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 and ts 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 the ts 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 the ts mutants as compared to the parental cells. In ts+ revertants isolated from the mutants, the ts phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.     

Inability of chlorophenols to induce 6-thioguanine-resistant mutants in V79 Chinese hamster cells

The induction of mutation at the hypoxanthine-guanine phosphoribosyl transferase locus and cytotoxicities of 6 different chlorophenols (2,4- and 2,6-dichlorophenol, 2,4,5- and 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol) were examined in V79 Chinese hamster cells without exogenous metabolic activation. The chlorophenols were cytotoxic to V79 cells, but failed to produce significant increases in the frequency of 6-thioguanine-resistant mutants.     

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.     

Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells

We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes--beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase--was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta-glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.     

A temperature-sensitive auxotropic variant of Chinese hamster ovary cells

We used a conventional procedure involving treatment with 5-bromodeoxyuridine and visible light to isolate a stable, temperature-sensitive, auxotrophic variant-TsNd-6-from its parental Chinese hamster ovary cell clone K₁. At the nonpermissive temperature of 39.5 °C, TsNd-6 requires thymidine, hypoxanthine, and glycine for growth. Folinic acid can substitute for hypoxanthine and glycine at the elevated temperature.     

X-ray induction of 6-thioguanine-resistant mutants in division arrested, G0/G1 phase Chinese hamster ovary cells

The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G₀/G₁ phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50–800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period of 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10–12 h. Cells irradiated at 5 h after serum addition, i.e. in G₁, show a similar does response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G₁, S or G₂, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G₀/G₁ phase allow more time for such interactions in the absence of semiconservative DNA replication.     

Recovery of function in Chinese hamster ovary cell mutants with temperature-sensitive defects in vacuolar acidification

After 4 h at 41 degrees C, B3853 and M311, temperature-sensitive Chinese hamster ovary cell End1 and End2 mutants, respectively, are pleiotropically defective in endocytosis and trans-Golgi network-associated activities (Roff, C. F., R. Fuchs, I. Mellman, and A. R. Robbins. 1986. J. Cell Biol. 103:2283-2297). We have measured recovery of function after return to the permissive temperature. Based on return of normal transferrin-mediated Fe uptake and sensitivity to diphtheria toxin both mutants had restored endosomal function at 10 h; based on delivery of endocytosed lysosomal enzymes to lysosomes and normal sensitivity to modeccin both had functional late endocytic organelles at 10-12 h; and based on retention of newly synthesized lysosomal enzymes and sialylation of secreted glycoproteins both had functional trans-Golgi network at 6 h. At 10 h, M311 had recovered almost all of its ability to endocytose lysosomal enzymes; B3853 required 30 h to recover fully its ability to endocytose lysosomal enzymes. Slow recovery of mannose 6-phosphate-dependent uptake in B3853 reflected altered trafficking of cation-independent mannose 6-phosphate receptors. Although B3853 had normal amounts of receptor at 6-8 h, it had greatly diminished amounts of receptor at the cell surface. Altered trafficking was also suggested by the finding that B3853 rapidly degraded receptor that had been present before the shift to the nonpermissive temperature.     

Inability of diethylstilbestrol to induce 6-thioguanine-resistant mutants and to inhibit metabolic cooperation of V79 Chinese hamster cells

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01–10 μg/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N′-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5–7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.     

Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells

Cytotoxicity, chromosome aberrations, and mutations to 6-thioguanine resistance were synergistically increased by incubating the ultraviolet light (UV)-irradiated Chinese hamster ovary (CHO) cells in medium containing sodium arsenite. However, the frequencies of sister-chromatid exchanges and mutations to ouabain resistance induced by UV were not synergistically increased by sodium arsenite. The synergistic effect of sodium arsenite on UV-induced chromosome aberrations varied with cell-harvesting time and decreased with increasing time intervals between UV and sodium arsenite treatments.     

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 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 degrees but will not grow at 39.5 degrees. 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 less than 10(-6). Preliminary chromosome analysis revealed no systematic chromasomal abnormality in the mutants. Mitotic accumulation is used to study the generation time of the parental cells and representative mutants at 34.5 degrees and 39.5 degrees. The uses of these mutants for genetic analysis of mammalian cells in culture is discussed.     

Fluctuation analyses of spontaneous mutations to 6-thioguanine resistance in Chinese hamster ovary cells in culture

Fluctuation analyses of the spontaneous appearance of 6-thioguanine (TG)-resistant mutants in cultured Chinese hamster ovary (CHO) cells were performed to investigate (1) whether the resistance is induced by the selective agent or is the result of a mutation which occurs prior to the TG selection and (2) to estimate the spontaneous mutation rate at the hypoxanthine—guanine phosphoribosyl transferase (hgprt) locus. The potential problem of phenotypic delay was minimized by allowing an adequate expression time through maintenance of the cultures in a division-arrested, viable state. The results demonstrate that the TG-resistant (TG^r) cells arise randomly in the cultures, independently of the selective agent, which is consistent with spontaneous mutations. The average values for mutation rate ± standard deviation, based on 4 independent determinations and 2 methods of calculation, are 3.4 ± 1.2 × 10^?7 (median method) and 5.1 ± 1.8 × 10^?7 (mean method) mutants/cell/generation.     

