Poly (ADP-ribose) polymerase inhibitor increases apoptosis and reduces necrosis induced by a DNA minor groove binding methyl sulfonate ester.

The poly(ADP-ribose) polymerase (PARP) is involved in cell recovery from DNA damage, such as methylation of N3-adenine, that activates the base excision repair process. In the present study we demonstrated that MeOSO(2)(CH(2))(2)-lexitropsin (Me-Lex), a methylating agent that almost exclusively produces N3-methyladenine, induced different modalities of cell death in human leukemic cell lines, depending on the presence of PARP inhibitor. Growth inhibition, provoked by the combination of Me-Lex and PARP inhibitor, was associated with a marked down-regulation of c-myc, increased generation of single strand breaks and apoptosis. When used as single agent, at concentrations that saturated cell repair ability, Me-Lex induced mainly cell death by necrosis. Surprisingly, addition of a PARP inhibitor enhanced apoptosis and reduced the early appearance of necrosis. Telomerase activity was completely suppressed in cells exposed to Me-Lex alone, by 24 h after treatment, whereas it did not change when Me-Lex was combined with PARP inhibitor. Thereafter, inhibition of telomerase was observed with both treatments. The results suggest new insights on different modalities of cell death induced by high levels of N3-methyladenine per se, or by the methylated base in the presence of PARP inhibitor.

hMSH3 overexpression and cellular response to cytotoxic anticancer agents.

Mutations or transcriptional silencing of mismatch repair genes have been linked with tumour cell resistance to O(6)-guanine methylating agents, 6-thioguanine, cisplatin, doxorubicin and etoposide. Recently, it has been demonstrated that overexpression of the MSH3 protein is associated with depletion of the mismatch binding factor MutSalpha, and then with a marked reduction in the efficiency of base/base mismatch repair. In the present study we evaluated sensitivity of the HL-60 cell line and its methotrexate-resistant subline HL-60R, which overexpresses the hMSH3 gene, to a panel of chemotherapeutic agents. Cell growth inhibition induced by temozolomide, 6-thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine was significantly lower in the hMSH3-overexpressing HL-60R cell line as compared with the HL-60 parental line. Moreover, HL-60R cells were more resistant than HL-60 cells to chromosome aberrations induced by either N-methyl-N'-nitro-N-nitrosoguanidine or temozolomide, and to apoptosis triggered by the latter drug. Both cell lines were equally susceptible to growth inhibition induced by cisplatin, etoposide or doxorubicin. In addition, HL-60 and HL-60R cells showed comparable sensitivity to the clastogenic and apoptotic effects of cisplatin and etoposide. These results further confirm that loss of base/base mismatch repair is the most important molecular mechanism involved in cell resistance to O(6)-guanine methylating agents and 6-thioguanine. However, the status of the mismatch repair system could still influence tumour cell sensitivity to cisplatin, etoposide and doxorubicin, depending on the specific component of the system that is lost, and on the genetic background of the cell.

Effects of single or split exposure of leukemic cells to temozolomide, combined with poly(ADP-ribose) polymerase inhibitors on cell growth, chromosomal aberrations and base excision repair components.

PURPOSE: To evaluate the antitumor activity of single versus split exposure of neoplastic cells to temozolomide (TZM) and poly(ADP-ribose) polymerase (PARP) inhibitor. METHODS: A leukemic Jurkat cell line and freshly isolated leukemic blasts were used. Jurkat cells are resistant to O6-methylguanine damage induced by TZM due to high levels of O6-alkylguanine-DNA alkyltransferase and to a functional defect in the mismatch repair system. Cells were treated with 3-aminobenzamide or with NU1025 to inhibit PARP activity. TZM was added to cell cultures immediately after PARP inhibitors. The concentrations of TZM used were 62.5 microM (corresponding to the peak plasma concentration in patients) or 125 microM. TREATMENT DESIGN: Cells were treated with 125 microM TZM plus PARP inhibitors (single exposure), or twice with 62.5 microM TZM plus PARP inhibitors with an interval of 24 h between treatments (split exposure). Tumor cell growth, clastogenicity and base excision repair gene transcripts or enzymatic activity were evaluated. RESULTS: The split exposure of Jurkat cells to TZM induced more pronounced and persistent growth inhibition and comparable chromosome damage in comparison with the single exposure. In addition, PARP inhibitors potentiated the cytotoxic effects induced by repeated treatment with TZM in fresh leukemic blasts. A marked decrease in X-ray repair cross-complementing 1 transcript and methylpurine glycosylase (MPG) transcript was detected in Jurkat cells subjected to the split exposure. In this case, a significant reduction in the corresponding enzymatic activity was also observed. CONCLUSIONS: Cytotoxicity induced by TZM and PARP inhibitors can be improved by a fractionated modality of drug treatment. The reduction in MPG transcript and function would presumably contribute to an increase in cell susceptibility to DNA damage induced by the methylating agent and PARP inhibitors.

