Isolation of complementary DNA clones for genes exhibiting reduced expression after treatment of mouse teratocarcinoma stem cells with a tumor-promoting phorbol ester.
For the study of the effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on early mammalian cell differentiation, a complementary DNA (cDNA) library was constructed on the poly(A)+RNAs extracted from undifferentiated F9 cells derived from a 129/Sv mouse teratocarcinoma OTT6050, and screening was done for the cNDA sequences corresponding to the mRNAs, the levels of which decreased significantly in the F9 cells after the TPA treatment. From about 80,000 clones screened, 3 different cDNA clones, pFT27, pFT43, and pFT60, were isolated and characterized. Levels of the RNAs hybridizable to these clones were decreased by fourfold to more than fiftyfold within 1-10 hours in the presence of TPA. Northern blotting experiments identified transcripts corresponding to these clones: pFT27 hybridized to 3.0 kb RNA, pFT43 hybridized to 1.5 kb RNA, and pFT60 hybridized to 1.0 kb RNA. The levels of these 3 transcripts were also decreased after treatment of the undifferentiated F9 cells with retinoic acid (RA) and dibutyryl cyclic AMP (cAMP). The TPA-induced as well as the RA- and cAMP-induced decreases in the RNAs hybridizable to pFT27 were regulated at the transcriptional level, whereas similar decreases in the RNAs hybridizable to pFT43 and pFT60 were regulated at the post-transcriptional level. These findings show that TPA treatment shares common effects with RA and cAMP on the undifferentiated F9 cells.
Miyata
A
A
Department of Biochemistry, Kumamoto University Medical School, Japan.
Akagi
J
J
Fukuda
M
M
Setoyama
C
C
Shimada
K
K
Akagi
M
M
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000302:isolation & purification*; D054278:Embryonal Carcinoma Stem Cells; D005786:Gene Expression Regulation / Q000187:drug effects*; D007700:Kinetics; D051379:Mice; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D009693:Nucleic Acid Hybridization; D012334:RNA, Neoplasm / Q000032:analysis; D013724:Teratoma / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic
D012334:RNA, Neoplasm; D004247:DNA; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate