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1.
Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants 总被引:3,自引:0,他引:3
Mouse lymphoma cells of the L5178Y TK+/- -3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Cells which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of NcoI-digested DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK+/- controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority (83/95) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb NcoI restriction fragment at the TK locus in the mouse lymphoma mutants analyzed. 相似文献
2.
TFT is an effective selective agent for TK-deficient mutants of L5178Y TK+/- -3.7.2C mouse lymphoma cells. Mutants can be classified by colony size into small colonies (many of which show readily observable chromosome abnormalities associated with chromosome 11--the location of the TK gene) and large colonies (which may represent events affecting only the expression of the TK gene). The precise nature of the induced damage causing the loss of the TK-enzyme activity for both mutant type is not known and is currently under investigation. The hypomethylating agent 5-azacytidine can be utilized to investigate the possibility that mutants might be the result of a suppressed rather than an altered TK gene. Mutant cell lines are treated with 5-azacytidine and then evaluated for re-expression of the TK enzyme as measured by resistance to THMG. In these studies, 11 mutants have been evaluated. None of the 11, including 10 small-colony mutants (6 with chromosome 11 translocations) and 1 large-colony mutant, show a high conversion to TK competency following 5-azacytidine treatment. 相似文献
3.
The effect of mixed mutagen exposures on the rate and type of induced mutants was studied in the L5178Y/TK+/-----TK-/- mouse lymphoma cell mutagenicity assay. In this assay, exposure to ethyl methanesulfonate (EMS) results in more mutants that form large colonies than small colonies. Exposure to methyl methanesulfonate (MMS) results in more mutants that form small colonies than large colonies. Other reports in the literature suggest that large colony TK-/- mutants appear to result from small-scale, perhaps single-gene mutations, and that small-colony TK-/- mutants appear to be associated with chromosomal mutations. Treating cells for 4 h with simple, 2-component mixtures containing 6.45 micrograms/ml MMS and either 261, 392, 560 or 712 micrograms/ml EMS resulted in synergism of mutants at each mixture level. The frequencies of total mutants were synergized 12, 20, 35 and 72%, respectively, in mixed exposures with graded doses of EMS, above the sums of the mixture components. Small colony mutants were synergized to a greater extent than large colony mutants. The frequencies of small colony mutants in mixed exposures were increased 31, 54, 73 and 123%, respectively, while the frequencies of large colony mutants were increased -7, -6, 11 and 39%. Statistical analyses provide strong evidence of synergism (within the limits of the assay) for total and small-colony mutants at all doses of EMS tested, and for large-colony mutants above 400 micrograms/ml EMS. Similar magnitudes of synergism resulted when other constant levels of MMS (4.30 or 8.60 micrograms/ml) were mixed with the same graded doses of EMS. The degree of synergism was dependent on EMS concentration but not on MMS concentration. 相似文献
4.
L5178Y/TK 3.7.2C cells are used for the assessment of chemical mutagenesis caused by presumptive TK gene mutations or multiple loci mutations affecting the TK locus that result in dose-related increases in resistance to the toxic thymidine analog, trifluorothymidine (TFT). This study was based upon our general observation that the incidence of TFTres in these cells could vary with the incubation temperature. As a result of these studies, we found that: (1) a substantial proportion of presumptive TK-/- variants produced by the mutagens 2-aminofluorene (2-AF), N-acetylaminofluorene (AAF), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3MCA), hycanthone methanesulfonate (Hyc), or methyl methanesulfonate (MMS) are more resistant to TFT at 37 degrees C than at 28 degrees C (or 39 degrees C than at 33 degrees C), (2) the loss of resistance to TFT was most notable in the small-colony variant population, (3) mutagen-derived variants become less resistant as the TFT concentration is increased from 4 micrograms/ml to 50 micrograms/ml, an effect that is more pronounced at 28 degrees C than at 37 degrees C, and (4) stock 3.7.2C cells develop a persistent TFTres due to sharply decreased TK activity when exposed to 40 degrees C for at least 24 h. These data demonstrate two different responses by these cells with respect to temperature stability at the TK locus and suggest that the degree of TFTres is influenced by both temperature and concentration of selective agent in this presumptive gene/chromosomal mutation assay. 相似文献
5.
