Chinese Hamster Cell Line Research Articles

Gibbon ape leukemia virus and the amphotropic murine leukemia virus 4070A exhibit an unusual interference pattern on E36 Chinese hamster cells

The gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV) 4070A, and the xenotropic mouse leukemia virus (X-MLV) exhibit wide but not identical species host ranges. However, most Chinese hamster cells resist infection by all three viruses. We have now determined that the Chinese hamster cell line E36 differs from other Chinese hamster cell lines in that it is susceptible to infection by wild-type GaLV, A-MLV, and X-MLV. Surprisingly, analysis of the interference pattern of GaLV and A-MLV in E36 cells indicated that GaLV and A-MLV interfere in a nonreciprocal fashion. E36 cells productively infected with GaLV were resistant to superinfection by both GaLV and amphotropically packaged recombinant retroviral vectors. In contrast, E36 cells infected with A-MLV were resistant to superinfection with an amphotropic vector but could still be infected by a GaLV vector. These results imply the existence of a receptor on E36 cells that interacts with both GaLV and A-MLV.

Spectrum of spontaneous mutation in animal cells containing an aphidicolin-resistant DNA polymerase α

We have used a polymerase chain reaction (PCR)-based exon screening assay to determine the spectrum of spontaneous hypoxanthine phosphoribosyltransferase (hprt) gene mutations occurring in an aphidicolin-resistant V79 Chinese hamster cell line (designated Aphr-4-2) that contains a mutant DNA polymerase-alpha and displays a spontaneous mutator phenotype. PCR analyses of 71 independent, 6-thioguanine (TG)-resistant sublines isolated from Aphr-4-2 or parental V79-743X cells using hprt exon 3- and exon 9-specific oligonucleotide primer pairs revealed the loss of exon 3 or 9 from 6 of 60 Aphr-4-2 derived-, and from 1 of 11 parental V79-derived, TG-resistant mutants. Exons 3 and 9 were both lost from 5 of 60 Aphr-4-2-derived mutants, while none of the 11 V79-derived mutants had lost both exons. The results of these PCR-screening assays were further corroborated by Southern and Northern blot hybridization analyses of 28 mutants: 22 of 28 mutants contained an intact hprt gene by Southern analysis; of these 22 mutants 6 of 11 Aphr-4-2-derived mutants contained either reduced or undetectable steady state mRNA levels in contrast to all 11 V79-derived mutants that contained normal amounts of a normal-sized hprt mRNA. The results of our PCR and blot hybridization analyses indicate that the rates of base substitution and deletion mutagenesis are elevated in Aphr-4-2 cells, and suggest that DNA polymerase-alpha may play a role in determining the rate of different molecular types of spontaneous mutations in vivo.

Mutagenic responses of nickel oxides and nickel sulfides in Chinese hamster V79 cell lines at the xanthine-guanidine phosphoribosyl transferase locus

Mutagenesis of several insoluble nickel compounds-crystalline nickel sulfide NiS, nickel subsulfide Ni 3S 2, nickel oxides (black and green) and soluble NiCl 2 was studied in three Chinese hamster cell lines - at the hrpt gene of the well-defined V79 cell line, and at gpt in two transgenic derivative cell lines G12 and G10. The transgenic cell line G12 responded very strongly to the insoluble Ni compounds, such that the gpt mutagenesis was at least 20 times higher than the spontaneous mutagenesis and in some experiments was even higher. In contrast the response of the G10 cells was much lower - the mutant frequencies only increased 2–3 times over the controls. In V79 cells, NiS and NiO (black) did not induce a mutagenic response at hprt. Soluble NiCl 2 also exhibited no mutagenic activity in V79 cells and induced considerably lower activity than the insoluble compounds in the transgenic G12 cells. Following vitamin E pretreatment of G12 cells for 24 h prior to nickel exposure, increased cell survival was observed for several insoluble Ni compounds whereas vitamin E had no effect on NiCl 2 cytoxicity. With vitamin E pretreatment, significantly lower mutagenic responses in G12 cells were also noted for some insoluble Ni compounds, while no such effect was observed for NiCl 2.

