Role of DNA repair in carcinogen-induced ras mutation.

In this contribution we discuss the gene- and cell type-specific repair of miscoding DNA alkylation products as a risk parameter in both mutation induction and malignant transformation by N-nitroso carcinogens. Upon exposure to N-nitroso compounds such as N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), about a dozen different alkylation products are formed in cellular DNA. Among these are O(6)-methylguanine (O(6)-MeGua) and O(6)-ethylguanine (O(6)-EtGua), respectively, which differ only by one CH(2) group in their alkyl residue and, when unrepaired, cause G:C-->A:T transition mutations by anomalous base pairing during DNA replication. We have analyzed the global and gene-specific repair of O(6)-MeGua and O(6)-EtGua in target cell DNA, ras gene mutation frequencies, and tumor incidence, in the model of mammary carcinogenesis induced in 50-day-old female Sprague-Dawley rats by a single application of MeNU or EtNU. Both carcinogens induce histologically indistinguishable mammary adenocarcinomas at high yield. In the target mammary epithelia, O(6)-MeGua is repaired at similar slow rates in both transcriptionally active genes (Ha-ras, beta-actin), silent genes (lgE heavy chain), and in bulk DNA, by the one-step repair protein O(6)-alkylguanine-DNA alkyltransferase (MGMT; low level of expression in the target cells). The slow repair of O(6)-MeGua translates into a high frequency of mutations at the central position of Ha-ras codon 12 (GGA) in MeNU-induced tumors. O(6)-EtGua, however, is removed approximately 20 times faster than O(6)-MeGua selectively from transcribed genes via an MGMT independent, as yet uncharacterized excision mechanism. Accordingly, no Ha-ras codon 12 mutations are found in the EtNU-induced mammary tumors. Neither MeNU- nor EtNU-induced tumors exhibit mutations at codons 13 and 61 of Ha-ras or at codons 12, 13 and 61 of Ki-ras. While a moderate surplus MGMT activity of the target cells - contributed by a bacterial MGMT transgene (ada) - significantly counteracts mammary tumorigenesis in MeNU-exposed rats, this is not the case in the EtNU-treated animals. Differential repair of structurally distinct DNA lesions in transcribed or (temporarily) silent genes thus determines the probability of mutation and, together with cell type-specific and interindividual differences in DNA repair capacity, influences carcinogenic risk.

DNA repair: counteragent in mutagenesis and carcinogenesis-- accomplice in cancer therapy resistance.

DNA-reactive carcinogens and anticancer drugs induce many structurally distinct mutagenic and cytotoxic DNA lesions. The varying capability of normal and malignant cells to recognize and repair specific DNA lesions influences both cancer risk and the relative sensitivity or resistance of cancer cells towards cytotoxic agents. Using monoclonal antibody-based immunoanalytical assays, very low amounts of defined carcinogen-DNA adducts can be quantified in bulk genomic DNA, in individual genes, and in the nuclear DNA of single cells. DNA repair kinetics can, thus, be measured in a lesion-, gene-, and cell type-specific manner, and the DNA repair profiles of malignant cells can be monitored in individual patients. Even structurally very similar DNA lesions may be repaired with strongly differing efficiency. The miscoding DNA alkylation products O(6)-methylguanine and O(6)-ethylguanine, for example, differ only by one CH(2) group. These lesions are formed in DNA upon exposure to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea, both of which induce mammary adenocarcinomas in female rats at high yield. Unrepaired O(6)-alkylguanines in DNA cause G:C-->A:T transition mutations via mispairing during DNA replication. O(6)-methylguanines are repaired at a similar slow rate in both transcriptionally active (H-ras, beta-actin) and inactive genes (IgE heavy chain; bulk DNA) of the target mammary epithelia (which express the repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) at a very low level). In contrast, O(6)-ethylguanines are repaired approximately 20 times faster than O(6)-methylguanines in both DNA strands of the transcribed genes selectively via an AGT-independent, as yet unclarified excision mechanism. Accordingly, G:C-->A:T transitions resulting from the misreplication of an O(6)-methylated guanine at the second position of codon 12 (GGA) of H-ras represent a frequent "signature" mutation in rat mammary adenocarcinomas that develop after exposure to N-methyl-N-nitrosourea. However, this mutation is not observed when these tumors are induced by N-ethyl-N-nitrosourea, due to the fast repair of O(6)-ethylguanines in the H-ras gene. The key importance of "conventional" and "conditional" gene knockout technology for resolving the intricacies of the complex network of DNA repair pathways is briefly discussed.

