Biomedical subjects

M Pirsel

Publications and source records attributed to M Pirsel.

At least 19 recordsLinked to original sources

PubMed1999

Lack of correlation between repair of DNA interstrand cross-links and differential sensitivity of G0 and proliferating CD4+ lymphocytes towards cisplatin.

G0 cells in a tumor are insensitive to the chemotherapeutical agents. The nature of this resistance is not completely understood. One of the factors modulating sensitivity of cells may be DNA repair of drug induced DNA damage. In this study we have compared gene-specific formation and repair of cisplatin-induced interstrand cross-links (ICL) in human G0 and proliferating CD4+ lymphocytes. Cisplatin killing of G0 CD4+ lymphocytes is inefficient, and these cells resemble those in a tumor. After exposure to cisplatin under similar conditions, the frequency of ICL introduced is twice as high in the proliferating compared to the resting lymphocytes. Repair of ICL was measured in the housekeeping gene, dihydrofolate reductase (DHFR), in the proliferation inducible c-myc gene, and in the inactive delta-globin gene. We observed similar relative rates and extent of ICL repair in all three genes studied, in G0 or proliferating CD4+ lymphocytes. The mechanisms responsible for the resistance of G0 CD4+ lymphocytes towards cisplatin are discussed.

Antineoplastic Agents↗

PubMed1997

The pre-UV nutritional stresses increase UV resistance, decrease UV mutagenesis and inhibit excision repair.

Nutritional stresses applied to E. coli prior to UV irradiation increase UV resistance and decrease UV mutagenesis. This effect is uvrA-dependent and might reflect a more efficient excision of pyrimidine dimers [1]. The data presented here, however, indicate that after prestarvation for glucose or amino acids pyrimidine dimer excision (PDE) was partly inhibited. It appears that the stress conditions stimulate a mode of uvr-dependent tolerance of lesions, efficient and precise. Possible modes of PDE inhibition and lesion tolerance are discussed.

Adenosine Triphosphatases↗

PubMed1997

The effect of the OmpT protease on excision repair in UV-irradiated Escherichia coli.

The extent of pyrimidine dimer excision (PDE) was inhibited in UV-irradiated E. coli KS272 (ompT+) cells when they were preinduced by a low UV predose preceded by a nutrition stress but not in the preinduced E.coli SF100 (ompT-) mutants. The preinduction, however, markedly inhibited PDE in the ompT- cells transformed with a multicopy plasmid carrying ompT gene. The data are consistent with the hypothesis that the inducible OmpT protease (controlled by rpoH) might terminate the SOS period of excision repair so that when cells are preinduced PDE might be inhibited prematurely.

DNA Damage↗

PubMed1996

Constitutive increase of RecA protein: its influence on pyrimidine dimer excision and survival of UV-irradiated Escherichia coli.

Transformation of E. coli with the plasmid pRA containing recA gene increased the constitutive level of RecA protein 50-67 fold. This slightly inhibited pyrimidine dimer excision and reduced cell survival in three investigated, UV-irradiated E. coli strains. Our data support the view that RecA protein prematurely present at a high level may mask the dimers. The masking subsequently reduces the dimer excision and switches off the inducing signal.

DNA, Bacterial↗

PubMed1995

Inducible stable DNA replication of Escherichia coli tolerates unexcised pyrimidine dimers in an uvr-dependent manner.

Damage-inducible DNA replication (iSDR) was followed in UV-irradiated E. coli uvr+ and uvr B5 cells. Owing to the inhibition of dimer excision in the former (caused by the metabolic treatment), both contained similar amounts of unexcised dimers. Since the iSDR took place in uvr+ but not in uvr B5 cells, it is concluded that the uvr system can tolerate unexcised dimers through the recombinogenic iSDR.

DNA Damage↗

PubMed1995

DNA repair in Escherichia coli: the dual function of uvr genes.

