Studies on temperature-sensitive mutants of Chinese hamster ovary cells affected in DNA synthesis

DNA synthesis in two mutants of Chinese hamster overy cells,ts 13A andts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and thets lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in thets mutants as compared to the parental cells. Ints ^* revertants isolated from the mutants, thets phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a universal hybridizer (10).     

Isolation and characterization of nitrogen mustard-sensitive mutants of Chinese hamster ovary cells

Three nitrogen mustard-sensitive lines of Chinese hamster ovary cells were isolated from mutagenized cultures using the procedure of Thompson et al. (1980). The lines, designated NM1, NM2 and NM3, were 2.1-, 17- and 6.8-fold more sensitive to nitrogen mustard, respectively, than their parent, wild-type, line as determined by the dose required to kill 90% of the cells, IC₉₀. Patterns of cross-sensitivity to other DNA-damaging agents including ultraviolet light, cis-diamminedichloroplatinum, and other alkylating agents were determined for each line. Analysis of these results suggests that the phenotypes of the mutant lines are different from those lines reported previously.     

Host cell reactivation of irradiated adenovirus in UV-sensitive Chinese hamster ovary cell mutants

In this study we have utilized the ability of rodent cells toreplicate viral DNA following semi-permissive infection by humanadenovirus (Ad) to examine the host cell reactivation (HCR)of radiation-damaged Ad in several UV-sensitive Chinese hamsterovary (CHO) cell mutants. A significant reduction in HCR ofviral DNA synthesis for UV-irradiated Ad was detected in a seriesof UV-sensitive mutants from complementation groups 1–6derived from parental CHO-AA8 cells. HCR for UV-irradiated Adin these CHO mutants varied from 18.8 to 48.0% of that in parentalAA8 cells. However, a significant reduction in HCR of viralDNA synthesis for UV-irradiated Ad could not be detected inseries of UV-sensitive PV mutants from complementation groups1, 5, 9 and 10 derived from parental CHO-K1 cells, which harbourrelatively small DNA repair deficiences. We also report a reducedHCR for gamma-irradiated Ad in UV-sensitive CHO cell mutantsfrom groups 1 and 4 derived from parental CHO-AA8 cells. ThisHCR technique for DNA synthesis of Ad can be employed to measurethe DNA repair capacity of both human and rodent cells and extendedto examine the repair of DNA damaged by a variety of differentphysical and chemical agents. ³To whom correspondence should be addressed at: Departments of Biology and Radiology, Life Sciences Building, Room 434, McMaster University, Hamilton, Ontario L8S 4K1, Canada     

Isolation and characterization of Chinese hamster ovary cell mutants with altered sensitivity to high doses of tunicamycin

A mutant, CTM422, resistant to low dose of tunicamycin (TM) was isolated from Chinese hamster ovary (CHO) cells, and it showed 7- to 10-fold higher resistance to TM than CHO. We further mutagenized CTM422, to isolate TM-high-resistant mutants which were resistant to about 100-fold higher dose of TM than CHO. The TM-high-resistant mutants (N101 and N102) acquired about 4-fold higher cross-resistance to 2-deoxy-D-glucose than CHO or CTM422, while both CHO and CTM422 showed similar sensitivity to 2-deoxy-D-glucose. TM-low-resistance appeared to be codominant, and TM-high-resistance was partially codominant against TM sensitivity, respectively. The transfer activity of N-acetylglucosamine from UDP-N-acetylglucosamine into the lipid fraction with CHO and CTM422 cell extracats was inhibited by TM to a similar extent, while the extract of N102 cells showed about 10-fold higher resistance to TM than CHO or CTM422.     

