Mutagenicity of 5-azacytidine and related nucleosides in C3H/10T 1/2 clone 8 and V79 cells

To determine whether 5-azacytidine (5-AzaCR)-induced transformation and/or differentiation of C3H/10T 1/2 clone B (10T 1/2) cells might have a mutational basis, we studied whether 5-AzaCR and structurally related nucleoside analogs could mutate 10T 1/2 and Chinese hamster V79 cells. In an assay for mutation to ouabain resistance in 10T 1/2 cells, which detects base substitution mutations but not frameshift mutations, 5-AzaCR and 6-azacytidine were not significantly mutagenic. 5-Aza-2'-deoxycytidine, 5-fluoro-2'-deoxycytidine, 5,6-dihydro-5-azacytidine, 5-fluoro-2'-deoxyuridine (FUdR), 5-bromo-2'-deoxyuridine (BUdR), and 1-beta-D-arabinofuranosylcytosine (ara-C) were only weakly mutagenic. In an assay for mutation to ouabain resistance in V79 cells, which also detects base substitution mutations but not frameshift mutations, 5-AzaCR, 5-aza-2'-deoxycytidine, FUdR, and ara-C were not detectably mutagenic, and BUdR was moderately mutagenic at highly cytotoxic concentrations. In an assay for mutation to 8-azaguanine resistance in V79 cells, which detects base substitution and frameshift mutations, 5-fluoro-2'-deoxycytidine and ara-C were weakly mutagenic, BUdR was moderately mutagenic at very cytotoxic concentrations, and 5-AzaCR, 5-aza-2'-deoxycytidine, FUdR, 6-azacytidine, and 5,6-dihydro-5-azacytidine were not significantly mutagenic. Therefore, 5-AzaCR and related cytosine analogs can be considered as negligibly mutagenic. This study does not provide support for a mutational basis for 5-AzaCR-induced differentiation in 10T 1/2 cells. Further, there was no correlation between the mutagenicity of the nucleosides 5-AzaCR, ara-C, BUdR, and FUdR studied here and their previously reported abilities to transform 10T 1/2 cells. The mutagenicities of 5-AzaCR and FUdR were so low that the biological significance of these effects is uncertain. Hence, it is not clear to what extent, if any, mutation contributes to the transformation caused by these two compounds, and other possible mechanisms of transformation should also be investigated.     

In vitro correlates of transformation in C3H/10T1/2 clone 8 mouse cells.

Various potential in vitro correlates of malignancy were studied in four chemically transformed C3H/10T1/2 Clone 8 mouse cell lines and were compared with controls cells. The degree of tumorigenicity was best predicted by the relative plating efficiencies of the morphologically transformed cells in soft agar. All transformed cells also showed an increase in extracellular fibrinolytic activity which may be an additional marker for transformation. Intracellular fibrinolytic activity and loss of 125I-labeled cell surface protein (M.W. 250,000) were not correlated with morphological transformation or tumorigenicity in these cells.     

Morphological correlates of transformation in cultured C3H/10T1/2 mouse embryo cells

In efforts to determine common and consistent morphological parameters of transformation in C3H/10T1/2 cells, 15 cell lines transformed by different carcinogens were examined with the scanning electron microscope (SEM) and compared to non-transformed cells. Cell lines were studied at different passages, at different cell densities, and after growth as anchorage dependent or independent cultures. The transformed cell lines could be distinguished in the SEM from non-transformed cultures by the expression of one or more of the following morphological characteristics: formation of mini- or macro-foci (random piling of cells on top of each other), pleiomorphism in cell size and shape, and cell surface complexity. The extent to which these characteristics were expressed varied widely in the different transformed cell lines. Light microscopic scoring for different types of foci also revealed broad variability among the different transformed lines. At the SEM level, cell lines could not be characterized as transformed on an individual cell basis. However, all transformed cell lines could be definitively characterized as transformed on a population basis due to the presence of mini-foci. The various transformed cell lines were classified semi-quantitatively into categories based on the extent of expression of the different morphological characteristics. There was broad correspondence between the morphological classification and the relative plating efficiencies of the cell lines in soft agarose.     

