Human plasma and amino acids as moderators of uptake and metabolic consequences of antifolates in WIL-2 and human leukemia cells.

Plasma and medium composition significantly affect cellular association of the lipid-soluble antifolate 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP). This was demonstrated by measuring association of labeled drug with cells and by assaying the antimetabolic effect of DDMP on incorporation of deoxyuridine into DNA. Uptake of both aqueous and lipid-soluble antifolates was substantially reduced in the human lymphoblastoid cell line WIL-2 when Eagle's minimum essential medium (EMEM) was substituted for a basal salts solution containing glucose [DDMP approximatley 50%, methotrexate (MTX) approximately 30%]. Uptake of DDMP, however, was inhibited by the amino acid fraction of EMEM or glutamine alone, whereas MTX uptake was unaffected by amino acids. Further studies with human leukemia cells showed that DDMP was only about 25% as effective an inhibitor of deoxyuridine incorporation in autologous human plasma when compared to its inhibitory effect in RPMI-1640 medium MTX inhibition of deoxyuridine incorporation in these cells was essentially unaffected by substitution of autologous human plasma for RPMI-1640 medium. Replacing EMEM with pooled human plasma resulted in a 60-70% decrease in DDMP uptake but had only a marginal effect upon MTX uptake. Thus the choice of medium is important in studies of lipid-soluble antifolates such as DDMP that have a high affinity for cellular and medium lipoprotein components.

Cytotoxicity and mutagenicity of frameshift-inducing agent ICR191 in mismatch repair-deficient colon cancer cells.

BACKGROUND: Deficiency of DNA mismatch repair is a common feature of cancers exhibiting instability of microsatellite DNA sequences. Cancers with microsatellite instability are recognizable by their high rate of spontaneous frameshift mutations within microsatellite sequences, their resistance to killing by cytotoxic agents, and their localization to specific tissues, e.g., the proximal colon and stomach. We hypothesized that the mismatch repair deficiency of these cancers would make them vulnerable to environmental or chemical frameshift-inducing agents. This study was undertaken to test whether exogenous frameshift-inducing agents selectively induce mutations in mismatch repair-deficient cells of mutagen-exposed tissues like the colon and whether cytotoxic doses of these agents would preferentially kill those cells. METHODS: Cytotoxicity of the acridine mutagen 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxy-acridine (ICR191), a DNA frameshift inducer, was determined in the mismatch repair-deficient human colon carcinoma cell line HCT116 versus the repair-reconstituted derivative HCT116+C3. Vulnerability to the mutagenic effects of ICR191 was determined by transfection of HCT116 or HCT116+C3 cells with a frameshift reporter vector, followed by treatment of the cells with ICR191. Alternatively, the reporter vector was reacted ex vivo with ICR191, and the derivatized vector was then transfected into HCT116 or HCT116+C3 cells. RESULTS: ICR191 proved to be fivefold to 10-fold more potent in inducing mutations in mismatch repair-deficient HCT116 cells than in mismatch repair-proficient HCT116+C3 cells. Moreover, at cytotoxic doses of ICR191, repair-deficient HCT116 cells proved to be fivefold more vulnerable to killing than did HCT116+C3 cells. CONCLUSIONS: Frameshift-inducing mutagens can selectively induce mutations in mismatch repair-deficient cells versus mismatch repair-proficient cells. Environmental exposures may, therefore, favor development of cancers with microsatellite instability in tissues like the gut. Frameshift-inducing agents can, however, also preferentially kill mismatch repair-deficient cancer cells and, thus, may be promising as model therapeutic compounds.

Mutational specificity of 1,3-bis-(2-chloroethyl)-1-nitrosourea in a Chinese hamster ovary cell line.

