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 kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus 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 cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited 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 inhibited 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 temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and 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, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.
Kitagawa
M
M
Banyu Tsukuba Research Institute, Merck Research Laboratories, Japan.
Higashi
H
H
Takahashi
I S
IS
Okabe
T
T
Ogino
H
H
Taya
Y
Y
Hishimura
S
S
Okuyama
A
A
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D016160:Retinoblastoma Protein / Q000378:metabolism*; D013936:Thymidine / Q000378:metabolism
D006657:Histones; D047428:Protein Kinase Inhibitors; D016160:Retinoblastoma Protein; C082868:butyrolactone I; D015107:4-Butyrolactone; D013936:Thymidine