Protein kinase C inhibitors arrest the C6 glioma cell cycle at a mid-G1 phase restriction point: Implications for the antiproliferative action of valproate

The teratogenic mechanism(s) of valproate (VPA) have been suggested to arise through inhibition of proliferation coupled with differentiation at a mid-G1 phase restriction point in the cell cycle. As protein kinase C (PKC) plays a pivotal role in cell proliferation and differentiation, the effect of inhibitors specific for the catalytic and regulatory domains on transit through the G1 phase of the cell cycle was determined. Calphostin C and bisindolylmaleimide GF 109203X produced a dose-dependent decrease in proliferation of C6 glioma with approximate 50% inhibitory concentration values of 10 nM and 1 mu M, respectively. Flow cytometric analysis indicated proliferative arrest to be in the G1 phase with the expected concomitant decrease of cells in the G2/M and S phases. Following release from drug-induced proliferative arrest, cells exhibited a synchronous entry into S phase as evidenced by an increase in [H-3]thymidine incorporation after approximately 6-8 hr, indicating the restriction point to be in the mid-G1 phase. Using mitotically synchronized cells continuously exposed to valproate (2 mM), PKC activity was found to be significantly reduced in the mid-G1 phase (5.5 hr) but not at an earlier (2.5 hr) time point, implying VPA to exert its effect at a site upstream to the point of proliferative arrest at 5-6 hr into the G1 phase which as yet, remains to be defined. (C) 1998 Elsevier Science Ltd.