Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human astrocytoma cells by the pyrrolo-1,5-benzoxazepine, PBOX-21

The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G I arrest in 1321N1 cells. PBOX-21-induced G I arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D-3 protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D-3-CDK4 leading to a decrease in the levels of active cyclin D-3-CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D-3. However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G I arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins. (C) 2003 Published by Elsevier B.V.