Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells.

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.     

Kinetics of UV-induced changes in deoxynucleoside triphosphate pools in Chinese hamster ovary cells and their effect on measurements of DNA synthesis

Measurements of dNTP pools following exposure of Chinese hamster ovary cells to ultraviolet radiation reveals a rapid accumulation of cellular dTTP and a rapid loss of cellular dCTP. Exposure to 3-, 10- or 20 Jm-2 results in a 3-, 4- or 5.4-fold increase in cellular dTTP, respectively, within the first 10 min after exposure. dTTP levels then decrease noticeably, approaching the control value 3 to 5 hr later. In contrast, dCTP levels decrease rapidly within 10 min after exposure, ultimately to 1/10 that observed in the unirradiated control population. Recovery to normal dCTP levels is slow, taking in excess of 12 hr. No change in dATP is observed for 1-2 hr; subsequently, a moderate decrease in dATP levels occurs which is then followed by recovery, beginning 8 hr after irradiation. These results contrast with changes in dNTP pools observed in Chinese hamster V-79 cells exposed to mutagens. Measurements of rates of DNA synthesis by pulse-labeling cells with [3H]thymidine are also apparently affected by UV-induced transient deviations in the endogenous radiospecific activity of the labeled precursor.     

Mechanism of UV-induced deoxynucleoside triphosphate pool imbalance in CHO-K1 cells

Several laboratories have reported that exposure of cells to UV radiation results in a significant imbalance in deoxynucleoside triphosphate pool concentrations. In our CHO-K1 cells, a rapid drop in dCTP is accompanied by a rapid increase in dTTP. Examination of enzyme activities associated with synthesis/degradation of these molecules suggests that UV transiently enhances a putative dCTPase, dCMP deaminase and CdR kinase activities. This results in accumulation of excess dUMP which is probably converted to dTMP, then to dTTP. The absence of dCMP deaminase in V79 cells prohibits this rapid response in those cells. Moreover, significantly different dCMP deaminase activities were observed in CHO-K1 cells obtained from other laboratories, suggesting they, too, may respond differently to irradiation.     

Mutagen-induced changes in cellular deoxycytidine triphosphate and thymidine triphosphate in Chinese hamster ovary cells

Deoxynucleoside triphosphate concentrations in Chinese hamster ovary cell lines, CHO-K1 and Mut 8–16, were examined following exposure of cells to UV or dimethylsulfate. Marked decreases in dCTP were observed 2 hr after exposure to both mutagens. In contrast, dTTP concentrations increased with increased cell killing after exposure to UV but not after exposure to dimethylsulfate. Examination of DNA synthesis in permeabilized cells in the presence of excess concentrations of dNTP substrates suggests that excess dCTP enhances replication while excess of dTTP inhibits replication. We therefore ask whether the increase in the $\text{dTTP}\text{dCTP}$ ration in mutagenized whole cells either contributes to or prolongs induced inhibition of replication. In addition we proposed that such an induced dNTP imbalance may also contribute to an increase in mutations by enhancing the probability for base-misincorporation.     

The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.     

Stable alterations at the cell membrane of Chinese hamster ovary cells resistant to the cytotoxicity of phytohemagglutinin.

Chinese hamster ovary (CHO) cells selected for resistance to the cytotoxicity of phytohemagglutin (PHA) have been found to exhibit stable alterations at their plasma membranes. The PHA-resistant (PhaR) cells bind markedly less 125I-PHA than do sensitive CHO cells and also exhibit an increased sensitivity to the cytotoxicity of concanavalin A, a lectin of different receptor specificity. Mutagenesis with ethylmethanesulfonate increases the proportion of PhaR cells 20- to 100-fold. PHA-resistant cells maintained for up to 8 months in continuous culture in the absence of the selective agent have retained the PhaR phenotype. These and other characteristics of the experimental system suggest that CHO cells selected for PHA resistance are authentic somatic cell mutants. The Pha marker appears to behave recessively in hybrids formed between PhaR and PhaS cells.     

Chromosome-mediated gene transfer with the Chinese hamster ovary cell line

Using an improved method of chromosome-mediated gene transfer, we have investigated transfer of the codominantly expressed methotrexate-resistant dihydrofolate reductase (MtxRIIIdhfr) gene into Chinese hamster ovary (CHO) cell recipients. The frequency of dhfr gene transfer with CHO cells varied considerably from clone to clone, ranging from 4 X 10(-7) to 5 X 10(-5). Using appropriate cell recipients we were able to test for linkage of several genetic markers available in the CHO cell line. For example, the mutation resulting in the auxotrophic glyB-CHO cell line has been reported by others to be linked to the dhfr gene. However, we could not demonstrate cotransfer of these two markers when glyB- recipient cells were treated with MtxRIII chromosomes and transformant clones were selected for either methotrexate-resistance (MtxR) or glycine prototrophy. We conclude that these two genes are not closely linked in the hamster genome. However, the genes for thymidine kinase (tk) and galactokinase (gk), which are known to be linked in mammalian genomes, were found to cotransfer into CHO recipients with a frequency of about 50%.     

