The DNA damage response network in the treatment of head and neck squamous cell carcinoma.

BACKGROUND: We sought to determine whether DNA damage response (DDR)-related aberrations predict therapeutic benefit in cisplatin-treated head and neck squamous cell carcinoma (HNSCC) patients and how DDR pathways are modulated after treatment with olaparib alone or in combination with cisplatin or durvalumab. PATIENTS AND METHODS: Oxidative stress, abasic sites and DDR-related parameters, including endogenous DNA damage, DNA repair mechanisms and apoptosis rates, were evaluated in HNSCC cell lines and peripheral blood mononuclear cells from 46 healthy controls (HC) and 70 HNSCC patients at baseline and following treatment with cisplatin-containing chemoradiation or nivolumab or enrolled in the OPHELIA phase II trial (NCT02882308; olaparib alone, olaparib plus cisplatin, olaparib plus durvalumab). RESULTS: HNSCC patients at diagnosis exhibited deregulated DDR-related parameters and higher levels of oxidative stress and abasic sites compared with HC (all P < 0.05). Accordingly, nucleotide excision repair (NER; ERCC1, ERCC2/XPD, XPA, XPC) and base excision repair (APEX1, XRCC1) genes were downregulated in patients versus HC whereas double-strand breaks repair (MRE11A, RAD50, RAD51, XRCC2) and mismatch repair (MLH1, MSH2, MSH3) genes were overexpressed. Corresponding results were obtained in cell lines (all P < 0.001). Excellent correlations were observed between individual ex vivo and in vivo/therapeutic results, with cisplatin non-responders showing higher levels of endogenous DNA damage, augmented oxidative stress and abasic sites, increased NER capacities and reduced apoptosis than responders (all P < 0.05). Also, longer progression-free survival correlated with lower NER capacity (P = 0.037) and increased apoptosis (P = 0.029). Interestingly, treatment with olaparib-containing regimens results in the accumulation of cytotoxic DNA damage and exerts an extra antitumor effect by elevating oxidative stress (all P < 0.05). Nivolumab induced no significant changes in the DDR parameters examined. CONCLUSIONS: Aberrations in DDR signals are implicated in the response to HNSCC chemotherapy and can be exploited as novel therapeutic targets, sensitive/effective non-invasive biomarkers as well as for the design of novel clinical trials.

Correction: Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased DNA double-strand breaks and enhanced apoptosis in patients with lupus nephritis.

OBJECTIVE: DNA double-strand breaks (DSBs) lead to mutations, genomic instability and apoptotic death, whereas accumulation of apoptotic cells results in excessive autoantigen presentation and autoantibody formation. We aimed to measure DSB levels in lupus nephritis, a severe complication of the prototypic systemic autoimmune disease. METHODS: The intrinsic DNA damage and the apoptosis induction/DSB levels were evaluated in peripheral blood mononuclear cells of six patients and 10 healthy controls following exposure to genotoxic agents (melphalan, cisplatin) ex vivo. DSBs were assessed using immunofluorescence quantification of γH2AX foci and comet assay. RESULTS: Intrinsic DNA damage was increased in lupus versus control cells in both assays (Olive Tail Moment units of 15.8 ± 2.3 versus 3.0 ± 1.4 in comet, p < 0.01; % γH2AX-positive cells: 13.6 ± 1.8 versus 4.6 ± 0.9, p < 0.01, respectively). Melphalan or cisplatin doses as low as 9.9 ± 4.8 or 29.8 ± 8.3 µg/ml, respectively, were sufficient to induce apoptosis in lupus cells; control cells required doses of 32.3 ± 7.7 and 67.7 ± 5.5 µg/ml, respectively. Drug-induced DSB levels were increased in lupus versus control cells, with the area under the curve (AUC) for melphalan-induced DSBs being 3050 ± 610 (% γH2AX-positive staining cells) × (drug dose) in patients and 1580 ± 350 in controls (p < 0.05); the corresponding values for cisplatin-induced AUC were 13900 ± 1800 for lupus and 4500 ± 750 for controls (p < 0.01). Interestingly, within either lupus patients or controls examined, the accumulation of DSBs correlated with apoptosis degrees (all p < 0.01). Results in lupus cells were not associated with individual disease activity level or treatment modalities at the time of the study. CONCLUSION: These findings suggest a novel mechanism by which increased accumulation of DSBs may render cells more sensitive to apoptosis, thus contributing to the induction of systemic autoimmunity.

Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma.

BACKGROUND: Melphalan is one of the most active chemotherapeutic agents in the treatment of multiple myeloma (MM). However, the mechanism underlying differential patient responses to melphalan therapy is unknown. METHODS: Chromatin structure, transcriptional activity and DNA damage response signals were examined following ex vivo treatment with melphalan of both malignant bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) of MM patients, responders (n=57) or non-responders (n=28) to melphalan therapy. PBMCs from healthy controls (n=25) were also included in the study. RESULTS: In both BMPCs and PBMCs, the local chromatin looseness, transcriptional activity and repair efficiency of the transcribed strand (TS) were significantly higher in non-responders than in responders and lowest in healthy controls (all P<0.05). Moreover, we found that melphalan-induced apoptosis inversely correlated with the repair efficiency of the TS, with the duration of the inhibition of mRNA synthesis, phosphorylation of p53 at serine 15 and apoptosis rates being higher in responders than in non-responders (all P<0.001). CONCLUSIONS: Our findings provide a mechanistic basis for the link between DNA repair efficiency and response to melphalan therapy. Interestingly, the observation of these phenomena in PBMCs provides a novel approach for the prediction of response to anti-myeloma therapy.

Progressive changes in chromatin structure and DNA damage response signals in bone marrow and peripheral blood during myelomagenesis.

The molecular pathways implicated in multiple myeloma (MM) development are rather unknown. We studied epigenetic and DNA damage response (DDR) signals at selected model loci (N-ras, p53, d-globin) in bone marrow plasma cells and peripheral blood mononuclear cells (PBMCs) from patients with monoclonal gammopathy of undetermined significance (MGUS; n=20), smoldering/asymptomatic MM (SMM; n=29) and MM (n=18), as well as in healthy control-derived PBMCs (n=20). In both tissues analyzed, a progressive, significant increase in the looseness of local chromatin structure, gene expression levels and DNA repair efficiency from MGUS to SMM and finally to MM was observed (all P<0.002). Following ex vivo treatment with melphalan, a gradual suppression of the apoptotic pathway occurred in samples collected at different stages of myelomagenesis, with the severity and duration of the inhibition of RNA synthesis, p53 phosphorylation at serine15 and induction of apoptosis being higher in MGUS than SMM and lowest in MM patients (all P<0.0103). Interestingly, for all endpoints analyzed, a strong correlation between plasma cells and corresponding PBMCs was observed (all P<0.0003). We conclude that progressive changes in chromatin structure, transcriptional activity and DDR pathways during myelomagenesis occur in malignant plasma cells and that these changes are also reflected in PBMCs.

Increased expression of the FoxP3 functional marker of regulatory T cells following B cell depletion with rituximab in patients with lupus nephritis.

B cell depletion may affect T cell activation and costimulation status in rituximab-treated patients with SLE. We examined whether rituximab administration in patients with active lupus nephritis is related to changes in mRNA expression of genes that define regulatory T cells (Tregs) in peripheral blood lymphocytes, measured by real-time PCR. At the early phase of B cell depletion mRNA levels of CD25, CTLA-4, GITR and the bona fide Treg functional marker FOXP3 increased significantly in all 7 patients examined. In contrast, mRNA levels of the costimulatory/activation T cell molecule CD40L were profoundly reduced, while mRNA levels of TGF-beta, a cytokine contributing to Treg induction, increased significantly in all. During follow-up, increased FOXP3 mRNA persisted in those patients in clinical remission, while in those patients with active disease subsequent decreases were noted. Further studies should examine whether modulation of Tregs by therapeutic B cell depletion contributes and/or predicts lupus disease remission.

Coexposure to ethanol with N-nitrosodimethylamine or 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone during lactation of rats: marked increase in O(6)-methylguanine-DNA adducts in maternal mammary gland and in suckling lung and kidney.

