INTERACTION OF RETINOBLASTOMA PROTEIN AND D-CYCLINS DURING CELL-GROWTH INHIBITION BY HEXAMETHYLENEBISACETAMIDE IN TM2H MOUSE EPITHELIAL-CELLS

To explore the regulation and function of D-type cyclins in breast can cer cells, the mouse mammary hyperplastic epithelial cell line TM2H wa s treated with 5 mM hexamethylenebisacetamide (HMBA), a polar differen tiation factor. The resulting growth-inhibitory effect of HMBA was com pletely reversible and was analyzed in terms of percent cells in G(1); association of D-type cyclins with cyclin-dependent kinase (cdk) 4 an d cdk6; G(1) kinase activity; association of retinoblastoma protein (p Rb) and phosphorylated pRb with D-type cyclins; and association of p16 (INK4a), p15(INK4b), and p27(Kip1) With cdk4 and cdk6. Synchronized TM 2H cells were examined at 0, 3, 5, 9, 12, and 24 h after exposure to 5 mM HMBA. Inhibition of DNA synthesis, as measured by thymidine uptake , was first observed at 5 h (40%) and peaked at 24 h (80%). Flow cytom etry at 9 h showed treated cells to be in G(1) arrest. Western blot an alysis showed weakly detectable cyclin D1 but readily detectable cycli n D2 and D3 proteins at 0 h; thereafter, cyclin D2 and D3 protein leve ls remained higher while cyclin D1 levels declined significantly in tr eated versus untreated cells. By 5 h (early G(1)), HMBA had markedly i nhibited cdk4 and cdk6 kinase activity (67% and 75%, respectively) in treated versus untreated cells. By 9 and 12 h, pRb levels had increase d 3.4-fold in treated versus untreated cells. At 5 h, cyclin D-associa ted pRb was totally hypophosphorylated in treated cells and hyperphosp horylated in untreated cells. The levels of pRb associated with cyclin D2 and D3 increased 2.89-fold and 4.6-fold, respectively, in treated versus untreated cells. At 5 h, treated cells showed a fivefold increa se in cdk4-associated p27(Kip1) and, at 9 h, a fourfold increase in cd k6-associated p27(Kip1) over control levels. In confirmation of these data, HMBA was found to inhibit the growth of Rb-positive Du/145Rb cel ls but not their Rb-negative parental Du/145 cells. The data suggest t hat HMBA-induced growth inhibition is due to multifactorial mechanisms involving decreases in total cyclin D1 and inhibition of cdk4 and cdk 6 kinase activities through elevation of levels of cdk4- and cdk6-asso ciated p27(Kip1 an)d concomitant increases in hypophosphorylated pRb a nd stable cyclin D2/pRb and cyclin D3/pRb complexes that help maintain pRb in a functional state. Mel. Carcinog. 22:728-143, 1998. (C) 1998 Wiley-Liss, Inc.