Expression and role of p27(kip1) in neuronal differentiation of embryonal carcinoma cells

We examined the expression and the regulation of p21(waf1) and p27(kip1) cd k inhibitors in P19 mouse embryonal carcinoma (EC) cells following treatmen t with all-trans retinoic acid (ATRA) to induce neuronal differentiation. T he levels of p27 mRNA and protein increased within 24 h of treatment with A TRA, reaching a plateau 4-5 days later prior to neurite formation. In contr ast, levels of p21 expression remained low until after neurites were extens ively formed. Induction of muscle differentiation from P19 cells by treatme nt with dimethyl sulfoxide caused only transient increases in p27 levels. I n a mutant P19 cell line, RAC65, treatment with ATRA induced neither p27 ac cumulation nor neuronal differentiation, but p21 mRNA expression increased markedly. In contrast, treatment of RAC65 cells with 9-cis retinoic acid in duced both p27 expression and neuronal differentiation. Correlation between p27 expression and neuronal differentiation was also observed in NT2/D1 hu man EC cells. Luciferase reporter assays showed that p27 promoter activity increased in ATRA-treated cells, consistent with the elevation of p27 mRNA levels. Arrest of neuronal differentiation of P19 cells by okadaic acid res ulted in inhibition of p27 expression, whereas p21 mRNA expression was grea tly enhanced. Conversely, inhibition of p27 expression by antisense p27 oli gonucleotides resulted in blockade of neuronal differentiation. Taken toget her, these results strongly suggest that the expression of p27 is indispens able for neuronal differentiation of EC cells. (C) 2000 Elsevier Science B. V. All rights reserved.