2-Amino-6-N-hydroxyadenine (AHA) treated L5178Y/TK (+/-)-3.7.2C mouse lymphoma cells were evaluated for mutations at the tk, hgprt, and Na+/K+ ATPase loci, as well as for gross chromosome aberrations and induction of micronuclei. In addition, AHA was evaluated for its ability to induce HGPRT mutants in CHO cells. AHA was found to induce mutations at all evaluated loci and in both cell types. The TK mutants were primarily large colonies although a few small colonies were also induced, particularly at the higher concentrations. Preliminary cytogenetic analysis of AHA-treated mouse lymphoma cells indicated that some gross aberrations but not micronuclei were induced. The 20 small-colony TK mutants evaluated by banded karyotype indicate that only a small fraction (2 of 20) showed chromosome 11 abnormalities. From these studies, it appears that AHA may be one of a very few chemicals that is capable of inducing multi-locus point mutations, with only slight clastogenic activity. Particularly at the higher concentrations, some of the mutants may contain multi-locus point mutations that result in slow growth. 相似文献
6.
The phenotypic stability of over 2000 large- and small-colony trifluorothymidine-resistant (TFTres) variants of L5178Y/tk(+/-)-3.7.2C cells has been examined. All except 4 of 488 spontaneously arising small-colony variants analyzed (0.8%) retained the TFTres phenotype when rechallenged with TFT after growth for several generations in its absence. All of 558 spontaneous large-colony variants, and 440 small-colony or 487 large-colony variants arising from 13 different mutagens showed similar stability. These results attest to the completeness of TFT selection in the mouse-lymphoma assay when used at 1 microgram/ml in Fischer's medium supplemented with heat-inactivated serum and, together with previous cytogenetic and molecular studies, justify considering essentially all such TFTres variants as stable mutants. The implications of these results for those versions of the mouse lymphoma assay that fail to optimize the recovery and scoring of small-colony mutants is discussed. 相似文献
7.
Micronucleus, chromosome aberration, and small-colony TK mutant analysis to quantitate chromosomal damage in L5178Y mouse lymphoma cells 总被引:4,自引:0,他引:4
In testing the hypothesis that the small-colony thymidine kinase-deficient mutants of L5178Y/TK+/- -3.7.2C mouse lymphoma cells represent an estimate of the clastogenicity of test chemicals, we have been performing gross aberration analysis. The present study was initiated to determine if the cytokinesis block method of micronucleus analysis could be performed in mouse lymphoma cells and to compare 3 different endpoints of clastogenicity: the number of metaphases with aberrations, number of binucleates with micronuclei, and small-colony TK mutant frequency. In this study, 12 compounds having varying clastogenic potencies were evaluated. As would be expected, the 3 endpoints vary in the relative magnitude of the quantitated response. This difference likely results from the types of clastogenic damage detected by each endpoint. Of the 3 endpoints tested, only the small-colony TK mutant frequency measures events compatible with long-term cell survival. 相似文献
8.
We have systematically evaluated the mouse lymphoma TK+/- leads to TK-/- mutagenesis assay to determine if this somatic-cell test system would be a useful addition to the routine screening battery already used in our laboratory for the detection of chemical mutagens. During these investigations we observed that, with certain modifications of the basic assay, mutagenicity data could be obtained in as little as 9 days once the relative cytotoxic properties of the test substance were known. By improving the culturing conditions, we were able to reduce the serum requirements by as much as 50--75% without appreciably altering either cell viability or the recovery of chemically-induced mutants. Phenotypic stability of test-derived trifluorothymidine resistant (TFTR) mutants was confirmed by demonstrating cross-resistance to bromodeoxyuridine and concomitant sensitivity to methotrexate (THMG) in TFTR cells grown for 20 generations under non-selective conditions. While reduced growth rates resulting from temporary cell-division delay in treated cells is probably not a contributing factor to the observed mutation frequencies, only TFTR colonies which formed large distinct colonies in the presence of trifluorothymidine were clearly phenotypically stable mutants when spontaneous mutants were isolated and verified. When a non-mutagen, a weak mutagen, and a well-established mutagen were compared at equitoxic doses under these modified conditions, clear quantitative differences were seen in the respective mutation frequencies induced by these 3 types of agents. With these technical modifications, we feel this assay is both reliable and amenable to the screening of diverse chemical compounds for point-mutational activity in a mammalian cell. 相似文献
9.
We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants. 相似文献
10.