Use of alcoholic beverages increases risk of cancer at several target sites, including the breast. Of several possible mechanisms for this effect, competitive inhibition by ethanol of hepatic clearance of nitrosamines, resulting in increased dose delivery to posthepatic tissues, gives the quantitatively most pronounced enhancement. We investigated whether this effect would pertain to the mammary gland, and to ethanol and nitrosamines delivered translactationally to sucklings. Ethanol (1.6 g/kg) was administered by gavage to nursing Sprague-Dawley rats 10 min before 5 mg/kg N-nitrosodimethylamine (NDMA) or 50 mg/kg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK); treatment was on postnatal days 1, 7, or 14. Tissues taken 4 h later for analysis of O(6)-methylguanine in DNA were liver, blood, and mammary glands from the mothers, and liver, lung, kidney, and blood from the sucklings. Ethanol cotreatment resulted in a marked, 10-fold increase in O(6)-methylguanine adducts from NDMA in mammary gland, as well as smaller but significant increases in this tissue from NNK and in maternal blood cells from both chemicals; adducts in maternal liver decreased slightly. In the sucklings, ethanol cotreatment also lowered adducts in liver after NDMA or NNK treatment. After NDMA, adducts were also detected in suckling lung and kidney and were increased five- to 10-fold after ethanol coexposure. Adducts from either chemical, with or without ethanol, decreased markedly in all suckling tissues with development from postnatal day 1 to day 14. Thus ethanol coexposure with nitrosamines increases O(6)-methylguanine DNA adducts in mammary gland and strongly influences adduct formation in suckling tissues after translactational delivery.

DNA adducts, mutant frequencies and mutation spectra in lambda lacZ transgenic mice treated with N-nitrosodimethylamine.

Groups of lambda lacZ transgenic mice were treated i.p. with N-nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in various tissues. Adduct induction in the liver exceeded by at least one order of magnitude than observed in the next nearest target tissue (lung), and was approximately linearly related to dose, except for O6-methylguanine after the first dose of 1 mg/kg which was lower than expected. Substantial induction of lambda lacZ mutagenesis was observed only in the liver, where the mutant frequency was already maximal within 7 days after 5 mg/kg NDMA and remained unchanged thereafter up to 49 days. Small but marginally significant increases in mutant frequency were consistently observed in the spleen after all three modes of treatment. A lack of proportionality between mutation induction and the administered dose or the corresponding adduct levels was observed, probably reflecting the importance of toxicity-related cell proliferation caused by NDMA at higher doses. Twenty eight days after a dose of 10 mg/kg (causing a 3.6-fold increase in mutant frequency), NDMA was found to increase the frequency of GC-->AT mutations (with a concomitant shift of their preferential location from CpG sites to GpG sites), which made up approximately 60% of the induced mutations. Surprisingly, NDMA also caused a significant increase in deletions of a few (up to 11) base-pairs (22%).

Effects of 2-chlorodeoxyadenosine and gold sodium thiomalate on human bcl-2 gene expression.

Aberrant expression of apoptosis-related genes, including the "cell death suppressor gene" bcl-2, may play an important pathogenetic role in cancer and autoimmune diseases, In vivo upregulation of bcl-2 mRNA in synovial lining cells of patients with rheumatoid arthritis but not in patients with osteoarthritis has been recently found. In the present study we investigated whether agents exerting beneficial effects in patients with rheumatoid arthritis, namely the long used Gold Sodium Thiomalate (GST) and the novel immunosuppressive, purine analogue 2-chlorodeoxyadenosine (2-CdA), a lymphocyte apoptosis-inducing agent interfere directly with induction of bcl-2 mRNA expression. The phytohemagglutinin (PHA)-induced in vitro proliferation of normal human peripheral blood lymphocytes was significantly inhibited by non-toxic concentrations of 2-CdA and GST which are within the range of in vivo plasma concentrations in patients receiving the respective treatment. Using mRNA dot-blot analysis and hybridization with an IL-2-specific probe we found that GST, similarly to dexamethasone that served as control, suppressed the PHA-induced IL-2 mRNA accumulation dose-dependently. In contrast, 2-CdA (0.1 microgram/ml) at concentrations that inhibit by 80-90% the PHA-induced proliferative responses of lymphocytes did not affect IL-2 mRNA accumulation. Hybridization with a bcl-2-specific probe showed that the activation-induced accumulation and kinetics of bcl-2 mRNA were not changed in the presence of a wide range of concentrations of either GST or 2-CdA. Similarly, the mRNA accumulation of the "house-keeping" control gene beta-action remained unchanged by both agents. These findings indicate that biosynthesis of bcl-2 is not specifically affected by GST and CdA, suggesting that the immunomodulating effects of these agents, including their efficacy in suppressing chronic arthritis, are not related with a bcl-2-dependent mechanism.