Induction of Recombinant Human γ-Glutamyl Transferase by Sodium Butyrate in Transfected V79 and CHO Chinese Hamster Cells

Sodium butyrate was used to enhance biosynthesis rates of recombinant human γ-gtutamyl transferase (GGT) expressed under the control of the SV40 or the cytomegalovirus immediate early promoter, respectively, in transfected V79 and CHO Chinese hamster cell lines. Maximal induction of GG specific activity in butyrate-treated cells ranged from 3 to 5-fold and resulted from a strong increase in the GGT mRNA ratio. We also observed that maximal transc~ption level in V79 cells occured within 12 hr of treatment, whilst the cell proliferation was transiently arrested. Despite its processing requirements, induced GGT exhibited unchanged catalytic and physico-chemical features relative to human serum or hepatoma enzyme, thus appearing as an excellent model for further studies on human GGT.

Induction of micronuclei in V79 Chinese hamster cells by hydroquinone and econazole nitrate

Two suspect aneugens (hydroquinone and econazole nitrate)_were examined for their ability to induce micronuclei in a number of V79 Chinese hamster cell lines which express rat cytochrome P-450 cDNAs. Hydroquinone elevated micronucleated cell frequencies in a dose-dependent manner in cell lines V79, XEM2 (expresses CYP1A1) and SD1 (expresses CYP2B1). Econazole nitrate was an effective inducer of micronucleic over a narrow dose range in cell lines V79, XEM2 and XEMd-MZ (expresses CYP1A2). The different cell lines showed similar responses to the test agents, indicating that hydroquinone is not a substrate for biotransformation by rat CYP1A1 or CYP2B1, nor is econazole nitrate biotransformed by rat CYP1A1 or CYP1A2.

A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens

Two in vitro cytogenetic assays were evaluated for their ability to detect aneugenic and polyploidy-inducing agents using a battery of 10 known or suspected aneugens as part of the EEC 4th Environmental Reseach and Development Programme. The compounds tested were colchicine, vinblastine, chloral hydrate, thiabendazole, hydroquinone, thimerosal, cadmium chloride, econazole nitrate, pyrimethamine and diazepam. The cell division aberration assay employed a differential chromosome/ spindle staining procedure to detect perturbations of the mitotic division apparatus. This assay was carried out in two pulmonary-derived Chinese hamster cell lines; the immortal DON:Wg3h culture and a low passage LUC2 culture. The second assay involved quantification of metaphase chromosomes, for which only the LUC2 cell line was used, due to the stability of its diploid karyotype. All the chemicals induced spindle disturbances in the immortal line. In addition, all the compounds except cadmium chloride yielded positive results in the LUC2 culture, although many were not as potent. In the low passage line, 8 of the compounds (colchicine, vinblastine, chloral hydrate, thiabendazole, thimerosal, econazole nitrate, pyrimethamine and diazepam) induced aneuploidy and/or tetraploidy. Cadmium chloride was negative in the chromosome enumeration assay and hydroquinone yielded inconclusive results. The study of cell division aberrations was much less time-consuming and technically complex than the counting of metaphase chromosomes. In addition, it provided a degree of mechanistic understanding of the mode of action of some aneugenic and polyploidy-producing agents. However, the enumeration of chromosomes provides a more definitive data set for the evaluation of a chemical's aneugenic potential.

Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross-linking of long and short single-stranded DNA molecules.

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.

Cytogenetic effects of promutagens in genetically engineered V79 chinese hamster cells expressing cytochromes P450

V79 Chinese hamster cell lines genetically engineered to express rat CYP2B1, CYP1A1, CYP1A2, and their parental cell lines V79-MZ, without acetyltransferase, and V79-NH, with acetyltransferase, were studied for chromosome aberrations and sister chromatid exchange induced by aflatoxin B 1, cyclophosphamide, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The parental V79 cell lines did not show clastogenic effects. Significant clastogenic effects were observed after an 18 h exposure to aflatoxin B 1 and cyclophosphamide in CYP2B1 expressing cells, to benzo[a]pyrene in CYP1A1 and CYP1A2 expressing cells, to 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine in cells, expressing CYP1A2 with or wothout acetyltransferase, and to cyclophosphamide in cells expressing both CYP1A2 and acetyltransferase. A significant sister chromatid exchange inducing effect was found after a 24 h exposure in each of the genetically engineered cell lines, except for benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in CYP2B1 expressing cells, and for benzo[a]pyrene in cells expressing both CYP1A2 and acetyltransferase. Thus, a battery of cell lines genetically engineered for metabolic competence may serve as a tool for investigating chromosomal changes induced by activated xenobiotics.

