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.

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.

A novel, sensitive assay for O6-methyl- and O6-ethylguanine in DNA, based on repair by the enzyme O6-alkylguanine-DNA-alkyltransferase in competition with an oligonucleotide containing O6-methylguanine.

A novel assay for O6-alkylguanine-type adducts in DNA is reported. It is based on the use of the suicide repair enzyme O6-alkylguanine-DNA-alkyltransferase (AGT) to repair such adducts in DNA in competition with an oligonucleotide containing a single residue of O6-methylguanine, end labeled to high specific activity. The stoichiometric mode of action of AGT results in decreased amounts of oligonucleotide being repaired in the presence of increasing levels of adducts in the competing DNA. The extent of oligonucleotide repair is determined by immunoprecipitation of the unrepaired form with rabbit antiserum directed against O6-alkyldeoxyguanosine and radiocounting. The amount of O6-alkylguanine in competing DNA is calculated by reference to a standard curve constructed using DNA of known alkylation. In view of its relatively wide spectrum of alkyl group specificity, use of AGT from rat liver permits the determination of both O6-methyl- and O6-ethylguanine (detection limits, 0.8 fmol and 3 fmol, respectively). On the other hand, the restricted specificity of Escherichia coli AGT to repair of O6-methylguanine makes the assay based on it specific for this type of lesion (detection limit, 0.5 fmol). The maximum amount of DNA which can be included in the assay is 15 micrograms and 10 micrograms for the rat liver and E. coli AGT-based assays, respectively, leading to a limit of sensitivity of 8 x 10(-8) mol O6-methylguanine/mol guanine (50 fmol/mg DNA) (both enzymes) and 3 x 10(-7) mol O6-ethylguanine/mol guanine (200 fmol/mg DNA) (rat liver AGT-based assay) and making this one of the most sensitive assays for these important precarcinogenic adducts. The new assay has been validated by assaying DNA from rat liver methylated in vivo with dimethylnitrosamine to a known extent and has been found to give results in close agreement with those of radioimmunoassay. Six h after i.p. administration of dimethylnitrosamine (0.01-1 mg/kg) to rats, O6-methylguanine was detectable by the competitive-repair assay in liver or lymphocyte DNA at levels of 0.14-14.4 mumol/mol guanine.

Accumulation of O6-methylguanine in human blood leukocyte DNA during exposure to procarbazine and its relationships with dose and repair.

O6-Methylguanine was measured by a competitive repair assay in blood leukocyte DNA of seven patients with Hodgkin's or non-Hodgkin's lymphoma during therapeutic exposure to procarbazine involving three daily p.o. doses (50 mg each) for 10 days (corresponding to 2.1 mg/kg/day for a 70-kg human). Adduct accumulation was observed in all seven cases, reaching levels up to 0.28 fmol/microgram of DNA (0.45 mumol/mol of guanine). In one individual, maximal levels of adduct were reached after 7 days of exposure, followed by a steady decline, whereas in all other individuals continuous accumulation was observed throughout the exposure period. In four individuals for which data were available for Day 11 (12 to 16 h after the final intake of procarbazine), decreased amounts of O6-methylguanine were observed relative to the last previous measurements. The accumulation of O6-methylguanine was linearly correlated (P less than 0.01) with the cumulative dose of procarbazine, with a slope of 0.011 fmol of O6-methylguanine/microgram of DNA per mg/kg of body weight or 2.68 x 10(-4) fmol of O6 methylguanine DNA per mg/m2. (Two h after the administration of single p.o. doses of 1 to 10 mg/kg of procarbazine to rats, O6-methylguanine formation in leukocyte DNA was just under half that in liver DNA and showed a linear relationship with dose with a slope of 0.017 fmol/microgram of DNA per mg/kg of body weight or 5.67 x 10(-4) fmol of O6-methylguanine/microgram of DNA per mg/m2. A negative correlation (P less than 0.05) between the rate of accumulation of O6-methylguanine in different individuals and lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) was observed, demonstrating a probable protective effect of AGT against the accumulation of O6-methylguanine during exposure to methylating agents. This observation supports the suggestion of a possible role of procarbazine-induced O6-methylguanine in the pathogenesis of acute nonlymphocytic leukemia appearing after treatment with chemotherapeutic protocols which include procarbazine, based on the finding of low lymphocyte AGT levels in patients with such therapy-related neoplastic disease (Sagher et al., Cancer Res., 48: 3084-3089, 1988). Lymphocyte AGT levels were mainly in the range of 5 to 10 fmol/micrograms of DNA and showed no consistent variation during procarbazine exposure.

