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B Kaina

Publications and source records attributed to B Kaina.

108 records · Page 6Linked to original sources
PubMed1987

Correction of alkylation hypersensitivity of CHO-W27-1 cells by transfection with human DNA.

Chinese hamster cells of the mutant strain W27-1 which is hypersensitive to UV and monofunctional alkylating agents were transfected with human DNA ligated to the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene. Selection was performed for resistance to mycophenolic acid and finally for survival after treatment with high doses of methyl methanesulfonate. A gpt+ transfectant was generated (T38-2-7) which acquired resistance to methyl methanesulfonate and cross-resistance to N-methyl-N'-nitro-N-nitrosoguanidine at levels comparable with the parental (wild-type) strain CHO-9. T38-2-7 cells were not more resistant, however, to UV, mitomycin C and N-hydroxyethyl-N-chloroethylnitrosourea than the mutant W27-1. The transfectant contains integrated human DNA and was shown to be deficient for the O6-methylguanine-DNA methyltransferase. The results indicate that the transfected DNA specifically complemented the defect underlying alkylation hypersensitivity of W27-1 cells or that a gene was transfected which is generally inactive in CHO cells and which causes alkylation resistance.

Alkylation↗
PubMed1987

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.

Adenine↗
PubMed1985

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction.

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.

Alkylating Agents↗
PubMed1983

Cross-resistance studies with V79 Chinese hamster cells adapted to the mutagenic or clastogenic effect of N-methyl-N'-nitro-N-nitrosoguanidine.

When V79 cells are exposed to a single low dose of MNNG or MNU they acquire resistance to the mutagenic or to the clastogenic effect of the agents. Here the effect of MNNG pretreatment on mutagenesis (6-thioguanine resistance) and aberration formation in cells challenged with various mutagens/clastogens is reported. MNNG-adapted cells were resistant to the mutagenic effects of MNU and, to a lower extent, of EMS. No mutagenic adaptation was observed when MNNG-pretreated cells were challenged with MMS, ENU, MMC or UV. Cells pretreated with a dose of MNNG which makes them resistant to the clastogenic effect of this compound were also resistant to the clastogenic activity of other methylating agents (MNU, MMS), but not so with respect to ethylating agents (EMS, ENU). Cycloheximide abolished the aberration-reducing effect of pretreatment. However, when given before the challenge dose of MNNG, MNU or MMS, it drastically enhanced the aberration frequency in both pretreated and non-pretreated cells. No significant enhancement of aberration frequency by cycloheximide was found for ethylating agents. The results indicate that clastogenic adaptation is due to inducible cellular functions. It is concluded that mutagenic and clastogenic adaptation are probably caused by different adaptive repair pathways.

Animals↗
PubMed1983

Studies on adaptation of V79 Chinese hamster cells to low doses of methylating agents.

Pretreatment of V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea (MNU) significantly reduced the frequency of mutations (6-thioguanine resistance) induced by a challenge dose of these agents. Mutagenic adaptation can be observed after exposure to pretreatment doses which have no measurable toxic or cytogenetic effects, and which do not change DNA synthesis as measured at the moment of challenge. The influence of pretreatment on challenge dose-induced cell killing, chromosomal aberrations, and sister chromatid exchanges has also been studied. The effect of the challenge with respect to these end points was not in the same way dependent on the pretreatment dose as the challenge dose-induced mutation frequency. Therefore it is concluded that mutagenic adaptation and adaptation with respect to the other end points studied have no identical underlying causes.

Animals↗
PubMed1983

Genetic and cytogenetic effects of 1,3-dimethyl-3-phenyl-1-nitrosourea in Salmonella typhimurium and Chinese hamster V79 cells.

