Chinese Hamster Cells In Culture Research Articles

Induction of chromosomal aberrations in active oxygen-generating systems I. Effects of paraquat in Chinese hamster cells in culture

A possible role for the superoxide anion radical (O 2 −) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O 2 −, but not to the formation of hydrogen peroxide by the dismutation reaction of O 2 − or of other active oxygen species including the hydroxyl radical and singlet oxygen.

Measurement of micronuclei by cytokinesis-block method in cultured Chinese hamster cells: comparison with types and rates of chromosome aberrations

The cytokinesis-block method of Fenech and Morley (1985) has been tested for the enumeration and characterization of micronuclei in exponentially growing Chinese hamster cells in culture. The consistent dose-response relations were obtained in cultures treated with mitomycin C, caffeine and colcemid. Comparison with the chromosome aberration frequencies indicated that approximately 30% of the acentric chromosomes are expressed as micronuclei in the mitomycin C and caffeine treated cells. The size distribution of the micronuclei suggested that the base-line frequency of micronuclei is mainly a reflection of mitotic dysfunction rather than chromosome structural aberrations.

Effect of surfactants on the induction of chromosomal aberrations in Chinese hamster cells in culture

Effect of surfactants on the induction of chromosomal aberrations in Chinese hamster cells in culture

The Role of Molecular Oxygen in the Photodynamic Effect of Phthalocyanines

Phthalocyanines are a class of mammalian cell photosensitizers which may be useful in photodynamic therapy for cancer. Chloroaluminum phthalocyanine tetrasulfonate was incubated with Chinese hamster cells in culture and exposed to white light at different concentrations of oxygen. The ability of the cells to form colonies served as an end point for the photobiological effect of the dye. The efficiency of photoinactivation of the sensitized cells decreased with decreasing oxygen concentration. Very little photoinactivation was observed when the atmosphere equilibrated with the cells was oxygen-free nitrogen. At an oxygen partial pressure of 2.5 mm Hg, photoinactivation was reduced by 50% compared to ambient atmosphere. In an attempt to understand the nature of the interaction between excited dyes and oxygen, the ability of several phthalocyanines to photogenerate singlet oxygen was measured. Thus phthalocyanines containing paramagnetic ions (copper, iron, vanadyl) do not generate 1O2 in contradistinction to diamagnetic metals (zinc and aluminum). The latter are efficient photosensitizers, while the former have little if any photobiological activity. In spite of this correlation, singlet oxygen may not be the intermediate involved in cytotoxicity. The reasons are discussed.

Spontaneous immortalization rate of cultured Chinese hamster cells.

Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation. The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity. Immortality was not related to rare, precommitted cells from the animals. The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells. On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal. In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization. This analysis yielded a rate of 1.9 X 10(6) per cell per generation.

Consequences of altered oncogene expression in rodent cells.

By using linkage to transcriptional enhancers to increase gene expression, it was shown that the introduction of the mutant Ha-ras gene from bladder carcinoma cells into primary cultures of Chinese and Syrian hamster cells, and Wistar rat cells, was sufficient to trigger malignant conversion. The normal Ha-ras gene when linked to enhancers did not trigger malignant conversion but gave rise to cell clones with extended growth capacity. We measured the levels of transforming growth factors (TGFs) released from cells containing the ras genes, and the availability of the receptor for epidermal growth factor (EGF) to which alpha-TGF also binds. The level of ras gene expression seemed to correlate with the level of TGF secreted, a reduction in available EGF receptors and the morphology of the transformed cells. However, when an immortalized cell line (rat 208F cells) was used as a recipient for the oncogene vectors a different result was obtained. TGF release was triggered to varying degrees by almost all of the ras and myc oncogene constructions tested, regardless of the level of gene expression. Enhanced expression of the two oncogenes led to increased anchorage-independence. Significantly, both normal ras and normal myc, when linked to transcriptional enhancers, caused the recipient cells to become tumorigeneic without inducing any marked morphological alterations in culture. Enhanced and constitutive expression of the mutant Ha-ras gene resulted in the recipient cells becoming tumorigeneic and the cells displayed a transformed phenotype in culture. The transfected fibroblasts with a markedly transformed phenotype displayed a relative paucity of EGF receptors which correlated with release of alpha-TGF, suggesting a down-regulation of EGF receptors on transformation. While enhanced expression of the normal Ha-ras gene did result in a decrease in EGF receptors on these transfectants, in contrast, the transfectants with enhanced expression of the c-myc and v-myc oncogenes did not display any significant changes in available EGF receptors although the cells were tumorigenic when tested in the nude mouse. These results are discussed in relation to a hypothesis implicating a role for oncogenes at various stages in the multi-stage process of carcinogenesis.

