Abbreviated cell cycle progression induced by the serine/threonine protein phosphatase inhibitor okadaic acid at concentrations that promote neoplastic transformation

We examined cell cycle-related effects of the phosphatase inhibitor okadaic acid (OA) in T51B rat liver epithelial cells under conditions chosen to mimic early stages of tumor promotion by this compound. Optimal transformation (colony formation in soft agar) was seen after prolonged culture of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiated T51B cells in 7 nM OA. Paradoxically, T51B cells treated with 2-10 nM OA showed decreased, rather than increased, proliferation in response to epidermal growth factor (EGF), as measured by 3H]thymidine incorporation. Complete inhibition was observed within 24 h at 10 nM OA. This response paralleled a loss of EGF-stimulated cdk2 kinase activity and an increase in association of the inhibitors p21 (cip-1) and p27 (kip-1) with cdk2. An increase in p53 phosphorylated on serine 15 accompanied the rise in p21 (cip-1). Both phosphorylation of the retinoblastoma protein and induction of cyclin A by EGF were blocked in cells treated with OA, but there was an increase in cyclin E. Resting cells treated with OA alone also showed elevated cyclin E levels, together with reduced levels of the E2F regulator pRb2/p130. Taken together, these observations indicate transforming levels of okadaic acid elicit a G(1)-trapping effect by facilitating cell cycle progression to the G(1)/S checkpoint, where cells are trapped by mechanisms that include p21 (cip-1)-mediated inhibition of cdk2. They support the premise that disruption of cellular processes regulating the transitions from G(0) to G(1) to S-phase is an important early step in tumor promotion by low levels of okadaic acid.