Isolation and characterization of Chinese hamster ovary cell mutants resistant to the amino acid analogβ-aspartyl hydroxamate

Chinese hamster ovary cell lines which are resistant to an amino acid analog, -aspartyl hydroxamate, have been isolated and characterized. Mutants resistant to 100–150 M -aspartyl hydroxamate arose from ethyl methane sulfonate-treated parental lines at frequencies of 3.4×10^–6 to 1.3 ×10^–7. The mutants fell into at least two genetic classes: 18% of the mutants behaved codominantly in hybrids, the others recessively. Complementation studies indicated that all the recessive mutants belonged to the same class. Mutants selected after one step of mutagenesis overproduce the enzyme asparagine synthetase constitutively with four- to sixfold increases in specific activities over the basal levels of the parental lines. -Aspartyl hydroxamate-resistant cell lines with up to 20-fold elevations in asparagine synthetase activity have been isolated after two steps of mutagenesis. In addition, highly resistant lines have been selected by long-term growth of a dominant mutant in increasing concentrations of the drug. Resistance in the latter appears to be due not only to overproduction of asparagine synthetase but also to an alteration in the affinity of the enzyme for -aspartyl hydroxamate.     

Isolation, characterization and mutation analysis of PEX13-defective Chinese hamster ovary cell mutants.

We isolated peroxisome biogenesis mutants ZP128 and ZP150 from rat PEX2 -transformed Chinese hamster ovary (CHO) cells, by the 9-(1'-pyrene)nonanol/ultraviolet method. The mutants lacked morphologically recognizable peroxisomes and showed a typical peroxisome assembly-defective phenotype such as a high sensitivity to 12-(1'-pyrene)dodecanoic acid/UV treatment. By means of PEX cDNA transfection and cell fusion, ZP128 and ZP150 were found to belong to a recently identified complementation group H. Expression of human PEX13 cDNA restored peroxisome assembly in ZP128 and ZP150. CHO cell PEX13 was isolated; its deduced sequence comprises 405 amino acids with 93% identity to human Pex13p. Mutation in PEX13 of mutant ZP150 was determined by RT-PCR: G to A transition resulted in one amino acid substitution, Ser319Asn, in one allele and truncation of a 42 amino acid sequence from Asp265 to Lys306 in another allele. Therefore, ZP128 and ZP150 are CHO cell lines with a phenotype of impaired PEX13.     

Three new complementation groups of temperature-sensitive Chinese hamster ovary cell mutants defective in the endocytic pathway

We describe here the results of complementation studies with six mutant Chinese hamster ovary cells expressing temperature-sensitive lesions affecting the endocytic pathway. The mutants were crossed with representatives of the End1 and End2 complementation groups identified previously by Robbins et al. (J. Cell Biol. 99:1296–1308, 1984). Two mutants, G.8.1 and 31.1, were members of the End1 complementation group. One mutant, 25.2, was a member of the End2 complementation group. The other three mutants each defined new complementation groups, which we have designated End3 (mutant G.7.1), End4 (mutant V.24.1), and End5 (mutant 42.2). Previous work on mutants of the Endl, End2, and End3 classes had shown that these mutants were defective in endosomal acidification. We prepared postnuclear supernatants from mutants harvested at the nonpermissive temperature and compared their acidification activities, assessed by ATP-stimulated quenching of acridine orange. Members of the End1, End2, and End2 groups had reduced acidification activity, correlating with the acidification defects known to be expressed by these mutants. Strain V.24.1 (End4) also expressed a 40% reduction in acidification activity, while strain 42.2 (End5) had no reduction of acidification activity.     

二氢叶酸还原酶缺陷型中华仓鼠卵巢细胞偏爱密码子的研究

目的研究二氢叶酸还原酶缺陷型中华仓鼠卵巢细胞（CHO dhfr-）的偏爱密码子。方法建立CHO dhfr-细胞高丰度mRNA的cDNA文库，对该文库进行签定以获得合格的编码蛋白质的cDNA序列，统计密码子使用频率并与CUTG数据库中的中华仓鼠比较，同时对所获得的cDNA序列进行对应统计分析。结果获得了50个合格的CHO dhfr-细胞高丰度蛋白的cDNA序列，与中华仓鼠密码子使用频率比较，除了Pro，Arg外，其余氨基酸的最高使用频率的密码子在CHO dhfr-细胞和中华仓鼠中都相同。同时根据对应分析确定了能解释最多变量（14．7％）的第一主成分，确定了22个同义密码子为CHO dhfr-细胞偏爱密码子。结论CHO dhfr-细胞存在自身偏爱的密码子，提示密码子的偏爱性是不同哺乳动物细胞功能差异的因素之一，也为外源基因在CHO dhfr-细胞中通过优化密码子而获得高表达提供了新思路。     