Quantitative Comparison of Genetic Effects of Ethylating Agents on the Basis of DNA Adduct Formation. Use of O 6 -Ethylguanine as Molecular Dosimeter for Extrapolation from Cells in Culture to the Mouse

DNA-adduct formation and induction of gene mutations were determined simultaneously after treatment with the four ethylating agents, ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), diethyl sulfate (DES), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Both, in E. coli K-12 (NAL-resistance) and in V79 Chinese hamster cells in culture (HPRT-deficiency), the frequencies of mutation induction by all chemicals were the same when plotted against the amount of O6-ethylguanine formed in DNA, suggesting that this DNA adduct can be used as a common dosimeter for the comparisons of the frequencies of gene mutations induced by ethylating agents in various mutagenicity assay systems. Using ENU, such a comparison was performed between mutation induction in V79 cells in vitro and in the specific-locus assay in the mouse. The data indicate that at equal levels of O6-ethylguanine in the DNA of V79 cells and in testicular DNA from male mice treated with ENU, the frequencies of induced mutants in both assay systems were quite similar. These results support the concept that the determination of premutagenic DNA adducts in vivo can be used to monitor exposure to chemical mutagens and that genetic risk estimations may ultimately be performed on the basis of such measurements and of comparative mutagenesis in vitro and in vivo.

Chromosomal aberrations and superoxide generating systems II. Effects of paraquat on Chinese hamster cells in culture

Chromosomal aberrations and superoxide generating systems II. Effects of paraquat on Chinese hamster cells in culture

Quantitative comparison of genetic effects of ethylating agents on the basis of DNA adduct formation. Use of O6-ethylguanine as molecular dosimeter for extrapolation from cells in culture to the mouse.

DNA-adduct formation and induction of gene mutations were determined simultaneously after treatment with the four ethylating agents, ethyl methanesulfonate (EMS), ethylnitrosourea (ENU), diethyl sulfate (DES), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Both, in E. coli K-12 (NAL-resistance) and in V79 Chinese hamster cells in culture (HPRT-deficiency), the frequencies of mutation induction by all chemicals were the same when plotted against the amount of O6-ethylguanine formed in DNA, suggesting that this DNA adduct can be used as a common dosimeter for the comparisons of the frequencies of gene mutations induced by ethylating agents in various mutagenicity assay systems. Using ENU, such a comparison was performed between mutation induction in V79 cells in vitro and in the specific-locus assay in the mouse. The data indicate that at equal levels of O6-ethylguanine in the DNA of V79 cells and in testicular DNA from male mice treated with ENU, the frequencies of induced mutants in both assay systems were quite similar. These results support the concept that the determination of premutagenic DNA adducts in vivo can be used to monitor exposure to chemical mutagens and that genetic risk estimations may ultimately be performed on the basis of such measurements and of comparative mutagenesis in vitro and in vivo.

A coordinate relationship between the GALK and the TK1 genes of the Chinese hamster.

Chinese hamster cells in culture were treated with various concentrations of thymidine, 5-bromodeoxyuridine, trifluorothymidine, and 2-deoxy-D-galactose. Selection was made for deficiencies in the activities of galactokinase and thymidine kinase. Selection in the presence of thymidine, 5-bromodeoxyuridine, and trifluorothymidine was expected to produce clones deficient in thymidine kinase only, whereas those deficient in galactokinase were expected to be selected in the presence of 2-deoxy-D-galactose. However, it was found that clones growing in the presence of these inhibitors were frequently deficient in both enzymes. Or if a clone was deficient in only one, the deficiency frequently was not expected according to the selection procedure. This indicates some sort of coordinate relationship between the two gene loci, GALK and TK1, which specify galactokinase and thymidine kinase, respectively. GALK and TK1 are linked in all primates and rodents in which linkage determinations have been made. It is therefore probable that this linkage has been conserved for a long period of time. It is suggested that the apparent relationship between the two genes shown by the data presented here, as well as by others, supports the conclusion that linkage has been conserved by natural selection and is therefore not fortuitous.

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).

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.

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.

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.

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.

Cell growth state determines susceptibility of repair DNA synthesis to inhibition by hydroxyurea and 1-beta-D-arabinofuranosylcytosine.

The effects of inhibitors of replicative DNA synthesis on repair DNA synthesis have been examined by autoradiography in several different cell types and in cells in different growth states. Hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), administered together, influence unscheduled DNA synthesis (UDS) in a manner which is independent of the status of the cell culture (normal or transformed) and of the species, but which is strongly affected by whether the cells are proliferating or quiescent. In proliferating human, Chinese hamster and Microtus cell cultures, UDS is not inhibited by HU and ara C, and may even appear to be stimulated. In quiescent cultures of these cells UDS is reduced by HU and ara C. In cells reseeded from a confluent culture and followed during proliferation and back to quiescence the effect of inhibitors parallels the growth pattern. The results are interpreted in terms of changes in the sizes of endogenous DNA precursor pools; they underline the potential problems associated with quantitating UDS in the presence of inhibitors.

