Hamster Cells In Culture Research Articles

Dose rate dependence of response of mouse lung to irradiation.

The dose-rate dependence of lung damage in mice has been studied using LD50/50-180 as an index of the incidence of radiation pneumonitis. Mean lethal doses for 60Co gamma radiation to the thorax delivered at 100, 25 and 6 cGy/min were 1403, 1923 and 2488 cGy respectively. There were statistically significant differences between values obtained at 6 and 25 cGy/min and between those obtained at 25 and 100 cGy/min. An isoeffect plot of this data on a log-log graph shows the sparing effect of dose rate reduction to be greater for the lung than for more rapidly responding systems (colony forming units of small intestine and Chinese hamster cells in culture).

A contact-insensitive subpopulation in Syrian hamster cell cultures with a greater susceptibility to chemically induced neoplastic transformation.

We previously have identified a subpopulation of contact-insensitive (CS-) cells which lacks density-dependent inhibition of cell division in primary and low-passage cultures of Syrian hamster embryonic (SHE) fibroblastic cells. Further, we have shown that the proportion of these CS- cells declines as a result of the stable phenotypic conversion of the CS- cells to contact-sensitive (CS+) cells. To determine whether these transient CS- cells are more sensitive to carcinogenic/mutagenic perturbation, the susceptibility to neoplastic transformation and somatic mutation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined in clonally isolated cell cultures containing various proportions of CS- cells (0.02-4%). The frequencies of morphological transformation, focus formation, and neoplastic transformation showed a positive correlation to the proportion of CS- cells in the treated cultures. In contrast, the frequency of MNNG-induced somatic mutation at the Na+,K+-ATPase locus was similar among cultures varying in their proportion of CS- cells. Thus, there is a transient subpopulation of CS- cells in primary SHE cell cultures that is more susceptible to neoplastic transformation although equally susceptible to induced point mutation. This dissociation between somatic point mutation and neoplastic transformation indicates a fundamental difference in the nature of these two phenomena. A possible relationship between the propensity of CS- cells (versus CS+ cells) to carcinogen-induced neoplastic transformation and the state of differentiation of the CS- cells is discussed.

Induction of 6-thioguanine-resistant mutants by hyperoxia and γ-irradiation: effect of compromising cellular antioxidant systems

Hyperoxia and γ-irradiation were found to be mutagenic in a transformed Syrian hamster cell line in a dose-dependent manner. The frequency of resistance to 6-thioguanine increased from 10 per 10 6 survivors after 48 h of growth in 70% O 2 to 32.6 (highly significant) after 75 h. Increasing the oxygen tension to 95% resulted in a significant mutagenic response in only 44 h. At equitoxic doses, γ-irradiation was 4 times more mutagenic than 70% O 2. After growth in hyperoxia, the cells showed an enhancement of catalase activity, glutathione peroxidase activity and glutathione levels but there was little effect on superoxide dismutase activity. Diethyldithiocarbamate (3 mM, 1.5 h) was mutagenic in normoxia and potentiated the mutagenic activity of both γ-irradiation and hyperoxia. Cells thus treated showed an 85% reduction in superoxide dismutase activity. When diethyldithiocarbamate was used in conjunction with a direct-acting alkylating agent, the mutagenic response was only additive. Depletion of cellular glutathione with buthionine sulfoximine (0.2 mM) or inhibition of catalase activity with aminotriazole (100 mM) was also effective in potentiating the mutagenic response of γ-irradiation and hyperoxia. The data demonstrates that endogenously produced activated oxygen species are mutagenic to hamster cells in culture and suggest that aerobic organisms are subject to an unavoidable background risk due to living in an oxygen atmosphore.

Genetic effects of chromium tannins.