Valinomycin-induced apoptosis in Chinese hamster ovary cells

Accumulating evidence endorses that excessive K(+) efflux is an ionic mechanism underlying apoptosis both in neuronal and non-neuronal cells. K(+) channels play important roles in mediating the pro-apoptotic K(+) efflux. Chinese hamster ovary (CHO) cells have been widely used for gene transfection experiments. These cells lack detectable endogenous voltage-gated K(+) channels. We were interested in knowing whether the absence of endogenous K(+) channels would render wild-type CHO cells more resistant to apoptotic death. We also wished to determine if direct stimulation of K(+) efflux would trigger apoptosis in these cells. Exposing CHO cells to hypoxia (1% O(2)) or to a typical apoptotic insult of serum deprivation for up to 24h did not affect cell survival. On the other hand, the K(+) ionophore valinomycin caused substantial cell death within 12h of its application. Valinomycin-treated CHO cells underwent several apoptotic events, including phosphatidylserine (PS) membrane translocation, caspase-3 activation, and mitochondrial membrane depolarization during the first few hours of exposure. Reducing K(+) efflux by elevating extracellular K(+) concentrations noticeably attenuated valinomycin-induced cell death. This study reinforces a K(+) efflux-mediated apoptotic mechanism in CHO cells and may help to explain the unique feature of their higher tolerance to apoptosis.     

Isolation and characterization of temperature-sensitive mutants of Chinese hamster ovary cells after treatment with UV and X-irradiation

The isolation of ten conditionally lethal temperature-sensitive mutants of the Chinese hamster ovary cell (CHO-Kl, pro^–) by the BUdR-visible light selection procedure is described. Treatment with radiation at doses known to cause single gene mutation in mammalian cells increases the mutation frequency by a factor of at least 14. These mutants will grow with normal plating efficiency at 34.5° but will not grow at 39.5°. Complementation analysis by two independent methods indicates that all mutants are recessive and allows the assignment of the mutants to six genetically independent complementation groups. Reversion analysis indicates that the TS-mutants are stable, spontaneous revertants arising at a frequency of <10^–6. Preliminary chromosome analysis revealed no systematic chromosomal abnormality in the mutants. Mitotic accumulation is used to study the generation time of the parental cells and representative mutants at 34.5° and 39.5°. The uses of these mutants for genetic analysis of mammalian cells in culture is discussed.     

Ouabain-resistant mutants of Chinese hamster ovary cells are not directly affected in Na+, K+-ATPase

The nature of biochemical lesions in a number of independent single-step ouabainresistant (Oua^R) mutants of Chinese hamster ovary cells has been examined. The cellular uptake of⁸⁶ Rb in the mutant cells was found to be resistant to inhibition by ouabain and correlated with the degree of resistance of the mutants to the drug. However, the plasma membrane Na⁺, K⁺-ATPase from all five of the Oua^R mutants examined showed similar sensitivity to inhibition by ouabain as seen for the enzyme from the parental sensitive cells. These results provide evidence that, contrary to the general assumption, Oua^R mutants of Chinese hamster ovary cells, which are widely used for genetic and quantitative mutagenesis studies, are not directly affected in the Na⁺, K⁺-ATPase.     