In vitro reversion of transformed phenotype in mouse C3H/10T1/2 cells

Spontaneous tumor regression is still one of the most puzzling events in human cancer. A cell culture model of malignant transformation designed to permit the study of this phenomenon in vitro was applied to examine reversion and re-expression of the transformed phenotype in two X-ray transformed mouse 10T1/2 cell clones. By alternating cell passages at low and high seeding density, the expression of cell contact inhibition and tumorigenic capacity were both reverted and restored. Growth of non-transformed wild-type cells was not affected by seeding density. This reversion of the transformed phenotype was associated with a modification in genomic 5-methylcytosine content. Initially, the transformed clones were hypomethylated, as occurs in most human tumors. After only four passages at low seeding density, the phenotype was reverted to that of non-transformed 10T1/2 cells and genomic 5-methylcytosine content was significantly increased to levels measured in non-transformed C3H/10T1/2 mouse cells. Thus, hypomethylation induced by ionizing radiation was not a permanent feature of malignantly transformed 10T1/2 cells. Although genomic 5-methylcytosine content returned to normal levels during low density passaging, the methylation pattern of the c-myc gene specifically was not associated with cell passages either at low or high seeding density. In an attempt to identify genes involved in this process, expression of the tumor suppressor gene p53 was measured. Western blot analysis failed to detect any correlation between expression of p53 protein and reversion of the transformed phenotype. The results of this study indicate that the transformed phenotype is not permanently associated with the malignant transformation of C3H/1OT1/2 cells, and can be modulated by growth conditions in vitro. We propose that modulation of genomic 5-methylcytosine levels may be involved in this process.     

Dose-rate effects in mammalian cells: V. Dose fractionation effects in noncycling C3H 10T 1/2 cells

Multiple gamma ray dose fractionation effects were studied using contact-inhibited C3H 10T 1/2 murine fibroblast cultures in an attempt to simulate conditions in tissues with low rates of cell turnover. Multifraction survival curves were determined for different doses per fraction using 12 hour interfraction intervals 7 days per week. Isoeffect curves were generated from the multifraction survival curves. For doses per fraction greater than approximately 1.0 Gy, these isoeffect curves were similar to those derived from tissue reactions in humans and experimental animals published previously. For doses per fraction below 1.0 Gy, the isoeffect curves became essentially flat, thus deviating appreciably from the total dose which would be predicted by extrapolating the NSD equation to achieve an isoeffect. The existence of a non-repairable component of gamma ray damage can be inferred from this finding, which has implications both for basic radiobiology, and radiotherapy.     

Increased thymidine uptake by methylcholanthrene-treated C3H/10T1/2 cells.

The uptake of 3H-thymidine in post-confluent cultures of methylcholanthrene-transformed C3H/10T1/I mouse embryo cells was markedly higher than in their non-transformed counterparts. In a reconstruction experiment as few as 2% transformed cells could be detected by increased thymidine uptake. Measurements made at various times up to 110 days in multi-chambered plates revealed that after 25 days methylcholanthrene-treated cultures incorporated significantly more thymidine than the acetone-treated controls. This increased uptake correlated with the appearance of Type III transformed foci.     

Antipain and radiation effects on oncogenic transformation and sister chromatid exchanges in Syrian hamster embryo and mouse C3H/10T1/2 cells

Depending on its time of addition to Syrian hamster embryo ormouse C3H 10T cells the protease inhibitor antipain (AP) canenhance, or reduce, radiation induced-oncogenic transformations.These opposing influences are not paralleled by changes in sisterchromatid exchanges in either cell system. A 24 h treatmentwith 10 µM AP prior to, and during irradiation, with removal10 min after irradiation results in greater than a two-foldincrease in transformants. Conversely, a 24 h treatment beginning10 min after irradiation results in about a two-fold decreasein transformants. The utility of AP as an agent for the reductionof tumorigenesis is brought into question, since quite shorttemporal differences in application can result in near five-folddifferences in the frequencies of oncogenic transformations.     

Oncogene-mediated multistep transformation of C3H10T1/2 cells

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.     

Tiadenol-mediated induction of peroxisomal enzymes in cultured C3H/10T1/2CL8 cells and in chemically transformed C3H/10T1/2 MCA16 cells