Cytotoxic and clastogenic effects of a DNA minor groove binding methyl sulfonate ester in mismatch repair deficient leukemic cells.

Mismatch repair deficiency contributes to tumor cell resistance to O6-guanine methylating compounds and to other antineoplastic agents. Here we demonstrate that MeOSO2(CH2)2-lexitropsin (Me-Lex), a DNA minor groove alkylating compound which generates mainly N3-methyladenine, has cytotoxic and clastogenic effects in mismatch repair-deficient leukemic cells. Moreover, MT-1 cells, which express p53 upon drug treatment and possess low levels of 3-methylpurine DNA glycosylase activity, are more susceptible to cytotoxicity induced by Me-Lex, with respect to p53-null and 3-methylpurine DNA glycosylase-proficient Jurkat cells. In both cell lines, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, which inhibits base excision repair capable of removing N-methylpurines, increases cytotoxicity and clastogenicity induced by Me-Lex or by temozolomide, which generates low levels of N3-methyl adducts. The enhancing effect is more evident at low Me-Lex concentrations, which induce a level of DNA damage that presumably does not saturate the repair ability of the cells. Nuclear fragmentation induced by Me-Lex + 3-aminobenzamide occurs earlier than in cells treated with the single agent. Treatment with Me-Lex and 3-aminobenzamide results in augmented expression of p53 protein and of the X-ray repair cross-complementing 1 transcript (a component of base excision repair). These results indicate that N3-methyladenine inducing agents, alone or combined with poly(ADP-ribose) polymerase inhibitors, could open up novel chemotherapeutic strategies to overcome drug resistance in mismatch repair-deficient leukemic cells.

An SRY-negative XX male with Huriez syndrome.

This report studies a 42-year-old 46,XX patient affected by palmoplantar keratoderma. clinically classified as Huriez syndrome. The patient showed a male phenotype with apparently normal male features including testicular development. Cytogenetic and chromosomal painting analysis excluded the presence of translocation of the Y chromosome. PCR analysis of genomic DNA failed to detect the presence of the testis-determining gene, SRY. The presence of other Y-chromosome genes, known to be involved in testicular maturation and spermatogenesis, has also been analyzed. The data suggest that the sex reversal in this 46,XX male patient is due to a defect on a yet unidentified autosomal or X-linked sex-determining gene. The relationship between the sex reversion and the presence of sclerotylosis is discussed.

Bleomycin-induced chromosome aberrations in lymphocytes derived from patients with lamellar ichthyosis.

Patients affected by some genetic skin defects, for example, dyskeratosis congenita or scleroderma, may present spontaneous or induced chromosomal fragility. Hence we performed a cytogenetic analysis in families of patients affected by lamellar ichthyosis, an autosomal recessive disease not yet fully characterized at the cellular and molecular levels. Chromosomal fragility was assayed in untreated lymphocyte cultures and in those supplemented with aphidicolin or bleomycin. Cells from some affected patients and some of their parents showed hypersensitivity to the radiomimetic agent bleomycin.

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents.

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.

Induction of apoptosis by bleomycin in resting and cycling human lymphocytes.

Bleomycin induces DNA and chromosome breakage. The differential sensitivity to the drug has been used in vitro to identify individuals at high risk of developing tumours. However, there are limited reports on the ability of bleomycin to induce apoptosis. In this study we tested induction of apoptosis in human peripheral lymphocytes by bleomycin at different concentrations and different culture times using various parameters, such as nuclear fragmentation and DNA fragmentation, evaluated either in situ with terminal transferase and labelled nucleotides (TUNEL) or by flow cytometry analysis. We demonstrate that bleomycin induces apoptosis without previous permeabilization of the cell membrane. Cell death occurs mainly by apoptosis and not by necrosis, with significant alteration of membrane lipoperoxidation (evaluated by luminescence).

In vitro effects of growth hormone (GH) and insulin-like growth factor I and II (IGF-I and -II) on chromosome fragility and p53 protein expression in human lymphocytes.