Seventeen tannins used in the hide and leather industry, most of which contain mainly Cr(III) sulphates, were tested for the ability to directly induce gene mutations in Salmonella typhimurium (TA 100 strain) and chromosomal effects (sister chromatid exchanges, SCE) in cultured hamster cells (CHO line). Total chromium [Cr(III) + Cr(VI)] content and contaminating Cr(VI) were determined spectrophotometrically by reaction with diphenylcarbazide. None of the tested compounds induced gene mutations, whereas eight tannins were able to increase significantly the frequency of SCE. A contamination with Cr(VI) was detected in four compounds (from 30 up to 100 parts of Cr(VI) per 10(6) parts of compound), insufficient to be revealed by the Salmonella assay but sufficient to account for the observed SCE increase. On the other hand, the increase of SCE induced by the other four tannins could not be explained by the level of Cr(VI) contamination, and can be ascribed to other impurities present in those industrial compounds. These four tannins did not induce gene mutations in the S. typhimurium assay even when strain TA 98 was used in addition to TA 100, independently of microsomal activation. By prolonging the time of the SCE assay from 30 to 48 h in order to facilitate Cr(III) endocytosis, a significant increase of the SCE frequency was induced by an analytical-grade Cr(III) reagent (chromium chloride), absolutely uncontaminated by Cr(VI), as well as by three Cr(III) tannins, otherwise inactive in the SCE assay.

The Reproductive Effects Assessment Group's report on the mutagenicity of 1,3-butadiene and its reactive metabolites.

A major data gap for assessing heritable risk from exposure to 1,3-butadiene is the lack of mammalian mutagenicity data. The data base on the mutagenic potential of 1,3-butadiene is limited to three bacterial studies from the same laboratory. Two of these studies were positive only in the presence of liver S9 mix from chemically pretreated animals. In vitro data suggest that 1,3-butadiene is metabolized to two epoxide intermediates. 3,4-Epoxybutene, one potential reactive metabolite of 1,3-butadiene, is a monofunctional alkylating agent and is a direct-acting mutagen in bacteria. In addition, unpublished data suggest that 3,4-epoxy-butene induces DNA damage and chromosomal aberrations in mice. Another potential reactive metabolite, 1,2:3,4-diepoxybutane, is a bifunctional alkylating agent and is mutagenic in a wide variety of organisms (bacteria, fungi, and the germ cells of Drosophila). This metabolite also induces DNA damage in mice and in cultured hamster cells, is clastogenic in fungi and cultured rat cells, and produces chromosome damage/breakage in Drosophila germ cells. These data, when combined with evidence that 1,3-butadiene is carcinogenic in rodent gonadal tissues and is associated with gonadal atrophy in mice, constitute suggestive evidence that 1,3-butadiene may be a human germ cell mutagen. However, because the mutagenicity of 1,3-butadiene has been studied only in bacteria, studies in mammalian test systems are needed to further characterize the mutagenic potential of 1,3-butadiene.

Enhancement of the cytotoxic effects of pepleomycin by dibucain.

The ability of dibucain, a local anesthetic, to enhance the cytotoxicity of and the repair of potentially lethal damage induced by pepleomycin or X-rays has been studied in exponential and plateau phase cultures of Chinese hamster cells. No enhancement of X-ray cytotoxicity or inhibition of repair from X-ray injury was observed, but dibucain produced very significant enhancement of pepleomycin effectiveness. In both cases the effects induced were larger in the exponentially growing cells than in the plateau phase cells. Greater than 20-fold increases in the cytotoxic effectiveness of pepleomycin, and greater than 10-fold differences in survival after the cells had been allowed to repair the potentially lethal damage induced by pepleomycin were observed when the cells were treated with dibucain. The possible mechanisms underlying this enhancement are discussed.

Benzo[a]pyrene dione-benzo[a]pyrene diol oxidation-reduction couples; involvement in DNA damage, cellular toxicity, and carcinogenesis.

