will consider the role of reactive oxygen species in the pathogenesis of three common tumors, carcinoma of the liver, lung, and prostate with consideration on the possible use of antioxidant preventive therapy to slow tumorigenesis sufficiently to prevent clinical presentation of these cancers during the life span of a patient.

We have determined the specificity of mutations produced by nickel(II), a known human carcinogen, in a forward mutation assay and also used a sensitive reversion assay to show that Ni(II), like iron and copper, can produce tandem double CC-->TT mutations, a hallmark of damage to DNA by either UV irradiation or oxygen free radicals. A reduction in mutation frequencies by the addition of oxygen radical scavengers also supports the involvement of reactive oxygen species in DNA damage and mutagenesis by Ni(II). Mutagenesis by Ni(II) is enhanced by the addition of both hydrogen peroxide and a tripeptide glycyl-glycyl-L-histidine. The enhancement of mutagenesis of Ni(II) by the tripeptide indicates that these complexes could serve to localize Ni(II) in nuclei and mediate DNA damage and mutagenesis via the generation of short-lived oxygen free radicals. These data suggest that Ni(II) carcinogenesis may proceed via the generation of active oxygen species and furthermore provide a model for nickel carcinogenesis based on the binding of Ni(II) to nuclear proteins.

Mutagenesis of protooncogenes has been postulated to contribute to the initiation and progression of human cancer. Activating mutations in the H-ras gene are predominantly single-base substitutions and are most frequently identified at codons 12, 13, and 61. We have analyzed the effects of DNA sequence context at specific codons that are hot spots for ras mutation with respect to abnormalities in copying by purified DNA polymerase alpha, a major eucaryotic replication enzyme. Exon 1 of H-ras gene was inserted into M13 mp19, single-stranded DNA constructs were isolated, and the progression of synthesis by polymerase alpha was measured. Strong termination sites were found in codons 12 and 13. Pausing at these codons is abolished when the template is mutated at the middle base of codon 12, the same alteration that converts H-ras into an activated oncogene. Resistance of codon 12 in double-stranded constructs to digestion with restriction enzymes and computer investigation of the ras sequence suggest that these termination sites are in a region of secondary structure. The frequency of sequence alterations within DNA chains that have been extended past codons 12 and 13 was found to be < 0.01. We consider a variety of mechanisms by which the potential secondary structure involving codons 12 and 13 may contribute to the pausing of DNA polymerase alpha and to the generation of clustered mutations at this site.

An important source of endogenous oxygen radicals are phagocytic cells such as neutrophils and macrophages. The human leukemia cell line HL-60 can be induced to differentiate into a neutrophil-like cell population. Among the properties of these differentiated cells is the ability to produce reactive oxygen species when stimulated by tumor promoters. Mutagenesis induced by HL-60-generated free radicals was assessed using the M13mp2 forward mutation assay. Single-stranded M13mp2 DNA was coincubated with phorbol ester-stimulated HL-60 cells, after which mutations were scored by transfecting the DNA into SOS-induced Escherichia coli. The mutation frequency was increased 6-fold above background in DNA incubated with HL-60 cells. The majority of the mutations were single-base substitutions. However, approximately 6% of the mutations were tandem double substitutions that occurred in runs of adjacent cytidines. Overall, the mutations were clustered at apparent "hot spots," many of which were similar to sites seen using iron to generate oxygen radicals. These results suggest that human cells able to produce oxygen radicals in response to tumor promoters might play a significant role in the generation of tumors.