1,3-Dimethyl-3-phenyl-1-nitrosourea (DMPNU) shows no spontaneous decomposition in aqueous buffered solution (pH 7.0) at 37 degrees C over a measuring period of 10 days, but it is directly genotoxic in all assays applied. This compound induces base substitutions in Salmonella typhimurium (especially in the plasmid harbouring strain TA 100), gene mutations at the HGPRT+ locus of Chinese hamster V79 cells, and is a very potent inducer of clastogenic damage and sister chromatid exchanges in V79 cells. Data on gene mutation induction by 1-methyl-1-nitrosourea are included for comparison. The findings on DMPNU are discussed in comparison with trialkylnitrosoureas. In contrast to these latter compounds, DMPNU is active without addition of S9 mix in vitro. It is assumed that its genotoxic activity is due to an intracellular catalytic degradation favoured by the electron-withdrawing effect of the phenyl group.

Animals↗
PubMed1982

Enhanced survival and reduced mutation and aberration frequencies induced in V79 chinese hamster cells pre-exposed to low levels of methylating agents.

Exposure of V79 Chinese hamster cells to a single very low (sub-toxic and sub-clastogenic) dose of MNU or MNNG made these cells resistant to the toxic, mutagenic and clastogenic activities of the same agents given 6 h later. Cell survival was increased nearly 2-fold under optimal conditions when compared with the non-pretreated controls. Aberration frequencies were reduced to nearly half the control values (cells not pretreated). This was observed for a wide range of pretreatment concentrations and at different recovery times. The effect of mutagen pretreatment was most pronounced with respect to the induction of TG resistance, which became drastically reduced. The data indicate the existence of an adaptive repair pathway in V79 cells which may be induced by very low levels of methylating agents and which is error-free in handling lesions responsible, at least partially, for reproductive cell death, mutations and chromosomal aberrations.

Animals↗
PubMed1979

Analysis of the Vicia faba genome by use of restriction endonucleases.

DNA of the broad bean, Vicia faba, was cleaved by the restriction endonucleases endoR . EcoRI, endoR . HindIII, endoR . HincII, endoR . BamI, and endoR . BspRI. Separation in agarose gels of the resulting fragments revealed, in addition to the bulk DNA, an enzyme-specific pattern of bands composed of restriction fragments of 300 to more than 30,000 base pairs in length. Bulk DNA was characterized by an unusual size distribution which significantly deviated from that expected according to the random fragmentation theory. It is argued that the observed distribution is due to the high proportion of repetitive DNA within this species (approximately equal to 75%). In all digests, a class of high-molecular-weight restriction fragments of more than 30,000 base pairs in length was observed which comprised 5-8% of the genome. It showed hybridization with highly repetitive DNA (c0t less than or equal to 2 x 10(-2) M . s) and included a fraction (2-3% of the genome) highly resistant to the activity of all the enzymes tested. The buoyant density in CsCl of this resistant DNA was not different from that of the total DNA (36% dG + dC). In endoR . EcoRI digests, the high-molecular-weight fragment class contained, in addition to the resistant DNA, a fraction of relatively high buoyant density (calculated dG + dC content: 61%) containing cleavage sites for the other enzymes used.

DNA↗
PubMed1977

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. I. Cell cycle inhibition and aberration induction in diploid and Down's fibroblasts.

The effect of N-methyl-N-nitrosourea (MNU) on the cell cycle, DNA synthesis and chromosomal sensitivity of cultivated diploid fibroblasts and fibroblasts with trisomy 21 was investigated in vitro. With the exception of the inhibition of G2, Down's cells proved to be more sensitive than diploid cells with respect to the decrease of the mitotic and labelling index, the inhibition of the progression of cells through the early and middle S and the frequency of induced chromosomal aberrations. The chromosomal sensitivity was dependent on the position of cells in the cell cycle during treatment with MNU. If treated during late S no differences concerning the S block and aberration frequencies were found between diploid and Down's cells. However, if MNU treatment took place in the middle and early S, Down's cells were more sensitive. The higher aberration frequencies in Down's cells resulted from elevated levels of chromatid breaks, multiple fragmentations and chromatid translocations. Possible reasons for the increased sensitivity of Down's cells are discussed.

Cell Division↗
PubMed1977

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. II. Non-random distribution of chromatid aberrations in diploid and Down's cells.