Three isomeric quinone metabolites of the environmental carcinogen benzo[a]pyrene undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrene diols and intermediate semiquinone radicals. Under anaerobic conditions, benzo[a]pyrene 1,6-dione, benzo[a]pyrene 3,6-dione, and benzo[a]pyrene 6,12-dione are readily reduced by mild biological agents such as NADH and glutathione. The benzo[a]pyrene diols, in turn, are very rapidly autooxidized to diones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autooxidations. The benzo[a]pyrene diol/benzo[a]pyrene dione interconversions proceed by one-electron steps; the corresponding semiquinone radicals were detected as intermediates when the reactions were carried out at high pH. Benzo[a]pyrene diones are electron-acceptor substrates for NADH dehydrogenase. Catalytic amounts of these metabolites, together with this respiratory enzyme, function as cyclic oxidation-reduction couples to link NADH and molecular oxygen in the continuous production of hydrogen peroxide. Benzo[a]pyrene diones induce strand scissions when incubated with T7 DNA. The damage is modified by conditions that indicate that reduced oxygen species propagate the reactions responsible for strand scission. Benzo[a]pyrene diones are cytotoxic at low concentrations to cultured hamster cells. The cytotoxic effect can be substantially reduced by depletion of oxygen from the growth medium and the atmosphere in which the cells are incubated. The results support the hypothesis that the biological activity of benzo[a]pyrene diones is due to the regenerative oxidation-reduction cycles involving quinone and hydroquinone forms; activated oxygen species and semiquinone radicals formed during these cycles are most likely responsible for the observed cytotoxic action. The role of activated oxygen species in carcinogenesis is discussed.

The nature of mutants by ionising radiation in cultured hamster cells: I. Isolation and initial characterisation of spontaneous, ionising radiation-induced, and ethyl methanesulphonate-induced mutants resistant to 6-thioguanine

A large number of thioguanine (TG)-resistant mutants of V79-4 Chinese hamster cells was isolated from untreated cultures and from cultures exposed to γ-rays, α particles or ethyl methanesulphonate (EMS). Selection condictions were chosen to optimise the survival of all types of TG-resistant mutant, and the isolation procedure ensured that each mutant originated independently of any other. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) enzyme activity was measured in cell-free extracts of each mutant, and compared with repeat measurements made on the parental V79-4 cells and on a series of non-mutant clones. Ionising radiation-induced mutants were found to be mostly (or perhaps entirely) of the ‘zero HGPRT activity’ type, but about 20% of EMS-induced mutants and 50% of spontaneously occurring mutants had significant HGPRT activity. However, none of the TG-resistant mutants were found to lack activity of another X-chromosome-linked enzyme, glucose-6-phosphate dehdrogenase. Few mutants were found with visible X-chromosome changes, but the incidence of hyperploidy was higher among spontaneous mutants than in the parental line and the induced mutants. Isoelectric focussing of cell extracts from those mutants which retained some HGPRT activity revealed several with shifts in the isoelectric points for HGPRT enzyme activity.

Characterization of a CMP-sialic acid:lactosylceramide sialyltransferase activity in cultured hamster cells

Previous studies have shown a strong correlation bteween reduced levels of G M3 ganglioside and an increase in the oncogenic transformation of cultured cells. CMP-sialic acid: lactosylceramide sialyltransferase, which catalyzes G M3 synthesis, was characterized in cultured hamster fibroblasts (NIL-8) with respect to substrate binding, pH optimum, detergent requirements, metal ion requirements, activity during cell cycle phases and activity during cell growth phases. The apparent K m values for CMP-sialic acid and lactosylceramide were 0.16 and 0.11 mM, respectively. The enzyme required Mn 2+ (15 mM) for maximal activity, but Mg 2+ and Ca 2+ were able to substitute to a lesser extent. Triton CF-54 (0.3%, w v ) compared to other nonionic detergents gave the greatest enzyme activation, while ionic detergents inhibited the enzyme. A broad pH optimum (4.5–8.0) was obtained, with maximum activity at pH 6.5 in cacodylate-HCl buffer. No buffer effects on enzyme activity were seen. Sialyltransferase activity was found to be highest in the M and G 1 phases of the cell cycle and in the contact-inhibited phase of cell growth.

A 61,000-dalton truncated large T-antigen is uniformly expressed in hamster cells transformed by polyomavirus.

Various polyomavirus-transformed hamster cell lines derived from tumors or from infected hamster cell cultures synthesized polyoma middle and small tumor (T)-antigens but no full-size large T-antigen. Instead, all cell lines produced the same or similar polyoma T-antigen-related proteins of ca. 61 kilodaltons (kDal). Like large T-antigen synthesized in lytically infected mouse cells, the 61-kDal proteins were phosphoproteins showing electrophoretic and charge heterogeneities. Chromatographic analysis of the methionine-containing tryptic peptides indicated that the 61-kDal proteins were truncated forms of large T-antigen comprising amino acid residues 1 to 485 (+/- 25). Analysis of viral DNA present in hamster chromosomal DNA of three independently isolated cell lines confirmed that synthesis of the 61-kDal proteins was due to a discontinuity in the large T-antigen coding sequence, most likely located between 7 and 8.9 map units on the polyoma DNA map. The three cell lines yielded essentially the same patterns of viral DNA-containing restriction enzyme fragments, suggesting that insertion of viral DNA into the hamster chromosomes took place at closely similar sites.