It has been shown earlier that the starvation of E. coli for both amino-acids and thymine applied prior to UV irradiation inhibits pyrimidine dimer excision without affecting cell survival after UV irradiation. In such cells pyrimidine dimers are tolerated by a rather error-free process that depends on the activity of uvrB, recA and lexA genes. Data presented here show: (a) that the efficient toleration of unexcised dimers requires also the uvrA gene; (b) that the starvation increases the level of RecA protein about 4.7 times; (c) that the effect of starvation on subsequent pyrimidine dimer excision is reversed by a 2 h incubation in complete medium before the cells are UV irradiated. The data suggest that the uvrA, uvrB, recA, lexA dependent nonexcisional repair may be a pathway temporarily functioning in repeatedly damaged cells.

Adenosine Triphosphatases↗

PubMed1993

Methyl methanesulfonate adduct formation and repair in the DHFR gene and in mitochondrial DNA in hamster cells.

Chinese hamster ovary CHO-B11 cells were exposed to methyl methanesulfonate (MMS) and the formation and repair of N-methylpurines were measured in the endogenous dihydrofolate reductase (DHFR) gene domain and in mitochondrial DNA by alkaline hydrolysis which generates strand breaks at apurinic (AP) sites formed after neutral depurination. The initial levels of damage in the transcriptionally active DHFR gene, 3'-flanking non-transcribed region and in mitochondrial DNA were slightly different; the highest level of damage was in mitochondrial DNA, the lowest in the DHFR gene. The rate of adduct removal was similar in all three DNA regions examined. We conclude that there is no preferential repair of MMS induced N-methylpurines under these conditions, and that these lesions are efficiently removed from mitochondrial DNA.

Animals↗

PubMed1993

Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents.

We have measured the DNA damage formation and repair in the ribosomal and the dihydrofolate reductase (DHFR) genes after treatment of hamster cells with different types of DNA damaging agents. In mammalian cells, the ribosomal DNA (rDNA) is transcribed by RNA polymerase I, whereas the DHFR is transcribed by RNA polymerase II, whereas the DHFR is transcribed by RNA polymerase II. Cells were treated with agents that induce different types of lesions, and that are known to be repaired via different pathways. We used UV (254 nm) irradiation, treatment with cisplatin and treatment with the alkylating agents nitrogen mustard (HN2) and methyl methanesulphonate (MMS). UV induced pyrimidine dimers were detected with the enzyme T4 endonuclease V, which creates nicks at the dimer sites; the breaks are then resolved and identified by denaturing electrophoresis and Southern blot. Intrastrand adducts formed by the alkylating agents HN2 and MMS were quantitated by generating strand breaks at abasic sites after neutral depurination. Interstrand crosslinks (ICL) formed by HN2 and cisplatin were detected by a denaturation-reannealing reaction before neutral agarose gel-electrophoresis. We find that the repair of the pyrimidine dimers is significantly less efficient in the RNA polymerase I transcribed rDNA genes than in RNA polymerase II transcribed DHFR gene at 8 and 24 h after irradiation. ICL and intrastrand adducts induced by HN2 are also removed more slowly from the rDNA than from the DHFR gene. In contrast, MMS induced intrastrand adducts and cisplatin induced ICL are repaired equally efficiently in the RNA polymerase I and RNA polymerase II transcribed genes. We conclude that for some types of DNA damage, there is less repair in the ribosomal genes than in the DHFR; but for other DNA lesions there is no difference. The difference in repair efficiency between the rDNA and the DHFR genes may reflect the different RNA polymerase involved in their transcription. It may, however, alternatively, reflect the different nuclear localization of these genes.

Alkylating Agents↗

PubMed1992

Overexpression of metallothionein in Chinese hamster ovary cells and its effect on nitrogen mustard-induced cytotoxicity: role of gene-specific damage and repair.

