X-ray-sensitive mutants of Chinese hamster ovary cell line isolation and cross-sensitivity to other DNA-damaging agents

A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D₁₀ values 5–10-fold of wild-type D₁₀ value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D₁₀ values less than 2-fold of wild-type D₁₀ value).The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks.     

Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC much greater than EMS greater than UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS greater than UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants.     

Radiation-induced chromosome damage in X-ray-sensitive mutants (xrs) of the Chinese hamster ovary cell line

The frequency of both spontaneous and X-ray- (95 rad) induced cytogenetical aberrations has been determined for 2 X-ray-sensitive strains (xrs-6 and xrs-7) of the Chinese hamster ovary cell line, and their wild-type parent (CHO-K1). Increased levels of spontaneous aberrations were not a general feature of the xrs strains, although xrs-7 did show a 2-fold increase in chromatid gaps. Unsynchronied populations of xrs cells, estimated to have been irradiated in late S and G2, showed a 3-5-fold increase in chromatid gaps, breaks and exchanges compared to CHO-K1. The irradiation of synchronised populations of xrs-7 and CHO-K1 in G1 demonstrated a 3-5-fold increase in chromosome breaks, gaps and exchanges in xrs-7. In addition xrs-7 displayed a large increase in chromatid-type aberrations, particularly triradials. These X-ray-sensitive strains have previously been shown to have a defect in double-strand break rejoining (Kemp et al., 1984), and an increased number of double-strand breaks (DBSs) remain in their DNA after irradiation compared to wild-type cells. The increased number of DSBs remaining in these strains 20 min after irradiation, correlates well with the increase in chromosome breaks.     

Differential gene expression in wild-type and X-ray-sensitive mutants of Chinese hamster ovary cell lines.

Complementary DNA cloning, differential screening and Northern hybridization techniques were used to study differential gene expression in the wild-type Chinese hamster ovary (CHO) K1 cell line and its two X-ray sensitive mutants, xrs-5 and xrs-6. 11 species of mRNAs were found underexpressed in the two independently isolated mutants. The steady-state levels of those mRNAs are 3-26-fold less in the two mutants, depending on the particular species. 6 of the underexpressed mRNAs have been identified by comparing the sequences of the cloned cDNAs to the known sequences in GenBank. 4 of them code for the structural proteins of ferritin heavy chain, nonmuscle myosin light chain 3nm, ribosomal protein S17 and L7, respectively. The other two have strong homology with mouse B2 or retroviral sequences. The remaining 5 mRNAs did not show significant homology with any of the known sequences and apparently represent newly isolated species. The effect of 137Cs gamma-rays on the expression of the 11 mRNAs has been studied. Radiation inhibited the expression of the B2-like gene in the mutants but not in the wild-type CHO cells. The levels of the other 10 mRNAs were not affected by radiation. The underexpression of this group of genes in both xrs-5 and xrs-6 mutants seems to be related to their radiation-sensitive phenotype, although the specific gene responsible has not been identified. Two models are proposed to explain the mechanism of underexpression. It is suggested that a cellular factor or/and chromosome structural changes are involved.     

Induction of beta-polymerase mRNA by DNA-damaging agents in Chinese hamster ovary cells.

Only a few of the genes involved in DNA repair in mammalian cells have been isolated, and induction of a DNA repair gene in response to DNA damage has not yet been established. DNA polymerase beta (beta-polymerase) appears to have a synthetic role in DNA repair after certain types of DNA damage. Here we show that the level of beta-polymerase mRNA is increased in CHO cells after treatment with several DNA-damaging agents.     

Isolation of Chinese hamster ovary cell pex mutants: two PEX7-defective mutants

We searched for novel Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by an improved method using peroxisome targeting signal 2 (PTS2)-tagged enhanced green fluorescent protein (EGFP). From mutagenized TKaEG2 cells, the wild-type CHO-K1 stably expressing rat Pex2p and PTS2-EGFP, cell colonies resistant to the 9-(1(')-pyrene)nonanol/ultraviolet treatment were examined for intracellular location of PTS2-EGFP. Of six mutant cell clones two, ZPEG227 and ZPEG231, showed cytosolic PTS2-EGFP, indicative of impaired PTS2 import, and numerous PTS1-positive particles. PEX7 expression restored the impaired PTS2 import in both mutants. Cell fusion with fibroblasts from a patient with PEX7-defective rhizomelic chondrodysplasia punctata did not complement PTS2 import defect of ZPEG227 and ZPEG231, confirming that these two are pex7 mutants. Mutation analysis of PEX7 by reverse transriptase (RT)-PCR indicated that ZPEG227-allele carried an inactivating nonsense mutation, Trp158Ter. Therefore, ZPEG227 is a pex7 mutant possessing a newly identified mutation in mammalian pex7 cell lines.     

Genetic analysis of mitomycin-C-sensitive mutants of a Chinese hamster ovary cell line

5 mutants of a Chinese hamster ovary (CHO) cell line, which exhibit similar levels of sensitivity to killing by mitomycin C, have been analysed genetically to determine whether they represent one or more genetic complementation groups. Hybrids were constructed by fusing cells carrying either the neo or the Ecogpt marker and selecting in medium containing G418 and mycophenolic acid. Selectable markers were introduced into the cells by DNA transfection using pSV5-neo or pSV5-gpt, which represents a quick and convenient method for generating resistant derivatives. Hybrids generated by crosses between any one mutant and the parental cell line exhibited near wild-type resistance to mitomycin C, indicating that the mutants are phenotypically recessive. Self-cross hybrids for all 5 mutants had D37 values for killing by mitomycin C of between 20 and 30 ng/ml. The values obtained for crosses between different mutants were 60-105 ng/ml, with the exception of 1 pairing which gave a value of 33 ng/ml. These results indicate that that the mutants represent at least 4 different genetic complementation groups, suggesting that cellular resistance to mitomycin C is mediated via a number of different mechanisms.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.     