The levels of peroxisomal enzyme activities in cultured C3H/10T1/2 CL8 cells and in chemically transformed C3H/10T1/2 MCA16 cells were studied after treatment with tiadenol and niadenate, two hypolipidemic drugs which are both carcinogenic and cause peroxisome proliferation in vivo. Administration of these peroxisome proliferators to the cells resulted in large increases in specific palmitoyl-CoA hydrolase, carnitine acetyl-transferase, and catalase activities. A reproducible induction of cyanide-insensitive palmitoyl-CoA oxidative activity was observed 5 and 9 days after initiation of tiadenol treatment. Basal activity was 0.16 nmole/min/mg protein compared to 0.95 nmole/min/mg protein in cells treated with 18 microM tiadenol (cytotoxicity of about 25%) for 9 days. The enzyme activities were more increased in the transformed MCA 16 cells than in the non-transformed cells and the order of increase in enzyme activities was: niadenate greater than tiadenol. In non-transformed cells, the specific activity of palmitoyl-CoA hydrolase was enhanced approximately 2.1-fold within 4 days after tiadenol treatment. During this time period the enzyme activity was constant in untreated cells, but decreased during longer incubation periods. The enhancement of palmitoyl-CoA hydrolase, carnitine acetyl-transferase and catalase activities was dose-related over a concentration range of 2 to 20 microM tiadenol, depending on the enzyme assayed. Tiadenol concentrations above 10 microM were increasingly cytotoxic, while 18 microM niadenate had no toxic effect on the C3H/10T1/2 C18 cells. Moreover, the stimulation of the 3 enzyme activities by the peroxisome proliferators were inhibited by cycloheximide. Neither of the two cell lines contained any appreciable urate oxidase activity. The responses of these cells to hypolipidemic drugs show that they constitute a useful system for studies on the role of peroxisomes in lipid metabolism and the relationship between hypolipidemic activity and carcinogenic potential of these drugs.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells

Continuous treatment of C3H/10T1/2 cells with low concentrations(>4pM) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhancedfocus production in cultures pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.Maximal enhancement occurred at 40 pM TCDD, a concentration10 000-fold lower than that required to produce an optimal responsewith 12-O-tetradecanoylphorboI-13-acetate. Single treatmentswith 0.06 nM-5 µM TCDD did not transform C3H/10T1/2 cellsor initiate the process of transformation in cultures subsequentlyexposed to the tumor promoter 12-Otetradecanoylphorbol-13-acetate.Promotion of transformation is thus the predominant effect ofTCDD in the C3H/101/2 cell transformation system.     

Mutation of Chinese Hamster V79 cells and transformation and mutation of mouse fibroblast C3H/10T1/2 clone 8 cells by aflatoxin B1 and four other furocoumarins isolated from two Nigerian medicinal plants

Mutation by aflatoxin B1 (AFB1), imperatorin, marmesin, chalepin, and 8-methoxypsoralen (MOP), with and without black light (BL; long-wavelength ultraviolet light) activation, was determined at the hypoxanthine-guanine phosphoribosyltransferase locus (8-azaguanine resistance) in Chinese hamster V79 cells and at the ouabain locus in mouse C3H/1OT1/2 cells. Transformation by these furocoumarins under the same activation conditions was also investigated in C3H/1OT1/2 cells. In V79 cells, AFB1 induced a 4-fold maximum mutation frequency over controls under BL activation at a concentration of 5 micrograms/ml; marmesin induced a 2-fold increased mutation frequency at 1.5 micrograms/ml; MOP induced a 19-fold increase at 10 micrograms/ml; chalepin induced a 3-fold increase at 5 micrograms/ml; and imperatorin induced a 20-fold increase at 10 micrograms/ml. Essentially no mutation was observed at the ouabain-resistant (Ouar) locus in C3H/1OT1/2 cells with any of these compounds. In the transformation assays, type II and type III foci were observed at a 1-microgram/ml addition of AFB1 with or without BL activation; while with MOP and imperatorin, these types of foci were observed only with BL activation. Marmesin, although relatively more cytotoxic than the other furocoumarins studied, with a 50% lethal dose of less than 0.5 micrograms/ml, was not as mutagenic or potentially carcinogenic as were AFB1, imperatorin, or MOP with BL activation. These furocoumarins are considered to be involved in the etiology of the high incidence of skin cancer in Nigeria. Our experiments reinforce that concept and suggest that exposure to these furocoumarins may constitute a real carcinogenic hazard.     

Suppression of X-ray induced transformation by vitamin E in mouse C3H/10T1/2 cells

Vitamin E (d-alpha-tocopherol) was shown to decrease X-ray induced transformation in mouse C3H/10T1/2 cells. The d-alpha-tocopherol was active in the form of succinate diluted in ethanol, but was inactive at the highest nontoxic concentration of the pure substance dissolved in oil and diluted in acetone. Vitamin E succinate was effective when present throughout the entire assay period or when treatments began after confluence was reached at day 12 post-irradiation. It was ineffective if present only for the early portion of the radiation transformation assay period, indicating that its effect may be reversible. Vitamin E did not suppress the growth and expression of transformed C3H/10T1/2 cells as foci when the transformed cells were surrounded by a monolayer of normal cells.     