DNA adducts and the mechanism of carcinogenesis and cytotoxicity of methylating agents of environmental and clinical significance.

DNA adducts are covalent complexes formed between genotoxic carcinogens and DNA bases, and constitute a critical early intermediate on the pathway of chemical carcinogenesis. Their accumulation in different tissues reflects the amount of activated carcinogen reaching DNA, and can therefore serve as an index of the biologically relevant dose reaching the target tissues or cells. Methylating agents are of interest in view of their occurrence in the environment and their use as cytotoxic drugs in cancer chemotherapy. Current evidence indicates that O6-methylguanine plays a particularly important role in the mutagenic, carcinogenic, and cytotoxic activities of methylating agents. O6-Methylguanine is repaired efficiently by the enzyme O6-alkylguanine-DNA alkyltransferase (AGT). Lack of this enzyme results in excessive accumulation of O6-methylguanine and recent evidence suggests that significant quantitative effects on adduct accumulation may be linked to conditions of very low AGT levels. This would be important from the point of view of clinical practice, since modulation of AGT is under investigation as a means of enhancing the therapeutic efficacy of clinical agents acting via the production of O6-methylguanine and related adducts, such as, for example, procarbazine, dacarbazine, and some nitrosoureas. The measurement of O6-methylguanine in human DNA has been employed as a tool to investigate the role of environmental methylating agents in human carcinogenesis. While the nature and origin of the methylating agents responsible for these adducts is currently unknown, recent studies in patas monkeys have shown that N-nitrosodimethylamine, a methylating carcinogen to which human exposure is well documented, is capable of efficiently generating O6-methylguanine in most tissues, including fetal tissues. Furthermore, it has been found that this damage is substantially enhanced by the coadministration of ethyl alcohol which acts by inhibiting the liver first-pass metabolism of the carcinogen, an observation which supports the hypothesis that alcohol consumption may act as a risk factor in human carcinogenesis by augmenting the action of nitrosamines.

A phase II study evaluating the effect of tamoxifen on DNA repair in melanoma patients treated with dacarbazine.

The addition of tamoxifen to dacarbazine containing chemotherapy regimens used in the treatment of melanoma, has been shown to increase response rates, but the mechanism of any interaction is uncertain. The object of this study was to determine whether the addition of tamoxifen to dacarbazine, would modify DNA repair in-vivo and cause an increase in O6-meG adducts in peripheral blood leucocytes. This would provide some insight into the nature of the interaction between these two drugs. Twenty three patients with metastatic malignant melanoma received dacarbazine (DTIC) 1 g/m2 every three weeks for a maximum of six cycles. Tamoxifen 20 mg daily, was started after the first cycle of chemotherapy and then taken continuously during the treatment. Adduct levels after the second cycle of treatment were significantly higher than those after the first cycle (p = 0.0001). A similar rise however, was also produced when a cohort of patients were given dacarbazine without tamoxifen during the second cycle of treatment. This study did not show an additional increase of O6-meG adducts when tamoxifen was administered and therefore this mechanism does not support a postulated interaction between tamoxifen and dacarbazine. This is in agreement with the recent randomised study which did not show any significant increase in response rate with the addition of tamoxifen.

Regulation of bcl-2 and fas expression in primary activation of human peripheral lymphocytes is not sensitive to dexamethasone or cyclosporin-A.