Do prostaglandins affect cellular radiosensitivity in vitro?

We have been unable to detect any change in the in vitro radiation response of mouse fibrosarcoma cells, HSDM1C1, which secrete 2 micrograms PGE2/mg cell protein/24 h, in the presence of the prostaglandin biosynthesis inhibitor flurbiprofen. Furthermore, addition of exogenous PGE1 or PGA2 to cultures of Chinese hamster cells was similarly without effect on radiation response. Although a high concentration of PGA2 inhibited the growth of Chinese hamster cells in vitro this effect disappeared upon removal of the prostaglandin. The implications of these results for radiotherapy are discussed.

Phototoxicity and mutagenicity of 4,5′-dimethylangelicin and long-wave ultraviolet irradiation in Chinese hamster cells and human skin fibroblasts

The phototoxicity and mutagenicity of 4,5′-dimethylangelicin (Ang) were assessed in cultures of hamster cells and human skin fibroblasts after long-wave ultraviolet irradiation. To obtain an impression of the clinical usefulness of Ang, we compared the phototoxicity and mutagenicity of this compound with those of 8-methoxypsoralen (8-MOP). To reach the same cell-killing rate in hamster cells and human skin fibroblasts, the concentration of Ang had to be 15 times higher than that of 8-MOP at comparable doses of radiation. Whether we used the same concentrations and an adjusted radiation dose or adjusted concentrations of Ang and 8-MOP at a constant radiation dose, the mutation induction in hamster cells and human skin fibroblasts was much higher after incubation with Ang than with 8-MOP

3-Hydroxy-3-methylglutaryl-CoA reductase: a transmembrane glycoprotein of the endoplasmic reticulum with N-linked "high-mannose" oligosaccharides.

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) is an abundant protein of the crystalloid endoplasmic reticulum of UT-1 cells, a line of cultured hamster cells that over-produces the reductase as a result of gene amplification. In the current studies, we show that reductase in UT-1 cells is a glycoprotein. The solubilized enzyme (Mr = 97,000) from UT-1 cells, Chinese hamster ovary cells, and rat liver was adsorbed quantitatively and specifically to concanavalin A-Sepharose. UT-1 cells incorporated [1,6-3H]glucosamine into the reductase; after release with endo-N-acetylglucosaminidase H most of the radioactivity was found in N-linked "high-mannose" chains, including Man6(GlcNAc)2, Man7(GlcNAc)2, and Man8(GlcNAc)2. The carbohydrate of the reductase was localized to a 30- to 35-kilodalton fragment that was separable proteolytically from a cytoplasmic 53-kilodalton fragment that contained the active site of the enzyme. We conclude that 3-hydroxy-3-methylglutaryl-CoA reductase is a transmembrane glycoprotein with an active site facing the cytoplasm and a carbohydrate-bearing site oriented toward the lumen of the endoplasmic reticulum.

The kinetics of dissociation of the inhibitor of nucleoside transport, nitrobenzylthioinosine, from the high-affinity binding sites of cultured hamster cells.

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site--NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.

Recovery from Lethal and Mutagenic Damage during Postirradiation Holding and Low-Dose-Rate Irradiations of Cultured Hamster Cells

Survival and mutation to thioguanine resistance were measured in V79-4 hamster cells grown to plateau phase without refeeding and irradiated with 60Co gamma rays. The effects of low-dose-rate irradiation and of postirradiation holding on recovery from gamma-ray damage leading to these two responses were also studied. The responses of these plateau (extended G1)-phase cells to acute irradiation were similar to those we previously found for exponentially growing cells, including the linear relationship between induced mutant frequency and (log) surviving fraction. Irradiation at low dose rate (0.34 rad/min) considerably reduced both the lethal and mutagenic effects of given doses of gamma rays, but the linear mutation-survival relationship was approximately the same as for acute irradiation. In contrast, cells given a 5-hr holding period after acute irradiation showed the anticipated recovery from potentially lethal damage but no recovery from damage leading to mutation. These results are discussed in terms of previously proposed cellular repair processes (sublethal damage repair and potentially lethal damage repair) and the possibility that the radiation damage leading to lethality is different from mutagenic damage.