Fast repair of O6-ethylguanine, but not O6-methylguanine, in transcribed genes prevents mutation of H-ras in rat mammary tumorigenesis induced by ethylnitrosourea in place of methylnitrosourea.

Differential repair of structurally distinct mutagenic lesions in critical genes may influence the cellular risk of malignant conversion. We have investigated rat mammary tumorigenesis induced by N-ethyl-N-nitrosourea (EtNU) versus N-methyl-N-nitrosourea (MeNU) with respect to tumor incidence, ras gene mutation, and gene-specific repair. Both carcinogens induced mammary adenocarcinomas at high yield. In mammary epithelia (very low expression of O6-alkylguanine-DNA alkyltransferase, MGMT), O6-methylguanine (O6-MeGua) was eliminated from transcribed (H-ras and beta-actin) and inactive genes (IgE heavy chain) at the same slow rate as determined for bulk genomic DNA. The persistence of O6-MeGua in DNA correlated with a high frequency of G:C --> A:T transition mutations at codon 12 of the H-ras gene in MeNU-induced tumors. Repair of O6-ethylguanine (O6-EtGua), too, was slow in the IgE heavy chain gene as in bulk DNA. Contrasting with O6-MeGua, however, O6-EtGua was removed approximately 20 times faster from the active H-ras and beta-actin genes via MGMT-independent mechanism(s). Accordingly, no H-ras codon 12 mutations were found in EtNU-induced tumors, and 5- to 8-fold surplus alkyltransferase activity of the mammary epithelia-via a bacterial ada transgene-did not significantly counteract tumorigenesis in EtNU-exposed contrary to MeNU-treated animals. Neither MeNU- nor EtNU-induced tumors exhibited mutations at codons 13 and 61 of H-ras or codons 12, 13, and 61 of K-ras. Fast repair of O6-EtGua, but not O6-MeGua, in transcribed genes thus prevents mutational activation of H-ras when rat mammary carcinogenesis is initiated by EtNU in place of MeNU.

Relevance of DNA repair to carcinogenesis and cancer therapy.

DNA-reactive carcinogens and anticancer drugs induce many structurally distinct cytotoxic and potentially mutagenic DNA lesions. The capability of normal and malignant cells to recognize and repair different DNA lesions is an important variable influencing the risk of mutation and cancer as well as therapy resistance. Using monoclonal antibody-based immunoanalytical assays, very low amounts of defined carcinogen-DNA adducts can be quantified in bulk genomic DNA, individual genes, and in the nuclear DNA of single cells. The kinetics of DNA repair can thus be measured in a lesion-, gene-, and cell type-specific manner, and the DNA repair profiles of malignant cells can be monitored in individual patients. Even structurally very similar DNa lesions may be repaired with extremely different efficiency. The miscoding DNA alkylation products O6-methylguanine (O6-MeGua) and O6-ethylguanine (O6-EtGua), for example, differ only by one CH2 group. These lesions are formed in DNA upon exposure to N-methyl-N-nitrosourea (MeNU) or N-ethyl-N-nitrosourea (EtNU), both of which induce mammary adenocarcinomas in female rats at high yield. Unrepaired O6-alkylguanines cause transition mutations via mispairing during DNA replication. O6-MeGua is repaired at a similar slow rate in transcribed (H-ras, beta-actin) and inactive genes (IgE heavy chain; bulk DNA) of the target mammary epithelia (which express the repair protein O6-alkylguanine-DNA alkyltransferase at a very low level). O6-EtGua, however, via an alkyltransferase-independent mechanism, is excised approximately 20 times faster than O6-MeGua from the transcribed genes selectively. Correspondingly, G:C-->A:T transitions arising from unrepaired O6-MeGua at the second nucleotide of codon 12 (GGA) of the H-ras gene are frequently found in MeNU-induced mammary tumors, but are absent in their EtNU-induced counterparts.

DNA damage and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation.

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters (s-values, O6/N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the Mexr-/Mexr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, kc, reaction at a critical nucleophilic strength, nc. For alkylating agents kc can be expressed as the second-order rate constant for hydrolysis, kH2O, and the substrate constant s:kH2OTD50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, ac, for 2 years exposure of the rodents. The TD*50 in mmol/kg x day) could then be written: [formula: see text] This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most SN2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be impro