The regulation of bcl-2 and fas (Apo-1/CD95) gene product expression plays a significant role in lymphocytes proliferation, survival, and apoptosis. Dexamethasone (Dex) and the immunosuppressive agent cyclosporin-A (CsA) inhibit primary activation of lymphocytes by distinct, though overlapping mechanisms that trigger undefined signals and can induce or prevent apoptosis in lymphoid cells in vitro. Here we demonstrate that Dex and CsA, at concentrations that markedly inhibit phytohemagglutinin (PHA)-induced proliferation of normal human peripheral blood lymphocytes, suppress the activation-dependent expression of interleukin 2 (IL-2) and the alpha-chain IL-2 receptor in a dose-dependent fashion without affecting the inducible accumulation and kinetics of either bcl-2 or fas mRNAs. Similar results were obtained when PHA-stimulated lymphocytes were cultured in the presence of the CsA analogue FK-506 or rapamycin. Moreover, the inducible maximal expression of either bcl-2 or fas protein levels on 48-h PHA-activated lymphocytes was not changed in the presence of either Dex or CsA. These findings show that the cell activation-induced biosynthesis of bcl-2 and fas proteins is not affected by immunosuppressive agents, suggesting that the expression of IL-2 and both bcl-2 and fas genes is regulated through independent mechanisms.

Biomonitoring human exposure to environmental carcinogenic chemicals.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.

Nitrosamines, alcohol, and gastrointestinal tract cancer: recent epidemiology and experimentation.

Recent epidemiological and experimental data continues to implicate nitrosamines in causation of gastrointestinal cancers. The evidence is strong for pharynx, esophagus, and stomach, and more problematic for liver, pancreas, and colorectum. Substantial levels of the promutagenic DNA adduct, Ob-methylguanine, in DNA from these organs in patas monkeys after a low dose of N-nitrosodimethylamine confirms the capacity for activation of environmental nitrosamines in these primate tissues. Alcohol is both an independent and a tobacco-interactive risk factor, influencing cancer incidence for oropharynx and esophagus strongly, and for stomach, colorectum, and liver more moderately. In a tabulation of experimental effects of ethanol potentially related to cancer-enhancing effects, toxicokinetic inhibition of hepatic first-pass clearance of nitrosamines is quantitatively greatest, and may be a major part of the mechanism of alcohol's effect on cancer risk for oropharnx, esophagus, and colon. Other operative mechanisms supported by experimental data are induction of activating enzymes, inhibition of DNA repair, and tumor promotion.

Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine.

The mutagenic, carcinogenic and cytotoxic activity of dacarbazine, a drug employed in cancer chemotherapy, may be related to the induction in DNA of O6-methylguanine (O6-meG), a quantitatively minor but biologically important lesion. In the present study the kinetics of O6-meG formation and repair in blood leukocyte DNA were examined in 20 Hodgkins lymphoma patients treated i.v. with 180 +/- 13 (mean +/- SD) mg/m2 dacarbazine and compared with those observed in various tissues of rodents treated with different doses of the drug. In Hodgkin's lymphoma patients adduct levels reached a value of 0.27 +/- 0.14 fmol/microgram DNA 2 h after dacarbazine administration, while the rate of subsequent loss suggested an adduct half-life of < or = 30 h. Measurement of adduct levels in the same individuals after successive courses of treatment spaced 3 weeks apart (up to 10 treatment courses) demonstrated a consistent individual response and statistical analysis of variance confirmed that intra-individual variation in adduct accumulation after a given dose of dacarbazine accounted for only 5% of the total variance observed. In contrast, inter-individual variation accounted for 70% of the observed variance, with adduct levels 2 h after drug treatment varying approximately 7.5-fold among adduct-positive individuals. No significant depletion of lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) occurred after patient treatment with dacarbazine. No significant relationship between adduct levels and clinical response to treatment was observed. In rats treated with single or multiple doses of dacarbazine causing varying degrees of AGT depletion the highest levels of O6-meG were seen in the liver, followed by the lymph nodes, bone marrow and blood leukocytes, which showed up to approximately 2-fold lower levels. A similar tissue distribution was also observed in mice and in a single rabbit. These observations suggest that O6-meG levels assayed in blood leukocytes of therapeutically treated humans reflect those present in the -lymph nodes (target tissue for chemotherapy) and the bone marrow (target tissue for leukaemogenesis) and may be utilized as a measure of the drug dose reaching these tissues. The quantitative data reported in this study show that under conditions of no depletion of AGT O6-meG accumulates in blood leukocyte DNA of humans at a rate similar to that observed in rats, suggesting that human susceptibility to any O6-meG-mediated genotoxic effects of dacarbazine may be comparable with that of the rat.