In order to investigate whether carcinogens induce alterations of the DNA polymerase alpha-primase complex we compared the physiochemical and catalytic properties and the fidelity of DNA synthesis of DNA polymerase alpha-primase complexes from carcinogen-treated and untreated Chinese hamster ovary cells. Complexes were purified by ion exchange or by immunoaffinity chromatography and both DNA-polymerizing activities and those of ancillary enzymes, such as RNA primase and exonuclease, were examined. Further characterization of the complexes included determination of the relative molecular masses, sedimentation coefficients, and diffusion coefficients, and measurements of the KmS for deoxynucleotide triphosphates and DNA templates, which were identical for the preparations from both carcinogen-treated and untreated cells. The fidelity of DNA polymerase alpha-primase complexes measured by the phi X174am3 reversion assay was also similar in carcinogen-treated and untreated cells. Thus, a carcinogen-mediated induction of a DNA polymerase alpha-primase complex with low fidelity was not observed within the detection limits of the phi X174 assay. RNA primase was found to be an ancillary enzyme activity of the DNA polymerase alpha from both carcinogen-treated and untreated cells; however, the RNA primase:DNA polymerase alpha activity ratio was significantly higher in DNA polymerase alpha-primase complexes from carcinogen-treated cells. These complexes also exhibited an at least 3 times greater velocity of synthesis with supercoiled or unprimed single-stranded DNAs as templates. Since the binding sites of DNA polymerase alpha-primase complexes for deoxynucleotide triphosphates and DNA templates were shown to be identical before and after treatment of cells with carcinogens (i.e., identical Km values for different DNA templates and Ki values for specific inhibitors), the increased synthesis catalyzed by the DNA polymerase alpha-primase complex from carcinogen-treated cells might be due to a carcinogen-induced alteration of an accessory protein of the complex.

The mutagenic potentials of ethylmethane sulfonate, N-methyl-N'-nitrosoguanidine, and benzo(a)pyrene diol-epoxide in human mitochondria were determined by cloning and nucleotide sequencing of mitochondrial (mt) DNA from HeLa cells treated with these mutagens. Mutagen concentrations that reduced cell survival to approximately 0.1% of untreated cultures were used. Mitochondrial DNA was prepared 2 to 3 weeks after mutagen treatment, at which time the treated cell population had regrown to 10 times the starting cell number. In one series of experiments, a portion of the D-loop region of mtDNA from treated or control HeLa cells was cloned into the bacteriophage vector M13mp19, and the nucleotide sequences of 102 independent clones were determined. Only a single G:C base pair deletion was observed in 1 of 12 clones derived from HeLa cells treated 6 times with ethylmethane sulfonate. From benzo(a)pyrene diol-epoxide-treated HeLa cells, G:C base pair deletions were found in 14 of 63 clones. All 14 of these G:C deletion mutations occurred at the same position in independent clones, however, and thus could be the progeny of a single mutational event. In a second series of experiments, a method for the selection of mtDNA mutants was utilized. Mutations in an "uncloneable" fragment of human mtDNA render the fragment cloneable and thus provide a selection for mutations in this region of human mtDNA. No enhancement in the cloning efficiency of this region of mtDNA was observed after exposure of cells to toxic concentrations of either MNNG or benzo(a)pyrene diol-epoxide. Moreover, the site and types of nucleotide sequence alterations observed after mutagen treatment were similar to those obtained in the absence of drug treatment. The results of both types of experiments suggest that mutagenesis of human mtDNA is an infrequent event, even after extensive treatment of HeLa cells with potent mutagens that can covalently modify mtDNA.

Nucleotide sequence variation in mitochondrial DNA isolated from human leukemic cells has been analyzed by recombinant DNA techniques. Three hundred eighty-seven independent recombinant DNA clones, each containing one of three defined segments of mitochondrial DNA isolated from the neoplastic cells of four leukemic patients, were analyzed. Partial nucleotide sequence determination of the 387 clones yielded a total of 81.7 kilobases of nucleotide sequence information. The only evidence of within-individual nucleotide sequence divergence consisted of three clones containing deletions of one or two nucleotides in one mitochondrial DNA region. These clones were three of 113 independent clones isolated from a patient with acute lymphocytic leukemia. The low level of nucleotide sequence divergence in the mitochondrial DNA population of neoplastic cells from individual leukemic patients suggests that a mechanism or mechanisms exist that limit the development of nucleotide sequence divergence in mammalian mitochondrial DNA. The results further suggest that this mechanism does not appear to be abrogated by neoplastic transformation in leukemic patients.