Overexpression of metallothionein in mammalian cells has been associated with protection from cytotoxic chemicals and acquired resistance of tumors to cytotoxic drugs. The mechanism of this effect, however, remains unclear. We have explored whether cytotoxicity of the bifunctional alkylating agent nitrogen mustard was correlated with the extent of DNA damage formation and repair in the metallothionein gene regions in Chinese hamster ovary cells. The DNA damage and repair were examined in metallothionein-overexpressing, cadmium-resistant Chinese hamster ovary cells, Cdr200T1, with or without zinc-induced transcriptional activation, and in the parental CHO-met- cell line. The zinc-induced Cdr200T1 cells tolerated significantly higher doses of nitrogen mustard than did the uninduced Cdr200T1 variant. The parental CHO-met- cells, which did not have any detectable metallothionein expression, were even more resistant to nitrogen mustard than the zinc-induced Cdr variants. Nitrogen mustard-induced N-alkylpurines were formed with a higher frequency in inactive genomic regions than in the active genes. The removal of N-alkylpurines was similar in the active MT I gene region in Cdr200T1 and the silent MT I gene region in the parental cells, and the expression of these genes was determined by Northern assay. The MT II gene-containing region was repaired less efficiently than the MT I gene, independently of zinc induction. Further, preferential repair of nitrogen mustard-induced N-alkylpurines were detected in a single copy of the essential active dihydrofolate reductase gene as compared to a downstream noncoding region. This preferential repair was unaffected by the presence of zinc. Neither damage formation nor repair kinetics in the MT gene regions seemed to parallel the observed spectrum of sensitivity to HN2.

Alkylation↗

PubMed1990

Polyomavirus-based shuttle vectors for studying mechanisms of mutagenesis in rodent cells.

We have constructed a series of polyomavirus-based shuttle vectors for analyzing mechanisms of mutagenesis in rodent cell systems. These vectors contain the supF suppressor tRNA gene which serves as the mutagenesis target; the pBR327 replication functions and ampr gene for replication and selection in bacteria; and the polyomavirus genome which permits replication in rodent cells. The polyoma genomes used in these vectors vary in their enhancer regions, causing varying efficiencies of replication in different types of rodent cells. One of the vectors (pPySLPT-2) which replicates particularly well in several different rodent cell types (i.e., Chinese hamster ovary, mouse hepatoma and mouse lymphoma) was used to compare mutation induction by UV radiation in UV repair-deficient mouse lymphoma L5178Y-R cells with mutagenesis in the related UV repair-proficient line, L5178Y-S. In both cell types, UV-induced mutants could be recovered at frequencies up to 50-fold higher than that of the spontaneous background. At a given UV fluence the L5178Y-R cells were more highly mutable than the L5178Y-S cells. Our results indicate that these new polyomavirus-based vectors should be useful for analysis of the molecular mechanisms of mutation induction in rodent cell systems, and in particular should allow detailed analysis of mutagenesis in the well characterized rodent somatic cell mutants.

Animals↗

PubMed1989

Transient appearance of photolyase-induced break-sensitive sites in the DNA of ultraviolet light-irradiated Syrian hamster fetal cells.

Syrian hamster fetal fibroblasts (HFC) were examined for photolyase-induced break-sensitive sites after ultraviolet light (UV) exposure and growth. These sites, observed in excision-defective human xeroderma pigmentosum (XP) cells, are due to cleavage of the internal phosphodiester bond of UV-induced pyrimidine dimers. Excision-inefficient HFC acquired photolyase-induced break-sensitive sites during incubation after UV (10 J/m2). However, these were observed transiently, with a maximum of 5% of the pyrimidine dimers at 9 h post UV; by 18 h they were undetectable. Caffeine (1 mM) delayed the peak of photolyase-induced break-sensitive sites by 2 h. In human XP cells photolyase-induced break-sensitive sites accumulate to a plateau level of about 20% of the pyrimidine dimers. The present results extend to rodent cells the observation that cleavage of the internal phosphodiester bond of pyrimidine dimers may be an early step in their excision repair. Furthermore, the data suggest that photolyase-induced break-sensitive sites might be necessary for replication bypass at pyrimidine dimers.

Animals↗

PubMed1984

DNA of Chinese hamster V79 cells newly synthesized after ultraviolet irradiation contains alkali-labile sites.

Pulse-labeled daughter DNA of UV-irradiated Chinese hamster V79 cells was denatured in alkaline or neutral conditions and analysed by sucrose gradient centrifugation. A comparison of the sedimentation profiles of DNA treated in alkaline or neutral conditions has shown that in UV-irradiated cells some alkali-labile sites are produced during replication of damaged templates.

Alkalies↗

PubMed1984

Error-free uvr+-dependent inducible DNA repair in Escherichia coli B/r Hcr+ cells.

