Evidence of a functional role for the cyclin-dependent kinase inhibitor p21(CIP1) in leukemic cell (U937) differentiation induced by low concentrations of 1-beta-D-arabinofuranosylcytosine

The functional role of the cyclin-dependent kinase inhibitor (CDKI) p21(CIP 1) in, differentiation of human myelomonocytic leukemia cells (U937) expose d to low concentrations of the antimetabolite 1-beta-D-arabino-furallosylcy tosine (ara-C) was examined utilizing a cell line stably expressing a p21(C IP1) antisense construct. Continuous exposure to 50 nM ara-C led to marked induction of p21(CIP1) at 48-72 h in empty-vector control cells but not in their antisense-expressing counterparts (p21AS/F4 and B8), Such treatment i nduced expression of the myelomonocytic differentiation marker CD11b in sim ilar to 35% of control cells, but no evidence of maturation was noted in an tisense-expressing lilies. However, antisense-expressing cells exposed to l ow concentrations of ara-C exhibited a reciprocal increase in apoptosis, ma nifested by the appearance of cells with classic morphologic features and h ypodiploid quantities of DNA, reduced mitochondrial membrane potential (Del ta psi(m)), an increase in cytochrome c release into the cytosol, cleavage! activation of procaspases-9 and -3, and degradation of PARP and p27(Kip1). Whereas empty-vector control cells exposed to 50 nM ara-C exhibited a decl ine in Bcl-2 expression, dephosphorylation of pRb, and an initial accumulat ion in S-phase, antisense-expressing cells not. However, c-Myc down-regulat ion induced by low concentrations of ara-C was, if anything, more complete in antisense-expressing cells. Exposure of control but not antisense-expres sing cells to ara-C led to phosphorylation/activation of MAP kinase at 24 h ; moreover, the specific MEK/MAP kinase inhibitor PD98059 enhanced low-dose ara-C-mediated apoptosis only in wild-type cells. Lastly, exposure to 50 n M ara-C for 72 h resulted in detectable levels of cytoplasmic p21(CIP1), a phenomenon associated with resistance to apoptosis, only in empty vector co ntrols. Collectively, these findings demonstrate a functional role for p21( CIP1), leukemic cell maturation induced by low concentrations of ara-C. The y also indicate that, as in the case of more conventional differentiation-i nducers such as phorbol esters, disruption of the p21(CIP1) response after exposure to low concentrations of the cytotoxic drug ara-C prevents leukemi c cells from engaging a maturation program, but instead directs them along an apoptotic pathway.