Disposition of endogenous homocysteine by mouse fibroblast C3H/10T1/2 Cl 8 and the chemically transformed C3H/10T1/2 MCA Cl 16 cells following methotrexate exposure

The tumorigenic cell line termed "MCA Cl 16" was derived from C3H/10T1/2 clone (Cl) 8 cells by chemical transformation in the presence of 3-methylcholanthrene [(MCA) CAS: 56-49-5]. Transformed (Cl 16) cells were more sensitive toward the cytotoxic effect of methotrexate (MTX) than their normal counterpart Cl 8 cells. The disposition of endogenous L-homocysteine (Hcy) was investigated in these two cell lines after MTX exposure. Both nonmalignant and transformed cells exported Hcy into the extracellular medium, and only small amounts were retained within the cells. The Hcy efflux from the malignant cells was markedly increased after MTX exposure (0.5-10 microM), and this effect was almost completely prevented by 5-formyl-tetrahydrofolate (THF), whereas treatment with thymidine plus hypoxanthine did not inhibit the MTX-dependent Hcy efflux. Cytotoxic concentration of MCA reduced rather than increased the Hcy efflux from these cells. High concentrations of MTX (greater than 10 microM) were required to increase the release of Hcy from nonmalignant cells. The enhancement of Hcy export from the malignant cells in the presence of MTX was not associated with cellular build-up of S-adenosyl-L-homocysteine (AdoHcy), indicating that the amount of intracellular Hcy was kept below the level required for inhibition or reversion of the AdoHcy hydrolase reaction. MTX-dependent Hcy efflux probably reflects cellular deficiency of 5-methyl-THF required for the salvage of Hcy to methionine and may therefore be a measure of lack of this reduced folate relative to the metabolic demand.     

Mutagenicity of nitrofluoranthenes, 3-aminofluoranthene and 1-nitropyrene in Chinese hamster V79 cells

1-Nitropyrene (1-NO₂Py), 3-nitrofluoranthene (3-NO₂Ft), 3-aminofluoranthene(3-NH₂Ft) and 8-nitrofluoranthene (8-NO₂Ft) were tested formutagenicity in cultured Chinese hamster V79 cells. Mutationsat the hypoxanthine-guanine phosphoribosyl transferase (HGPRT)gene locus were quantified. 1-NO₂Py had marginal direct-actingmutagenicity which was enhanced by a mixture of Aroclor 1254-and 1242-induced liver homogenates (S9) in the treatment medium.1-NO₂Py was more mutagenic with S9 activation than 3-NO₂Ft,3-NH₂Ft or 8-NO₂Ft. However, 8-NO₂Ft, 3-NO₂Ft and 3-NH₂Ft weremore mutagenic than 1-NO₂Py when a post-microsomal liver supernatant(S100) was used for metabolic activation. The results of theseinvestigations strongly support activation of some nitratedpolycyclic aromatic hydrocarbons to proximate mutagens by asequence of reductions and possible formation of polycyclicaromatic hydroxylamines.     

Maintenance of adult rat hepatocytes on C3H/10T1/2 cells.

A procedure is described for maintaining primary cultures of adult rat hepatocytes on a layer of irradiated C3H/10T1/2 cells. These hepatocytes were capable of metabolizing the liver carcinogen N-2-acetylaminofluorene to water-soluble products and after 14 days in culture could still metabolize approximately 70% of the Day 1 level. Hepatocytes maintained on the C3H/10T1/2 cells were inducible for the liver-specific enzyme tyrosine aminotransferase, and exhibited approximately a 4-fold induction by hydrocortisone during a 10-day culture period. Morphologically, these hepatocytes retained many characteristics of hepatocytes in vivo. By contrast, hepatocytes maintained on plastic lost both N-2-acetylaminofluorene-metabolizing ability and tyrosine aminotransferase activity by Day 5. This was presumably due to degeneration of the hepatocytes and an overgrowth by fibroblasts. The maintenance of morphologically and biochemically functional hepatocytes in culture on feeder cells may provide a valuable approach for studying drug metabolism and liver cell transformation in vitro.     

Suppression of x-ray induced transformation by Valium and aspirin in mouse C3H10T1/2 cells

Two commonly used drugs, Valium (diazepam) and Aspirin (acetylsalicyclic acid), were shown to suppress X-ray induced transformation in mouse C3H/10T1/2 cells. Valium was studied in an ethanol solution. Aspirin, which is soluble in both water and ethanol, was active only in the ethanol solution. Both drugs were effective only when present throughout the entire assay period.     

