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Transcriptional regulation of differentiation of embryonal carcinoma and malignant melanoma cells: The Role of Proteins p21 (WAF1) and MITF
Šestáková, Blanka ; Vachtenheim, Jiří (advisor) ; Černý, Radim (referee) ; Pavel, Stan (referee)
Summary. F9 cells (embryonal carcinoma) can be induced to differentiate with retinoic acid and dibutyryl-cAMP into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation. Clones of F9 cells stably expressing ectopic p21 revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter, implying that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. Whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. As differentiation of F9 cells into parietal endoderm-like cells...
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