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 Chinese hamster cells.

A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed "growth inhibiting factor" (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity of GIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.

Increased resistance of the Chinese hamster mutant irsl cells to monofunctional alkylating agents by transfection of the E. coli or mammalian N3-methyladenine-DNA-glycosylase genes

Irs1 cells are mutants of the Chinese hamster cell line V79-4, and exhibit cross-sensitivity to various DNA-damaging agents, especially to the alkylating compounds methyl methanesulfonate and ethyl methanesulfonate. To test whether this sensitivity was due to the persistence of alkylated residues in DNA, we have transfected irs1 cells with the pMSG expression vector containing two coding sequences for enzymes of different origin, either the E. coli AlkA gene, coding for 3-methyladenine-DNA-glycosylase II, or rat APDG cDNA, encoding alkylpurine-DNA-glycosylase. The two coding sequences for the repair enzymes were ligated in the pMSG vector, under the control of the MMTV-LTR promoter, which is responsive to glucocorticoid regulation. The presence of the AlkA gene or of the APDG cDNA in the transfected cells was detected by Southern blot analysis and the transcription of these foreign sequences was checked by Northern hybridization of the cellular RNA. The transfected irs1 cells treated with [3H]dimethylsulfate removed the 3-methyladenine residues more efficiently from their DNA than the control cells. Irs1 cells harboring the AlkA or the APDG gene become about 2- and 3-fold more resistant to the toxic effect of methyl methanesulfonate, respectively. However, a 3-fold resistance to ethyl methanesulfonate was only observed in irs1 cells harboring the mammalian APDG cDNA.

Chromosomal aberrations induced in vitro by 3,7- and 3,9-dinitrofluoranthene

The chromosomal aberration test using a Chinese hamster cell line (CHL) was carried out with 3,7- and 3,9-dinitrofluoranthene (DNF) with and without exogenous metabolic activation (rat liver S9 mix). The highest dose tested was limited to 20 μg/ml because of the compounds' insolubility in dimethyl sulfoxide. Both DNFs induced chromosomal aberrations in the absence of S9 mix; the frequency was not very high. Results were reproducible, but without clear dose-response relationships. Neither DNF induced chromosomal aberrations in the presence of S9 mix. Both DNFs did not induce polyploid cells under any conditions.

Gene amplification and gene deletion are induced at different rates in V79/AP4 Chinese hamster cells.

The effect of mitomycin C (MMC) treatment on gene amplification and gene deletion induction in a V79/AP4 Chinese hamster cell line was investigated. Spontaneous and induced cellular variants resistant to N-phosphonacetyl-L-aspartate (PALA), which selects for carbamyl-P-synthetase, aspartate transcarbamylase and dihydroorotase (CAD) gene amplification events, and to intermediate concentrations of 2,6-diaminopurine (DAP), which selects for adenine phosphoribosyl transferase (aprt) gene deletion events, were isolated. The molecular analysis of spontaneous and induced PALA- and DAP-resistant clones showed that while in the former the CAD gene was amplified, in the latter both mutations and deletions occurred at the aprt gene. These results indicate that MMC favored the rise of gene amplification rather than gene deletion events, and suggest that aberrant DNA replication processes were responsible for the observed gene amplification.

Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.

Direct Selection for Mutations Affecting Specific Splice Sites in a Hamster Dihydrofolate Reductase Minigene

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.

A human cell line proficient in O6-methylguanine-DNA-methyltransferase and hypersensitive to alkylating agents.