Induction and expression of mutations at multiple drug-resistance marker loci in Chinese hamster ovary cells

We have observed quantitative and qualitative differences in the mutability and mutagen-specificity of various drug-resistance marker loci in Chinese hamster ovary (CHO) cells, which suggest that mammalian gene loci may differ in their relative mutability by a given mutagenic agent. We have used the CHO-AT3–2 multiple-marker mutagenesis assay system to examine the dose-dependent induction and kinetics of expression of mutations at four well-characterized, drugresistance marker loci, after treatment with chemical agents which produce various types of DNA damage. The CHO-AT3–2 subline allows simultaneous quantitation and direct comparison of induced mutation frequencies at the hgprt, oua (Na+/K+ATPase), aprt, and tk loci. The agents tested in this study included ethyl methanesulfonate, methyl methanesulfonate, mitomycin C, ICR-191, benzo[a]pyrene, and dimethylnitrosamine. The expression kinetics and optimal expression times for each drug-resistance marker were determined in dose-response experiments in which cells from mutagen-treated populations were plated at 1-2-day intervals over a period of 10 days following mutagenesis. Comparison of induced mutation frequencies for each drug-resistance marker after mutagen treatments yielding equivalent cell survivals (equitoxic doses resulting in relative cell survivals of 0.37) revealed locus-specific differences in the relative mutagenicities of the agents tested. These results indicate that the apparent mutagenicity of a particular agent at a single genetic locus may not necessarily be an accurate indicator of that agent's mutagenic potential for the genome as a whole.     

Respiration-deficient Chinese hamster cell mutants: Genetic characterization

We present here genetic experiments with a series of Chinese hamster cell mutants defective in oxidative energy metabolism. The mutations were all shown to be recessive in intraspecies hybrids. Thirty-five mutants were sorted into eight complementation groups, but one of these mutants failed to complement representatives of two distinct complementation groups. The possibility was raised that this is a cell carrying two mutations or a deletion. Because of the greatly different frequencies with which such mutants could be isolated from two different Chinese hamster cell lines, CCL16 (DON) and V79, the stability of representatives from each cell line was examined, and it was found that revertants could be obtained after treatment with mutagens, while spontaneous revertants appeared at unmeasurable or extremely low frequencies, with one exception. The mutant with a very noticeable frequency of spontaneous reversion was defective in mitochondrial protein synthesis, and the question arose whether the mutation was on the mitochondrial genome. A detailed fluctuation analysis of reversion rate and comparison with rates for other mutations was consistent with a nuclear mutation. This conclusion was supported by experiments involving fusions with cytoplasts.     

Respiration-deficient Chinese hamster cell mutants: Biochemical characterization

We have previously classified 35 of our respiration-deficient mutants into seven complementation groups and one overlapping mutant which does not complement mutants from groups I and II. In this paper we report on the biochemical characterization of representatives of complementation groups I, II, VII, and the overlapping mutant. We show that these mutants all have a defect in complex I of the electron-transport chain. The general features of these mutants are: (1) a low rate of O₂ consumption in whole cells; (2) a low rate of release of¹⁴CO₂ from [2-¹⁴C]pyruvate, [1-¹⁴C]pyruvate, and [3-¹⁴ C]-hydroxybutyrate; (3) a low rate of release of¹⁴CO₂ from [5-¹⁴C]glutamate and [1-¹⁴C]glutamate in mutants from groups II, VII, and the overlapping mutant, whereas a significant amount of¹⁴CO₂ is released in mutants from group I; (4) a substantial rate of release of¹⁴CO₂ from [U-¹⁴C]aspartate; (5) in isolated mitochondria, succinate and -glycerol phosphate stimulate O₂ consumption whereas substrates which generate NADH, such as malate, do not; and (6) there is little or no rotenone-sensitive NADH oxidase activity in isolated mitochondria.     

Altered form of subunit 6 of mitochondrial ATP synthase complex in oligomycin-resistant mutants of Chinese hamster ovary cells

Using an antiserum generated against a synthetic peptide predicted from the DNA sequence of the ATPase 6 gene of the mitochondrial DNA, we demonstrate that mitochondria from two oligomycin-resistant Chinese hamster ovary cell lines with a defined mutation in the ATPase 6 gene synthesize an altered ATPase 6 gene product. This altered gene product migrates in sodium dodecyl sulfate-polyacrylamide gels as if it has a molecular mass that is larger by 1000 daltons than the wild-type ATPase 6 gene product. We also demonstrate that mitochondria from four other independently isolated oligomycin-resistant Chinese hamster ovary mutant cell lines contain a similar altered ATPase 6 gene product. These results suggest that all six oligomycin-resistant cell lines have a similar mutation in the ATPase 6 gene of the mitochondrial DNA that encodes subunit 6 of the ATP synthase complex.     

