Effects of antisense p21 (WAF1/CIP1/MDA6) expression on the induction of 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 to compare the effects of p21(MDA6) dysregulation on the response of myeloid leukemia cells to differentiating and cytotoxic agents. Antisense-expressing cells (HL-60/AS5) treated with 5 nM PMA for 24 h exhibited attenuated induction of p21(MDA6) compared to empty vector controls (HL-60/V2). This phenomenon was accompanied by a reduction in the percentage 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 differences in the phosphorylation status of the retinoblastoma protein (pRB), in E2F complex formation, or in p27(kip1) induction following PMA exposure, inhibition of activity of cyclin-dependent kinase-2 was attenuated in the antisense-expressing line. A 24-h exposure to 5 nM PMA also reduced the cloning efficiency of HL-60/V2 cells to a significantly 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 disparate 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 inhibition 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 cells interferes with PMA-related G(1) arrest, CDK-2 inhibition, differentiation, and loss of clonogenic survival in the absence of obvious alterations in pRB phosphorylation status or E2F complex formation. They also provide functional evidence that p21(MDA6) induction does not appear to be required for Ara-C-induced apoptosis, G, arrest, or the resulting reduction in the self-renewal capacity of HL-60 cells.