A.R. Peterson

The incorporation of 5-fluoro-2′-deoxyuridine into DNA of mammalian tumor cells

Summary Treatment of L1210 mouse leukemia cells with 10 −6 M [ 3 h]5-fluoro-2′-deoxy-uridine resulted in the incorporation of radioactivity into DNA. The DNA hydrolysate was shown to contain [ 3 h]5-fluoro-2′-deoxyuridylic acid by an unequivocal assay involving the formation of a tightly-bound complex with thymidylate synthetase in the presence of 5,10-methylenetetrahydrofolate. The extent of incorporation was estimated to be 1 molecule of 5-fluoro-2′-deoxyuridylic acid per 10 DNA nucleotides.

Effect of cholesterol epoxides on the inhibition of intercellular communication and on mutation induction in chinese hamster V79 cells

Cholesterol, cholesterol-5 alpha, 6 alpha-epoxide, cholesterol-5 beta, 6 beta-epoxide and cholestane-3 beta,5 alpha,6 beta-triol were tested for their ability to induce mutations at the Na+/K+-ATPase loci of the Chinese hamster V79 cells. None of these compounds induced ouabain-resistant mutations compared to the background mutation frequency in the control cells. These compounds were further tested for their ability to inhibit intercellular communication, using the Chinese hamster V79 cell metabolic cooperation assay. The diastereomeric epoxides and cholestane-triol, but not cholesterol, were found to be inhibitors of intercellular communication in a manner similar to other known tumor promoters.

Induction of mutations by 5-fluorodeoxyuridine: A mechanism of self-potentiated drug resistance?

In medium containing concentrations of deoxycytidine that occur in vivo, 5-fluorodeoxyuridine induced mutation frequencies 6-90 fold greater than spontaneous mutant frequencies at two genetic loci in Chinese hamster cells. In medium lacking deoxycytidine, 5-fluorodeoxyuridine was more cytotoxic but induced no mutants. Hence, the effectiveness of cancer therapy with 5-fluorodeoxyuridine may be limited by self potentiated development of 5-fluorodeoxyuridine-resistant mutants and enhanced and prolonged by manipulating deoxycytidine metabolism.

Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells.

SummaryA reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol.

Mechanisms of ascorbic acid-induced inhibition of chemical transformation in C3H/10T1/2 cells

Ascorbate irreversibly inhibited morphological transformation induced by methylcholanthrene in C3H/10T1/2 cells. To determine the mechanisms of this inhibition, we studied ascorbate uptake, redox potential, matrix proteins, and lipid composition of 10T1/2 cells. Ascorbate (16.8 nmol/dish) saturated cells and reduced the NADH-to-NAD+ ratio. Daily treatments with ascorbate, 28 nmol/dish, maintained intracellular ascorbate and reduced NADH by half. Matrix collagen and glycoproteins were stimulated by ascorbate, Iso-ascorbate, and dehydroascorbate in a dose-dependent manner. Both ascorbate and dehydroascorbate reduced total lipids with time; neutral lipids increased but were released into the media, phospholipids were modified, cholesterol-phospholipid ratios declined, and an inverse relationship between unsaturation index and cholesterol-phospholipids was apparent. Lipophilic bodies gradually accumulated. Our data suggest that inhibition of transformation by ascorbate, Iso-ascorbate, or dehydroascorbate may be associated with regulation of the redox potential, glycoproteins, and lipids in 10T1/2 cells.

Deoxyribonucleoside-Induced Selective Modulation of Cytotoxicity and Mutagenesis

Treatment of Chinese hamster V79 cells with dThd, dCyd, or dThd plus dCyd increased MNNG-induced AGr-, TGr-, and Ouar-mutant frequencies but did not significantly increase background mutant frequencies. All the AGr colonies that were isolated possessed phenotypes characteristic of HGPRT-deficient mutants, and the deoxyribonucleosides did not selectively affect the growth of the mutants, nor the selecting efficiency of AG, and did not significantly enhance background mutagenesis. These data show that both dThd and dCyd facilitated MNNG-induced mutagenesis. This facilitation was maximal when cells were exposed to the deoxyribonucleosides throughout the first doubling time (24 h) after treatment with MNNG and for 4 more doubling times prior to mutant selection with AG. This indicates that one round of DNA replication was sufficient for mispairing of methylated bases in the DNA with the C and T provided by the deoxyribonucleosides, and that 4-6 doublings prior to mutant selection with AG were necessary to deplete pre-existing hypoxanthine: guanine phosphoribosyl transferase in newly mutated cells. The dCyd facilitated mutagenesis by FdUrd, which was not mutagenic without dCyd, indicating that increased dCTP:dTTP ratios were mutagenic. Treatment with FdUrd plus dCyd also induced FdUrdr cells, suggesting that inhibition of dCyd utilization may prevent the development of FdUrd-resistance in cancer chemotherapy. Although dCyd and dThd facilitated mutagenesis in cells treated with monofunctional alkylating agents that methylate DNA oxygens, facilitation of mutagenesis did not occur in cells treated with BCNU, which cross links DNA, nor with benzo(a)pyrene and aflatoxin B1, which are frame shift mutagens, nor with MMS, which produces barely detectable levels of O-methylation in DNA. Virtually non toxic concentrations of dThd potentiated the cytotoxicity of MNNG more than 10-fold but that of MMS was potentiated only about 2-fold showing that O-alkylation of DNA was associated not only with the facilitation of mutagenesis but also with the potentiation of cytotoxicity. The potentiation of MNNG-induced cytotoxicity was maximal in V79 and L1210 cells after only 2 h treatment with dThd, showing that not even one round of DNA replication was necessary for this potentiation. Moreover, dCyd abolished the potentiation, and, at equitoxic concentrations, MNNG induced higher mutant frequencies than did MMS. These data show that the mechanisms by which methylating agents plus dThd induce mutagenesis are fundamentally different from their mechanisms of cytotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Genetic and Probability Aspects of Cell Transformation by Chemical Carcinogens