BACKGROUND: We have reported previously that growth hormone (GH) therapy increases cell radiosensitivity; in this study we tested whether GH itself or IGFs induce chromosome aberrations and investigated the expression of p53 protein in response to DNA damage. METHODS: Human peripheral blood lymphocytes were incubated with GH [100 and 1000 microg L(-1)], insulin-like growth factor I [IGF-I; 150 and 1000 microg L(-1)] and IGF-II [600 and 1200 microg L(-1)] for 24 h. The radiomimetic agent bleomycin [BLM; 5 microgm L(-1)] was added in the last 3 h. Cytogenetic analysis was performed by assessing the percentages of damaged cells (%DC) and chromosome aberrations (%CA). The expression of p53 was investigated by flow cytometric assay using the monoclonal antibody DO-7, and expressed as percentage positive cells and mean fluorescence intensity. RESULTS: BLM significantly increased both percentage DC and percentage CA and p53 expression (P < 0.01). The %DC was unaffected by the tested peptides. IGF-I [150 microg L(-1)] increased spontaneous percentage CA (P < 0.01). All peptides further increased the BLM-induced chromosome breakage: GH 100 and 1000 microg L(-1) by 30% and 73% respectively, IGF-I 150 and 1000 microg L(-1) by 41% and 96% respectively and IGF-II 600 and 1200 microg L(-1) by 89% and 45% respectively. The spontaneous and BLM-induced expression of p53 was unaffected by GH, whereas it was significantly increased by IGFs (P < 0001). CONCLUSIONS: These results indicate that the DNA-damaging effect of BLM is amplified by GH and, more markedly, IGF-I and -II. IGF-I and -II also stimulate p53 protein expression that, taking part in DNA repair, may counteract the IGF action on genome stability.

Sister chromatid exchanges and DNA topoisomerase II inhibitors: effect of low concentrations of etoposide (VP-16) in ataxia telangiectasia lymphoblastoid cell lines.

The correlation between etoposide (VP-16) cytotoxicity and the induction of sister chromatid exchanges (SCEs) suggested that the promotion of DNA recombination events may be crucial for the activity of antitopoisomerase drugs. To further evaluate this hypothesis, we investigated the correlation between VP-16 induction of SCEs, chromosomal aberrations and cell cycle alterations in lymphoblastoid cell lines derived from patients affected by ataxia telangiectasia (AT), whose cells are known as hypersensitive to the cytotoxic and clastogenic activity of DNA topoisomerase II inhibitors. Our present study has shown that AT homozygous and heterozygous cell lines exposed to low VP-16 concentrations, although hypersensitive to the induction of chromosomal aberrations, exhibit an induction of SCEs comparable to that found in normal cell lines. Moreover, while the clastogenic effect of the drug was directly correlated to the reduction of the mitotic index, the enhancement of SCE frequencies, obtained over the same range of VP-16 concentrations, was not paralleled by a modification of proliferation index. Thus, these results suggest that etoposide retains in AT cells a strong clastogenic and cytostatic activity which is independent from DNA recombination events and which may be important for the induction of cell death by this kind of drug.

Cytogenetic effects in lymphocytes from children exposed to radiation fall-out after the Chernobyl accident.

In a previous paper we reported that a group of children exposed to ionizing radiation following the Chernobyl accident exhibited an appreciable number of chromosome breaks and rearrangements reflecting the persistence of a radiation-induced damage. The results suggested that the children were still exposed to radioactive contamination through consumer foodstuff and life styles. In the present paper, 31 exposed children have been considered together with a control group of 11 children with the aim to confirm previous results. All children underwent whole-body counter (WBC) measures and conventional cytogenetic analysis. The frequency of chromosome aberrations detected by conventional cytogenetics in the group of children chronically exposed to low doses of ionizing radiation resulted in significant differences with respect to the control group. The present work suggests that, for these groups of children, even if the frequency of aberrations is very low and the observation of statistically significant differences is consequently a problem, a persistently abnormal cytogenetic picture is still present several years after the accident.

Do human lymphocytes exposed to the fallout of the Chernobyl accident exhibit an adaptive response? III. Challenge with bleomycin in lymphocytes from children hit by the initial acute dose of ionizing radiation.

In the present paper, we report data on the possible adaptive response, induced in vivo by exposure to ionizing radiation to a challenge treatment with the radiomimetic glycopeptide bleomycin (BLM). Lymphocytes from children living in Pripjat at the time of the Chernobyl accident, and thus hit by the initial acute dose of ionizing radiation, were treated for the last 5 h of culture with 0.004 U/ml BLM. Significantly lower chromosome damage was found only in lymphocytes from children who, independently of the initial acute exposure to ionizing radiation, still showed a 137Cs internal contamination, due to persistent continuous exposure to low doses of radiation. The present results indicate that past exposure to acute high dose of ionizing radiation does not interfere with resistance to BLM which is related to internal contamination.

