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Biomedical subjects

M Bignami

Publications and source records attributed to M Bignami.

At least 19 recordsLinked to original sources
PubMed1992

Fidelity of replication of the leading and the lagging DNA strands opposite N-methyl-N-nitrosourea-induced DNA damage in human cells.

Semi-conservative replication of double-stranded DNA in eukaryotic cells is an asymmetric process involving leading and lagging strand synthesis and different DNA polymerases. We report a study to analyze the effect of these asymmetries when the replication machinery encounters alkylation-induced DNA adducts. The model system is an EBV-derived shuttle vector which replicates in synchrony with the host human cells and carries as marker gene the bacterial gpt gene. A preferential distribution of N-methyl-N-nitrosourea (MNU)-induced mutations in the non transcribed DNA strand of the shuttle vector pF1-EBV was previously reported. The hypermutated strand was the leading strand. To test whether the different fidelity of DNA polymerases synthesizing the leading and the lagging strands might contribute to MNU-induced mutation distribution the mutagenesis study was repeated on the shuttle vector pTF-EBV which contains the gpt gene in the inverted orientation. We show that the base substitution error rates on an alkylated substrate are similar for the replication of the leading and lagging strands. Moreover, we present evidence that the fidelity of replication opposite O6-methylguanine adducts of both the leading and lagging strands is not affected by the 3' flanking base. The preferential targeting of mutations after replication of alkylated DNA is mainly driven by the base at the 5' side of the G residues.

Animals↗
PubMed1992

Expression of the endogenous O6-methylguanine-DNA-methyltransferase protects Chinese hamster ovary cells from spontaneous G:C to A:T transitions.

We have investigated whether the presence of a DNA repair enzyme, O6-methylguanine-DNA-methyltransferase (MGMT), affects the nature of spontaneous mutations in a mammalian cell line. We compared spontaneous mutations in the adenine phosphoribosyl transferase gene of a Chinese hamster ovary (CHO) cell line that expressed 14,000 MGMT molecules/cell with those in the parental CHO cells lacking this DNA repair activity. The mutation rate/cell/generation of the two CHO cell lines did not differ significantly. However, DNA sequence analysis of spontaneous mutations in the MGMT-proficient CHO cell line revealed a complex picture. No significant difference from the parental CHO cells was found in the number or type of deletions, frameshifts, multiple substitutions, or insertions. The frequency of G:C to T:A transversions was elevated in MGMT-proficient CHO cells. Expression of the enzyme considerably reduced G:C to A:T transitions (25% versus 8.3%). This latter result is the first evidence that this protein is active on an endogenous source of O6-methylguanine that is normally responsible for spontaneous G:C to A:T transition mutations.

Adenine Phosphoribosyltransferase↗
PubMed1992

Non-phenotypic selection of N-methyl-N-nitrosourea-induced mutations in human cells.

The distribution of mutations in a particular gene as detected by a selective mutation assay could be affected by the structural properties of the target protein. To investigate this, we have analysed N-methyl-N-nitrosourea (MNU)-induced mutations in two restriction recognition sequences of a target gene for mutation analysis and compared these data with what previously observed in a phenotypic mutation assay. DNA base changes in the Ncil and EcoRV sites of the gpt gene maintained in human cells by a shuttle vector system were measured by restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) technique. After MNU-treatment of human cells, mutations were detected in the Ncil recognition sequence but not in the EcoRV site. DNA sequencing analysis revealed that all Ncil-resistant mutations were GC to AT transitions located over four bases of the Ncil recognition sequence. Only one of these mutations drastically affected the functionality of the GPT protein. The Ncil-resistant mutations were randomly distributed in both DNA strands of the gpt gene and were preferentially targeted at guanine residues flanked 5' by a guanine. Our results indicate that the structure of the GPT protein is the main contributor to the strand-specificity of MNU-induced mutations previously reported by using a phenotypic mutation assay. The potential use of the RFLP/PCR technique as a general tool for mutation detection is also discussed.

Base Sequence↗
PubMed1992

Methoxyamine modification of abasic sites protects CHO cells from the cytotoxic and mutagenic effects of oxygen alkylation.

