Abbreviated cell cycle progression induced by the serine/threonine proteinphosphatase 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 mi mic early stages of tumor promotion by this compound. Optimal transformatio n (colony formation in soft agar) was seen after prolonged culture of N-met hyl-N ' -nitro-N-nitrosoguanidine (MNNG)initiated T51B cells in 7 nM OA. Pa radoxically, T51B cells treated with 2-10 nM OA showed decreased, rather th an increased, proliferation in response to epidermal growth factor (EGF), a s measured by [H-3]thymidine incorporation. Complete inhibition was observe d within 24 h at 10 nM OA. This response paralleled a loss of EGF-stimulate d 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 se rine 15 accompanied the rise in p21 (cip-1). Both phosphorylation of the re tinoblastoma 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 trea ted with OA alone also showed elevated cyclin E levels, together with reduc ed levels of the E2F regulator pRb2/p130. Taken together, these observation s indicate transforming levels of okadaic acid elicit a GI-trapping effect by facilitating cell cycle progression to the G(1)/S checkpoint, where cell s are trapped by mechanisms that include p21 (cip-1)-mediated inhibition of cdk2. They support the premise that disruption of cellular processes regul ating the transitions from G(0) to G(1) to S-phase is an important early st ep in tumor promotion by low levels of okadaic acid.