The mutational specificity of the alkylating agent 1-3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) was analyzed at the endogenous hemizygous adenine phosphoribosyl transferase gene of the Chinese hamster ovary cell line D422. A 1-h treatment of the Chinese hamster ovary cells with 50 microM BCNU resulted in a toxicity level of 62% and induced mutation at this target with a frequency of 32.1 mutants/10(6) survivors (6-fold above background). Analysis of 49 BCNU-induced mutants at the DNA sequence level revealed that BCNU induced primarily base substitutions. The predominant BCNU-induced mutations were G:C----T:A transversions, which comprised 51% (25 of 49) of the mutations; while G:C----A:T transitions, expected from miscoding of O6-alkylguanine, represented only 16.3% (8 of 49) of the mutants recovered. This result was not anticipated, since Chinese hamster ovary cells are deficient in O6-alkylguanine-DNA alkyltransferase, which should render them especially sensitive to O6-alkylguanine-mediated mutations. It was also notable that two "hotspots" for BCNU-induced G:C----T:A transversions were observed, which involved different surrounding DNA sequences but similar helix parameters when analyzed by an application of Calladine's Rules. Possible mechanisms for the observed BCNU-induced mutations are presented.

Paradoxical effect of BW 301U, a lipophilic antifolate, on methotrexate-inhibitable deoxyuridine incorporation by human hematopoietic cells.

The ability of methotrexate and BW 301U, a lipophilic folate antagonist, to inhibit tritiated deoxyuridine incorporation into acid-precipitable material by human bone marrow cells was evaluated before and after five sequential daily infusions of BW 301U. After in vivo BW 301U therapy, bone marrow cells from five of the six patients exhibited significantly reduced inhibition by 1 microM methotrexate in vitro, whereas the response to 1 microM BW 301U remained unchanged. Megaloblastic marrow morphology and decreased myeloid progenitor cloning efficiency were also observed following five daily BW 301U infusions of 21 and 71 mg/sq m, respectively. A similar reduction in the ability of methotrexate to inhibit tritiated deoxyuridine incorporation was also seen in HL-60 cells, a human acute promyelocytic leukemia cell line, after incubation in vitro with cytostatic concentrations of BW 301U for 3 days. Concomitant changes in the response to BW 301U did not occur. While it is premature to infer clinical significance from this preliminary observation of BW 301U-induced asymmetry in the response to subsequent antifolates, our results augment a growing body of evidence which suggests that lipophilic folate antagonists might be effective in the treatment of methotrexate-resistant neoplasms.

Induction of myeloid differentiation of HL-60 cells with naphthalene sulfonamide calmodulin antagonists.

The naphthalene sulfonamide calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, both induce limited myeloid differentiation of the human promyelocytic cell line, HL-60. In addition, these inhibitors augment the differentiation observed when HL-60 cells are induced with retinoic acid, dimethyl sulfoxide, or dibutyryl cyclic adenosine monophosphate. The dose-response curve for HL-60 differentiation was consistent with the published 50% inhibitory dose for inhibition of calmodulin-activated phosphodiesterase and with the calmodulin drug-binding potential of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide and their less active congeners, N-(6-aminohexyl)-1-naphthalenesulfonamide and N-(4-aminobutyl)-2-naphthalenesulfonamide. These effects, of the naphthalene sulfonamide calmodulin antagonists, are consistent with a regulatory role for calmodulin in cell differentiation, but parallel effects on protein kinase C cannot be excluded.

DNA sequence specificity of doxorubicin-induced mutational damage in uvrB- Escherichia coli.