In vitro infection of leukemic bone marrow with HTLV-I generates immortalized cell lines expressing T or myeloid cell phenotype.

Leukemic bone marrow cells ( > 90% blasts) of a patient with acute myeloblastic leukemia (AML), non-treated or pretreated in vitro with a mutagenic triazene compound, were infected with HTLV-I by coculture with irradiated virus-donor cells. Immortalized, HTLV-I+, double-positive CD4/CD8 euploid T cell lines, expressing HLA class I/II monomorphic determinants, and inappropriate myeloid and progenitor cell markers (ie CD13, CD14, CD15 and CD33 antigens) were obtained. In one out of 10 triazene-pretreated samples, HTLV-I infection resulted in the appearance of a rapidly growing triploid cell line (ie MTLC1 line) showing: (1) myeloid but not lymphoid phenotype; (2) beta and delta T cell receptor in germline configuration; (3) integrated, complete and incomplete HTLV-I provirus genome (also detected in a number of MTLC1 clones); (4) a high percentage of cells positive for non-specific cross-reacting antigen (a CEA-related molecule present in myeloid cells) under the influence of gamma-interferon; (5) absence of HLA class I/II antigen expression; (6) absence of tax gene transcription. Blast cell proliferation was marginal or absent when leukemic marrow was not subjected to retroviral infection. These results show that exposure of leukemic bone marrow to HTLV-I can be followed by immortalization of T and myeloid cells. Although no data are available to establish whether tax expression played a role in the early phase of the immortalization process of MTLC1 line, tax gene product was not required for maintaining long-term growth of MTLC1 cells.

Do human lymphocytes exposed to the fallout of the Chernobyl accident exhibit an adaptive response? 1. Challenge with ionizing radiation.

Several studies suggest that cells appear to become less susceptible to the induction of radiation damage, and in particular of chromosome and chromatid aberrations in short-term cultures of human lymphocytes, when a challenge exposure to ionizing radiation is preceded by a low 'adaptive' dose. Contradictory results have been reported on the conditions under which the phenomenon can be evidenced. In the present work, circulating lymphocytes of 13 children contaminated from the fallout after the Chernobyl accident were tested for their capability to exhibit an adaptive response in experiments in which the challenge dose was administered to stimulated lymphocytes in the S-G2 phase. Furthermore, the possible influence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, was also investigated. Our results indicate that, at least in the instance of the end-point here used (chromosome and chromatid aberrations, the former resulting possibly from the Cs burden), human lymphocytes, chronically exposed to low doses from fallout, do not exhibit any decreased susceptibility to ionizing radiation. However, as reported in the accompanying paper, the same samples appear to show an 'adaptive' response when exposed to a challenge treatment with bleomycin (B. Tedeschi et al., 1995, this issue).

Do human lymphocytes exposed to the fallout of the Chernobyl accident exhibit an adaptive response? 2. Challenge with bleomycin.

The present study concerns the possible adaptive response, induced in vivo by a continuous exposure to ionizing radiations, to a challenge treatment with the radiomimetic glycopeptide bleomycin (BLM). Lymphocytes from children contaminated as a consequence of Chernobyl accident were treated for the last 5 h of culture with 2.5 micrograms/ml BLM. The induced chromosome damage was significantly lower than that found with the same treatment in lymphocytes from control children. This hyposensitivity to BLM was still present if, 1 h after the addition of the drug, inhibitors of the enzymes involved in DNA repair, such as 3-aminobenzamide (2 mM), or aphidicolin (0.4 microM) or 3-dideoxythymidine (5 mM) were added to the cultures. The resistance to BLM in lymphocytes from contaminated children seems to be related to a mechanism upstream in respect to the activities of enzymes involved in the DNA repair and specifically linked to the action of this drug. This is consistent with the different response found when the cells were challenged with ionizing radiation in vitro, as reported in the accompanying paper (L. Padovani, L. et al. (1995) Mutation Res., this issue).

Characteristic chromosomal fragility of human embryonic cells exposed in vitro to aphidicolin.