Publisher Summary This chapter discusses the genetic and probability aspects of cell transformation by chemical carcinogens. In C3H/lOT1/2 cell system with 3-methylcholanthrene as the carcinogen, nothing, including replating, affects the validity of the probabilistic equations when n is greater than 5. The peak in the transformation curve at n = 4 to 5 cannot per se explain the high transformation frequencies obtained at low cell numbers. Possible explanations for this paradox might be: (1) that more than one gene mutation is required for transformation; (2) that the event resulting in transformation is not a simple gene mutation; or (3) that another nonmutational process requiring cell divisions is involved in transformation. Such a process might be categorized as “promotion” or possibly the production of chromosomal aberrations.

Oncogenic transformation of C3H/10T1/2 Cl 8 mouse embryo fibroblasts by inhibitors of nucleotide metabolism.

Morphological or oncogenic transformation of mouse embryo, C3H/10T1/2 Cl 8 fibroblasts was induced by methotrexate, 5-fluorouracil and 5-fluorodeoxyuridine. It is known that these compounds cause inhibition of thymidylate synthetase and, hence, depletion of deoxythymidine triphosphate (dTTP) and an increased ratio of deoxycytidine triphosphate (dCTP) to dTTP in the deoxyribonucleotide pools that are used for DNA synthesis in mammalian cells. This ratio is, in effect, increased by treating mammalian cells with arabinosyl cytosine and 5-azacytidine, which are converted into analogs of dCTP in mammalian cells and also induce oncogenic transformation of C3H/10T1/2 cells. By contrast, trifluorothymidine, 5-bromodeoxyuridine and 5-iododeoxyuridine, which are analogs of thymidine that in effect reduce the dCTP:dTTP ratio, did not induce oncogenic transformation. Moreover, thymidine was selectively lethal to tumorigenic C3H/10T1/2 cells and inhibited oncogenic transformation in cells treated with 5-fluorodeoxyuridine. These observations suggest that treatments that effectively increase the dCTP:dTTP ratio in mammalian cells facilitate oncogenic transformation of C3H/10T1/2 cells, whereas treatments that have the effect of decreasing this ratio inhibit transformation. However, dCyd did not induce oncogenic transformation of C3H/10T1/2 cells, although it has been shown to increase the dCTP:dTTP ratio in mammalian cells. Thus, increasing this ratio may not be sufficient to cause the transformation.

Association between the cytotoxicity of thymidine and tumorigenicity of clones derived from C3H10T12 mouse embryo fibroblasts

Abstract We have measured the cytotoxicity of thymidine to C3H 10T 1 2 mouse embryo fibroblasts derived from morphologically transformed foci of cells from cultures exposed to chemical carcinogens. Four of these cell lines have previously been shown to be tumorigenic in irradiated syngeneic hosts and were all more sensitive to the lethal effects of thymidine than were the non-transformed cells. Strikingly, the most tumorigenic of the cell lines were most sensitive to thymidine. Differences in plating efficiencies or growth rates of the various cell lines were not associated with differences in thymidine sensitivity.

FACILITATION BY PYRIMIDINE NUCLEOSIDES AND HYPOX-ANTHINE OF MNNG MUTAGENESIS IN CHINESE HAMSTER CELLS

Publisher Summary This chapter examines the facilitation by pyrimidine nucleosides and hypoxanthine of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis in Chinese hamster cells. In the experiment described in the chapter, hypoxanthine (Hx), thymidine (TdR), and deoxycytidine (CdR), at concentrations of 10 M, increased the yield of 8-azaguanine resistant (AzG ) mutants induced by MNNG in cultured Chinese hamster V79 cells. The cytotoxicity of MNNG was increased twofold in the presence of Hx, and tenfold in the presence of TdR. This effect of TdR was abolished by equal concentrations of CdR, which by itself did not increase the cytotoxicity of MNNG. The nucleosides did not affect the growth or expression time of the hypoxanthine guanine phosphoribosyl transferase deficient (HGPRT) mutants, and the same extent of alkali-labile DNA damage occurred in cells treated with alkylating agents in the presence and absence of TdR and CdR. The increase in mutation frequency in the presence of these nucleosides occurred not only with MNNG but also with ethylating agents and not only at the HGPRT locus but also at that of the ouabain sensitive sodium–potassium ATPase.