FLAVOPIRIDOL INDUCES G(1) ARREST WITH INHIBITION OF CYCLIN-DEPENDENT KINASE (CDK)2 AND CDK4 IN HUMAN BREAST-CARCINOMA CELLS

Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G(1), or G(2) and is a po tent cyclin-dependent kinase (CDK) 1 inhibitor, In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb posit ive and contain CDM-cyclin D1 and MDA-MB-468 breast carcinoma cells th at are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigat e the G(1) arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (K-iapp, 65 nM), competitive w ith respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavopiridol) had an ap proximately 3-fold increased kinase activity compared with untreated c ells. Cyclin D and CDK4 levels were not different at 3 h, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphoryla tion state of pRb was shifted from hypercoincident to hypocoincident w ith the development of G(1) arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G(1) and G(2) by Flavopir idol. Flavopiridol inhibited the in vitro kinase activity of CDK2 usin g an immune complex kinase assay (IC50, 100 nM at 400 mu M ATP). Immun oprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cel ls exposed to Flavopiridol (300 nM) for increasing time showed an init ial increased activity (approximately 1.5-fold at 3 h) compared with u ntreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cycl in E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol . Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol c an therefore account for the G(1) arrest observed after exposure to Fl avopiridol.