The involvement of O6-methylguanine (O6-meGua) in mutagenesis is well established, while the toxic effect of these residues is still controversial. In this study, we compare the cytotoxicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) on three cell lines of different origin, which have different abilities to repair O6-meGua residues (Mer phenotype): a human hepatoma cell line (LICH cells, Mer+), a rat hepatoma cell line (H4 cells, Mer+) and a Chinese hamster cell line (CHO cells, Mer- phenotype). LICH and CHO cells show the same sensitivity to the killing effect of MNNG and MNU and are approximately 5-fold more sensitive than H4 cells. However, LICH and H4 cells share similar sensitivities to the toxic effect of 1,3-bis(2-chloroethyl)-1-nitrosourea. O6-meGua residues are removed at the same rate from the DNA of [3H]MNU-treated LICH and H4 cells, which also do not differ in the rate of removal of N3-methyladenine residues nor in overall DNA repair synthesis. The results suggest that MNNG and MNU produce a lethal lesion that is repaired by a process that does not involve the alkyltransferase.

Relationships between DNA damage and the survival of radiosensitive mutant Chinese hamster cell lines exposed to gamma-radiation: Part 2. Effect of cellular redox status.

To characterize further the nature of the defects underlying the differential radiosensitivities of the Chinese hamster ovary cell lines NM2, EM9, and UV41, we compared the abilities of anoxia and of the thiol WR-1065 to protect these mutants and their parent cell line, AA8, from the lethal effects of gamma-radiation. Wide differences in oxygen enhancement ratios (OERs) for cell killing were observed in the different cell lines, those for UV41 and NM2 cells (1.8 and 2.1, respectively) being reduced and that for EM9 cells (3.3) being slightly (although significantly) increased compared with wild-type AA8 cells (2.9). These OER data support the hypothesis that repair-deficient mutants are hypersensitive to radiation under redox conditions that favour the formation of the particular lesions that correspond to their repair defect, and also support the earlier suggestion that the underlying molecular bases of the radiosensitivity of EM9 and NM2 cells are very different. In contrast to protection by anoxia, a 30-min preirradiation treatment with WR-1065 (4 mmol dm-3) protected aerated AA8, EM9, NM2, and UV41 cells to a similar extent with respect to both cell killing and the efficiency of DNA double-strand break (dsb) induction as measured by neutral elution. This observation is in marked contrast with reports of a greatly reduced protection by thiols for some repair-deficient cell lines and with the above-mentioned anoxia data. Thus, the particular types of mutations characteristic of NM2, EM9, and UV41 cells that give rise to their unusual OERs have little impact on the ability of WR-1065 to modify either cell killing or dsb induction, supporting radiochemical evidence that the types of deoxyribose radicals modifiable by oxygen and thiols are qualitatively different. Furthermore, because the extent of protection of these CHO mutants by thiols and anoxia show no correlation, oxygen depletion cannot be a major component of protection of aerated cells by thiols under these conditions.

Mechanism of hyperoxia‐induced chromosomal breakage in chinese hamster cells

Exposure of cell cultures to hyperoxia, i.e., an atmosphere containing more than 20% O2, results in various genotoxic effects. The most prominent effect of hyperoxia is its clastogenicity. In this paper, earlier published data, obtained from research devoted to the mechanism of hyperoxia-induced clastogenesis, are reviewed. In addition, new data are presented concerning the hyperoxia-sensitivity of the DNA-repair deficient Chinese hamster cell lines xrs1, irs1, and EM9. None of these ionizing radiation-sensitive mutants showed hypersensitivity to hyperoxia, as measured by chromosomal aberration induction and loss of clonogenic cell survival. From the normal hyperoxia-sensitivity of xrs1, it may be concluded that DNA double strand breaks, of the type that are induced by ionizing radiation, do not play a role in chromosomal aberration formation by hyperoxia. In addition, since xrs1 is hypersensitive to drugs that inhibit topoisomerase II, it seems rather unlikely that exposure to hyperoxia affects topoisomerase II activity. Based on circumstantial evidence we hypothesize that perturbation of poly(ADP-ribose) metabolism may play a critical role in the mechanism of hyperoxia-induced clastogenesis.

Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test

The in vitro micronucleus (MN) test was carried out simultaneously with the conventional chromosomal aberration (CA) test on 11 clastogenic chemicals or spindle poisons with different modes of action using a Chinese hamster cell line (CHL). The method of slide preparation for the MN test was the same as that for the conventional metaphase analysis, except that 1% acetic acid in methanol was used as the cell suspension medium for air-drying (to preserve the cytoplasm around the nucleus). All chemicals tested induced micronuclei reproducibly and dose-dependently in good agreement with the results of metaphase analysis ( r = 0.99). Since the MN test methodology is simple and the observation of MN is less subjective than that of CA, we conclude that the in vitro MN test would be a good alternative to the conventional CA test for screening the genotoxicity of chemicals.

Characterization of adriamycin-resistant and radiation-sensitive Chinese hamster cell lines

A series of cell lines derived from Chinese hamster V79 cells by selection in increasing concentrations of Adriamycin® (ADRM) was developed to study the mechanisms of drug resistance and its relationship to radiation response. Survival studies revealed that selection in increasingly higher concentrations of ADRM positively correlated with increased cellular drug resistance. Increased cellular resistance correlated positively with amplification of the hamster multidrug-resistance gene ( pgp 1) as detected with dot blot analysis using the pCHP1 probe. Southern blot analysis of restriction endonuclease digested DNA ( Eco RI, Hind III, Pst I, or Bam HI) showed that (1) some fragments were preferentially amplified compared to others in the ADRM-resistant lines; and (2) no major gene rearrangement appeared to have occurred during the selection for greater ADRM resistance. Levels of pgp 1 gene expression assayed with dot blot and Northern analysis showed a parallel increase of mRNA with gene amplification and increased ADRM resistance. The amounts of the pgp 1 gene product, P-glycoprotein (P-gp), in the cell membrane of the ADRM-resistant cells correlated with the amount of gene amplification/expression. However, levels of P-gp only correlated with degree of drug resistance as measured by cell survival in earlier selection stages (77A and LZ-3). In later selection stages (LZ-8 and LZ-24), higher levels of ADRM resistance were achieved but levels of P-gp did not increase beyond -20% of plasma membrane proteins. These results suggest that (1) the LZ cell plasma membrane may have a physical limit as to the amount of P-gp it can accommodate and/or there is a cellular mechanism for regulating the amount of P-gp in the plasma membrane, and (2) additional resistance mechanisms are present in LZ-8 and LZ-24 cells. Microscopic observations of intracellular drug distribution in these cell lines revealed that (1) ADRM appeared to be sequestered in cytoplasmic vesicles, and (2) the amount of sequestration (number of vesicles) exhibited correlated with the degree of drug resistance attained by the cell lines. These results suggest that drug sequestration is another mechanism of resistance in LZ cells in addition to P-gp-mediated drug efflux.

DNA base sequence changes in spontaneous and ethyl methanesulfonate-induced mutations of a chromosomally-integrated gene in Chinese hamster ovary cells

A series of spontaneous and ethyl methanesulfonate-induced 6-thioguanine-resistant mutants were isolated in the CHO-10T5 cell line. This cell line was constructed by the introduction of a shuttle vector containing the Escherichia coli gpt gene into a hypoxanthine-guanine phosphoribosyltransferase deficient derivative of the Chinese hamster cell line CHO-K1. Shuttle vector sequences were recovered from many of the mutant cell lines by the COS cell fusion technique and the DNA base sequence of the gpt genes was determined whenever possible. The base sequences were determined for gpt genes recovered from 29 spontaneous mutants. Of these 29 mutants, 9 have single base substitutions, 1 has a small duplication, 17 have simple deletions, 1 has a deletion with additional bases inserted at the deletion site, and 1 has no change in the gpt coding sequence. Many of the deletions were less than 20 basepairs in length and several occurred in a region previously observed to be a hotspot for spontaneous deletions. The generation of the deletion/insertion mutation may have involved a quasi-palindromic intermediate. A total of 59 ethyl methansesulfonate-induced mutants were isolated and vector sequences were recovered from 50 mutants. All 50 mutants sequenced had single base substitutions and most (45) were G:C to A:T transitions. While there were no strong hotspots in this collection of mutations, the site distribution was obviously nonrandom. Many of the G:C to A:T transitions either produced a nonsense codon or occurred at glycine codons.