Abstract
Phorbol-12-myristate-13-acetate (PMA) induces p21WAF-1 expression in human myeloid leukaemic HL-60 cells. We show that this induction is specifically mediated by protein kinase C (PKC). In addition, the PKC inhibitor Ro 31-8220 with predominant PKC-alpha isoform specificity almost completely inhibited PMA-induced up-regulation of p21WAF1 in HL-60 cells as well as in the myelomonocytic leukaemic U937 cells. Pretreatment of HL-60 cells with Ro 31-8220 also inhibited PMA-induced activation of c-raf-1, a known PKC alpha target. In the phorbol ester-tolerant HL-60 subline (PET) with PKC-beta isoform deficiency PMA or bryostatin-1 induced p21WAF1 expression, but to a lesser extent than in wild-type HL-60 cells. In PET cells, Ro 31-8220 also inhibited PMA and bryostatin-1-induced up-regulation of p21WAF1 expression. Our findings indicate that at least in HL-60 cells up-regulation of p21WAF-1 is specifically activated by PKC. We suggest that PKC isoforms other than beta, presumably the PKC-alpha isoform, are involved in this process.
Highlights
Cancer is characterized by profound alterations both in cell cycle control and in cellular differentiation
We investigated the expression of c-raf-1, a protein serine-threonine kinase required for p21WAFI induction and up-stream regulation of mitogenactivated protein (MAP) kinase (Blagosklonny et al, 1995)
To distinguish between protein kinase C (PKC)-specific receptor-mediated and non-specific effects, HL-60 cells were treated with 4ax-phorbol
Summary
USA), were dissolved in acetone to a stock solution of 1 mm and stored at -20°C. Ro 31-8220 ('compound 3"; provided by Dr G Lawton, Roche Research Centre, Welwyn Garden City, Herts, UK), and bryostatin-1 (provided by Dr GR Pettit, Cancer Research Institute, Tempe, AZ, USA) were dissolved in dimethylsulphoxide (DMSO; Merck Chemical, Darmstadt, Germany) to stock solutions of 1 mM. Normal diploid lung fibroblasts (WI-38; ATCC), expressing high levels of p21WAFI, and a human breast cancer cell line MCF7 (provided by Dr A Ziemiecki, Department of Clinical Research, University of Berne, Switzerland), expressing high levels of c-raf-1, served as positive controls (Blagosklonny et al, 1995; Schwaller et al, 1995). Human cDNA probes were: p21WAFI (2.1 kb; BamHI-HindIII) from pCEP (El-Deiry et al, 1993), and 3-actin (0.7 kb; EcoRIBamHI, from pHF-A-3'UTR (Schwaller et al, 1995)
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