Effects of tumor promoters on the differentiation of C3H/10T 1/2 mouse embryo fibroblasts.
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Adipose Tissue↗
Biomedical subjects
C Heidelberger
Publications and source records attributed to C Heidelberger.
Induction of cytotoxicity, mutation, cytogenetic changes, and neoplastic transformation by benzo(a)pyrene and derivatives in C3H/10T1/2 clone 8 mouse fibroblasts.
Benzo(a)pyrene (BaP), a series of its metabolic derivatives, and benzo(e)pyrene, a very weakly carcinogenic isomer, were tested for their biological effects on transformable C3H/10T1/2 cells. These cells were used as targets in a series of assays designed to measure oncogenic transformation, mutation to ouabain resistance, cytotoxicity, and induction of cytogenetic changes, as evidenced by chromosomal aberrations and sister chromatid exchange. Of all the compounds tested, only the parent hydrocarbon, BaP, an (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene were found to be significantly active in producing transformation and cytogenetic alterations. BaP, (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, and (+/-)-7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, however, were all effective inducers of mutation in C3H/10T1/2 cells. (+/-)-7 alpha, 8 beta-Dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was the most potent agent in tests for cytotoxicity, Benzo(e)pyrene was inactive in all assays examined. Among the compounds tested, there was a correlation between the ability to induce cytogenetic changes and the ability to produce mutation and transformation. These results support the demonstrated role of (+/-)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene as a proximal carcinogenic form of BaP and illustrate the utility of the C3H/10T1/2 cell system as an important tool for the detection of genotoxic damage by carcinogenic chemicals.
Animals↗
Metabolic activation of benzo(a)pyrene by transformable and nontransformable C3H mouse fibroblasts in culture.
Incubation of 2.5 microM benzo(a)pyrene (BaP) with C3H/10T 1/2 or CVP3SC6 (CVP) mouse fibroblasts for 48 hr resulted in the metabolism of 36 to 42% of the BaP to organic soluble derivatives, which cochromatographed with 7,8-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene, 9,10-trans-dihydroxy-9,10-dihydrobenzo(a)pyrene, 3-hydroxybenzo(a)pyrene, and 9-hydroxybenzo(a)pyrene, or to water-soluble derivatives. The formation of both organic and water-soluble metabolites during the 48-hr period increased proportionally with time, except in the case of BaP phenols, which increased initially but then remained the same or decreased. The distribution of organic soluble metabolites in the extracellular culture medium consisted primarily of BaP diols and was significantly different from that found inside the cells. The intracellular profile of organic soluble metabolites produced by both cell lines consisted predominantly of BaP phenolic derivatives and was qualitatively similar to the spectrum of metabolites produced by the incubation of BaP with C3H/10T 1/2 or CVP cell microsomes. The nature of the BaP water-soluble derivatives produced by the C3H/10T 1/2 and CVP cell lines was investigated by hydrolysis of culture medium with beta-glucuronidase and arylsulfatase. Although sulfation was not a major conjugation pathway for BaP in these cells, glucuronidation of BaP phenols was found to account for 30% of the total water-soluble derivatives. The similarity in the kinetics and qualitative nature of the metabolism of BaP by C3H/10T 1/2 and CVP cells indicates that both cell lines are equally capable of biosynthesizing the proximal carcinogen, 7,8-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene. Analysis of the water-soluble metabolites produced by these cells suggests further that the nonresponsiveness of the CVP cells to BaP-induced transformation cannot be accounted for on the basis of an increased detoxication of 7,8-trans-dihydroxy-7,8-dihydrobenzo(a)pyrene.
Animals↗
Isolation of fluoropyrimidine-resistant murine leukemic cell lines by one-step mutation and selection.
The effectiveness of the clinically useful fluoropyrimidines in the treatment of human cancer is often limited by the development of resistance to the drugs by the tumor. In order to systematically study the mechanisms of resistance to 5-fluorouracil and its nucleoside derivatives, several cell lines resistant to these drugs have been derived from murine leukemia cells by a one-step mutation and selection procedure. Logarithmically growing suspension cultures of L1210 and P388 cells were treated with ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, or ICR-191 at concentrations which result in 20 to 30% cell survival. After a 10-day expression time, mutagenized cells were plated into soft-agarose medium that contained 10(-5) M 5-fluorouracil, 10(-5) M 5-fluoro-2'-deoxyuridine, or 10(-6) M 5-fluorouridine. Twenty stable clones were isolated and found to be 5- to 28-fold resistant to growth inhibition by 5-fluorouracil, 4,000- to 25,000-fold resistant to 5-fluoro-2'-deoxyuridine, or 8- to 220-fold resistant to 5-fluorouridine. The clones retain their drug-resistant phenotype after repeated passaging in the absence of selection. Since the biochemical changes responsible for resistance to one drug can render the cells collaterally sensitive to other drugs, the growth-inhibitory effects of antimetabolites that inhibit other steps in pyrimidine metabolism were examined in the wild-type cells and in the fluoropyrimidine-resistant sublines. Although cross-resistance to 5-azacytidine was found in L1210 cells selected for resistance to 5-fluorouridine, none of the cell lines tested demonstrated collateral sensitivity to methotrexate, 1-beta-D-arabinofuranosylcytosine, 5-azacytidine, or N-(phosphonacetyl)-L-aspartate.