Human peripheral blood mononuclear cells from normal healthy volunteers were exposed to elevated temperatures of 41-43 degrees for up to 6 hr. Thereafter, the cells were stimulated with phytohemagglutinin in vitro in order to measure indirectly the surviving fraction. DNA replication in heated cells in response to phytohemagglutinin was found to be a sensitive indicator of thermal injury. Exposure to even 40 degrees for 2 hr lowered thymidine incorporation at early time points after phytohemagglutinin stimulation, but the cells were able to recover from thermal injury after exposure for up to 4 hr at 42 degrees. At 43 degrees, exposure for even 1 to 2 hr caused irreversible damage. The changes in thymidine incorporation were not due to changes in endogenous nucleotide pools since parallel changes were observed in DNA polymerase activity. Thus, the heat sensitivity of normal human lymphocytes could be a limiting factor for use of hyperthermia as an adjunct to radiotherapy and chemotherapy of human cancer.

The effect of modifying phi chi 174 viral DNA by the chemical carcinogens beta-propiolactone, N-acetoxyacetylaminofluorene and anti-benzo[a]pyrene diol-epoxide was investigated by transfecting the modified DNA into Escherichia coli spheroplasts. Modification of the DNA in vitro by each of these agents was mutagenic for the phi chi 174 amber mutants am3 and am86. Mutagenicity depended on the induction of the "SOS" response in the host spheroplasts. Heating beta-propiolactone-treated DNA at neutral pH caused strong inactivation such that the number of lethal hits was increased 4-fold. Sucrose gradient analysis showed the induction of alkali-labile sites in the heated DNA. The "nicked circle assay" with double-stranded phi chi 174 DNA showed greater than 70% of these sites to be apurinic sites. Concomitantly with the production of these new sites, a strong increase in the mutation frequency was observed. This mutagenesis also depended upon the induction of the error-prone SOS response in the spheroplasts, as was previously shown to be the case for mutagenesis at putative apurinic sites induced directly by acid-heat treatment. These results suggest that depurination may be of importance to the mechanism of mutagenesis by beta-propiolactone and other carcinogens.

This position paper summarizes the overwhelming evidence that tobacco smoking is the cause of 30 to 40% of deaths from cancer. The focus is on lung cancer because of the sheer magnitude of this disease in males and the likelihood of a similar epidemic in females. There are two categories of evidence that indicate smoking to be the major cause of human lung cancer. Without exception, epidemiological studies have demonstrated a consistent association between smoking and lung cancer in men and now suggest a similar association in women. Chemical analyses of cigarette smoke reveal a multitude of known mutagens and carcinogens. Moreover, these chemicals are absorbed, are metabolized, and cause demonstrable genetic changes in smokers. Two consequences of smoking are evaluated. The results of treatment of lung cancer are not encouraging; despite vigorous therapy, the 5-year survival rate remains less than 10%. The social and economic costs of lung cancer and the smoking habit impinge on the productiveness of our society.

An assay has been developed to measure the ability of human lymphocytes to repair damage to DNA. In this assay, purified human lymphocytes are exposed to graded doses of radiation and then stimulated with phytohemagglutinin to undergo DNA replication. The rate of incorporation of thymidine in irradiated lymphocytes during the second and subsequent rounds of DNA replication is taken to be indicative of the ability of the cells to repair damage to DNA. In lymphocytes from normal individuals, X-irradiation with doses of 100 to 800 rads was found to inhibit phytohemagglutinin-stimulated thymidine incorporation proportionally to the dose of radiation without curtailing the induction of DNA polymerase. The response to phytohemagglutinin of lymphocytes from a patient with xeroderma pigmentosum after exposure to graded doses of X-irradiation was found to be similar to that of the normal controls, whereas the response after ultraviolet irradiation was markedly impaired. In contrast, lymphocytes from patients with ataxia telangiectasia were hypersensitive to X-irradiation. The data on these clinical syndromes support the idea that this assay measures DNA repair and indicates the feasibility of using this method for screening individuals for genetic deficits in DNA repair.