The frequency and distribution of aphidicolin (APC)-induced common fragile sites (cfs) were analyzed in human embryonic cells of different origins. Embryonic lung fibroblasts (MRC-5), amniocytes (AMINO) and embryonic retina cells (HERO790) are as sensitive to the APC-induced clastogenic effect as peripheral lymphocytes, whereas embryonic kidney cells (HEK) seem more resistant to the induction of chromosomal gaps and breaks by the drug. Analysis of the distribution of fragile sites confirmed that the expression of specific APC-induced cfs varies in different cells and that the embryonic cell strains show a greater similarity among themselves than to lymphocytes. In addition, HEK, MRC-5, HERO790 and AMINO cells show specific APC induction of the cfs at the 1p31.2 chromosomal band, which seems to be a distinctive feature of the embryonic stage of cells.

Subregional localization of 14 yeast artificial chromosomes to human chromosome region 1p by fluorescence in situ hybridization.

We have sublocalized to the region between 1p22 and 1p33 a total of 14 yeast artificial chromosomes previously assigned to a broader area of human chromosome 1p. Our purpose was to map DNA sequences that could be used for the molecular characterization of the two common fragile sites present in bands 1p31.2 and 1p32, the expression of which is increased in patients with neuroblastomas.

Cytogenetic study in lymphocytes from children exposed to ionizing radiation after the Chernobyl accident.

The present study concerns the monitoring of children from the Byelorussian, Ukrainian and Russian republics exposed to the fall-out of the Chernobyl accident. Cytogenetic analyses have been performed on 41 children coming from different areas and exhibiting varying amounts of 137Cs internal contamination, as evaluated by whole-body counter (WBC) analysis. On a total of 28,670 metaphases scored, radiation-induced chromosome damage is still present, although at a very low frequency. Due to the very low fraction of dicentrics, because of the time elapsed from the accident and the relatively low doses of exposure, radiobiological dosimetry is not possible for these children. However, considering that the WBC data indicate that the children are still exposed to 137Cs contamination, the observed occurrence of stable chromosome rearrangements and breaks may represent the persisting effect of continuous low doses of radiation. The present study also indicates that the parallel use of internal contamination dosimetry and cytogenetics could be usefully employed to monitor individual exposure to radiation and to define further management measures.

Increased chromosome fragility in lymphocytes of short normal children treated with recombinant human growth hormone.

A few years ago it was reported that some growth-hormone-deficient children had developed leukemia following therapy with human growth hormone. This raised concern that this therapy may stimulate tumor development. Since it is known that the tendency to develop cancer is closely related to chromosome breakage, we decided to investigate whether recombinant human growth hormone (rhGH) therapy can increase chromosome fragility. Ten short normal children were studied during their first year of treatment. Lymphocytes were collected at 0, 6 and 12 months of rhGH therapy, and we assessed the rate of spontaneous chromosome aberrations, the frequency of sister chromatid exchanges, the proliferative rate indices, the expression of common fragile sites induced by aphidicolin, and the sensitivity towards the radiomimetic action of bleomycin. At 6 months of therapy, there was a significant increase in bleomycin-induced chromosome aberrations, which remained unchanged after 1 year of treatment. An increase in spontaneous chromosome rearrangements at 6 and 12 months of therapy was also observed. These findings are further supported by data obtained from the analysis of 16 short normal children already on rhGH therapy.

Incidence of chromosome abnormalities and clinical significance of karyotype in de novo acute myeloid leukemia.

Cytogenetic studies with high-resolution banding were performed on specimens from 132 consecutive patients with de novo acute myeloid leukemia (AML). All patients were treated according to therapeutic protocols in the same institution. Clonal abnormalities were detected in 97 of the 124 patients in whom an adequate number of mitoses was obtained (78.2%). Neither sex, FAB classification, WBC, or the extent of bone marrow infiltrate affected the rate of chromosomal aberrations, whereas patients younger than 40 years had a greater proportion of normal karyotypes (p = 0.047). Two different chromosomal classifications were evaluated: the presence of normal and abnormal metaphases (NN-AN-AA classification), and a classification in cytogenetic categories, the latter being based on the frequency of cytogenetic abnormalities. Both classifications were found to correlate significantly with the clinical outcome. They also showed independent prognostic significance when age, sex, and FAB morphology were considered in a multivariate analysis. Two abnormalities were closely associated with specific clinical-pathologic subsets of AML. All the 15 patients with t(15;17) had acute promyelocytic leukemia; this translocation was not found in any other subset of AML. Eight of the nine patients presenting rearrangements at 11q23 belonged to a FAB subset with monocytic differentiation (M4 and M5). Our data suggest that cytogenetic findings should influence the therapeutic approach to AML. In particular, young patients with karyotypes associated with poor responses may be considered for more eradicating treatments, including allogenic bone marrow transplantation.