Evidence of a Functional Role for p21WAF1/CIP1Down-Regulation in Synergistic Antileukemic Interactions between the Histone Deacetylase Inhibitor Sodium Butyrate and Flavopiridol

Abstract

The functional significance of disruption of p21^WAF1/CIP1 induction by flavopiridol (FP) in human leukemia cells (Jurkat) exposed to the histone deacetylase (HDAC) inhibitor sodium butyrate (SB) was investigated. Coexposure of leukemic cells to FP blocked SB-mediated induction of p21^WAF1/CIP1 and resulted in a marked increase in mitochondrial injury, activation of procaspases-3 and -8, Bid cleavage, and PARP degradation. Enforced expression of p21^WAF1/CIP1 (i.e., in Jurkat cells inducibly expressing p21^WAF1/CIP1 under the control of a doxycycline-responsive promoter) partially but significantly reduced cytochrome c and apoptosis-inducing factor release, loss of mitochondrial membrane potential, caspase-3 and -8 activation, Bid cleavage, poly(ADP-ribose)polymerase (PARP) degradation, and apoptosis in response to SB/FP. Furthermore, increasing expression of p21^WAF1/CIP1 (i.e., by culturing cells in the presence of higher concentrations of doxycycline) rendered cells more resistant to SB/FP-mediated lethality. Enforced expression of p21^WAF1/CIP1 did not modify SB/FP-mediated JNK activation or generation of reactive oxygen species. Consistent with these results, Jurkat cells stably expressing a p21^WAF1/CIP1 nuclear localization mutant (p21ΔNLS) were also resistant to SB/FP-mediated mitochondrial injury, activation of procaspases-3 and -8, PARP cleavage, and apoptosis. Finally, enforced expression of full-length or ectopic expression of ΔNLS p21^WAF1/CIP1 increased the amount of p21^WAF1/CIP1 coimmunoprecipitating with procaspase-3. Together, these findings suggest that interruption of HDAC-mediated p21^WAF1/CIP1 induction by FP plays a significant functional role in potentiating apoptosis, possibly by preventing the formation of a procaspase-3/p21^WAF1/CIP1 complex.