Oncogenic transformation of C3H/10T1/2 clone 8 mouse embryo cells by halogenated pyrimidine nucleosides.

Oncogenic transformation has been induced in vitro in the C3H/10T1/2 clone 8 line of mouse cells by exposure to 5-fluoro-2'-deoxyuridine (FUdR) or 5-fluorouracil. This transformation is both dose and time dependent and can be markedly decreased by simultaneous exposure of the cells to thymidine. The transformation induced by 5-fluorouracil is probably due to its intracellular conversion to FUdR or its monophosphate. Transformation by FUdR was found to be cell cycle dependent with maximum sensitivity to transformation occurring in early S phase. Cell lines that produced sarcomas in antithymocyte-treated syngeneic mice were isolated from FUdR-transformed cultures. Trifluorothymidine, 5-bromo-2'-deoxyuridine, and 5-iodo-2'-deoxyuridine induced no transformed foci in the C3H/10T1/2 clone 8 cell line. Thus, not all mutagens produce oncogenic transformation nor does the lack of mutagenicity, as classically measured, completely exclude the possibility that a given agent is oncogenic. Also, there was no evidence of the "switch on" of oncornaviral information in the FUdR-transformed cell lines.     

Phorbol ester induced 1,2-diacylglycerol accumulation and protein phosphorylation in C3H/10T1/2 mouse embryo cells

Within 10 min of addition of the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate(TPA) to C3H/10T1/2 mouse embryo fibroblasts, there is a two-foldincrease in the level of cellular 1,2-diacylglycerol levelscompared to controls. This increase in 1,2-diacylglycerol isdependent on the concentration of TPA added to the cell culturemedium. The ability of macrocyclic diterpenes to induce 1,2-diacylglycerolaccumulation correlated with their tumor promoting activityexcept for mezerein. The accumulation of 1,2-diacylglycerolin response to TPA was not blocked by a concentration of cycloheximidesufficient to inhibit protein synthesis by 95%. These data supportour previous suggestion that TPA activates a phospholipase C.During the same time period, TPA increased protein phosphorylationin both quiescent and growing cells. Proteins of mol. wt. 50000, 45 000, 35 000 and 27 000 are markedly phosphorylated inresponse to TPA in both growing and quiescent cultures. Therelationship of these phosphorylated proteins to a Ca²⁺ phospholipidactivated protein kinase remains to be determined.     

Vanadate enhances transformation of bovine papillomavirus DNA-transfected C3H/10T1/2 cells.

Bovine papillomavirus (BPV) DNA-transfected C3H/10T1/2 cells respond to tumor promoters by enhanced production of transformed foci. Vanadate, a suspected carcinogen, is a mitogen, generates active oxygen species and alters phosphorylation of proteins. We investigated whether vanadate would enhance transformation of BPV DNA-transfected C3H/10T1/2 cells. Transformed foci in BPV DNA transfected C3H/10T1/2 cells exposed continuously to vanadate for 21 days increased in a dose-dependent manner to 50-fold at 4 microM vanadate. This increase was not due to enhanced uptake of BPV DNA post transfection. Neither catalase nor superoxide dismutase inhibited the vanadate-mediated increase in transformed foci but this does not necessarily rule out the involvement of intracellular active oxygen species. At vanadate concentrations greater than 6 microM, cells lost adherence to the Petri plates. We conclude that vanadate is capable of enhancing BPV DNA-mediated cell transformation. Possible mechanisms may involve active oxygen species or altered patterns of protein phosphorylation.     

Effects of promoters on DNA synthesis in C3H/10T1/2 mouse fibroblasts

The synthesis of DNA has been studied by autoradiography and by measurements of tritiated thymidine ([3H]TdR) incorporation in cultured C3H/10T1/2 mouse embryo fibroblasts. The cells were first treated with 3-methylcholanthrene as an initiator and then with promoters according to schedules that produce oncogenic transformation. The levels of 3-methylcholanthrene used did not affect the growth or [3H]TdR incorporation of the cells. Treatment during the log phase of growth with 12-O-tetradecanoyl-phorbol-13-acetate, phorbol didecanoate, or 4alpha-phorbol didecanoate produced a transient inhibition of [3H]TdR incorporation with the maximum at 12 hr after treatment. This resulted in a temporary delay of growth followed by recovery of the normal cell-doubling time. Phorbol did not produce these effects, suggesting that the inhibition of DNA synthesis is associated with the process of promotion. Although treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate during stationary phase resulted in a 2- to 3-fold stimulation of [3H]TdR incorporation, multiple treatments spanning log and stationary phases were found to be necessary for promotion.