PON1 increases cellular DNA damage by lactone substrates.

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme that by hydrolysing exogenous and endogenous substrates can provide protection against substrate induced toxicity. To investigate the extent to which PON1 provides protection against lactone induced DNA damage, DNA damage was measured in HepG2 cells using the neutral Comet assay following lactone treatment in the presence and absence of exogenous recombinant PON1 (rPON1). Low dose lactones (10 mM) caused little or no damage while high doses (100 mM) induced DNA damage in the following order of potency: α-angelica lactone > γ-butyrolactone ~ γ-hexalactone > γ-heptalactone ~ γ-octaclactone ~ γ-furanone ~ γ-valerolactone > γ-decalactone. Co-incubation of 100 mM lactone with rPON1, resulted in almost all cells showing extensive DNA damage, particularly with those lactones that decreased rPON1 activity by > 25%. In contrast, with the lactones that are poor rPON1 subtrates (γ-decalactone and γ-furanone), rPON1 did not increase DNA damage. DNA damage induced by a 1 h co-treatment with 10 mM α-angelica lactone and rPON1 was reduced when cells when incubated for a further 4 h in fresh medium suggesting break formation was due to induced DNA damage rather than apoptosis. Preincubation (1-6 h) of α-angelica lactone with rPON1 in the absence of cells, decreased cellular DNA damage by around 40%  in comparison to cells treated without preincubation. These results suggest that in addition to its well-recognised detoxification effects, PON1 can increase genotoxicity potentially by hydrolysing certain lactones to reactive intermediates that increase DNA damage via the formation of DNA adducts.

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents.

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and ß, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin.

A phase I study of the safety and tolerability of olaparib (AZD2281, KU0059436) and dacarbazine in patients with advanced solid tumours.

BACKGROUND: Poly adenosine diphosphate (ADP)-ribose polymerase (PARP) is essential in cellular processing of DNA damage via the base excision repair pathway (BER). The PARP inhibition can be directly cytotoxic to tumour cells and augments the anti-tumour effects of DNA-damaging agents. This study evaluated the optimally tolerated dose of olaparib (4-(3--4-fluorophenyl) methyl-1(2H)-one; AZD2281, KU0059436), a potent PARP inhibitor, with dacarbazine and assessed safety, toxicity, clinical pharmacokinetics and efficacy of combination treatment. PATIENTS AND METHODS: Patients with advanced cancer received olaparib (20-200 mg PO) on days 1-7 with dacarbazine (600-800 mg m(-2) IV) on day 1 (cycle 2, day 2) of a 21-day cycle. An expansion cohort of chemonaive melanoma patients was treated at an optimally tolerated dose. The BER enzyme, methylpurine-DNA glycosylase and its substrate 7-methylguanine were quantified in peripheral blood mononuclear cells. RESULTS: The optimal combination to proceed to phase II was defined as 100 mg bd olaparib with 600 mg m(-2) dacarbazine. Dose-limiting toxicities were neutropaenia and thrombocytopaenia. There were two partial responses, both in patients with melanoma. CONCLUSION: This study defined a tolerable dose of olaparib in combination with dacarbazine, but there were no responses in chemonaive melanoma patients, demonstrating no clinical advantage over single-agent dacarbazine at these doses.

Dietary variables associated with DNA N7-methylguanine levels and O6-alkylguanine DNA-alkyltransferase activity in human colorectal mucosa.

Components of human diets may influence the incidence of colorectal adenomas, by modifying exposure or susceptibility to DNA-damaging alkylating agents. To examine this hypothesis, a food frequency questionnaire was used to assess the diet of patients recruited for a case-referent study where biopsies of normal colorectal mucosa were collected during colonoscopy and subsequently analysed for DNA N7-methylguanine (N7-MeG) levels, as an indicator of exposure, and activity of the DNA repair protein O6-alkylguanine DNA-alkyltransferase (MGMT), as an indicator of potential susceptibility. Cases with histologically proven colorectal adenomas (n = 38) were compared with referents (n = 35) free of gastrointestinal neoplasia. The case group consumed significantly more red meat (4.5 versus 3.4 servings/week, P < 0.05), processed meats, (4.7 versus 3.2 servings/week, P < 0.05) and % food energy as fat (34.9 versus 30.7%, P < 0.001). N7-MeG [mean: 95% confidence interval (CI)] levels were significantly lower in the group that consumed the highest proportion of dietary fibre/1000 kcal in comparison with the group with the lowest intake (0.61; 0.35-0.86 versus 1.88; 0.88-2.64 micromol/mol dG, P < 0.05). N7-MeG levels were also inversely associated with folate consumption (P < 0.05). MGMT activity (mean; 95% CI) was significantly higher in the group with the lowest consumption of vegetables than in the group with the greatest vegetable consumption (7.02; 5.70-8.33 versus 4.93; 3.95-5.91 fmol/microg DNA, P < 0.05). Our results are consistent with the hypothesis that dietary factors may modify exposure or susceptibility, respectively, to DNA damage by alkylating agents.