The effect of three environmentally important metals, arsenic, selenium, and chromium, on the accuracy of DNA synthesis in vitro has been analyzed. The addition of arsenic to fidelity assays did not significantly alter accuracy. Selenium did not alter fidelity under normal conditions of magnesium activation, nor did it affect the mutagenicity of manganese. Chromium in the form of Cr(III) as well as Cr(VI) diminished the fidelity by which Escherichia coli DNA polymerase I copies polynucleotide templates. Nearest-neighbor analysis of the product synthesized in the presence of chromium indicates that the misincorporated in the presence of chromium indicates that the misincorporated bases are present as single-base substitutions. Chromium was also mutagenic using the recently developed phi chi 174 assay, which measures the fidelity of DNA synthesis with a natural DNA template.

The in vitro accuracy of DNA replication has been investigated through the measurement of the frequency with which noncomplementary nucleotides were incorporated during polynucleotide replication. The effect of beta-propiolactone treatment of deoxynucleotide templates, ribopolynucleotide templates, and the DNA polymerase from avian myeloblastosis virus was determined. Treatment of the deoxynucleotide template, poly(dA) (see article) oligo(dT) 12-18, by beta-propiolactone resulted in an increased frequency of noncomplementary nucleotide incorporation during DNA polymerization. Carcinogen treatment of the ribonucleotide templates, poly(rA) (see article) oligo(dT) 12-18, and poly(rC) (see article) oligo(dG) 12-18, and carcinogen treatment of avian myeloblastosis virus DNA polymerase did not alter the frequency of noncomplementary nucleotide incorporation. This suggested that carcinogen-induced error incorporation during DNA synthesis was restricted solely to the treatment of a deoxynucleotide template.

We have analyzed colon carcinomas by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki-ras-2 gene. DNA was chemically extracted from 50-microns sections of paraffin-embedded colon carcinomas and amplified in vitro, and mutations were documented directly by DNA sequencing. Enrichment for tumor cells was obtained histologically and by sorting nuclei on the basis of DNA content differences. Mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms. We have demonstrated the feasibility of utilizing DNA from tissues treated with different fixatives, including methyl carnoys, formalin, and Hollande's solution. This procedure allows one to retrospectively reconstruct the temporal relationship between the occurrence of mutations and sequential morphological changes during tumorigenic progression.

Human peripheral blood lymphocytes are well-differentiated cells. Ordinarily, they do not divide and are considered to be in the G0 stage of the cell cycle. These cells can be stimulated to undergo DNA replication in culture by mitogens such as phytohemagglutinin. In the present study, we have examined cellular and biochemical events that occur after exposure of lymphocytes to X-irradiation. Irradiation with up to 100 rads, prior to stimulation with phytohemagglutinin, did not interfere with DNA replication. At later periods, DNA replication was inhibited proportionally to the amount of radiation. In comparison to DNA synthesis, the effect of X-irradiation on RNA and protein synthesis in phytohemagglutinin-stimulated lymphocytes was less marked. Furthermore, X-rays did not inhibit either the induction or the continual synthesis of DNA polymerase-alpha or -beta in response to phytohemagglutinin. Kinetic studies with different nucleotide substrates suggest that cellular pools of nucleotides are not significantly altered by X-irradiation. Thus, the inhibition of DNA synthesis in irradiated cells is likely to be due to damage to the cellular DNA template. The inhibition of DNA synthesis was accompanied by accumulation of cells in the G2 and M stages of the cell cycle, suggesting that inhibition of DNA replication by X-irradiation is a postmitotic event.