Chinese Hamster Lung Cell Line Research Articles

Mechanisms affecting peplomycin sensitivity of Chinese hamster cell lines.

Chinese hamster lung cell line V79 was ca. 13 times more resistant to peplomycin (PEP), and 6 times more resistant to bleomycin (BLM)-A2 than Chinese hamster ovary (CHO) cell line. The natural resistance of V79 cells to PEP or BLM was attributed to higher levels of BLM hydrolase activity and lower cellular uptake of the antibiotic. The sensitivity to PEP of a mutant clone CHO/O-2 T-1 was similar to that of CHO. A hybrid clone of CHO/O-2 T-1 X V79 showed an intermediate sensitivity to PEP between those of both parental cell lines, suggesting that the gene responsible for the natural resistance to PEP appears codominantly in the hybrid. The BLM hydrolase activity of the hybrid was also found intermediate between those of both parental cells. Mutant clones CHO/O-2 T-5 and CHO/O-2 T-6 were 8.3-9.0 times more sensitive to PEP than CHO cells. Hybrid clones CHO/O-2 T-5 X V79 and CHO/O-2 T-6 X V79 displayed PEP sensitivity similar to that of V79, suggesting that the gene responsible for the PEP supersensitivity (PEPss) behaves recessively in the hybrids. Both PEPss clones showed levels of BLM hydrolase and cellular uptake of [3H]PEP similar to the parental CHO cells, suggesting that the PEPss is due to neither BLM hydrolase nor cellular uptake of the antibiotic. Increased PEP-induced DNA cleavage and decreased DNA repair in the PEPss clones were demonstrated by alkaline sucrose density gradient sedimentation method. The results suggest that the PEPss of these mutant clones is attributed to decreased DNA-repairing activity and/or increased DNA-breaking activity.

Analysis of a Chinese hamster temperature-sensitive cell cycle mutant arrested in early S phase

E36 ts24 is a temperature-sensitive cell cycle mutant which has been derived from the Chinese hamster lung cell line E36. This mutant is arrested in phase S when incubated at the restrictive temperature (40.3 °C) for growth. At this temperature, proliferation of the mutant cells ceases after 10 h. About 2 h earlier, DNA synthesis is arrested. These kinetic studies indicate that the execution point of the mutant cells is in early S phase well beyond the G1/S boundary. The pattern of replication bands in E36 ts24 cells grown for 9 h at 40.3 °C strengthen the kinetic studies and map the execution point to early S phase. The exact point of arrest of the mutant cells in phase S was mapped in early S phase near the execution point. At the point of arrest the cells continue to synthesize DNA at a high rate but practically all of the newly synthesized DNA is degraded. This high rate of DNA degradation is limited to nascent DNA at the point of arrest. In the presence of 5-bromodeoxyuridine (5-BudR), the last E36 ts24 cells which reach mitosis at the restrictive temperature for growth show asymmetric replication bands which illustrate DNA degradation and resynthesis occurring in these cells at 40.3 °C.

Partial purification and characterization of the mRNA complementing a temperature-sensitive S-phase cell cycle mutation.

E36ts24 is a temperature-sensitive (ts) cell cycle mutant derived from the Chinese hamster lung cell line E36. At the restrictive temperature for growth (40.3 degrees C), the mutant cells are arrested at early S phase. We have microinjected poly(A)+ RNA isolated from the wild-type E36 cells into the cytoplasm of E36ts24 cells arrested at early S phase after 24 hr of incubation at 40.3 degrees C. The ts mutation was transiently complemented in a significant fraction of the microinjected cells as evidenced by the incorporation of [3H]thymidine assayed by autoradiography. Microinjection of mRNA fractionated by methylmercuric hydroxide/agarose gel showed that the mRNA capable of transiently complementing the mutation in E36ts24 contains about 940 nucleotides. Hence, it can code at most for a protein containing about 230 amino acids. We estimate that the partial purification by fractionation of the mRNA active in the transient complementation is on the order of 100- to 200-fold.