A phase I study of extended dosing with lomeguatrib with temozolomide in patients with advanced melanoma.

Lomeguatrib, an O(6)-methylguanine-DNA methyltransferase inactivator, was evaluated in an extended dosing regimen with temozolomide, designed according to pharmacodynamic data from previous studies. Patients with unresectable stage 3 or 4 cutaneous or unknown primary melanoma metastases were treated with lomeguatrib 40 mg, b.i.d. for 10 or 14 days and temozolomide 75-100 mg m(-2) on days 1-5. Drugs were administered orally with cycles repeated every 28 days, for up to six cycles. A total of 32 patients were recruited to the study. Lomeguatrib for 10 days with temozolomide 75 mg m(-2) was established as the optimal extended lomeguatrib dosing schedule, with haematological toxicity being dose limiting. There were two partial responses to treatment giving an overall response rate of 6.25%. Extending lomeguatrib administration beyond that of temozolomide requires a reduced dose of the latter agent. Only limited clinical activity was seen, suggesting no advantage for this regimen over conventional temozolomide administration in the treatment of melanoma.

O(6)-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib.

We evaluated the pharmacodynamic effects of the O(6)-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O(6)-methylguanine (O(6)-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O(6)-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O(6)-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O(6)-meG in PBMC DNA during treatment.

A phase II trial of lomeguatrib and temozolomide in metastatic colorectal cancer.

To evaluate the tumour response to lomeguatrib and temozolomide (TMZ) administered for 5 consecutive days every 4 weeks in patients with metastatic colorectal carcinoma. Patients with stage IV metastatic colorectal carcinoma received lomeguatrib (40 mg) and TMZ (50-200 mg m(-2)) orally for 5 consecutive days every 4 weeks. Response was determined every two cycles. Pharmacokinetics of lomeguatrib and TMZ as well as their pharmacodynamic effects in peripheral blood mononuclear cells (PBMC) were determined. Nineteen patients received 49 cycles of treatments. Despite consistent depletion of O(6)-methylguanine-DNA methyltransferase in PBMC, none of the patients responded to treatment. Three patients had stable disease, one for the duration of the study, and no fall in carcinoembryonic antigen was observed in any patient. Median time to progression was 50 days. The commonest adverse effects were gastrointestinal and haematological and these were comparable to those of TMZ when given alone. This combination of lomeguatrib and TMZ is not efficacious in metastatic colorectal cancer. If further studies are to be performed, emerging data suggest that higher daily doses of lomeguatrib and a dosing period beyond that of TMZ should be evaluated.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice.

Type 1 diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta-cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta-cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in APNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta-cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(+/+) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

L-ß-N-methylamino-l-alanine (BMAA) nitrosation generates a cytotoxic DNA damaging alkylating agent: An unexplored mechanism for neurodegenerative disease.

BACKGROUND: L-ß-N-methylamino-l-alanine (BMAA) is a non-proteinic amino acid, that is neurotoxic in vitro and in animals, and is implicated in the causation of amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC) on Guam. Given that natural amino acids can be N-nitrosated to form toxic alkylating agents and the structural similarity of BMAA to other amino acids, our hypothesis was that N-nitrosation of BMAA might result in a toxic alkylating agent, providing a novel mechanistic hypothesis for BMAA action. FINDINGS: We have chemically nitrosated BMAA with sodium nitrite to produce nitrosated BMAA (N-BMAA) which was shown to react with the alkyl-trapping agent, 4-(p-nitrobenzyl)pyridine, cause DNA strand breaks in vitro and was toxic to the human neuroblastoma cell line SH-SY5Y under conditions in which BMAA itself was minimally toxic. CONCLUSIONS: Our results indicate that N-BMAA is an alkylating agent and toxin suggesting a plausible and previously unrecognised mechanism for the neurotoxic effects of BMAA.

Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.