The biological effects of the interaction of methoxyamine (MX) with apurinic/apyrimidinic (AP) sites produced in CHO cells by treatment with alkylating agents were examined. A decrease in cytotoxicity was observed after a 10 min treatment with the SN1 alkylating agents ethylnitrosourea (ENU), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and N-methyl-nitrosourea when MX was present in the culture medium. Furthermore MX reduced the number of mutations to 6-thioguanine resistance induced by ENU and ENNG and the number of sister chromatid exchanges induced by ENU. In contrast, no protective effect of MX on survival was observed after a 10 min treatment with the SN2 alkylating agents diethylsulfate (DES), ethyl methane sulfonate and methyl methane sulfonate. A 3 h exposure to MX abolished the protective effect of MX on ENU-induced cytotoxicity and increased the cytotoxicity of DES. In vitro studies with synthetic oligonucleotides containing a single AP site opposite a normal guanine or O6-methylguanine showed that MX inhibits the cleavage of AP sites by the CHO AP endonuclease(s). A model is proposed in which different DNA lesions are involved in AP site formation after treatment with SN2 or SN2 alkylating agents. The involvement of specific alkylation products in cytotoxicity and mutagenesis is also discussed.

Alkylating Agents↗
PubMed1991

Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo.

In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.

Base Sequence↗
PubMed1991

Site-dependent inhibition by single O6-methylguanine bases of SV40 T-antigen interactions with the viral origin of replication.

The effects of O6-methylguanine on the reactions involved in initiation of DNA replication were investigated by measuring the interactions of SV40 T antigen with oligonucleotides substituted with the methylated base. O6-Methylguanine residues were positioned in either binding site I or binding site II of the SV40 origin of replication. Binding of purified T antigen, measured by both nitrocellulose filter binding and delayed oligonucleotide migration, was unaffected by the presence of seven methylated bases in binding site II. Single substitutions within binding site I were sufficient to inhibit T-antigen binding, and the extent of inhibition was dependent on the position of O6-methylguanine in the DNA sequence. Unwinding by T antigen was analyzed by measuring displacement of a single-stranded oligonucleotide from similarly substituted, partially duplex substrates. The presence of three O6-methylguanine residues in binding site I facilitated the helicase activity of T antigen. In contrast, single O6-methylguanine bases inhibited unwinding. A correlation was observed between the position of the methylated base and the inhibition of both binding and unwinding by T antigen.

Animals↗
PubMed1990

Tolerance to O6-methylguanine and 6-thioguanine cytotoxic effects: a cross-resistant phenotype in N-methylnitrosourea-resistant Chinese hamster ovary cells.

The biochemical and genetic characteristics of a clone of Chinese hamster ovary cells displaying resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (6-TG) were analyzed. The initial level of 7-methylguanine, 3-methyladenine, and O6-methylguanine formation and the repair rates for these alkylated bases were the same in the resistant and in the parental cell line, indicating that the resistance to alkylation damage is not due to differences in DNA alkylation. After exposure for 24 or 48 h to 6-TG (0.6 micrograms/ml) in culture medium, the resistant clone in contrast to them, was able to replicate the DNA containing the base analogue during the following 24 h. These data are in agreement with the hypothesis that resistant cells tolerate both O6-methylguanine and 6-TG present in DNA. The tolerance to MNU and 6-TG also included chromosomal damage induced by these two agents, and MNU-resistant cells incurred less sister chromatid exchanges after treatment with either MNU or 6-TG. 6-TG-resistant cells, selected by growth in 6-TG, exhibited cross-resistance to MNU but not to methyl methanesulfonate, confirming that a common pathway of tolerance is responsible for resistance to 6-TG and O6-methylguanine.

Alkylation↗
PubMed1990

O6-methylguanine in the SV40 origin of replication inhibits binding but increases unwinding by viral large T antigen.

To study the effect of the potentially cytotoxic base O6-methylguanine (O6-meG) on the initiation of DNA replication, double-stranded oligonucleotides corresponding to the SV40 origin of replication were constructed in which O6-meG replaced guanine in one strand. Out of 14 methylated residues, 10 were present in the Binding sites for T antigen (3 in Binding Site 1 and 7 in Binding Site 2). Binding of purified T antigen to the substituted oligonucleotide was considerably reduced in comparison to the unsubstituted one, as measured by nitrocellulose filter binding. Both the ATP-dependent and ATP-independent binding of T antigen were affected by the presence of the methylated base. Band shift analysis revealed an altered pattern of delayed-migrating complexes of T antigen with the O6-meG-containing oligonucleotide. Competition experiments, in which unmodified oligonucleotides containing Binding Site 1 or 2 were included in the binding assays, indicated that the affinity of T antigen for the O6-meG modified sites was reduced. When partially duplex oligonucleotides containing either Binding Site 1 or Site 2 of the origin of replication were used as substrates for the helicase activity of T antigen, the presence of O6-meG increased the extent of T antigen catalysed displacement of single-stranded DNA fragments.