Rat liver homogenate (S9)-mediated binding of benzo[ a]pyren to DNA in V79 cells, V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[ a]pyrene (B[ a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[ a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy- t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[ a]P ( anti-B[ a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[ a]pyrene (9-OH-B[ a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[ a]PDE-DNA adduct to the 9-OH-B[ a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[ a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[ a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[ a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[ a]P in the presence of the S9 activation system. These results demonstrate that the relative abilities of various reactive bbenzo[ a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.

Isolation and characterization of vitamin A-sensitive Chinese hamster lung cell lines.

Retinyl acetate (RA)-sensitive variants (RAs-2 and RAs-3) of V79 cell line were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The variants were stable and showed a 3- to 4-fold increase in sensitivity to RA compared to parental V79 cells. The RAs-2 clone was also sensitive to retinol and retinol palmitate. The RA-sensitivity behaves as a recessive trait in all hybrids of RAs-2 and V79. A number of physiological parameters were indistinguishable in V79 and RAs-2 cells, including the extent of uptake of [3H]retinol, the release of K+ from the cells induced by RA, and the levels of retinol and retinoic acid binding proteins. However, one possible correlation with the RA-sensitive phenotype was observed: Gomori acid-phosphatase staining of RA-treated RAs-2 and V79 cells indicated that lysosomal membrane of RAs-2 cells was more labile than those of the parental V79 cells.

Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid

Interspecific somatic cell hybrids were constructed between a Chinese hamster lung cell line deficient in hypoxanthine phosphoribosyltransferase and two lymphoblastoid cultures (GM 4025 and GM 3200) from unrelated males affected with the fragile (X) syndrome. Thirteen independent colonies survived selection in hypoxanthine-azaserine, while only one colony survived selection in hypoxanthine-aminopterin-thymidine. One hybrid formed from GM 4025 was found to contain a human X chromosome as the only detectable human chromosome in the majority of cells analyzed. Induction of fragile (X) expression in this hybrid at frequencies up to 20% was achieved by treatments with 5-fluoro-2'-deoxyuridine (5 X 10(-8) M or 1 X 10(-7) M) or methotrexate (5 X 10(-6) or 1 X 10(-5) for 12 h. Use of the somatic cell hybrid system may allow study of the fragile (X) from different patients on a homogeneous xenogeneic background and may provide a better system for characterization of the fragile (X) at the biochemical and molecular level.

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

Biochemical evidence for multiple independent emetine resistance genes in Chinese hamster cells.

Hybridization-complementation studies indicated that mutations in multiple genes can render Chinese hamster cells resistant to the alkaloid translation inhibitor emetine. Two of the genes, emtA and emtB, recognized in Chinese hamster lung and ovary cell lines, respectively, are known to affect the ribosomes of the cells directly. Although mutations in a third gene, emtC, affect the translation apparatus of Chinese hamster peritoneal cells in vitro (Wasmuth et al., Mol. Cell. Biol. 1:58-65, 1981), the molecular product of the emtC locus remains to be determined. To study the molecular basis for genetic complementation among emetine-resistant Chinese hamster cell mutants, we analyzed ribosomal proteins elaborated by complementing, emetine-sensitive hybrid clones (EmtB X EmtA and EmtB X EmtC) and by emetine-resistant clones that segregated from the hybrids. The electrophoretic forms of ribosomal protein S14 (the emtB gene product) elaborated by these clones indicated that the EmtA and EmtC phenotypes are independent of the emtB locus and that the emtA and emtC loci are not chromosomally linked to emtB.

Isolation by tritium suicide of uridine-cytidine kinase-defective mutants in Chinese hamster V79 cells

Tritium suicide was shown to be a highly effective method for isolating mutants defective in uridine-cytidine kinase in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of three kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]uridine for 10 min, followed by storage of 3H-labelled cells at −70 °C for 4–7 days. Nine clones that survived the third kill cycle were tested for incorporation of [ 3H]uridine and for uridine kinase activity in extracts. Eight of these clones were defective in whole-cell uridine incorporation and in uridine kinase activity. A kinetic study was made on the uridine-cytidine kinase activity of three of the mutants. The apparent V max of the mutants was reduced approx. 10-fold when either uridine or cytidine was used as substrate. In contrast, the apparent K m of uridine was reduced approx. 12-fold in the mutants with only a 2-fold (probably insignificant) reduction in K m's for cytidine or for ATP.