The frequency of suppressor (tryptophan reversions) and of true (streptomycin-resistant and dependent) mutations has been followed in E. coli cells irradiated with a single dose or two separate doses of ultraviolet (U.V.) radiation. Under these conditions dimers were efficiently excised after a single dose, while about 40 per cent of the dimers remained unexcised after two doses. Although the level of unexcised dimers in the latter case increased proportionally with the second U.V. dose, the mutation frequency increased by 1.5-2-fold, but did not continue to increase with the level of unexcised dimers. A comparison of excision-proficient and excision-deficient cells containing similar amounts of persisting dimers has shown that proficient cells can tolerate a high level of dimers without an adequate increase in mutation frequency. Our results suggest the existence of an error-free uvr+-dependent inducible repair in E. coli B/r Hcr+ cells.

Cell Survival↗

PubMed1982

Inducible nature of tolerance to lesions produced by ultraviolet light in DNA of Chinese hamster V79 cells.

Chinese hamster V79 cells irradiated by split doses of ultraviolet light (2.5 + 5.0 J m-2) synthesize higher molecular weight DNA molecules than those irradiated by a single dose (5.0 or 7.5 J m-2). A second dose applied 8 h following the first one results in a higher portion of higher molecular weight molecules both in relative and absolute terms. Molecular weight of DNA pulse-labeled 8 h after a dose of 2.5 J m-2 is moderately higher than that of pulse-labeled DNA in unirradiated cells, even after the DNA synthesis in preirradiated cells has been fully recovered. Since the amount of DNA synthesized during the 8 h time interval between the two doses is not lower than that of DNA synthesized during an equal period in unirradiated cells, the synthesis of higher molecular weight molecules is not likely to represent elongation of part of the replicons, the other part being blocked. It is supposed that the induction produced by the first dose is involved in the replication of the damaged template.

Animals↗

PubMed1978

Uv-inducible repair II: its role in various defective mutants of Escherichia coli K-12.

Involvement of UV-inducible protein(s) in repair of various E. coli K-12 cell strains has been investigated using a procedure of double UV irradiation and postincubation with chloramphenicol. From the course of dose survival curves the following conclusions concerning significance of a UV-inducible protein have been drawn: 1. It is a very important for wild type cells; in these cells its early occurrence is necessary to prevent killing. 2. It is involved in repair of excision-deficient cells; however, its action early after UV is less urgent. 3. It is not involved at all in repair of lex mutant cells; 4. It exhibits some effect on survival of recA as well as recB mutant cells. We conclude that the protein is involved in excision repair as well as in resumption of DNA replication.

Bacterial Proteins↗

PubMed1977

Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec mutant of Escherichia coli K12.

A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA-) or by treatment with chloramphenicol (CAP) prior to UV irradiation (2.5 Jm-2) increased the resistance of the strain Escherichia coli K12 SR19 to UV radiation more than ten-fold. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation and a higher stability of both parental and newly synthesized DNAs could be demonstrated.

Amino Acids↗

PubMed1977

Replication of DNA in UV-irradiated Escherichia coli in the absence of amino acids.

Since pyrimidine dimers are considered to be the cause of the synthesis of short DNA segments, normalization of DNA replication after UV irradiation should be in a temporal correlation with their removal. This correlation holds in exponentially growing excision-proficient Escherichia coli cells. However, when these cells are preincubated and postincubated without amino acids, synthesis of short segments continues although dimers are efficiently excised.

Amino Acids↗

PubMedDate not supplied

Expression of the Escherichia coli recA gene in the yeast Saccharomyces cerevisiae.

The isolation of the protein coding region of the recA gene from Escherichia coli by extensive Bal31 digestion is described. The structural recA gene was ligated into an extrachromosomally replicating yeast expression vector, downstream of the yeast alcohol-dehydrogenase gene promoter region, to produce pADHrecA plasmid. The pADHrecA plasmid was transformed into the wild-type and the repair deficient strains of Saccharomyces cerevisiae. The crude protein samples were extracted from the individual yeast transformants. A 38 kDa protein was present in all transformants containing the recA gene on plasmid. Thus the recA gene from E coli was successfully expressed in cells from a lower eukaryote.

Alcohol Dehydrogenase↗