In the absence of excision repair, doxorubicin caused a striking (41-fold) increase in the frequency of large deletion mutations extending from the lac operator (lacO) into the lac repressor gene (lacI) of Escherichia coli. In contrast, there was only a 2-fold increase in the frequency of small deletions despite a 3-fold increase in overall mutation frequency. The 5'-endpoints of doxorubicin-induced lacO and lacI/lacO deletions occurred at the DNA sequence 5'-pyTAA or 5'-AATpy (where py is pyrmidine) (16%), at runs of purines or pyrimidines (41%) and adjacent to 5'-dGdC or 5'-dCdG doublets (34%). Ninety % (27 of 30) of the doxorubicin-induced deletions involving the region of the lacO palindrome had 3'-endpoints within the palindrome sequence as compared with 40% (4 of 10) spontaneous deletions in an untreated set. Doxorubicin-induced single base substitutions were highly focused at one site (4 of 6) in the i-d region of lacI, in contrast to the spontaneous distribution of point mutations, where 16 mutants were recovered at 12 different sites. An increased frequency (3-fold) of highly focused base substitutions was also observed at 2 sites in the lac operator region (at lacO +6, which is a transition "hotspot" in the spontaneous spectra of both wild type and uvrB- organisms and at the adjacent +5 site). Notably, the frequency of 1- and 2-base frameshifts did not increase in the doxorubicin-induced spectrum, relative to the spontaneous mutation spectrum. These in vivo observations in E. coli suggest that in the absence of excision repair, doxorubicin causes highly focused deletions and base substitutions. These mutations occur adjacent to DNA sequences identified in previous in vitro studies as preferential sites of doxorubicin binding.

New dose-time relationships of folate antagonists to sustain inhibition of human lymphoblasts and leukemic cells in vitro.

In this study, three methods are utilized to analyze toxicity produced by methotrexate and the lipid-soluble antifolate, 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, in human lymphoblasts (WIL-2) and leukemic cells. These methods detect increasingly severe metabolic damage; inhibition of deoxyuridine incorporation into DNA, the reversibility of inhibition of deoxyuridine incorporation by supplementation with formyltetrahydrofolate as Ca2+ leucovorin, and the ability of cells to form clones in soft agarose. The critical dose and exposure time for establishing and maintaining the metabolic toxicity of methotrexate is examined in detail. It is shown that, if an initial loading dose of methotrexate is of high enough concentration or is maintained for a sufficient period to achieve greater than 98% inhibition of deoxyuridine incorporation, this inhibition can be sustained by low concentrations of 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine or methotrexate. Concentrations of methotrexate or 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine that equal or exceed 0.1 microM are sufficient for maintenance of inhibition by an initial loading dose of methotrexate but escape from inhibition that occurs if lower levels of drug are used. The possible implications of these observations for in vivo protocols are discussed.

Differences in calmodulin levels of normal and transformed cells as determined by culture conditions.

Several studies have suggested that calmodulin (CAM) levels increase in cells as a consequence of transformation by RNA tumor viruses. This study examines factors affecting CAM levels in normal and transformed chick embryo fibroblasts. Significant differences in CAM levels of normal and transformed cells were observed as cells grew from subconfluent to confluent densities. These changes were not cell cycle dependent, nor did they correlate with the growth rate of the cultures. The most significant difference between normal and transformed cultures was a lack of down-regulation of CAM levels in transformed cells as compared to normal chick embryo fibroblasts. This decrease in CAM levels in normal cells occurred in high density cultures that were allowed to grow undisturbed for several days without trypsinization and reseeding. These experiments do not support the contention that differences in the growth potential of cycling cultures of normal and transformed cells are regulated through modulation of CAM levels.

Specificity of bischloroethylnitrosourea-induced mutation in a Chinese hamster ovary cell line transformed to express human O6-alkylguanine-DNA alkyltransferase.

