SELECTIVE INDUCTION OF CELL-CYCLE REGULATORY GENES CDK1 (P34(CDC2)), CYCLINS A B, AND THE TUMOR-SUPPRESSOR GENE RB IN TRANSFORMED-CELLS BY OKADAIC ACID/

Genes encoding cdk1 (p34(cdc2)), cyclin A, cyclin B, and the tumor sup pressor gene Rb are fundamental regulators of cell cycle progression w hich associate as a complex with the transcription factor E2F. Express ion of many of these proteins has previously been shown to be represse d by okadaic acid, a specific inhibitor of protein phosphatases 1/2A ( PP1/PP2A), resulting in growth arrest in nontransformed but immortiliz ed cells. We have investigated levels of mRNA encoding cdk1 (p34(cdc2) ), cyclin A, cyclin B, Rb, GAPDH, c-myc, and histone H4 genes for sens itivity to okadaic acid in HeLa cells to determine if transformation a ltered their regulation. Serum starvation slowed growth and diminished mRNA levels for all genes tested except c-myc and GAPDH. When starved cells were subsequently exposed to 19 nM okadaic acid or refed 10% se rum, mRNA levels of cyclin A, cyclin B, cdk1, and Rb dramatically incr eased while mRNA levels for c-myc and GAPDH were largely unaffected. H istone H4 mRNA levels and the rate of DNA synthesis were greatly enhan ced by serum addition but not affected appreciably by okadaic acid. Ok adaic acid was also effective in blocking proliferation of exponential ly growing HeLa cells at G2/M and S phase. Despite the cell cycle phas e-specific block, elevated mRNA levels for cdk1, cyclin A, cyclin B, R b, and suppression of H4 mRNA levers were detected and persisted for a t least 12 hr following okadaic acid removal. The results demonstrate that cell cycle progression is blocked and several cell cycle regulato ry genes, encoding transcription factor E2F-associated proteins, exper ience elevation of mRNA levels through mechanisms sensitive to okadaic acid likely through a PP1/PP2A-sensitive mechanism. Data from transfo rmed cells contrast with data from immortalized but nontransformed cel ls in which okadaic acid also blocks cell cycle progression during G2/ M phase but suppresses expression of these genes. Such contrasts may b e correlated with reduced growth factor dependence and transformation. (C) 1995 Wiley-Liss, Inc.