pRb and Cdk regulation by N-(4-hydroxyphenyl)retinamide

The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) can inhibit the growth and induce apoptosis of tumor cells. In this study we analysed the growth suppressive effect of HPR on human breast canc er cell lines in vitro and the role of the retinoblastoma protein (pRb) in this response. Treatment of MCF7, T47D and SKBR3 for 24-48 h with 3 mu M HP R, a concentration attainable in vivo, resulted in growth inhibition and ma rked dephosphorylation of pRb involving Ser(612), Thr(821), Ser(795) and Se r(780), target residues for cyclin-dependent kinase 2 (Cdk2) the former two , and Cdk4 the latter two. Interestingly, this dephosphorylation of pRb occ urred in S-G2-M phase cells, as revealed by experiments on cells fractionat ed by FACS according to the cell cycle phase, hence suggesting that the ret inoid interferes with the regulation of pRb phosphorylation, The in vitro p hosphorylation of a GST-pRb recombinant substrate by Cdk2 immunocomplexes f rom MCF7, T47D and SKBR3 was markedly suppressed after HPR treatment, where as that by Cdk4 complexes was suppressed in T47D and SKBR3 but not in MCF7, The steady-state levels of Cdk2, Cdk4 and Cyclin A proteins were unaffecte d by HPR, while those of Cyclin D1 were significantly reduced in all three cell lines. Interestingly, Cyclin D1 downregulation by HPR correlated with transcriptional repression, but not with enhanced proteolysis of Cyclin D1 typically elicited by other retinoids, Collectively, our data suggest that the antiproliferative activity of HPR arises from its capacity to maintain pRb in a de-phosphorylated growth-suppressive status in S-G2/M, possibly th rough Cyclin D1 downregulation and inhibition of pRb-targeting Cdks.