The cyclin-dependent kinase inhibitor (CDKI) flavopiridol disrupts phorbol12-myristate 13-acetate-induced differentiation and CDKI expression while enhancing apoptosis in human myeloid leukemia cells

Interactions between the cyclin-dependent kinase inhibitor (CDKI) flavopiri dol (FP) and phorbol 12-myristate 13-acetate (PMA) were examined in U937 hu man leukemia cells in relation to differentiation and apoptosis, Simultaneo us, but not sequential, exposure of U937 cells to 100 nM FP and 10 nM PMA s ignificantly increased apoptosis manifested by characteristic morphological features, mitochondrial dysfunction, caspase activation, and poly(ADP-ribo se) polymerase cleavage while markedly inhibiting cellular differentiation, as reflected by diminished plastic adherence and CD11b expression. Enhance d apoptosis in U937 cells was associated with an early caspase-independent increase in cytochrome c release and accompanied by a substantial decline i n leukemic cell clonogenicity, Moreover, PMA/FP cotreatment significantly i ncreased apoptosis in HL-60 promyelocytic leukemia cells and in U937 cells ectopically expressing the Bcl-2 protein. In U937 cells, coadministration o f FP blocked PMA-induced expression and reporter activity of the CDKI p21(W AF1/CIP1) and triggered caspase-mediated cleavage of the CDKI p27(KIP1). Co exposure to FP also resulted in a more pronounced and sustained activation of the mitogen-activated protein kinase kinase/extracellular signal-regulat ed protein kinase cascade after PMA treatment, although disruption of this pathway by the mitogen-activated protein kinase kinase I inhibitor U0126 di d not prevent potentiation of apoptosis. FP accelerated PMA-mediated dephos phorylation of the retinoblastoma protein (pRb), an event followed by pRb c leavage culminating in the complete loss of underphosphorylated pRb (approx imate toM(r) 110,000) by 24 h. Finally, gel shift analysis revealed that co administration of PP with PMA for 8 h led to diminished E2F/pRb binding com pared to the effects of PIMA alone, Collectively, these findings indicate t hat FP modulates the expression/activity of multiple signaling and cell cyc le regulatory proteins in PMA-treated leukemia cells and that such alterati ons are associated with mitochondrial damage and apoptosis rather than matu ration. These observations also raise the possibility that combining CDKIs and differentiation-inducing agents may represent a novel antileukemic stra tegy.