thioguanine-anhydrous and Teratoma

From among a series of stable, aphidicolin-resistant mutant strains of mouse teratocarcinoma, derived from a multipotent parental line (PSA-1-80), three were selected for further study on the basis of their comparatively high degrees of resistance and elevated frequencies of spontaneous forward mutation to 6-thioguanine and ouabain resistance. Fluctuation tests confirmed that they were mutator strains. Since each of the three mutants was isolated after multiple rounds of selection, and since a variety of biochemical abnormalities were observed, it is likely that a number of mechanisms, probably consisting of overlapping subsets, determine the phenotypes. Abnormalities in the metabolism of the nucleotide substrates for polymerization are likely to be of major importance in mutants designated Aph-2 and Aph-3, as there were marked alterations in the dCTP and dATP pool sizes. The specific activity of DNA polymerase alpha was also increased. For the case of Aph-3, which exhibited the greatest (400-fold) increase in resistance to aphidicolin, a mutation in the structural gene for DNA polymerase alpha may be an additional important component, since in vitro assays revealed that the isolated enzyme was resistant to aphidicolin. For the case of Aph-1 however, only minor alterations in dNTP pools were observed, and there was no increase in the specific activity of DNA polymerase alpha or in the aphidicolin resistance of the isolated DNA polymerase alpha, suggesting yet another mechanism(s) underlying the aphidicolin resistance/mutator phenotype. All three mutants formed subcutaneous tumors in syngeneic mice; both Aph-1 and Aph-2 were multipotent; whereas Aph-3 was nullipotent.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aphidicolin; Cell Line; Chimera; Diterpenes; DNA-Directed DNA Polymerase; Drug Resistance; Mice; Mutation; Ouabain; Teratoma; Thioguanine

Metabolic cooperation may be associated with the processes of compaction and subsequent differentiation in aggregates of embryonal carcinoma cells (ECC). To determine if the gap junctions present in loose and compacted aggregates of H6 ECC are active in metabolic cooperation, aggregates of each type containing a mixture of 5-bromodeoxyuridine- and 6-thioguanine-resistant H6 cells were exposed to HAT medium, 6-thioguanine, or [3H]thymidine. These three methods indicated that some crossfeeding occurred through the small clusters of gap junctions in loose aggregates and more crossfeeding occurred through the larger clusters of gap junctions in compacted aggregates.

Topics: Animals; Cell Aggregation; Cell Communication; Cell Line; Intercellular Junctions; Mice; Teratoma; Thioguanine

A mutator strain [AraCr (1.5)4], isolated from mutagenized cultures of multipotent mouse teratocarcinoma cells (embryonal carcinoma stem cells), exhibited a dNTP pool imbalance, with more than a 10-fold relative increase in the intracellular concentration of dCTP. The increase in the spontaneous rate of mutation for 6-thioguanine resistance was 3.6-fold and for ouabain resistance, 7.9-fold. Normalization of the dCTP/dTTP ratio by addition of thymidine and deoxycytidine to the media was associated with normalization of the mutation rates. AraCr (1.5)4 cell retained its multipotency (including chimerization potential) when injected into blastocysts. Moreover, its differentiated progeny expressed the dNTP pool imbalance and mutator phenotype in vitro. The preliminary finding of an increased frequency of morphologically abnormal embryos derived from a series of transplanted blastocysts injected with AraC2 (1.5)4 stem cells is consistent with significant phenotypic effects in vivo.

Topics: Animals; Cell Line; Chimera; Cytarabine; Deoxycytosine Nucleotides; Drug Resistance; Embryonal Carcinoma Stem Cells; Mice; Mutation; Neoplasm Transplantation; Neoplastic Stem Cells; Ouabain; Phenotype; Teratoma; Thioguanine; Transplantation, Isogeneic

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10 -tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 X 10(4) cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4-6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5'-monophosphate (IMP)/min/microgram protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polycyclic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 microM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10(6) colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.

Topics: Biotransformation; Breast Neoplasms; Carcinogens; Cells, Cultured; Drug Resistance; Humans; Hypoxanthine Phosphoribosyltransferase; Mutagenicity Tests; Mutation; Teratoma; Thioguanine

Cells of the teratocarcinoma-derived line P19S1801A1 (01A1) are pluripotent embryonal carcinoma cells and can be induced to differentiate when aggregated and exposed to dimethyl sulfoxide. Many nonneural cell types appear in dimethyl sulfoxide-treated cultures, cardiac and skeletal muscle being the most easily identified. We have used immunofluorescence procedures with monoclonal antibodies directed against muscle myosin to confirm and quantitate the number of muscle cells formed. A monoclonal antibody reactive with an embryonal carcinoma-specific surface antigen was used to confirm the disappearance of undifferentiated cells after dimethyl sulfoxide treatment. Cardiac muscle cells developed within 4 to 5 days of drug exposure, but skeletal muscle cells did not become evident until 7 to 8 days. We have isolated a mutant cell line (D3) which appears to be incapable of muscle development but which does form neurons and glial cells when exposed to high retinoic acid concentrations. We propose that this system will be useful for investigation of the means by which pluripotent cells become committed to development along the striated muscle lineages.

Topics: Animals; Antibodies, Monoclonal; Butyrates; Butyric Acid; Cell Differentiation; Cell Line; Dimethyl Sulfoxide; Embryonal Carcinoma Stem Cells; Heart; Mice; Muscles; Mutation; Myosins; Neoplastic Stem Cells; Neurons; Stem Cells; Teratoma; Thioguanine

Topics: Animals; Cell Differentiation; Drug Resistance; Ethyl Methanesulfonate; Genes, Dominant; Hybrid Cells; Male; Mice; Neoplasms, Experimental; Ouabain; Phenotype; Teratoma; Thioguanine