Clastogenic potential of heavy oil extracts and some aza-arenes in Chinese hamster cells in culture

Chromosomal aberration tests in vitro, with a Chinese hamster fibroblast cell line, CHL, were carried out on B class heavy oil fractions, obtained by silica-gel column chromatography and a liquid-liquid extraction method. The original oil and basic nitrogen-containing fractions induced structural chromosomal aberrations in the CHL cells in the presence of rat-liver microsome fraction (S9 mix). 8 tricyclic or pentacyclic aza-arenes, which possibly exist in the positive fractions, were also examined for their clastogenic activities in the system with or without S9 mix. Acridine, benzo[ a]quinoline, pyrenoline and pyrenoline 4,5-oxide induced chromosomal aberrations both with and without S9 mix. Benzo[ h]quinoline was positive without S9 mix, whereas benzo[itc,h]acridine and benzo[ a,j]acridine were only positive with S9 mix. The results suggest that the clastogenic effects of the heavy oil may in part be due to the presence of nitrogen-containing polycyclic hydrocarbons such as aza-arenes.

Zinc-induced resistance to alkylating agents: Lack of correlation between cell survival and metallothionein content

Suspension cultures of line CHO Chinese hamster cells and three derived sublines which differ in their capacity to induce synthesis of metallothionein (MT) by at least a factor of 40-fold were exposed to 100 μ m ZnCl 2 prior to treatment with the alkylating agent iodoacetate, and cytotoxicity was determined by measuring colony-forming ability. Three of the four types of cells exhibited an 8- to 10-fold enhancement in survival when pretreated with zinc, but there were two lines of evidence that suggested that the enhanced survival was not attributable to metallothionein. The only cell in the series which failed to exhibit increased survival when pretreated with zinc was the Cd r2ClO cell which is readily induced to synthesize MT when treated with ZnCl 2. An even stronger indication that metallothionein did not play a major role in the protective response was provided by the observation that CHO cells were protected by pretreatment with zinc, even though they are unable to induce synthesis of MT as a result of exposure to that metal. These results suggest the existence of a novel, zinc-inducible mechanism which protects cells against the toxic effects of alkylating agents.

Newly synthesized hypoxia-mediated drugs as radiosensitizers and cytotoxic agents

Chinese hamster cells in culture were used to compare the radiosensitizing efficiency and cytotoxicity of misonidazole with several 2, 4 and 5 substituted nitroimidazoles. The two substituted compounds (SR 2508 and SR 2555) are similar to misonidazole in radiosensitizing effectiveness, but are significantly less toxic to hypoxic cells. This reduced cytotoxicity may result from either slower drug penetration or slower removal of non-protein sulfhydryl compounds (NPSH) The compound MJLr1-191-VII ∗ (a 4-nitroimidazole) is a much more effective radiosensitizer than would be predicted from its electron affinity. It appears to sensitize by two mechanisms, the first resulting from its electron affinity and the second a consequence of its rapid removal of endogeneous cellular NPSH; which are naturally occurring radioprotective substances.

Hydroxyurea revisited: A decade of clinical effects studies

Over a decade ago hydroxyurea was shown to selectively kill cells in the S phase in a proliferating cell population and to block cells at the G 1-S border. Consequently, blocked cells became sensitized to irradiation and were further sensitized when the drug was present after exposure. These features were identified initially in Chinese hamster cells in culture. They seemed to indicate that, in circumstances where tumor cells are proliferating more rapidly than the surrounding normal tissues, hydroxyurea might be an effective agent for use in combination therapy with X or gamma radiation. In the ensuing decade, many in vivo studies on hydroxyurea have confirmed that the main properties of hydroxyurea identified in the dish are also evident in vivo. During a period of about ten years, a considerable number of clinical studies have been performed, the results of which have been mixed, ranging from indeterminate to encouraging, depending to some extent on the site treated and whether careful randomized studies were done. Some clinicians have abandoned hydroxyurea in favor of other drugs or procedures, but others find the agent useful and perhaps especially so in the treatment of carcinoma of the cervix. Definitive, quantitative information on the value of the drug in combination therapy seems to be available in only a few instances. Thus, its value is still debatable. The question then arises whether the clinical studies have represented adequate tests, by laboratory standards, of the likely effectiveness of the drug in clinical circumstances. The clinical studies with hydroxyurea have been examined in this light, since the results might also bear on the use of many other such agents in combination therapy. This examination revealed that no attempts have been made to determine the concentration of hydroxyurea in the tumor and other relevant tissues as a function of time or to assess the cell kinetic features of the tumor and thus estimate the appropriate dose regimen. It would seem that a wide gap still exists between laboratory research and clinical application. While the difficulties of the latter are well known, it may be more important to adequately exploit, in the clinic, the laboratory properties of the drugs we know rather well than to continue searching for a miracle panacea while repeating similar errors in translating from laboratory to clinic.