The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell 总被引:3,自引:0,他引:3
The mutagenic potential of the cytidine analog, 5-azacytidine (Aza Cyd), was tested at the thymidine kinase (TK) gene locus of L5178Y mouse lymphoma cells. 3-h exposure to as little as 20 ng/ml Aza Cyd yielded a substantial increase in TK-deficient L5178Y cells as measured by drug-induced resistance to trifluorothymidine (TFTres) 48 h later. This mutagenic effect was diminished up to 75% when Aza Cyd was tested in the presence of either enzymatically active or heat-denatured 9000 X g supernatant prepared from rat liver homogenate. The mutagenicity of Aza Cyd was also decreased in the presence of 1-5 X 10(-3) M thymidine and eliminated in the presence of greater than 1 X 10(-5) M cytidine. Two L5178Y TK-deficient cell lines had no selective survival advantage compared to TK-competent L5178Y cell stock when plated in soft-agar medium that contained Aza Cyd. Four other specific inhibitors of scheduled DNA synthesis in mammalian cells, deoxyadenosine, aphidicolin, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea were also L5178Y/TK mutagens. These data along with other published results suggest that chemicals known to disrupt nucleotide biosynthesis, alter deoxyribonucleotide pools, or directly inhibit DNA polymerase can cause stable, heritable increases in TFT resistance through mechanisms dependent upon altered replicative DNA synthesis, yet not necessarily dependent upon DNA incorporation or the binding of these mutagenic agents to nuclear DNA. 相似文献
11.
12.
Genotoxicity of gamma-irradiation in L5178Y mouse lymphoma cells 总被引:1,自引:0,他引:1
The ability of gamma-irradiation to induce gene mutation at the thymidine kinase locus and gross chromosome aberrations in L5178Y TK+/- 3.7.2C mouse lymphoma cells was evaluated. Positive results were obtained for both end-points. The majority of mutants were found to be small-colony mutants which correlated with the induction of gross chromosome aberrations. 相似文献
13.
Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells 总被引:1,自引:0,他引:1
The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective. 相似文献
14.
Mutagenesis assays at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells frequently yield mutant colonies with a bimodal size distribution. The objectives of this study were to determine whether a relationship exists between mutant colony size and chromosomal aberrations and whether the colony-size distributions obtained from this assay can indicate the clastogenic activity of a test chemical. Cells from 8 different types of L5178Y mouse lymphoma cell colonies were examined for chromosomal abnormalities within 10 cell generations after colony isolation. The colonies included small (sigma) and large (lambda) unselected cell (UC) and trifluorothymidine-resistant (TFTr) colonies derived from TK +/- cell cultures treated with the solvent dimethyl sulfoxide (DMSO) or hycanthone methanesulfonate (HYC). Chromosome abnormalities were present in cells from 12% (7/60) of the UC colonies, but there was no apparent relationship between colony diameter and the presence of chromosomal abnormalities. Abnormalities affecting chromosome 11, which is believed to be the site of the TK gene, were not observed in cells from UC colonies. Abnormalities affecting chromosome 11 were observed only in cells from sigma-TFTr colonies irrespective of whether they were spontaneous (5/15 colonies) or induced by HYC (4/15 colonies). Overall, 30% (9/30) of sigma-TFTr colonies had cells with an abnormal chromosome 11 and 10% (3/30) had abnormalities affecting other chromosomes. Abnormalities affecting chromosome 11 were not observed in cells from lambda-TFTr colonies (0/30 colonies). The observation of only 30% of sigma-TFTr colonies with chromosome damage affecting chromosome 11 indicates that other mechanisms, in addition to chromosome damage at the level of resolution used in this study (i.e., 200-300 chromosome bands). contribute to small TFTr colony size. 相似文献
15.
Actinomycin D was clastogenic and mutagenic in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. The majority of the mutants were small colonies, indicating that actinomycin D acts primarily by a clastogenic mechanism. 相似文献
16.
17.
《Mutation Research/DNA Repair Reports》1984,131(2):71-79
The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective. 相似文献
18.