Animals↗
Biochemical characterization of fluoropyrimidine-resistant murine leukemic cell lines.
Twenty clones stably resistant to 5-fluorouracil, 5-fluoro-2'-deoxyuridine, or 5-fluorouridine have been isolated from L1210 or P388 murine leukemia cells by a one-step mutation and selection procedure. The activities of enzymes of the pyrimidine salvage pathway relevant to the activation of these drugs have been determined in order to elucidate the mechanisms of resistance in these cells. Cell line resistant to 5-fluorouracil have 7 to 50% of the pyrimidine phosphoribosyltransferase activity found in the wild-type cells, with 5-fluorouracil, uracil, or orotate as substrate. Cells selected for resistance to 5-fluoro-2'-deoxyuridine have no detectable thymidine kinase activity. 5-Fluorouridine-resistant cells have 3 to 25% of the uridine kinase activity measured in the wild-type cell lines. No significant changes were observed in the activities of thymidylate synthetase, nucleoside phosphorylases, or 5-fluorouridylate kinase in any of the resistant cell lines. These findings have relevance to the treatment of human cancer, since pyrimidine phosphoribosyltransferase, thymidine kinase, or uridine kinase could be assayed in tumor biopsies in order to predict whether the fluoropyrimidines would be effective in individual patients.
Animals↗
Effects of praziquantel, a new antischistosomal drug, on the mutation and transformation of mammalian cells.
Praziquantel (PQ), a tetrahydroquinoline derivative, is a new and clinically effective antischistosomal drug, which has been shown to lack or to possess very weak mutagenic activity. However, in bacteria, this compound can act as a weak comutagen that increases the mutagenicity of several chemical mutagens and carcinogens. We have found that PQ can act as a very weak comutagen in animal cells. At 10 to 50 micrograms/ml, PQ increased the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine and 2-methoxy-6-chloro-9-[3-(2-chloroethyl)amino-propylamino]acridine dihydrochloride about 2-fold in Chinese hamster V-79 cells. In C3H/10T 1/2 mouse embryo cells, PQ exhibited only negligible comutagenic activity. PQ did not oncogenically transform C3H/10T 1/2 cells but had a pronounced effect on 3-methylcholanthrene-induced transformation of these cells. When PQ was coadministered with or added after 3-methylcholanthrene treatment, the number of type III foci produced was about 5-fold lower than in cultures treated with 3-methylcholanthrene alone. Therefore, PQ can inhibit type III focus formation in C3H/10T 1/2 cells.
Animals↗
The incorporation of 5-fluoro-2'-deoxyuridine into DNA of mammalian tumor cells.
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Animals↗
Development of two cloned epithelial cell lines from normal adult mouse and rat ventral prostates.
Two epithelial cell lines were established, one from adult C3H mouse and one from adult Fischer rat ventral prostate. These cell lines were obtained from explant cultures, using Ham's F12 medium supplemented with HEPES, insulin, testosterone, hydrocortisone, epidermal growth factor, and 7.5% fetal bovine serum. A low concentration of trypsin and EDTA in Ca++- and Mg++-free phosphate buffer was used for passaging the cells. The rat cell line was established following implantation of prostate tissue in nude mice. These cell lines stained positively for acid phosphatase and were dependent upon epidermal growth factor for growth. Morphological studies, including electron microscopy, revealed a highly characteristic epithelial morphology of both cell lines. These cell lines have hypotetraploid chromosome number and are capable of metabolizing benzo(a)pyrene. We propose the application of these cells as models for the study of prostate carcinogenesis.
Acid Phosphatase↗
Interaction of 5-ethynyl-2'-deoxyuridylate with thymidylate synthetase.