The human O6-alkylguanine-DNA alkyltransferase complementary DNA was transfected into the alkyltransferase-deficient Chinese hamster ovary cell line, D422, in an effort to dissect the underlying mechanisms of bischloroethylnitrosourea (BCNU)-induced mutations. The alkyltransferase-transformed cell line exhibited 100-fold protection against BCNU-induced toxicity and an overall decrease in mutation frequency to 25% of that observed in the parental cell line at the hemizygous adenine phosphoribosyl transferase gene target. The frequency of the predominant mutation in the parental cell line, the G:C-->T:A transversion, was reduced from 16 x 10(-6) to 0.7 x 10(-6) in the O6-alkyltransferase-transformed cell line. Likewise, the G:C-->A:T transitions, the second most common BCNU-induced mutation in the parental cell line, was reduced in frequency from 5.2 x 10(-6) to 0.9 x 10(-6) in the alkyltransferase-transformed Chinese hamster ovary cells. These findings suggest that both the G:C-->T:A transversions and G:C-->A:T transitions were O6-alkylguanine-mediated mutations. In the alkyltransferase-transformed Chinese hamster ovary cell line, T:A-->G:C transversions, comprising 45% (23 of 51) of the recovered mutations, emerged as the most common base substitution. In summation, in the absence of alkyltransferase-dependent DNA repair, mutations resulting from O6-alkylation of guanine underlie both the cytotoxic and mutagenic activity of BCNU. In cells expressing high levels of alkyltransferase activity, the cytotoxic and mutagenic actions of BCNU are greatly reduced and mutations resulting from A:T base pair modifications appear to be the major genotoxic lesions induced by the drug.

Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.

Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer.

Hereditary Non-Polyposis Colon Cancer (HNPCC) tumors and some sporadic colon cancers acquire somatic changes in the length of microsatellite sequences. We hypothesized that this 'replication error' (RER) phenotype in these cancers reflects a more general defect which should result in hypermutability of expressed genes. To test this hypothesis mutations of hprt were studied in RER and non-RER tumor cell lines. Increased mutation rates of greater than 100-fold were found in RER compared to non-RER lines. Heterogeneity within the RER group suggests the likely existence of different classes of RER tumors. One non-RER cell line demonstrated a greater than 10-fold increase in mutation rate, suggesting that a novel mutator phenotype may exist in some non-RER tumors.

Chromosome number and structure both are markedly stable in RER colorectal cancers and are not destabilized by mutation of p53.

Fourteen colorectal cancer cell lines, categorized according to the presence or absence of microsatellite instability, were further analysed for chromosomal stability by karyotyping. NonRER (microsatellite stable) cell lines typically displayed highly aberrant karyotypes with alterations not only of chromosome number but also of chromosome structure including chromosomal deletions, inversions, and translocations. RER (microsatellite unstable) cell lines, in contrast, displayed significantly fewer alterations of chromosome number. Moreover, RER cell lines also displayed significantly fewer cytogenetically evident alterations of chromosome structure. Compared to NonRER colon cancers, RER colon cancers are significantly less likely to have undergone chromosomal gain, loss, or breakage. Characterization of p53 gene status by gene sequencing was performed in an attempt to determine if p53 gene status correlated with the chromosomal stability of the RER cancers. Gene mutations in p53 were present in all of the NonRER colon cancers. However, p53 gene mutations were also found present in four of nine of the RER colon cancers. Unexpectedly, RER colon cancers bearing mutant p53 demonstrated the same stability of chromosome number, and the same stability of chromosome structure, as the RER colon cancers with wild-type p53. Therefore, in RER colon cancers specific p53 independent mechanisms actively maintain the stability of both chromosome number and structure.

Increased transversions in a novel mutator colon cancer cell line.

We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

Somatic mutation of hPMS2 as a possible cause of sporadic human colon cancer with microsatellite instability.

Inactivation of DNA-mismatch repair underlies the genesis of microsatellite unstable (MSI) colon cancers. hPMS2 is one of several genes encoding components of the DNA-mismatch repair complex, and germline hPMS2 mutations have been found in a few kindreds with hereditary nonpolyposis colorectal carcinoma (HNPCC), in whom hereditary MSI colon cancers develop. However, mice bearing null hPMS2 genes do not develop colon cancers and hPMS2 mutations in sporadic human colon cancers have not been described. Here we report that in Vaco481 colon cancer the hPMS2 gene is inactivated by somatic mutations of both hPMS2 alleles. The cell line derived from this tumor is functionally deficient in DNA mismatch repair. This deficiency can be biochemically complemented by addition of a purified hMLH1-hPMS2 (hMutLalpha) complex. The hPMS2 deficient Vaco481 cancer cell line demonstrates microsatellite instability, an elevated HPRT gene mutation rate, and resistance to the cytotoxicity of the alkylator MNNG. We conclude that somatic inactivation of hPMS2 can play a role in development of sporadic MSI colon cancer expressing the full range of cancer phenotypes associated with inactivation of the mismatch repair system.

Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability.

Colon cancer and an increasing number of other cancers have been found to exhibit instability of DNA microsatellite sequences. Such tumors have been designated as replication errors (RER) tumors. However, as microsatellites are only rarely found within coding regions of the genome, instability of these sequences cannot directly contribute to carcinogenesis. Recently, we have shown RER colon cancers also demonstrate a marked 100-fold increase in mutation rates measured within an expressed gene, hprt, suggesting the mutator phenotype in these tumors extends beyond microsatellite sequences. To determine whether the RER phenotype indeed destabilizes non-repetitive DNA sequences we have sequenced hprt gene mutations recovered from the RER colon cancer cell line RKO. Greater than 10% of hprt mutants proved to be a single 3 bp deletion located in a nonrepetitive ATTAT sequence motif. Additionally, 1-4 bp deletions or insertions were found to be randomly located throughout the hprt gene. Lastly, one third of hprt mutations proved to be transitions or transversions. The microsatellite instability demonstrated in RKO is thus a global mutator phenotype which destabilizes DNA sequences both inside and outside of repetitive sequence elements and which augments base substitutions as well as frameshifts. These findings extend the characteristics of mutations associated with RER tumors and suggest additional mechanisms by which mutator phenotypes may alter oncogenes and tumor suppressor genes.

Effect of isopropyl-beta-D-thiogalactopyranosid induction of the lac operon on the specificity of spontaneous and doxorubicin-induced mutations in Escherichia coli.

Previous studies of doxorubicin-induced mutations employing F' lacl/lacO as an endogenous gene target have focused on properties of large deletions with 3' endpoints residing in the lacO region of the target gene. This study considers the influence of Lac repressor binding on the distribution of these deletions. Results of the DNA sequence level analysis of spontaneous and doxorubicin-induced i-d and lacO mutations in Escherichia coli uvrB- are reported for mutants isolated under conditions where Lac repression is relieved by isopropyl-beta-D-thiogalactopyranosid (IPTG; an inducer that prevents repressor binding to lacO). The location of deletions isolated from doxorubicin-treated cultures in the presence and absence of IPTG suggests that doxorubicin preferentially focuses deletion endpoints adjacent to its binding sites in lacO and that the distribution of these deletion endpoints is not modulated by Lac repressor binding. In contrast, spontaneous deletion endpoints are preferentially clustered in the loop away from the palindromic sequences under conditions of repression. However, when the Lac repressor/lacO binding complex is dissociated by IPTG, the spontaneous 3'-deletion endpoints distribute proportionally between the putative stem and loop of the lacO palindrome. The single most striking effect of IPTG induction of the Lac operon was elimination of a "hot spot" for T:A-->C:G transitions at position +6 in lacO. This base substitution "hot spot," which accounted for 17.6% of total doxorubicin-induced mutants and 16.4% of spontaneous mutants in repressed bacterial cultures, accounted for approximately 1% of total mutations in similar experiments carried out in the presence of IPTG. A large number of mutations at the +6 position are induced only by doxorubicin in the absence of IPTG, however, suggesting that both doxorubicin-induced and spontaneous mutation at this transition "hot spot" are mediated by Lac repressor binding to lacO.

Overcoming methotrexate resistance by a lipophilic antifolate (BW 301U): from theory to models to practice.

We have provided a rationale for the clinical use of a new lipid-soluble folate antagonist, BW 301U, in terms of its potential for killing several classes of methotrexate-resistant cells. As part of a Phase I evaluation of this agent we studied normal bone marrow from cancer patients and their metabolic susceptibility to either BW 301U or to MTX and then repeated the observations at the end of five days of BW 301U infusions. Both inhibitors were roughly comparable at equimolar concentrations prior to therapy, but a relative resistance developed to MTX after BW 301U treatment. Such findings were replicated in an in vitro HL-60 cell culture system that was exposed to BW 301U. Some possible mechanisms for this unusual collateral resistance are discussed.