Chromatid gaps, breaks and aberrations involved in interchanges induced by N-methyl-N-nitrosourea (MNU) were found non-randomly distributed on individual chromosomes and chromosome segments (G bands) both in human diploid fibroblasts with trisomy 21 cultured in vitro. Aberration events were located exclusively in pale G bands. Considering cells in the first post-treatment mitosis, the pattern of aberration distribution, as revealed by the position of hot spots, varied with recovery time and was different in diploid and Down's cells. In comparison with diploid cells, the X chromosomes of Down's cells were not involved in aberrations. Despite the higher aberration frequencies of Down's cells, the number of hot spots and the proportion of aberrations located in hot spots were not increased in this cell type. Therefore, the increased chromosomal sensitivity to MNU of Down's cells does not reflect an increased sensitivity of special chromosomes or chromosome sites.

Cell Division↗
PubMedDate not supplied

Cell and tissue-type specific expression of Ras-related GTPase RhoB.

Ras-homologous (Rho) GTPases are involved in the regulation of a variety of cellular processes such as the organization of the actin cytoskeleton, malignant transformation and genotoxic stress-induced signaling. Here we show that, among the family of Rho GTPases, specifically rhoB mRNA expression is rapidly induced upon UV-irradiation, whereas the level of rac 1 and cdc42 mRNA is not affected. Increase in rhoB mRNA was accompanied by a approximately 4-fold increase in the amount of membrane-bound RhoB protein. Basal expression of rhoB mRNA appears to be cell-type specific with low amounts in rodent NIH 3T3, V79, H4IIE, CHO and human HaCat cells and comparably high amounts in monkey COS, human HeLa and HepG2 cells. In rabbit tissues, exceptionally high levels of rhoB mRNA and RhoB protein were found in lung whereas its expression was quite low in heart, liver, spleen and kidney. Variations in rhoB mRNA expression level are not due to cell-type specific differences in rhoB mRNA stability as shown by inhibitor experiments. However, transiently transfected rhoB promoter CAT construct was expressed at significantly higher level in HeLa and HepG2 as compared to NIH 3T3 and CHO cells. Thus, cell-type specific differences in the level of rhoB mRNA are likely to be due to variations in the transcriptional activity of the rhoB gene. The data indicate that, among the family of Rho GTPases, only the expression of rhoB is rapidly stimulated by genotoxic stress. Furthermore basal rhoB expression appears to be regulated in a cell and tissue-type specific manner. This may be related to yet unknown tissue-specific physiological function of RhoB.

3T3 Cells↗
PubMedDate not supplied

Overexpression of cDNA encoding FANCC, SPHAR, MPG, SNM1 or HA 3611 does not render CHO cells more resistant to DNA crosslinking agents.

DNA crosslinking agents (DCA) are commonly used cytostatic drugs, whose efficiency in tumor therapy is limited due to the appearance of drug resistant tumor cells. In an effort to modulate the resistance of cells to DCA, we transfected into Chinese hamster cells various cDNAs whose loss of function was previously shown to render cells more sensitive to crosslinking agents. We show that overexpression of FANCC, SPHAR, MPG, SNM1 or HA 3611 (a human homologue of the yeast crosslink DNA repair gene SNM1) does not alter the level of resistance of CHO cells to clinically relevant DCA, such as mafosfamide, melphalan and mitomycin C. Therefore, DCA resistance frequently observed in tumor cells is not likely to be the result of up-regulation of either one of these genes, but a more complex phenomenon. Also, the data suggest that protection of normal cells from toxic side effects of DCA cannot easily be accomplished by transfer of either one of these genes.

Animals↗
PubMedDate not supplied

Detection of O6-methylguanine-DNA methyltransferase-expression in human tumors by means of in situ hybridization.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a main determinant of resistance of tumor cells towards nitrosoureas that are in use as cytostatic drugs. It therefore appears of importance to determine its expression in tumors before the utilization of nitrosoureas for tumor treatment. We have determined the level of expression of MGMT in mammary carcinomas and brain tumors by means of in situ hybridization, using digoxygenin-labeled RNA driven by MGMT expression vector in sense and antisense orientation. It is shown that the intensity of the hybridization signal correlates with the MGMT activity of a given tumor. The results demonstrate the applicability of in situ hybridization for determination of MGMT expression in tumor sections.

Brain Neoplasms↗