The interaction of 5-ethynyl-2'-deoxyuridylate (5-ethynyl-dUMP; 1) with thymidylate (dTMP) synthetase has been investigated. The compound was an inhibitor of the enzyme, competitive with 2'-deoxyuridylate (dUMP) when the reaction was initiated by addition of enzyme (Ki = 2.7 X 10(-6) M). However, upon preincubation of 1 with dTMP synthetase, the inhibition pattern became noncompetitive. The time course of the enzyme reaction in the presence of 1 was nonlinear, indicating an increase in binding with time. Irreversible inactivation of the enzyme did not occur. The compound did not appear to become altered structurally as a result of interaction with the enzyme. A ternary complex was formed among dTMP synthetase, compound 1, and 5,10-methylenetetrahydrofolate, which was stable enough to survive Sephadex G-25 filtration but dissociated upon denaturation of the enzyme.
Binding, Competitive↗
Oncogenic transformation and mutation of C3H/10T 1/2 clone 8 mouse embryo fibroblasts by alkylating agents.
Two-hr treatments with N-methyl- and N-ethyl-N'-nitro-N-nitrosoguanidines and ethyl methanesulfonate induced ouabain-resistant mutants in C3H/10T 1/2 cells. The alkylnitronitroso-guanidines gave linear dose-response curves and were more potent mutagens than were ethyl methanesulfonate and methyl methanesulfonate. These differences in potency were largely due to differences in the half-lives of the alkylating agents in culture medium. Differences in mutation frequencies at equitoxic concentrations of the alkylating agents are considered to reflect differences in the chemical mechanisms of alkylation and mutagenesis by the compounds. However, the frequencies of mutations produced at equitoxic concentrations were not uniformly associated with the nucleophilic selectivities of the compounds as expressed by their published Swain-Scott substrate constants. Whether or not followed by repeated replating, the yield of oncogenically transformed foci of asynchronous cells after treatment with the alkylating agents was so low that we could not obtain dose-response curves, and the yield may not be significant. By contrast, in previous experiments with N-methyl-N'-nitro-N-nitrosoguanidines and polycyclic hydrocarbons in Syrian hamster embryo fibroblasts and with ultraviolet light and polycyclic hydrocarbons in C3H/10T 1/2 cells, transformation occurred to an equal or greater extent than mutation measured in the same cells.
Alkylating Agents↗
On the rational development of a new drug: the example of the fluorinated pyrimidines.
The specific rationale for the development of the fluorinated pyrimidines and the predictions of their mode of action have been described. The mode of action of 5-FU involves incorporation into RNA and metabolic activation to 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), which inhibits the essential enzyme for DNA synthesis, thymidylate synthetase. I believe that the latter effect is responsible for the major chemotherapeutic activity. Our studies on the mechanism of the inhibition of that enzyme have led to the demonstration that a ternary covalent complex of enzyme, inhibitor, and cofactor is formed that results in essentially irreversible inhibition. The nature of this ternary complex has enabled us to devise ultrasensitive competitive ligand binding assays for thymidylate synthetase, FdUMP, and dUMP, the substrate of the enzyme. These determinants will be studied in needle biopsy specimens obtained from cancer patients undergoing chemotherapy with 5-FU in a prospective clinical investigation.
Animals↗
Association between the cytotoxicity of thymidine and tumorigenicity of clones derived from C3H/10T1/2 mouse embryo fibroblasts.
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Animals↗
Inhibition of nucleoside phosphorylase cleavage of 5-fluoro-2'-deoxyuridine by 2,4-pyrimidinedione derivatives.
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Animals↗
Further evidence that ouabain-resistant variants induced by chemical carcinogens in transformable C3H/10T1/2 Cl 8 mouse fibroblasts are mutants.
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Acetoxyacetylaminofluorene↗
Probabilistic view of the transformation of cultured C3H/10T1/2 mouse embryo fibroblasts by 3-methylcholanthrene.
When C3H/10T1/2 cells are treated with a given concentration of a chemical carcinogen, the transformation frequency can vary over 4 orders of magnitude, depending primarily upon the number of cells plated. To explain this phenomenon, we have developed a probabilistic theory of the formation of transformed foci in this system. We define p1 as the probability that a cell will be activated by carcinogen treatment, p2 as the probability per cell generation that an activated cell will be transformed, and p3 as the probability per cell generation that an activated cell will be deactivated. The equation we have derived: log (F/N) = log [2p1p2(1 -- p3)/2(1 -- p3) -- 1]+ n log (1 -- p3) describes focus formation; F is mean number of loci per dish after carcinogen treatment, N is number of cells in a dish at confluence, and n is number of cell generations to confluence. This equation has been verified experimentally; p3 = 0.24 and p1p2 = 3.8 X 10(-6) at a single concentration of 3-methylcholanthrene. This relationship explains previously inexplicable effects of cell density on transformation frequency.