O6-methylguanine DNA adduct formation and modulation by ethanol in placenta and fetal tissues after exposure of pregnant patas monkeys to N-nitrosodimethylamine.

Perinatal nitrosamine exposures may contribute to childhood cancer risk. To test primate fetal susceptibility to formation of cancer initiation-related DNA adducts from nitrosamines, pregnant patas monkeys were given 1.0 or 0.1 mg/kg N-nitrosodimethylamine. Appreciable levels of the promutagenic O6-methylguanine adduct occurred in placental and fetal liver DNA after both doses and were lower but detectable in other fetal tissues after the higher dose. Coadministered ethanol (1.6 g/kg) reduced adducts in placenta and fetal liver by one-half and increased levels in other fetal tissues to the same degree. Thus, primate placenta and fetal tissues have a significant, ethanol-modulated capacity to activate N-nitrosodimethylamine, supporting implication of nitrosamines in human perinatal carcinogenesis and of alcohol as a modulating factor.

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.

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.

Accumulation of O6-methylguanine in human DNA after therapeutic exposure to methylating agents and its relationship with biological effects.

O6-Methylguanine has been measured in peripheral blood leukocytes of 14 patients during one or more cycles of treatment with procarbazine (daily treatment for 10 days) and in 12 patients during one or more cycles of treatment with dacarbazine (single dose per cycle). Adduct formation at levels up to about 0.4 fmole/microgram DNA was detected in all procarbazine- and all but one dacarbazine-treated patients at some point after treatment. O6-Methylguanine accumulated during procarbazine treatment in a dose-related manner (mean rate of accumulation 2.8 x 10(-4) fmole/microgram DNA per mg/m2 dose) and appeared to approach a plateau by the end of the cycle (above 600 mg/m2 cumulative dose). The average rate of O6-methylguanine formation 2 hr after dacarbazine treatment was 11 +/- 8 x 10(-4) fmole/microgram DNA per mg/m2 dose. Individuals examined on more than one treatment cycle with either drug showed broadly similar methylation responses. The rate of adduct accumulation showed a nonsignificant, negative correlation with the pretreatment lymphocyte levels of the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) in the case of procarbazine and no correlation in the case of dacarbazine. No consistent lymphocyte AGT depletion was noted as a result of treatment with either drug. No correlation between O6-methylguanine formation and hematological toxicity was observed. In eight patients showing full remission after treatment with dacarbazine, the value of O6-methylguanine (averaged over all the cycles) was 0.252 +/- 0.120 fmole/microgram DNA while in four patients showing partial or no response it was 0.087 +/- 0.110 fmole/microgram DNA (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Alcohol-related cancer risk: a toxicokinetic hypothesis.

Consumption of alcoholic beverages is an accepted social custom world-wide. This makes its involvement in events contributing to human cancer risk very important. Although it is neither tumorigenic nor genotoxic in animals, ethanol can potentiate the carcinogenic risk associated with certain environmentally present agents. The reasons for such a synergistic action are speculative, but among theories postulated may be ethanol's ability to modify the toxicokinetics/dynamics of carcinogen metabolism. Experiments conducted with rodents and primates support this hypothesis, demonstrating increased exposure of posthepatic organs to nitrosamines when given in combination with ethanol, followed by enhancement of DNA adduct formation and, at least in rodents, of tumor development. In addition, ethanol may induce enzymes responsible for carcinogen activation, including hepatic cytochrome P450 2E1 in rodents and humans, and in lung, kidney, and brain in rodents. Studies have also shown that these effects can extend to the next generation via maternal and in utero fetal exposure. What impact such ethanol-induced modulations have on tumorigenesis during childhood and later stages of life needs to be investigated further.

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.

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.

Suppression of interleukin-2 and interleukin-2 receptor biosynthesis by gold compounds in in vitro activated human peripheral blood mononuclear cells.

OBJECTIVE: To further investigate the mechanism of action of gold compounds by studying their effects on interleukin-2 (IL-2) and IL-2 receptor (IL-2R) biosynthesis. METHODS: We cultured phytohemagglutinin- or anti-CD3 antibody-activated normal peripheral blood mononuclear cells (PBMC), as well as the erythroleukemic K562 cell line, in the presence of gold sodium thiomalate or auranofin. Tritiated thymidine incorporation assays, cytotoxicity assays, immunofluorescence analysis, enzyme-linked immunosorbent assay, Northern blot, and RNA dot-blot hybridization were used. RESULTS: Gold compounds, at concentrations attainable in vivo, inhibited the proliferation of normal PBMC, with no evidence of direct cytotoxicity. This inhibitory effect was associated with a dose-dependent suppression of both IL-2 and IL-2R messenger RNA accumulation. In contrast, the same concentrations of gold compounds failed to inhibit the spontaneous proliferation of the IL-2-independent K562 cells. CONCLUSION: Our findings suggest an IL-2/IL-2R-mediated mechanism for suppression of lymphocyte proliferation by gold compounds, which might account for the immunomodulatory effects of gold in patients with rheumatoid arthritis.