Cellular survival following ionising radiation-mediated damage is primarily a function of the ability to successfully detect and repair DNA double-strand breaks (DSBs). Previous studies have demonstrated that radiosensitivity, determined as a reduction in colony forming ability in vitro, may be related to the incorrect repair (misrepair) of DSBs. The novel rapid dual fluorescence (RDF) assay is a plasmid-based reporter system that rapidly assesses the correct rejoining of a restriction-enzyme produced DSBs within transfected cells. We have utilised this novel assay to determine the fidelity of DSB repair in the prostate tumour cell line LNCaP, the bladder tumour cell line MGH-U1 and a radiosensitive subclone S40b. The two bladder cell lines have been shown in previous studies to differ in their ability to correctly repair plasmids containing a single DSB. Using the RDF assay we found that a substantial portion of LNCaP cells [80.4 +/- 5.3(standard error)%] failed to reconstitute reporter gene expression; however, there was little difference in this measure of DSB repair fidelity between the two bladder cell lines (48.3 +/- 3.5% for MGH-U1; 39.9 +/- 8.2% for S40b). The RDF assay has potential to be developed to study the relationship between DSB repair fidelity and radiosensitivity as well as the mechanisms associated with this type of repair defect.

Nitrosated glycine derivatives as a potential source of O6-methylguanine in DNA.

The nitrosated bile acid conjugate N-nitrosoglycocholic acid reacts with DNA to give, rise to several adducts including O6-carboxymethylguanine and, unexpectedly, O6-methylguanine (O6-MG). O6-MG is well established as a toxic and promutagenic lesion and is a substrate for the DNA repair protein O6-alkylguanine-DNA-alkyltransferase. In contrast, O6-carboxymethylguanine is not repaired by this protein. Similar results have been obtained for other nitrosated glycine derivatives, which suggests that O6-MG, which has been observed in DNA from human gastrointestinal tissues, may be derived from intragastric nitrosation of glycine or related compounds.

O6-alkylguanine-DNA alkyltransferase depletion and regeneration in human peripheral lymphocytes following dacarbazine and fotemustine.

O6-Alkylguanine-DNA alkyltransferase (ATase) levels were measured in peripheral blood lymphocytes of 13 patients with advanced malignant melanoma treated with sequential dacarbazine (DTIC) and fotemustine. Wide interindividual variation in the pretreatment levels and in depletion and regeneration of ATase activity was noted. Depletion of ATase was seen within the first h after DTIC administration with values ranging from 44 to 92% of pretreatment levels. In 10 patients, progressive depletion of ATase activity occurred with nadir activity occurring at about 4 to 6 h with values ranging from 0 to 67% of pretreatment activity; at 18 h after DTIC infusion. ATase activity varied from 6 to 81%. No significant difference was seen between the rates of ATase depletion or regeneration between the two groups of patients receiving either 500 or 800 mg/m2 of DTIC with the same dose of fotemustine (100 mg/m2). In one patient, maximum depletion occurred within 1 h and no ATase activity was detectable over the next 18 h. In another patient, maximum depletion occurred at 2 h after DTIC followed by recovery of ATase activity to 71% at 18 h. In 2 patients who returned for subsequent cycles of chemotherapy, an increase in pretreatment ATase activity was seen. Overall, the extent of depletion of ATase following DTIC/fotemustine was directly proportional to the initial ATase level.

Interaction of the carcinogen 3,3-dimethyl-1-phenyltriazene with nucleic acids of various rat tissues and the effect of a protein-free diet.

The methylation by 3,3-[14C]dimethyl-1-phenyltriazene of nucleic acids in various rat tissues was investigated. Following a single s.c. injection of 3,3-[14C]dimethyl-1-phenyltriazene (50 mg/kg), approximately 40% of the radioactivity was subsequently exhaled as 14CO2. Expiration of 14CO2, metabolic 14C labeling of liver proteins, and formation of 7-[14C]methylguanine, the major reaction product with nucleic acid bases, were completed within about 15 hr. Minor alkylation products detectable in DNA were O6-methylguanine and 3-methyladenine. In cytoplasmic RNA, 1-methyladenosine, 3-methylcytidine, and O6-methylguanosine were present in addition to 7-methylguanosine. Concentrations of 7-methylguanine were highest in nucleic acids of kidney and liver. Among the other organs investigated (brain, lung, spleen, small intestine), 7-methylguanine levels showed little variation but were 4 to 7 times lower than those in liver and kidney. Feeding of a protein-free diet prior to 3,3-[14C]dimethyl-1-phenyltriazene administration reduced the formation of 7-methylguanine in liver and kidney RNA, whereas in the remaining organs the extent of methylation was markedly increased. The results are discussed with respect to the significance of methylation at specific sites in nucleic acids for the initiation of malignant transformation and the possible role of 3-methyl-1-phenyltriazene as the systemically distributed proximate carcinogen of 3,3-dimethyl-1-phenyltriazene.