Downregulation of interleukin-2 and apha-chain interleukin-2 receptor biosynthesis by cisplatin in human peripheral lymphocytes.

To further investigate the mechanisms by which the antineoplastic agent cisplatin interferes with immune function, we studied its effect on the biosynthesis of interleukin-2 (IL-2) and its alpha-chain receptor (IL-2Ralpha). Normal human peripheral blood lymphocytes (PBL) were activated in vitro with phytohemagglutinin (PHA), and anti-CD3 antibody in the presence of various concentrations of cisplatin. Purified T cells were also cultured with anti-CD3 antibody and costimulated by CD80 (B7-1, B7/BB1)-transfected P815 mastocytoma cells in the presence of cisplatin. Tritiated thymidine incorporation assays, an enzyme-linked immunosorbent assay for soluble IL-2Ralpha determination, and RNA dot-blot analysis and hybridization with IL-2- and IL-2Ralpha-specific probes were used. PHA-induced and anti-CD3 antibody-induced proliferation of PBL were significantly inhibited by cisplatin at concentrations attainable in vivo. This inhibition was not due to direct cell death as shown by the absence of trypan blue uptake in the presence of high concentrations of cisplatin. Therapeutic concentrations of cisplatin (1 microgram/ml) also inhibited the IL-2-dependent proliferation of purified T cells, mediated via the CD28-CD80 costimulatory pathway. In addition, the amount of soluble IL-2Ralpha released in the T cell culture supernatants was decreased by cisplatin in a dose-dependent fashion, suggesting that inhibition of cell proliferation was associated with a parallel decrease in IL-2Ralpha production. These effects correlated with a specific cisplatin-induced downregulation of both IL-2 and IL-2Ralpha messenger RNA accumulation in PHA-stimulated PBL that was dependent on the concentration of the drug. These findings suggest that the immunomodulatory effects of cisplatin may result in part from its capacity to directly downregulate the IL-2/IL-2R system in activated lymphocytes.

N-nitrosodimethylamine-derived O(6)-methylguanine in DNA of monkey gastrointestinal and urogenital organs and enhancement by ethanol.

N-nitrosodimethylamine (NDMA) is a human cancer initiator suspect. Ethanol, a cancer risk factor, may synergize with nitrosamines by suppressing hepatic clearance, to increase internal exposure. A limitation to these hypotheses is lack of activation of NDMA by many rodent tissues. However, systemtic primate studies are lacking. Patas monkeys were utilized to investigate NDMA activation by primate tissues in vivo, generating the promutagenic DNA lesion 0(6)-methylguanine (0(6)-meG). Adult monkeys received 0. 1 mg/kg NDMA by gavage, in some cases preceded by ethanol. Four hours after NDMA only, 0(6)-meG was detected in DNA from all tissues. Levels were highest in gastric mucosa and liver and were only about 50% lower in DNA from white blood cells, esophagus, ovary, pancreas, urinary bladder and uterus. With ethanol co-exposure, amounts of 0(6)-meG increased at least 2-fold in all tissues except liver. The largest effect was in esophagus (17-fold increase), followed by ovary, large intestine, urinary bladder, spleen and cerebellum (9- to 13-fold increases), and uterus, cerebrum and brain stem (7- to 8-fold increases). Alkylguanine alkyltransferase activities varied over a 30-fold range and were highest in liver and stomach. Thus primate tissues, especially those of the gastrointestinal and urogenital organs, are sensitive targets for DNA adduct damage due to NDMA, and ethanol co-exposure leads to striking increases in adducts. Our data support epidemiology implicating nitrosamines in causation of cancers of stomach and other organs, and alcohol as enhancing internal exposure to nitrosamines.

