aphidicolin and Teratoma

Several differentiation-specific genes, including those for collagen IV and laminin, are induced by retinoic acid (RA) in mouse F9 teratocarcinoma cells. Dibutyryl cAMP can enhance the effect of RA in these cells, but dibutyryl cAMP alone does not induce these genes. Inhibition of RNA synthesis with 5-6-dichloro-1-B-D-ribofuranosylbenzimidazole prevents the induction of these genes by RA; inhibition of DNA synthesis with aphidicolin does not prevent the induction. In vitro transcription studies (Wang et al., Dev. Biol., 107:75-86, 1985) demonstrate that these differentiation-specific genes are regulated by RA at least partially at the level of transcription. To determine whether the regulation of transcription of these differentiation-specific genes is a primary effect of RA, we measured the sensitivity of the induction of mRNAs specific for these RA-inducible genes to inhibitors of protein synthesis. RNA was isolated from F9 cells that had been treated for 20 hr with RA (with or without dibutyryl cAMP) in the presence or absence of either cycloheximide or puromycin. We then hybridized the 32P-labeled recombinant plasmids collagen IV (alpha 1) (pcI5), laminin B1 (pcI56), and pcJ6 to RNA from the treated cells. Both cycloheximide and puromycin inhibited the RA induction of the collagen IV (alpha 1), laminin B1, and J6 mRNAs. In contrast, in a control experiment, a 20-hr treatment with cycloheximide did not inhibit the accumulation of metallothionein I-specific mRNA in response to zinc in F9 cells. Thus protein synthesis is required for the expression of the collagen IV (alpha 1), laminin B1, and J6 genes, and this result suggests that the transcriptional regulation of these genes by RA is indirect.

Topics: Animals; Aphidicolin; Bucladesine; Cell Line; Collagen; Dichlororibofuranosylbenzimidazole; Diterpenes; Gene Expression Regulation; Laminin; Protein Synthesis Inhibitors; Puromycin; Retinol-Binding Proteins; RNA, Messenger; Teratoma; Tretinoin

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

With the long-range goals of elucidating the biochemical genetics and the phenotypic expression, both in vitro and in vivo, of mutator strains of mammalian cells, we have isolated and partially characterized a series of drug-resistant mutants from a subline a feeder-dependent parental mouse teratocarcinoma line (PSA-1) known to be capable of chimerizing host blastocysts via the injection of inner cell mass. Two series of stable mutants were isolated--one (Aphr) selected on the basis of resistance to aphidicolin (Aph), a specific inhibitor of DNA polymerase-alpha, and the other (AraCr) selected by resistance to arabinofuranosyl cytosine, an analogue of cytosine. Irrespective of the method of selection, most of the 32 mutants isolated were resistant to both agents, although to different degrees, and with variations in growth characteristics, deoxynucleoside sensitivities, and sensitivities to mutagens (5-bromodeoxyuridine, N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet light). A protocol of multi-step selection provided stable mutants highly resistant to aphidicolin and only mildly resistant to AraC, even in the absence of prior mutagenesis, and is thus recommended as an approach to the isolation of candidate mutator strains of multipotent teratocarcinomas.

Topics: Animals; Aphidicolin; Cell Line; Cell Separation; Cytarabine; Diterpenes; Dose-Response Relationship, Drug; Drug Resistance; Methylnitronitrosoguanidine; Mice; Teratoma