Increased excision of O6-methylguanine from rat liver DNA after chronic administration of dimethylnitrosamine.

Male BDIV rats were given dimethylnitrosamine (2 mg/kg daily) by stomach tube on weekdays for a total of 9 weeks, the final dose being of 14C-labeled material. Control rats received only the labeled dimethylnitrosamine. Liver DNA was isolated at various times later (from 2 to 12 hr), and normal and alkylated purines were determined after hydrolysis in mild acid by chromatography on Sephadex G-10. The levels (measured as dpm/micronmol of parent base) of 7-methylguanine in the DNA of the pretreated rats were the same as or slightly higher than those of the control animals, and the persistence of this product was similar in both groups. This was also true for 3-methyladenine. In contrast, the initial amount of O6-methylguanine in the liver DNA of the pretreated rats was one-third of the amount found in the control rats, and the rate of loss of this product from DNA was higher in the pretreated animals. These differences were reflected in the alkylation product ratios: the 3-methyladenine:7-methylguanine ratios were closely similar in the two groups of animals at all times, whereas the O6-methylguanine:7-methylguanine ratio was initially 3 times higher in the control animals and fell more slowly. DNA synthesis (as measured by the incorporation of [3H]thymidine) was higher in the liver, kidney, and lung of rats receiving dimethylnitrosamine pretreatment. These findings are discussed with respect to the hepatocarcinogenicity of chronically administered dimethylnitrosamine.

Dimethylnitrosamine-induced inhibition of hepatic protein synthesis in vitro and the effect of pretreatment with cystamine or pregnenolone-16alpha-carbonitrile.

Hepatic protein synthesis was investigated using a postmitochondrial supernatant system derived from the livers of rats that were given injections of a single dose of dimethylnitrosamine (DMN), 30 mg/kg. The time course and extent of DMN-induced inhibition in vitro were identical to those reported for the incorporation of amino acids into liver proteins in vivo, maximum inhibition being about 70% at 5 hr. Addition of specific inhibitors of chain initiation (polyinosinic acid and aurin tricarboxylic acid) to the postmitochrondrial supernatant system from DMN-treated rats caused only a slight additional inhibition, indicating that DMN predominantly affects translation by a block of initiation. Treatment with cystamine prior to DMN administration completely abolished the depression of protein synthesis and reduced by more than 90% the methylation by [14C]DMN of purine bases in liver DNA. Pretreatment with pregnenolone-16alpha-carbonitrile stimulated protein synthesis in controls but had no preventive effect in DMN-treated rats and did not reduce the extent of DNA alkylation in vivo.

Effect of acute doses of 2-acetylaminofluorene on the capacity of rat liver to repair methylated purines in DNA in vivo and in vitro.

Male Wistar rats were given various doses of 2-acetylaminofluorene (AAF) at doses of 0.74, 2.22, 6.67, or 20 mg/kg i.p. 24 hr before administration of [14C]dimethylnitrosamine (1 or 2 mg/kg i.p.). Analysis of liver DNA isolated from animals killed 5 hr later showed variations between groups treated with different amounts of AAF in the amounts of 3-methyladenine, 7-methylguanine, and O6-methylguanine (O6-mGua). However, the relative amounts of these products were unchanged by AAF pretreatment except after 20 mg/kg when the reduced O6-mGua:7-methylguanine ratio indicated enhanced O6-mGua repair. Specific enhancement of O6-mGua repair was also found 5 hr after administration of [14C]methylnitrosourea (11.5 mg/kg) to animals pretreated with AAF (20 mg/kg), while the amounts of O6-mGua in liver ribosomal RNA afer [14C]dimethylnitrosamine were unaffected by this AAF dose. Pretreatment of rats with AAF 29 hr earlier increased the capacity of cell-free liver extracts to remove O6-mGua from [3H]methylnitrosourea-methylated DNA in vitro. The increase was detectable after 2.22 mg/kg and reached a maximum 3.5-fold increase after AAF, (60 mg/kg). 7-Methylguanine and 3-methyladenine-DNA glycosylase activities were also increased, but this was independent of the dose of AAF. AAF pretreatment produced a slight (3- to 4-fold) increase in incorporation of [3H]thymidine or labeled one-carbon breakdown products of [14C]dimethylnitrosamine into liver DNA which appeared to parallel in vitro O6-mGua repair enhancement, but the increased [3H]thymidine uptake was statistically significant only after the 60-mg/kg dose.