Genetic analysis of mitomycin C-hypersensitive Chinese hamster cell mutants

The genetic diversity of Chinese hamster cell mutants exhibitinghypersensitivity to the bifunctional alkylating agent mitomycinC has been examined. The eight mutants irs3, VH4, UV1, MC5,MMC1, MMC3, MMC4 and MMS2, are between 4- and 30-fold more sensitiveto mitomycin C than their respective wildtype parental lines.A number of the mutants show phenotypic similarities to culturedcells from the human cancer-prone syndrome Fanconi's anaemia.Hybrids were formed between pairs of mutants using the thioguanine/ouabainresistant (TOR) hybridization and hypoxanthine/aminopterin/thymidine(HAT)/ouabain selection system and the mitomycin C responseof pooled populations of hybrids assessed by constructing survivalcurves. In every case, hybrids formed between pairs of mutantsexhibited a mitomycin C response indistinguishable from thatof wildtype cells, indicating complemlenation. Therefore, theeight mutant lines examined represent eight distinct complementationgroups for mitomycin C-hypersensitivity. The results are incontrast to the complementation analysis of UV-sensitive Chinesehamster cell mutants and indicate that the response of mammaliancells to mitomycin C-induced DNA damage is complex and involvesa large number of genes.     

Biochemical genetics of the mammalian oxidative phosphorylation system: Analysis of the difference in the sensitivity of various Chinese hamster cell lines to inhibitors of the mitochondrial ATP synthase complex

Seven different Chinese hamster cell lines were found to vary greatly in their sensitivity to inhibitors of the mitochondrial ATPase. In plating-efficiency experiments, Chinese hamster lung V79 and bone marrow M3-1 cells were approximately 10,000-fold more resistant to oligomycin, 100-fold more resistant to efrapeptin, and 10-fold more resistant to ossamycin and leucinostatin than were ovary CHO or peritoneal B14 cells. In vitro experiments indicated that the increased resistance of V79 versus CHO cells to these inhibitors was due to an increased resistance of the mitochondrial ATPase. Heat-inactivation experiments indicated that there was a difference in the structure of the mitochondrial ATPase of V79 and CHO cells. Genetic experiments indicated that the difference in the sensitivity of V79 and CHO cells to inhibitors of the ATPase and the difference in the structure of the mitochondrial ATPase of V79 and CHO cells was due to a difference in both a nuclear and a cytoplasmic gene.     

Mechanisms determining sensitivity to cisplatin in three mutant Chinese hamster ovary cell lines

To gain insight into factors determining the response of tumours to cisplatin, we studied pathways involved in resistance to cisplatin: drug uptake, cytoplasmic detoxification and DNA repair, in three cisplatin-sensitive Chinese hamster ovary (CHO)² mutant cell lines. The mutant lines, CHO-MMC6, CHO-MMC1, CHO-MMS2, displayed inherent sensitivity to cisplatin (2.2, 4.1 and 10.6-fold, respectively) compared to the CHO-K1 line from which they were derived. CHO-MMS2 was the only mutant to show sensitivity to UV and this was slight (<2-fold). None of the mutants displayed increased sensitivity to X-irradiation. The CHO-MMS2 cell line appeared to have multiple mechanisms involved in its sensitivity to cisplatin, including increased drug accumulation, decreased levels of glutathione and a decreased capacity for DNA repair. The CHO-MMC1 mutant demonstrated reduced ability for DNA repair in a host cell reactivation assay, but no difference in drug accumulation or glutathione levels compared to the parent. The CHO-MMC6 cell line was not defective in any of the mechanisms studied. These three mutant cell lines demonstrate that similar mechanisms may account for inherent sensitivity or resistance to cisplatin, and suggest that multiple mechanisms may determine the sensitivity of human tumours to cisplatin.