¹Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA, USA

² Department of Pathology, Columbia-Presbyterian Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, NY, USA

³ Department of Urology, Columbia-Presbyterian Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, NY, USA

⁴Department of Microbiology, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA, USA

⁵Department of Pharmacology, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA, USA

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 n _M 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 G₁ arrest (67.6 ± 4.7 vs 82.9 ± 1.3; P 0.01) and expressing the monocytic maturation marker cd11b (35.5 ± 2.8 vs 50.5 ± 2.4; P 0.005). Although HL-AS5 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 5nM 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 0.01). In contrast to the disparate responses to PMA, HL-60/AS5 and HL-60/V2 cells treated with the antimetabolite 1-- _D-arabinofuranosylcytosine (Ara-C; 10 M for 6 h) displayed equal susceptibility to G₁ arrest, apoptosis, and inhibition of clonogenicity, phenomena unaccompanied by p21^MDA6 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 G₁ 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, G₁ arrest, or the resulting reduction in the self-renewal capacity of HL-60 cells.

p21; MDA-6; apoptosis; Ara-C; cell cycle arrest; HL-60 cells