Isolation and characterization of temperature-sensitive mutants in a Chinese hamster cell line

A replica plating method was used for the isolation of temperature-sensitive (ts) mutants after treatment of Chinese hamster cells with ethyl methanesulfonate (EMS). No significant increase in ts mutants was found after this treatment. The limitations and advantages of the replicating procedure to detect such differences, as well as an alternative method, are discussed.Mutants isolated were classified into two general groups—density-dependent and clear-cut—as measured by survival at low and high cell densities at the restrictive temperature. The density-dependent mutants may be truly “leaky”, losing a metabolite to the medium at an excessive rate at the restrictive temperature. On the other hand, the one clear-cut mutant analyzed extensively dies at a rate determined by its ability to utilize one or more components from the medium. It shows an inverse density relationship in rate of death, as inferred from rates of macromolecular synthesis, as opposed to its growth rate at the permissive temperature.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors

Summary Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase ase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent K _m for dCTP and the apparent K _i for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.Abbreviations ara-A 9--d-arabinofuranosyl adenine - ara-C 1--d-arabinofuranosyl cytosine - aph aphidicolin     

Isolation of NPC1-deficient Chinese hamster ovary cell mutants by gene trap mutagenesis

Chinese hamster ovary cell mutants defective in the NPC1 gene (NPC1-trap) were generated by retrovirus-mediated gene trap mutagenesis from a parental cell line JP17 expressing an ecotropic retrovirus receptor. Insertion of the gene trap vector in the NPC1 gene and the absence of the gene product were verified by 5'RACE and immunological analyses, respectively. NPC1-trap cells showed intracellular accumulation of low-density lipoprotein (LDL)-derived cholesterol and had an increased level of unesterified cellular cholesterol. Cholesterol biosynthesis through the mevalonate pathway was upregulated in the mutant cells as assessed by [(14)C]acetate incorporation into cellular sterols. When JP17 cells were depleted of lipoproteins and then loaded with LDL, cell surface LDL receptors were promptly downregulated and the mature form of the sterol regulatory element-binding protein-1 disappeared from the nucleus. These responses to LDL were obviously retarded in NPC1-trap cells, suggesting an impaired response of the cholesterol-regulatory system to LDL. NPC1-trap cells will be a useful tool to study the regulation of cellular cholesterol homeostasis and the pathogenesis of Niemann-Pick disease type C.     

Isolation of Chinese hamster ovary cell mutants requiring the continuous presence of taxol for cell division

Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two-dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor.     

Chinese hamster ovary cell mutants defective in myo-inositol transport

By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.     

Gamma-ray- and UV-sensitive strains of a radioresistant cell line: isolation and cross-sensitivity to other agents

Two gamma-ray-sensitive and two ultraviolet (UV)-sensitive variants were isolated from the gamma-ray- and UV-resistant TN-368 lepidopteran insect cell line. The isolation was performed by inducing mutations in the TN-368 cells using ethyl methanesulfonate, growing them for an expression period, irradiating with 137Cs gamma rays or 254-nm UV radiation, allowing cells to incorporate 5-bromodeoxyuridine (BrdU) in the presence of hydroxyurea (DNA repair synthesis), and finally irradiating with 365-nm UV radiation to cause DNA strand breakage at sites of BrdU incorporation with the intent of killing those cells that have undergone DNA repair synthesis and sparing those cells which, for a variety of reasons, did not. The survival of the Cs2 and Cs7 variants exposed to X rays is significantly different from the parent TN-368 line at the P less than 0.0001 level. The survival of the UV10 and UV19 variants exposed to UV radiation is different from the parent at the P less than 0.0001 and P less than 0.003 levels, respectively. In cross-sensitivity testing of the gamma-ray-sensitive variants, only Cs2 is more sensitive to 254-nm UV and only Cs7 is more sensitive to 44 degrees C heating; both are sensitive to PUVA. The UV-sensitive mutants are both sensitive to X irradiation, PUVA, and mitomycin C. However, UV10 is not sensitive to 44 degrees C heating while UV19 is, making UV19 the only variant strain sensitive to all agents examined. Despite the isolation procedure which was intended to select for DNA repair-deficient cells, the results suggest that a more general mechanism is responsible for the sensitivity of the variant cells to the agents tested.     

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.     

Isolation and preliminary characterization of bleomycin-resistant mutants from Chinese hamster ovary cells

Stable mutants resistant to an anticancer antibiotic, bleomycin-A2, were selected in Chinese hamster ovary (CHO) cell either spontaneously or after ethylmethane sulfonate mutagenesis. Fluctuation analysis showed that bleomycin resistance occurs in CHO at a rate of 6.50--6.58 x 10(-7) mutations per cell per generation. Bleomycin-A2-resistant cell lines exhibited increased resistance to bleomycin analogs--bleomycin-A5, -B2, -B4, and pepleomycin. Colchicine, mitomycin C, and ultraviolet light irradiation inhibited colony formation equally in CHO cells and in bleomycin-resistant mutants. Cell-cell hybridization tests showed that bleomycin-resistance behaves as a dominant trait. Bleomycin-inactivating activity in the mutant cell extracts was three to fourfold higher than that in extracts of the parental CHO cell.     

Isolation and characterization of tunicamycin resistant mutants from Chinese hamster ovary cells

Stable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross-resistance for ConA but are slightly cross-resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.