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.     

Characterization of a Chinese hamster ovary cell line resistant to uncouplers

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.     

Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells.

Six X-ray-sensitive (xrs) strains of the CHO-K1 cell line were shown to revert at a very high frequency after treatment with 5-azacytidine. This suggested that there was a methylated xrs+ gene in these strains which was structurally intact, but not expressed. The xrs strains did not complement one another, and the locus was autosomally located. In view of the frequency of their isolation and their somewhat different phenotypes, we propose that the xrs strains are mutants derived from an active wild-type gene. However, there is in addition a methylated silent gene present in the genome. Azacytidine treatment reactivated this gene. We present a model for the functional hemizygosity of mammalian cell lines, which is based on the inactivation of genes by de novo hypermethylation. In contrast to results with xrs strains, other repair-defective lines were found not to be reverted by azacytidine.     

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.     

A Chinese hamster ovary cell mutant resistant to aphidicolin

A triple (aphr ara-Ar and araCr) mutant (AP7) of Chinese hamster ovary cells resistant to DNA polymerase inhibitors is described. The aphidicolin-resistance of the mutant was stable and inherited as a dominant genetic trait. The DNA polymerase alpha from the wild type (aphs) and the mutant (aphr) cells differed in their elution profiles on DEAE-cellulose chromatography and in their molecular weights which were 192,000 for the wild type (CHO-K-1, AC6a) and 165,000 for the mutant (AP7) enzymes.     

DNA nucleotide excision-repair synthesis is independent of perturbations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [3H]TTP from [3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [3H]TTP pool was formed within 30 min of the addition of [3H]thymidine and remained relatively constant for the next 6 h. Addition of 2-10 mM hydroxyurea (HU) caused a gradual 2-4-fold increase in the [3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [3H]TTP pool size in cells exposed to 20 J/m2 and unirradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8-10-fold reduction in the size of the [3H]TTP pool relative to the initial studies. In the UV excision-repair studies the presence of hydroxyurea did not alter the specific activity of [3H] thymidine 5'-monophosphate incorporated into parental DNA due to repair replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair synthesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [3H]TTP pool.     

DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [³H]TTP from [³H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [³H]TTP pool was formed within 30 min of the addition of [³H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [³H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [³H]TTP pool size in cells exposed to 20 M/m² and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [³H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [³H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [³H]TTP pool.     

Surface polypeptides of the cultured Chinese hamster ovary cell.

The organization of the plasma membrane of logarithmically growing Chinese hamster ovary (CHO) suspension cells has been probed using surface label techniques in conjunction with subcellular fractionation and sodium dodecyl sulfate gel electrophoresis. Five components of apparent molecular weights 137,000, 121,000, 97,000, 67,000, and 57,000 have been shown to be exposed at the outer surface of the cell. These components fully meet the criteria of being (a) reactive with two or more surface label reagents, (b) enriched in a purified plasma membrane fraction, and (c) sensitive to proteolytic digestion of intact cells. Three other components of molecular weights 200,000, 44,000 and 30,000 are also reactive with certain surface label reagents, but fail to meet other criteria for cell surface components. Two polypeptides of molecular weights 180,000 and 37,000 are substantially enriched in the plasma membrane fraction, but are unreactive with surface label reagents. The organization of the CHO cell membrane and the applicability of surface label techniques to cultured cell systems are discussed.     

Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.     

Increased sensitivity of a Chinese hamster ovary cell line to alkylating agents after overexpression of the human metallothionein II-A gene.

The elevation of intracellular levels of metallothionein has been associated with the development of resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms underlying metallothionein associated drug resistance we transfected the alkylating agent sensitive CHO cell line MMC-1 (Robson et al., 1985) with an episomally replicating plasmid coding for the human metallothionein II-A gene. Two transfectants were isolated which carried about 10 copies of the plasmid. The basal expression of metallothionein was increased from undetectable levels in the recipient cell line MMC-1 to between 22 and 26 micrograms metallothionein per 10(7) cells in the transfectants. Treatment with cadmium salts increased metallothionein levels a further 1.7-fold. Cytotoxicity assays revealed that MTII-A transfection increased the sensitivity of the parental MMC-1 cells to N-methyl-N'-nitro-N-nitrosoguanidine and N-nitroso-N-methylurea. These results suggest that metallothionein does not act as a simple scavenger of alkylating agents, but interacts with unknown factor(s) in host cells.