Animals↗
Isolation of mutants temperature-sensitive for expression of the transformed state from chemically transformed C3H/10T1/2 cells.
58 MCA Cl 16 is an oncogenic methylcholanthrene-transformed variant of the non-transformed mouse embryo fibroblast cell line, C3H/10T1/2 Cl 8. Using two different protocols, we have isolated six temperature-sensitive mutants from N-methyl-N'-nitro-N-nitrosoguanidine treated cultures of 58 MCA Cl 16. C3H/10T1/2 Cl 8, 58 MCA Cl 16 and the six mutant lines were characterized with respect to several properties associated with the transformed state: morphology, saturation density, anchorage independence, cell surface morphology and growth in medium containing 1% fetal calf serum. In general, C3H/10T1/2 cells behaved as non-transformed, whether grown at 33 degrees C or 39.5 degrees C. The transformed parental line and all six mutants behaved as transformed cells at 33 degrees C. At 39.5 degrees C, only the parental transformed line retained the transformed phenotype. Three of the mutants revert towards non-transformed behavior at 39.5 degrees C for all of the properties tested. The remaining mutants are temperature-sensitive for some, but not all, transformed characteristics. Thus, while the expression of these transformed properties is sometimes coupled, we have been able to dissociate the expression of traits such as saturation density, anchorage independence and transformed morphology from each other. These mutants should prove to be valuable tools in the study of the mechanisms which underly the expression of the chemically-induced transformed state.
Animals↗
Inhibition of induced differentiation of C3H/10T 1/2 clone 8 mouse embryo cells by tumor promoters.
C3H/10T 1/2 cells were induced to differentiate into muscle cells by treatment with 5-azacytidine, and the effects of tumor promoters, nonpromoters, and inhibitors of tumor promotion on this induced differentiation were investigated. Cell morphology was dramatically changed within 30 min after treatment with phorbol ester-related tumor promoters and mezereine but not with other tumor promoters. There was a good correlation between the tumor-promoting activity of the compounds and their inhibitory action on differentiation except in case of phenobarbital; this promoter of liver carcinogenesis did not inhibit differentiation. Inhibitors of tumor promotion, dexamethasone, fluocinolone acetonide, retinoic acid, and antipain, also inhibited the 5-azacytidine-induced differentiation. When the cells were treated after induction of differentiation with 12-O-tetradecanoylphorbol-13-acetate, a potent tumor promoter, and simultaneously with one of those inhibitors of tumor promotion, the inhibitory action of 12-O-tetradecanoylphorbol-13-acetate was not affected. From these facts, it is evident that using a single phenomenon produced by one class of tumor promoter as a criterion for screening environmental tumor promoters is not justifiable.
Animals↗
Specificity of pyrimidine nucleoside phosphorylases and the phosphorolysis of 5-fluoro-2'-deoxyuridine.
Isoelectric focusing and studies with 1-(2'-deoxy-beta-D-glucopyranosyl)thymine (GPT), a specific inhibitor of uridine phosphorylase activity, were used to determine the substrate specificities of mammalian pyrimidine nucleoside phosphorylases and their cleavage of 5-fluoro-2'-deoxyuridine (FdUrd). Isoelectric focusing profiles for the cytosol fractions from Ehrlich ascites cells and from Novikoff hepatoma cells each consisted essentially of one peak of nucleoside phosphorylase activity [isoelectric points (pl) 5.4 and 5.8, respectively] that cleaved both uridine and thymidine (dThd), as well as FdUrd. By contrast, cytosol fractions from HeLa (S3) cells, mouse liver, and normal human leukocytes each exhibited a major peak of activity (pl 4.6, 6.5, and 4.9, respectively) that cleaved only dThd and FdUrd, while mouse liver exhibited a second peak (pl 5.2) that cleaved primarily uridine. To distinguish clearly between (a) uridine phosphorylases that cleave primarily uridine and that are inhibited by GPT and (b)dThd phosphorylases that cleave only deoxynucleosides and that are not inhibited by GPT, we propose the term "uridine-deoxyuridine phosphorylases" to define those pyrimidine nucleoside phosphorylases that cleave both uridine and dThd and that are inhibited by GPT. On the basis of this definition and studies with GPT in nonfocused cytosol preparations, we conclude that FdUrd is cleaved to 5-fluorouracil by uridine-deoxyuridine phosphorylase activity in Ehrlich ascites cells and in Novikoff hepatoma cells, and by dThd phosphorylases in mouse liver, in normal human leukocytes, and in HeLa (S3) cells.
Animals↗