Methyl DNA adducts, DNA repair, and hypoxanthine-guanine phosphoribosyl transferase mutations in peripheral white blood cells from patients with malignant melanoma treated with dacarbazine and hydroxyurea.

Dacarbazine (DTIC) is a DNA-methylating drug used in the treatment of malignant melanoma. Among the DNA dducts induced by DTIC are N7-methylguanine (N7-meG) and O6-methylguanine (O6-meG). The latter adduct, in particular, may be important in the mutagenic as well as the cytotoxic activity of DTIC. Repair of O6-meG is carried out by the enzyme O6-alkylguanine-DNA-alkyltransferase (AGT) by a process which results in its autoinactivation. N7-meG is lost from DNA partly spontaneously and partly by enzymatic depurination followed by excision repair of the resulting apurinic site. The purpose of this study was to determine the in vivo kinetics of formation and repair of O6-meG and N7-meG and the changes in AGT in peripheral WBCs with repeated doses of DTIC, and to determine the effects on these processes of concomitant administration of hydroxyurea. In addition, we examined the induction of mutations at the HPRT gene locus. Thirty-four patients with malignant melanoma received 1.0 g/m2 DTIC i.v. every 3 weeks. Hydroxyurea was added to the second and subsequent doses of DTIC in 19 patients. The concentrations of O6-meG, N7-meG, and AGT in peripheral blood lymphocytes were determined up to 24 h after each of the first two doses of DTIC. Mutations at the HPRT gene locus were determined using the T-cell clonal assay. Peak O6-meG levels were detected 1 and 4 h after the first and second dose of DTIC, respectively. AGT concentrations declined to 56.7% (range, 40.3-76.9%) and 55.0% (range, 45.4-58.9%) of pretreatment levels 24 h after the first and second doses of DTIC, respectively, and were still approximately 25%below their initial levels just prior to administration of the second dose of DTIC. An increase in formation of O6-meG was observed at all time points after the second dose of DTIC (P = 0.0001), which was not affected by cotreatment with hydroxyurea (P > 0.5). There was a negative correlation between pretreatment AGT levels and the O6-meG concentration at 24 h after therapy (r = -0.554, P = 0.014). N7-meG levels peaked at 6 h after DTIC therapy and were not significantly influenced by the cycle number. Cotreatment with hydroxyurea tended to be associated with lower levels of N7-meG (P = 0.08). There was no correlation between either O6-meG or N7-meG levels and the grade of neutropenia. On the basis of a limited series of blood samples analyzed, there was no firm evidence that chemotherapy with DTIC resulted in induction of HPRT mutations in lymphocytes. In conclusion, repeated administrations of DTIC resulted in higher concentrations of O6-meG, probably due to reduction in cellular AGT. Hydroxyurea did not significantly influence the kinetics of O6-meG, and N7-meG adduct formation. There was no significant induction of HPRT gene mutations with DTIC. This study suggests that sequencing of DTIC doses should be evaluated using the time course of cellular AGT depletion and DNA adduct formation to achieve higher cytotoxic efficiency.

N7-Methylguanine and O(6)-methylguanine levels in DNA of white blood cells from cancer patients treated with dacarbazine.

Abstract Methyl-DNA adducts are induced by a number of lifestyle, environmental and occupational carcinogens, however knowledge about their kinetics is scarce. Here, N7-methylguanine (N7-MeGua) and O(6)-methylguanine (O(6)-MeGua) levels were determined in the DNA of white blood cells from eight cancer patients treated hr with the antitumour drug dacarbazine (DTIC). Five of the patients were treated with the drug as a single agent (a single dose of 800 mg m(-2)) and three on three successive days with dacarbazine (225 mg m(-2) day(-1)) in combination with other drugs. The data indicate that maximum adduct levels are reached at 4-8 h after treatment and that the amount of N7-MeGua is at least 20-fold higher than that of O(6)-MeGua. The half-life of N7-MeGua is 40-96 h and that of O(6)-MeGua 25-27 h. Following treatment on three consecutive days, an accumulation of N7-MeGua was observed but not of O(6)-MeGua. The data show substantial interindividual differences in adduct levels but not in the ratio of N7/O(6)-MeGua. This may reflect differences in the metabolism of dacarbazine or in repair capacities.