Suppression of interleukin-1 beta and tumour necrosis factor-alpha biosynthesis by cadmium in in vitro activated human peripheral blood mononuclear cells.

Cadmium is a highly toxic element responsible for acute and chronic toxicity in man. There is evidence that cadmium induces pathophysiological effects by modulating components of the immune system. Cytokines are being increasingly recognized as essential mediators of normal and pathologic immune responses. Cadmium at concentrations varying from 1.0 x 10(-4) to 3.3 x 10(-6) M inhibited the phytohemagglutinin induced production of interleukin-1 beta and tumour necrosis factor-alpha, in in vitro activated human peripheral blood mononuclear cells. The messenger RNA levels of interleukin-1 beta and tumour necrosis factor-alpha were examined during a 24-h culture period, at different time points. The decreased messenger RNA levels at the time points of the maximum expression of interleukin-1 beta and tumour necrosis factor-alpha indicate that cadmium suppresses their production at the transcriptional level.

Differential effects of procarbazine and methylnitrosourea on the accumulation of O6-methylguanine and the depletion and recovery of O6-alkylguanine-DNA alkyltransferase in rat tissues.

The kinetics of accumulation of the premutagenic DNA adduct O6-methylguanine (O6-meG) in the liver, blood leukocytes, lymph nodes and bone marrow of rats was examined and compared after single or multiple doses of procarbazine, a methylating cytostatic drug employed in the treatment of Hodgkin's lymphoma patients, and methylnitrosourea (MNU), an experimental methylating agent and carcinogen. Maximal O6-meG levels occurred 1-2 h after administration of single doses of procarbazine (10 mg/kg) or MNU (1 mg/kg), thereafter decreasing with half-lives of approximately 20-45 h, depending on the tissue. A relatively uniform tissue distribution was observed with both agents, with the liver generally showing highest adduct levels, followed by the lymph nodes, bone marrow and blood leukocytes which contained broadly similar amounts of O6-meG. During daily, oral administration to rats of procarbazine for 10 days at dose rates of 2.5, 5, 10 or 20 mg/kg/day (treatment analogous to that of the MOOP chemotherapy protocol for Hodgkin's lymphoma) followed by animal death on different days (in each case 24 h after the last treatment), a biphasic mode of O6-meG induction was observed: an initially steep build-up during the first 3-4 days was followed by a transient decline in the rate of accumulation, in turn followed by a second wave of accumulation and then a further slow-down. During the same treatment, liver O6-methylguanine-DNA alkyltransferase (AGT) declined in a dose-related manner. AGT recovery after the end of treatment was slow, taking nearly 20 days after the end of the high-dose treatment to return to control levels, despite the fact that all detectable adducts had been lost from DNA within 3 days after the end of treatment. A similar depletion and slow recovery of AGT in the liver, blood lymphocytes, bone marrow and lymph nodes was observed after treatment with a single dose of 100 mg/kg procarbazine. In contrast to these observations, O6-meG accumulated smoothly during a 10 day administration of MNU (1 or 10 mg/kg/day) to reach a steady-state within 5-6 days, while liver AGT was partially depleted after the high dose and recovered fully within 72 h of cessation of treatment. Similarly, a single dose of MNU (35 mg/kg) resulted in AGT depletion followed by rapid recovery in all four tissues examined. It is concluded that procarbazine (but not MNU) causes a decrease in cellular AGT concentrations by a mechanism additional to suicide repair of O6-meG.(ABSTRACT TRUNCATED AT 400 WORDS)

The urea-soluble low molecular weight cuticle proteins from the different developmental stages of Dacus oleae.

The cuticle proteins of the insect Dacus oleae have been isolated by extraction with a solution of 7 M urea. The affinity properties of cuticle proteins, isolated from the third instar larvae (L3DCPs 1-7), to chitin have been studied. Purified cuticle antigens were polymerized by glutaraldehyde and used for raising antibodies. The developmental appearance of the cuticle proteins has been studied by two-dimensional electrophoresis.