p21 Cip1/WAF1 inhibits cell-cycle progression by binding to G 1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N-and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21 Cip1/WAF1 is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21 Cip1/WAF1 in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21 Cip1/WAF1 , and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21 Cip1/WAF1 demonstrated G 1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21 Cip1/WAF1 , concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21 Cip1/WAF1 forms a complex with the apoptosis signalregulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21 Cip1/WAF1 lacking the nuclear localization signal (ΔNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21 Cip1/WAF1 itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21 Cip1/WAF1 , which are determined by its intracellular distribution and are dependent on the stage of differentiation.

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