Spontaneous Mutation Rates in Mammalian Cells: Effect of Differential Growth Rates and Phenotypic Lag 总被引:2,自引:0,他引:2
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We calculated a spontaneous rate of 26-37 x 10(-6) mutations per cell division for L5178Y MOLY (mouse lymphoma) cells at the thymidine kinase locus (tk+/(-)----tk-/-) using a procedure that isolated and segregated cells during expression. This rate was 50 times higher than when cells expressed the mutant phenotype in suspension. The higher mutation rates obtained with the in situ procedure suggest that many of the mutants, whether expressed or unexpressed, grew more slowly than wild-type cells prior to selection with trifluorothymidine (TFT), implying that the slow growth phenotype is expressed earlier than the TFTr (TFT-resistant) phenotype. The loss of mutants was not restricted to cells forming small colonies; the mutation rate for cells forming large colonies was more than ten times higher using the in situ procedure. In this new procedure, the cells expressed spontaneous mutations while growing in semisolid medium for up to 3 days without TFT. Mutants were then selected in situ by adding an overlay of TFT and the visible colonies were analyzed after 11 days. Cells with spontaneous mutations at the tk locus required approximately 30 hr for the more rapidly expressing cells with new mutations to be detected. Most of the TFTr colonies selected after 60 hr of growth in semisolid medium represented independent mutations that had accumulated during the first 30 hr.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
3-Aminobenzamide (3-AB) interferes with DNA repair and enhances lethality in growing MMS (methyl methane sulfonate)-treated human fibroblasts. This sensitivity to 3-AB disappears slowly; MMS-treated cells are sensitive to 3-AB for up to 36 hours (Boorstein and Pardee, 1984). Evidence is now presented that 3-AB potentiates the effects of MMS primarily during S phase. When cells were synchronized at the G1/S boundary, released, and then treated with MMS, 3-AB caused very substantial lethality in only 4 hours, and a 12-hour treatment gave maximum lethality. These cells also lost sensitivity to 3-AB within 12 hours of growth minus 3-AB. In contrast, MMS-treated quiescent (G0) cells did not lose sensitivity to 3-AB nor did 3-AB cause lethality during G0. Enhanced lethality occurred when damaged G0-arrested cells were subsequently allowed to proceed through S phase in the presence of 3-AB; this 3-AB sensitivity was removed only during growth in the absence of 3-AB. The lethality of 3-AB to a population of asynchronously cycling cells treated with MMS is thus the summation of effects on the cells as they traverse S phase. Aphidicolin prevented lethality of 3-AB to cells released from G1/S and treated with MMS. It also inhibited the loss of sensitivity to added 3-AB later. Correlation with the inhibition of DNA synthesis by this drug suggests that DNA synthesis is essential for the lethality enhancement by 3-AB. Cells treated first with MMS and then with 3-AB accumulated in G2. This G2 arrest depended on S-phase events and correlated with cell lethality. Cells treated with a nonlethal dose of MMS at the G1/S boundary were delayed briefly (3 hours) in their passage through S and G2. These cells, when also exposed to 3-AB, were delayed 6-9 hours in S and they became arrested in G2. There was no G2 arrest when 3-AB was added only after these cells had reached G2. Treatment with 3-AB during S phase thus resulted in both enhanced lethality and G2 arrest. 3-AB inhibited repair of DNA single-strand damage, shown by alkaline elution analysis, in both S-phase and quiescent cells. Aphidicolin inhibited disappearance of breaks and eliminated the difference between 3-AB-treated and untreated cells. Lethality did not correlate well with the measured single-strand damage.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
20.
Thymidine kinase activity and trifluorothymidine resistance of spontaneous and mutagen-induced L5178Y cells in RPMI 1640 medium 总被引:1,自引:0,他引:1
L5178Y/TK+/- cells were treated with 3-methylcholanthrene (3MC) in order to obtain thymidine-kinase-deficient mutants (TK-/-) which were resistant to trifluorothymidine (TFTr). Clones of TK-/- cells were harvested from soft agar and adapted to growth in suspension culture. The phenotype of the TK-/- and TK+/- clones was confirmed by measuring thymidine kinase activity. These studies were undertaken with cells from 16 3MC-induced large colony clones (lambda TK-/-), 21 3MC-induced small colony clones (sigma TK-/-), and 51 spontaneous sigma TK-/- clones. Thymidine kinase activity was absent in all of the lambda TK-/- and sigma TK-/- 3MC-induced clones and also in 49 of 51 sigma TK-/- spontaneous clones. After at least 50 generations in suspension culture, TFTr was retained by 80% of the 3MC-induced lambda TK-/- cells, by 75% of the 3MC-induced sigma TK-/- cells, and by 89% of the spontaneous sigma TK-/- cells. The collective results showed that 86 of the 88 TFTr colonies examined lacked thymidine kinase activity and indicate that at least 98% of all TFTr colonies seen in the L5178Y assay are true TK-/- mutants. 相似文献