A CYCLIN-DEPENDENT KINASE INHIBITOR, BUTYROLACTONE-I, INHIBITS PHOSPHORYLATION OF RB PROTEIN AND CELL-CYCLE PROGRESSION

Butyrolactone I is a selective inhibitor of the cyclin-dependent kinas e (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely af fects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-t yrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We stu died the effects of butyrolactone I on the cell cycle as well as on ph osphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibit ed phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by bacul ovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the c ell cycle after serum stimulation. pRB was phosphorylated after 10 h s erum stimulation. Incorporation of [H-3]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibi ted by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inh ibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression, tsFT210 cells, a tempera ture-sensitive cdc2 mutant cell line, were synchronized at G2/M at a n onpermissive temperature, butyrolactone I inhibited the cell cycle pro gression of these cells at G2/M at the permissive temperature. Thus bu tyrolactone I, a cyclin-dependent kinase family inhibitor, which preve nted the phosphorylations of the cell cycle-regulating proteins pRB an d H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, al though it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.