The occurrence and consequences of deoxyuridine in DNA.

Deoxyuridine can become resident in the DNA of prokaryotic and eukaryotic cells via two general mechanisms - deamination of cytosine to uracil, and nucleotide pool changes that lead to misincorporation of deoxyuridine in place of thymidine. In this paper we have examined the chemical basis of deamination reactions in DNA and discussed a possible mechanism for an increased rate of deamination by means of cross-strand protonation of cytosine by alkylated guanine. In addition, we have examined the genetic and drug-induced conditions that lead to dUMP misincorporation into DNA in place of thymidine and have presented experimental evidence indicating that the antifolate-induced lesion is a general drug-dose dependent lesion of human blood cells. Finally, the toxic and genetic impact of this lesion has been evaluated within the context of a review of the repair mechanisms elicited by dUMP in DNA.

Deoxyuridine misincorporation causes site-specific mutational lesions in the lacI gene of Escherichia coli.

Spontaneous forward mutation in lacI was analyzed by DNA sequencing in a Dut- strain of E. coli. Hyperuracil incorporation into DNA due to the defect in deoxyuridinetriphosphatase caused a 5-fold increase in mutation frequency. Deletion, duplication and base-substitution frequencies were all enhanced in the Dut- strain. However, the analysis of the specificity of mutation revealed a remarkable site- and class-specificity. For example, base substitutions at a single site, a G:C = greater than A:T transition (Ochre 34) accounted for 55% of the base substitutions recovered. The spontaneous A:T = greater than G:C hotspot at position +6 at the lac operator was also recovered at an enhanced frequency in the Dut- strain where it accounted for 25% of the base substitutions. Many of the deletion and duplication events were recovered more than once; most had endpoints in A/T rich regions. The spontaneous frameshift hotspot involving the gain or loss of 5'-CTGG-3' in a region where this tetramer is tandemly repeated 3 times, was also greatly enhanced. No frameshifts involving a single base pair nor IS1 insertions were identified among the 86 lacI mutants sequenced. The analysis of these events reveals them to be generally consistent with a mechanism involving AP sites generated by the removal of misincorporated uracil by uracil-N-glycosylase. Considering the number of potential AP sites (approximately 1 per 170 base pairs) E. coli is remarkably refractory to mutational consequences of deoxyuridine misincorporation in place of thymidine.

Excision repair reduces doxorubicin-induced genotoxicity.

LacI mutations induced by doxorubicin in a wild-type, uvr(A)BC repair-proficient E. coli strain were analyzed by DNA sequencing. These mutations were contrasted with mutations previously recovered from doxorubicin-treated uvrB- organisms in order to assess the role of excision repair in doxorubicin-induced genotoxicity. After a 30-min exposure of wild-type E. coli to 330 microM doxorubicin, survival was 34% and the overall lacI mutation frequency increased 1.8-fold to 340 x 10(-8). The distribution of doxorubicin-induced mutants among subclasses of mutation involving the i-d and lac operator regions differed significantly between repair-proficient and -deficient strains. Distributional differences appeared to result both from a decrease in deletions involving the lac operator and an increase in base substitutions involving the i-d region in repair proficient organisms. However, elements of the doxorubicin-induced mutation spectrum in uvrB- E. coli are still discernable in wild-type organisms. These elements include the remarkable shift of 3'-deletion endpoints to palindromic sequence within the lac operator and the recovery of multiple isolates of T:A-->A:T transversions at position 96 in doxorubicin-treated cultures. These observations suggest that components of the uvr(A)BC nucleotide excision repair system function through a general mechanism prior to fixation of mutations to reduce, but not completely eliminate, the genotoxic effects of doxorubicin.