Gene amplification accompanies low level increases in the activity of dihydrofolate reductase in antifolate-resistant Chinese hamster lung cells containing abnormally banding chromosomes.

Three independently-derived, antifolate-resistant Chinese hamster lung cell lines that exhibit low level increases in dihydrofolate reductase (DHFR) activity, i.e., three- to fivefold vs. controls, have been compared with drug-sensitive cells to determine relative DHFR gene content. With a solution hybridization technique that makes use of genomic DNA and a cloned double-stranded Chinese hamster DHFR cDNA probe, it has been found that the enzyme activity increases are associated with an approximately proportionate amplification of DHFR genes. Trypsin-Giemsa staining techniques and hybridizations in situ further show that the amplified DHFR genes are located within abnormally banding regions along chromosome 2q and also suggest that, in each subline, only one chromosome 2 homolog is initially involved in the amplification process.

Mutagenesis by chemical agents in V79 Chinese hamster cells: A review and analysis of the literature: A report of the gene-tox program

This report reviews and evaluates the current literature (about 125 primary publications) on chemically induced specific locus mutations in the V79 Chinese hamster lung cell line. The V79 cell is convenient to use for mutagenesis studies since f it has a rapid growth rate, high plating efficiency, and a stable karyotype. Mutation can be easily measured at either the hypoxanthine-guanine phosphoribosyl transferase or the Na +/K + ATPase locus, both of which have been well characterized. Other less-studied markers are also described. We discuss the protocols for quantitative mutation studies including measurements of cytotoxicity, mutant expression times, mutant selection agents, cell densities during selection, and the stability and verification of mutant phenotypes. Mutations in the V79 cells by chemicals that require activation can be tested after their metabolism by cell homogenates or by intact cells, and the results with each type of activation are compared. For purposes of analysis, we classified a compound as mutagenic if it induced a mutation frequency that is at least 3 times higher than the spontaneous mutant frequency reported for that specific experiment. By this criterion two-thirds of the chemicals analyzed were mutagenic—; 11% with and 55% without metabolic activation. Of the 191 chemicals examined; 119 were polycyclic aromatic hydrocarbons; 25 were nitro or nitroso compounds, 9 were alkyl halides; 7 were purine or pyrimidine derivatives and the remaining 31 were from other chemical classes. We also defined mutagenic potency as the concentration of a compound that increases the mutant frequency by 10 times the spontaneous frequency. Mutagenic potencies of the compounds examined varied over a range of 5 × 10 6. We have also found large interlaboratory variations in the mutagenic potencies. Such variation in potency could be reduced by normalizing the results to a standard mutagen such as N-methyl- N′-nitro- N-nitrosoguanidine. The role of the V79 assay in mutagenicity and carcinogenicity testing is discussed and recommendations are suggested for future investigation.

Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]hypoxanthine for 5 or 10 min, followed by storage of 3H-labelled cells at −70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [ 3H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.

Isolation and characterization of hybrids between mouse and Chinese hamster cell lines

Eleven clonal hybrid cell lines between an 8-azaguanine (AG)-resistant FC-1 line derived from mouse mammary carcinoma and a Chinese hamster lung cell line (CHL) were isolated by fusion using ultraviolet (UV)-irradiated Hemagglutinating Virus of Japan (HVJ), followed by cultivation in a selection medium and subsequent cloning. They have maintained the state of active proliferation in vitro for more than 1 year. Chromosome analysis revealed that these hybrid lines were derived from fusion between two or more mouse and one or less Chinese hamster cells, in spite of large differences in karyotype of each clonal hybrid line. Morphologically, they fell into three types: ( A) adherent and fibroblastic, ( F) floating and round, and ( M) mixed of the above two types. The growth rates of these three hybrid lines examined were almost the same and their doubling time was about 20 h. FC-1 cells had 13-fold higher activity of glutamic-pyruvic transaminase (GPT) than CHL cells and two lines among four hybrids assayed were intermediate between the parental lines in the enzyme activity, whereas the other two lines had the same high level of activity as the mouse cells. In addition, it was indicated that there was a close correlation between chromosome constitution and morphological characteristic as well as GPT activity in each hybrid line.