Correlation of the structure of amino-substituted anthraquinones to cytotoxicity in cultured Chinese hamster cells.

A number of substituted anthraquinones (SAQs) are currently being tested as cancer chemotherapeutic agents because of their structural similarity to Adriamycin (ADR) and other DNA-intercalating antibiotics. In this study, the effect of SAQs on the induction of cytotoxicity of asynchronous Chinese hamster cells in culture was studied and compared to those produced by ADR and dihydroxyanthraquinone (DHAQ), a SAQ previously shown to be more effective than ADR. SAQs produced cytotoxicity that was dependent upon the concentration and duration of drug exposure. A correlation was noted between the activity of a compound (the concentration required to produce a certain level of cell kill) and the presence of a particular triangular arrangement of one nitrogen atom and two oxygen atoms. There was also a correlation between chemical structure and antitumor activity in the murine P388 leukemia model system. This correlation between chemical structure and biological activity may aid in the development of new SAQs with greater potential as cancer chemotherapeutic agents.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and its mRNA in rat liver as studied with a monoclonal antibody and a cDNA probe.

A monoclonal antibody directed against 3-hydroxy-3-methylglutaryl Coenzyme A reductase and a cDNA to reductase mRNA were used to study the subunit structure of the enzyme and the regulation of its mRNA in rat liver. Although the monoclonal antibody and the cDNA were made with materials from cultured hamster cells, the two reagents cross-reacted with reductase protein and mRNA from rat liver. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibody, the subunit molecular weight of rat liver reductase was 90,000. When the enzyme was solubilized from microsomes by freeze-thawing, the subunit molecular weight was reduced to 52,000-58,000, owing to proteolysis. This proteolysis was inhibited by EGTA and leupeptin. The cDNA probe for reductase, radiolabeled with 32P, hybridized to restriction fragments of genomic DNA from rat liver, as visualized by Southern blot analysis. In the livers of control rats, no reductase mRNA was detected when the 32P-cDNA was blot-hybridized to poly(A+) RNA. Hepatic reductase activity was increased 45-fold when rats were fed cholestyramine and mevinolin. Under these conditions, the amount of immunodetectable reductase protein rose by 33-fold, and the reductase mRNA became visible by blot hybridization as a band of approximately 4 kilobases in length. When the mevinolin/cholestyramine-treated rats were fed cholesterol, reductase activity and immunodetectable protein declined markedly and the reductase mRNA was reduced to barely detectable levels. We conclude that treatment with cholestyramine and mevinolin increases the amount of reductase protein in rat liver by elevating the amount of its mRNA and that cholesterol feeding to such induced rats lowers the amount of hepatic reductase protein by decreasing the level of its mRNA.

Genetic effects of chromium compounds

Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA polα from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [ 3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.

The effect of methionine on the accumulation of ornithine by chinese hamster cells in culture

Ornithine was found to be toxic to cells that lack ornithine aminotransferase (EC 2.6.1.13). These cells also accumulated substrantial amounts of ornithine. The addition of methionine to these protected them from the toxic effects of ornithine and alos diminished their accumulation of ornithine. Cells that have an active ornithine aminotransferase were resistant to the toxic effects of ornithine, and accumulated less of this compound.

Biological effects of pion therapy beams: I. Cultured cells

The results of cell killing by pion beams of different peak widths, ranges and sizes used in therapy are reported. Cultured hamster cells (V79) suspended in gelatin were used. The results indicate that: 11 there are no significant differences in cell-killing between pion beams of different ranges but of the same peak width; 21 there is a slight decrease in biological effectiveness with increasing peak width; and 31 the range-modulation functions used to produce uniform cell-killing are satisfactory for intermediate-range pion beams, but slight corrections may be required for shorter- and longer-range pion beams.