Adenosine Triphosphate↗
PubMed1990

Evidence for AP site formation related to DNA-oxygen alkylation in CHO cells treated with ethylating agents.

DNA single-strand breaks (ssb) induced by N-ethyl-N-nitrosourea (ENU) in CHO cells are quickly resealed within 10 min after treatment. This rapid repair kinetics is not explained by the rate of base excision repair which removes the main ethyl products with a half-life in the order of hours. We have explored the potential use of methoxyamine (MX), a chemical that reacts at neutral pH with AP sites in DNA in vitro, to clarify the origin of ENU-induced ssb. The presence of 50 mM MX during cell treatment with diethyl sulfate (DES) caused selective inhibition of the repair of AP sites generated during base excision repair and inhibited alkaline cleavage at these sites. The treatment of CHO cells with ENU in the presence of MX clearly showed that the burst of ssb observed immediately after treatment was due to AP site formation. Plasmid DNA treated in vitro with ENU did not present AP endonuclease-sensitive sites; therefore, the AP sites produced in CHO cells by ENU treatment are not due to the chemical hydrolysis of a very unstable ethyl adduct but rather are intermediates of an as yet undefined enzymatic pathway. This process occurs specifically after treatment with SN1-type ethylating agents (ENU and N-ethyl-N'-nitro-N-nitrosoguanidine) suggesting an association between this phenomenon and DNA-oxygen alkylation. We suggest that these breaks are generated by a mechanism of O6-ethylguanine processing without removal of the modified base.

Animals↗
PubMed1989

Tolerance to methylnitrosourea-induced DNA damage is associated with 6-thioguanine resistance in CHO cells.

Clones (13 and B) of O6-methylguanine-DNA-methyl-transferase-proficient (MT+) CHO cells showing different levels of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) but similar MT activity, were found to be sensitive to methyl methanesulphonate and resistant to N-methyl-N-nitrosourea (MNU). A 2.8-fold increase in resistance to MNU-induced cytotoxicity was observed in clone 13 and a 16-fold increase in clone B. A slight increase in survival (1.5-fold) after N-ethyl-N-nitrosourea treatment was observed in clone B. These data indicate that the resistant phenotype is specific for agents that preferentially methylate O atoms in DNA. The survival of MNNG- and MNU-resistant clones as well as of the parental CHO cell line was analysed after exposure to purine analogues substituted in different positions, 8-azaguanine (8-AG), 8-azaadenine (8-AA) and 6-thioguanine (6-TG). A 6-fold increase in resistance to 6-TG was found in clone B, although the hypoxanthine guanine phosphoribosyltransferase gene is functional in these cells. The same cytotoxicity was found in all the lines after treatment with 8-AG and 8-AA. These data are in agreement with the previous observation that clone 13 and clone B belong to two different classes of resistance, clone 13 resistance being explained by MT levels. The finding that clone B is cross-resistant to 6-TG is discussed in the light of a mechanism of tolerance to modifications at specific positions of guanine.

Animals↗
PubMed1989

O6-methyltransferase-deficient and -proficient CHO cells differ in their responses to ethyl- and methyl-nitrosourea-induced DNA alkylation.

The mutagenic and cytotoxic effects of N-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) were compared in two isogenic Chinese hamster ovary (CHO) cell lines differing for the expression of the repair function for O6-methylguanine (O6-meGua), the O6-methyl-DNA-methyltransferase (MT). Survival and ouabain resistance (ouar) mutation frequency were similar in the two cell lines after treatment with ENU while both effects were strongly reduced in the MT-proficient (MT+) CHO cells after exposure to MNU. The slow repair kinetics of O6-ethylguanine (O6-etGua) when compared to O6-meGua, i.e. 25% versus 88% removal at 20 h after treatment, could still account for the similar mutational curves reported in the two cell lines after ENU treatment. The number of ENU-induced sister chromatid exchanges (SCE) was slightly reduced in the MT+ as compared to MT-deficient CHO cells suggesting a role for O6-etGua in SCE formation. Comparison of survival after exposure to ENU and MNU showed that, at similar levels of O6-alkylguanine on DNA, the ethyl- is more tolerated than the methyl-adduct. These data focus the attention on the importance of DNA damage processing in the cytotoxic response to alkylating agents.

Alkylation↗
PubMed1989

Quantitative evaluation of genotoxic effects by molecular dosimetry.

