EFFECTS OF ANTISENSE P21 (WAF1 CIP1/MDA6) EXPRESSION ON THE INDUCTIONOF DIFFERENTIATION AND DRUG-MEDIATED APOPTOSIS IN HUMAN MYELOID-LEUKEMIA CELLS (HL-60)/

The p21(MDA6) gene product induces cell cycle arrest in p53-null human leukemic cells exposed to differentiation stimuli. We employed an HL- 60 cell line stably transfected with a p21(MDA6) antisense construct t o compare the effects of p21(MDA6) dysregulation on the response of my eloid leukemia cells to differentiating and cytotoxic agents. Antisens e-expressing cells (HL-60/AS5) treated with 5 nM PMA for 24 h exhibite d attenuated induction of p21(MDA6) compared to empty vector controls (HL-60/V2). This phenomenon was accompanied by a reduction in the perc entage of cells undergoing G(1) arrest (67.6 +/- 4.7 vs 82.9 +/- 1.3; P less than or equal to 0.01) and expressing the monocytic maturation marker cd11b (35.5 +/- 2.8 vs 50.5 +/- 2.4; P less than or equal to 0. 005). Although HL-ASS and HL-60/V2 cells did not exhibit obvious diffe rences in the phosphorylation status of the retinoblastoma protein (pR B), in E2F complex formation, or in p27(kip1) induction following PMA exposure, inhibition of activity of cyclin-dependent kinase-2 was atte nuated in the antisense-expressing line. A 24-h exposure to 5 nM PMA a lso reduced the cloning efficiency of HL-60/V2 cells to a significantl y greater extent than HL-60/AS5 cells (ie to 30.1 +/- 7.0 vs 57.2 +/- 5.6 of controls; P less than or equal to 0.01). In contrast to the dis parate responses to PMA, HL-60/AS5 and HL-60N2 cells treated with the antimetabolite 1-beta-D-arabinofuranosylcytosine (Ara-C; 10 mu M for 6 h) displayed equal susceptibility to G(1) arrest, apoptosis, and inhi bition of clonogenicity, phenomena unaccompanied by p21(MDAS) and p27( kip1) induction, or pRB dephosphorylation. These observations indicate that dysregulation of p21(MDA6) in p53-null human myeloid leukemia ce lls interferes with PMA-related G(1) arrest, CDK-2 inhibition, differe ntiation, and loss of clonogenic survival in the absence of obvious al terations in pRB phosphorylation status or E2F complex formation. They also provide functional evidence that p21(MDA6) induction does not ap pear to be required for Ara-C-induced apoptosis, G, arrest, or the res ulting reduction in the self-renewal capacity of HL-60 cells.