Mechanisms of Retinoid Inhibition of Cell Proliferation in Neuroblastoma

Abstract

Cyclin/cdk kinases stimulate progression through the G₁ phase of the cell cycle while members of the CIP/KIP gene family function to inhibit cdk kinase activity and cell cycle progression. To study the mechanism of retinoid induced decrease in NB cell growth, the expression and activity of cyclin/cdk kinases, and their inhibitors was evaluated in KCNR NB cells arrested in G₁ by treated with all trans-retinoic acid (RA). After 2 days in 5uM RA and coincident with the arrest of cells in G₁, there is a 10 and 5-fold decrease in CDK2 and CDK4 kinase activity, respectively. The decrease in kinase activity occurs despite the fact that the protein levels of the components of the cyclin- cdk kinases are unchanged during this time. RA stimulates a 5-fold increase in p27^KIP steady-state levels and a comparable increase in p27^KIP complexed to cdk2 and cdk4 compared to untreated cells. The levels of other CIP/KIP genes are unchanged. RA does not stimulate an increase in p27^KIPmRNA suggesting an increase in the stability of p27^KIP or other post-translational modifications. The mechanism by which RA increases p27^KIP may be by decreasing N-myc levels. We find that over-expression of N- myc leads to decreases in p27^KIP and increases in cyclin/cdk kinases activity and an increase in cells in G₁. Increases in p27^KIP are important in inhibiting the growth of NB, since infection of NB with adenoviruses containing p27^KIP arrested cells in G₁ and decreased cyclin/cdk kinase activity. These data suggest a model in which the RA induced arrest of NB cells is mediated by a decrease in N-myc levels leading to a increase in p27 that binds and inhibits cyclin/cdk kinase activity causing an arrest of cells in G₁.