The covalent binding of alkylating agents with DNA is considered a critical event in the mutagenic and carcinogenic effect of these chemicals. Several studies have indicated that only certain DNA alkylation products contribute to the mutagenic or carcinogenic activity of these agents. In this paper we summarize data obtained in Chinese hamster ovary (CHO) cells after treatment with two ethylating agents, N-ethylnitrosourea and diethylsulfate, which are known to alkylate DNA sites with different efficiency. A correlation study between DNA adduct formation and induction of mutations at two gene loci, i.e. hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, has been performed. The influence of DNA repair processes on the final yield of mutation is specifically discussed.

Alkylating Agents↗
PubMed1989

The use of in vitro assays for neoplastic transformation and tumor promotion.

We investigated growth control in mixed cultures of normal and oncogene-transformed mouse fibroblasts. NIH/3T3 transformed by v-myc, polyoma large T, polyoma middle T, v-ras and v-src showed comparable cloning efficiencies in agarized medium. However, when cultivated with an excess of normal cells (Balb/3T3, C3H10T1/2 or primary rat and hamster embryo cells) ras, src, and middle T-transformed cells were able to form "foci" of transformation on the layer of density arrested normal cells, whereas myc- and polyoma large T-transformed cells lacked this ability. Addition of the phorbol ester tumor promoter, phorbol-12,13-didecanoate, rescued proliferation and focus-formation by these nuclear oncogenes-transformed cell lines. Evidence is presented and discussed supporting a main role of intercellular communication between normal and transformed cells in modulating suppression or expression of the transformed phenotype.

Animals↗
PubMed1989

The origin of DNA single strand breaks induced by ethylating agents in mammalian cells.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.

Alkylating Agents↗
PubMed1988

Specific viral oncogenes cause differential effects on cell-to-cell communication, relevant to the suppression of the transformed phenotype by normal cells.

We have studied growth regulation in mixed cultures of normal and oncogene-transformed 3T3 cells. The NIH 3T3 cells transformed by myc, src, and ras showed comparable cloning efficiency in semisolid medium. However, when they were plated on plastic with an excess of normal mouse embryo fibroblasts, BALB/c 3T3 ClA31-1-1, ras- and src-transformed cells were able to form distinct foci on the layer of density-arrested normal cells, whereas myc-transformed cells lacked this ability. In order to determine whether suppression or expression of the transformed phenotype could be correlated with the ability of the different cell populations to communicate, gap-junctional intercellular communication (IC) was measured by the Lucifer yellow dye transfer assay in coculture of normal and transformed cells. The dye was observed to spread from BALB/c 3T3 to myc-NIH 3T3 cells, indicating the presence of IC between these two cell types. In contrast no passage of Lucifer yellow was observed between src-NIH 3T3 or ras-NIH 3T3 and BALB/c 3T3. Addition of a phorbol ester tumor promoter, phorbol-12,13-didecanoate, efficiently rescued proliferation and focus formation by myc-transformed cells. The tumor promoter was able to inhibit IC in BALB/c 3T3 cells, although this response greatly varied among the different oncogene transformed clones. Tumorigenicity in nude mice strongly correlated with growth behavior in vitro: myc-transformed cells were either nontumorigenic or slowly tumorigenic, and src- and ras-transformed cells were highly tumorigenic. These data suggest an important role of IC in modulating abnormal growth behavior in vitro and in vivo.

Animals↗
PubMed1988

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells.

DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.

Alkylating Agents↗
PubMed1988

Isolation of clones displaying enhanced resistance to methylating agents in O6-methylguanine-DNA methyltransferase-proficient CHO cells.

O6-Methylguanine-DNA methyltransferase (MT)-proficient Chinese hamster ovary cells were grown in the presence of low, gradually increasing levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with the aim of selecting MNNG-resistant cell lines. Six resistant clones with two levels of resistance were isolated. A 3-fold increase in survival was observed in clones 13, 14 and 15 and a greater than 10-fold increase in clones A, B and C. Cross resistance to N-methyl-N-nitrosourea but not to mitomycin C was observed. By comparison with the parental MT-proficient cells, MT activity was doubled in two resistant clones (13 and B) irrespective of their resistance levels. DNA glycosylase activity responsible for the removal of 7-methylguanine and 3-methyladenine showed similar levels in resistant clones 13 and B, in the MT-proficient cells and in the original MT-deficient cells. Alkylation-induced DNA damage, as measured by alkaline elution at the same MNNG dose, was higher in clones 13 and B than in the parental cells. The induction of sister chromatid exchanges by MNNG was inversely related to the resistance levels, thus paralleling the induction of cytotoxicity. These results suggest the existence in Chinese hamster ovary cells of at least two independent functions which control resistance to methylating agents, one possibly being the capacity to repair O6-methylguanine.

Alkylating Agents↗