Induction of mutations resistant to 6-thioguanine by fast neutrons in cultured Chinese hamster cells

A study was made of induction of mutations, resistant to 6-thioguanine (TGr), and reproductive death of Chinese hamster cells after irradiation by fission-spectrum fast neutrons (mean energy of 0.75 MeV) with doses of 10-130 cGy. A high relative biological effectiveness (RBE) of fast neutrons was shown. The maximum RBE values (13-16) were within the dose range inducing minimum mutagenic and lethal effects. RBE decreased with the dose increase. Inspite of high mutagenic effectiveness of neutrons, estimated according to TGr mutation frequency per cell per dose unit, their relative mutagenic effectiveness, estimated per cell per one lethal event, did not substantially differ from that of X-radiation.     

Inverse dose-rate effect for the induction of 6-thioguanine-resistant mutants in Chinese hamster V79-S cells by 60Co gamma rays

Chinese hamster V79-S cells capable of growing in suspension culture were exposed to 60Co gamma rays at a high dose rate (84 Gy/h), low dose rates (200, 50, and 39 mGy/h), and a spectrum of very low dose rates (between 29 and 4.5 mGy/h). Following time for appropriate expression the cultures were assayed for the induction of 6-thioguanine-resistant mutants. For a given dose, a decrease in mutation induction occurred as the dose rate was reduced from high dose rates to low dose rates. However, further reduction in dose rate resulted in a reverse dose-rate effect, and an increase in the frequency of mutants was observed. The contribution of background mutation frequency to this reverse dose-rate effect was studied, both by examining fluctuations of mutation frequency in nonirradiated culture and by its impact upon the dose-rate-independent nature of the reversed effect, and it was found to be negligible. The physiological state of the suspension culture under periods of protracted exposure to very low dose rates was also investigated. The effect of doubling time, plating efficiency, cell cycle distribution, and sensitivity on survival and mutation were examined. In no case was a change apparent during the very low-dose-rate exposures. The results are discussed in terms of the possible expression of cryptic radiation damage after prolonged culture times and/or the involvement of an error-free repair system which requires a certain amount of radiation damage to become active.     

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.     

Myristic acid utilization in Chinese hamster ovary cells and peroxisome-deficient mutants.

Chinese hamster ovary (CHO) cells convert [9,10-3H]myristic acid ([3H]14:0) to several lipid-soluble, radioactive metabolites that are released into the medium. The main products are lauric (12:0) and decanoic (10:0) acids. Some of the 12:0 formed also is retained in cell lipids. Similar metabolites are not synthesized from palmitic (16:0), oleic (18:1), or arachidonic (20:4) acids, and the addition of these fatty acids does not reduce the conversion of [3H]14:0 to 12:0. Two peroxisome-deficient CHO cell lines do not convert [3H] 14:0 to any polar metabolites, but, they elongate, desaturate, and incorporate [3H]14:0 into intracellular lipids and proteins normally. While BC3H1 muscle cells convert some [3H]14:0 to 12:0, they also produce at least nine lipid-soluble polar products from [3H]12:0. These findings suggest that a previously unrecognized function of myristic acid is to serve as a substrate for the synthesis of 12:0, which can be either secreted into the medium or converted to other oxidized metabolites. The absence of this peroxisomal oxidation pathway, however, does not interfere with other aspects of myristic acid metabolism, including protein myristoylation.     

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.     

X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining

Six CHO mutants have previously been described as being sensitive to ionizing radiation and bleomycin treatment, with little or no cross sensitivity to UV-radiation (Jeggo and Kemp, 1983). Their ability to rejoin single- and double-strand breaks has been examined here. Using two techniques, gradient sedimentation and alkaline elution, no difference could be observed between wild-type and mutant strains in the initial number of single-strand breaks induced, the rate of rejoining, or the final level of single-strand breaks rejoined. Thus, a major inability to rejoin single-strand breaks is not the basis for sensitivity in these mutants. In contrast, all 6 mutants showed a decreased ability to rejoin the double-strand breaks induced by gamma-irradiation as measured by neutral elution. Rejoining of half of the breaks occurred in 37 min in wild-type cells and reached a maximum level of 72% after 2 h. All the mutants showed a decreased rate of rejoining, and the final level was 17% of that observed in the wild-type in the most defective mutant, and ranged from 35 to 69% in the other 5 mutants. These are the first mammalian cell mutants to be described with a defect in double-strand break rejoining.     

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.     

Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells.

6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.