Relationships among cell survival, O6-alkylguanine-DNA alkyltransferase activity, and reactivation of methylated adenovirus 5 and herpes simplex virus type 1 in human melanoma cell lines.

O6-Alkylguanine-DNA alkyltransferase (ATase) activity and host cell reactivation (HCR) of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC)-methylated viruses were compared in human melanoma cell lines that were sensitive or resistant to killing by the antitumor DNA-methylating agent MTIC. Enhanced HCR of adenovirus 5 (defined as the Mer+ phenotype) generally showed a semiquantitative correlation with the natural or induced resistance of the host cells to the toxic effects of MTIC and to the level of ATase activity. However, one MTIC-resistant cell line was found (MM170) which had a low level of ATase and intermediate HCR of adenovirus. The HCR of herpes simplex virus type 1 (HSV-1) was enhanced in the Mer+ cells that had natural resistance to MTIC compared with Mer- cells. On the other hand, HCR of HSV-1 in Mer+ cells with induced resistance to MTIC was similar to that in Mer- cells. Neither adenovirus 5 nor HSV-1 infection induced ATase activity in Mer- cells. This indicates that resistance to the toxic effects of methylating agents is not invariably associated with high levels of ATase activity in human melanoma cells. Furthermore, while induction of the Mer+ phenotype from Mer- cells was usually accompanied by the recovery of ATase activity, induced Mer+ cells had less proficient repair than natural Mer+ cells, as judged quantitatively by slightly lower cellular resistance and qualitatively by deficient HCR response for HSV-1. These results suggest that the Mer- and induced Mer+ cells lack an ATase-independent DNA repair mechanism. No differences in MTIC-induced DNA repair synthesis or strand breaks were found between the Mer-, natural Mer+, and induced Mer+ phenotypes. However, UV-induced DNA repair synthesis was higher in the natural Mer+ than in the Mer- or induced Mer+ cells, both of which had increased cellular sensitivity to the antimetabolites methotrexate and hydroxyurea. These differences may be related to the effects observed with MTIC. A wide range of ATase activities was found in human melanoma biopsy material.

Relationships between the formation of O6-methyldeoxyguanosine by 1-p-carboxyl-3,3-dimethylphenyltriazene in DNA and O6-alkylguanine-DNA alkyltransferase in human peripheral leukocytes.

There is increasing experimental evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain antitumor alkylating agents and that the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide interindividual variation in the depletion and subsequent regeneration of ATase in peripheral blood lymphocytes of patients treated with 24-h continuous infusion of 1-p-carboxyl-3,3-dimethylphenyltriazene (CB10-277) for metastatic melanoma. We have now measured the formation of O6-MedG in the DNA of peripheral leukocytes of nine patients receiving this treatment regimen. This lesion could be detected in DNA within 1 h and a progressive increase in adduct levels occurred during the CB10-277 infusion and for 24 h after completion. Considerable interindividual variation was observed in the peak O6-MedG levels, with values ranging from 3.0 to 23.8 mumol O6-MedG/mol deoxyguanosine (mean, 12.3 +/- 6.4 mumol O6-MedG/mol deoxyguanosine) following the first treatment cycle, possibly as a consequence of differences in the capacity of patients to metabolize CB10-277 to a methylating agent. There was, nevertheless, a clear temporal relationship between the progressive formation of leukocyte O6-MedG and lymphocyte ATase depletion. Repeated-measures regression showed that this was statistically significant (P < 0.001) during the CB10-277 infusion. A significant inverse correlation was also seen between pretreatment lymphocyte ATase activity and peak O6-MedG levels in leukocyte DNA (r = -0.73) and the area under the leukocyte O6-MedG concentration-time curve (r = -0.76). Metabolism of CB10-277 to a methylating agent could be one factor that combines with DNA repair capacity to determine clinical response, because the two responses observed in this series occurred in the two patients with the highest leukocyte O6-MedG levels and also the lowest pretreatment ATase activity. Hematological toxicity developed in the same two patients.