Inhibition of G1 cyclin expression in normal rat kidney cells by inostamycin, a phosphatidylinositol synthesis inhibitor

We previously reported that inostamycin, an inhibitor of CDP-DG:inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G₁ phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G₁ progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G₁ arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.