title: Effects of antisense p21 (WAF1/CIP1/MDA6) expression on the induction of differentiation and drug-mediated apoptosis in human myeloid leukemia cells (HL-60).
abstract: The p21MDA6 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 p21MDA6 antisense construct to compare the effects of p21MDA6 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 p21MDA6 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 < or = 0.01) and expressing the monocytic maturation marker cd11b (35.5 +/- 2.8 vs 50.5 +/- 2.4; P < or = 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 p27klp1 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 < or = 0.01). In contrast to the disparate responses to PMA, HL-60/AS5 and HL-60/V2 cells treated with the antimetabolite 1-beta-D-arabinofurano-sylcytosine (Ara-C; 10 microM for 6 h) displayed equal susceptibility to G1 arrest, apoptosis, and inhibition of clonogenicity, phenomena unaccompanied by p21MDA6 and p27klp1 induction, or pRB dephosphorylation. These observations indicate that dysregulation of p21MDA6 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 p21MDA6 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.
authors.affiliation: Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298-0230, USA.
authors.forename: A J
authors.initials: AJ
authors.lastname: Freemerman
authors.forename: J A
authors.initials: JA
authors.lastname: Vrana
authors.forename: R M
authors.initials: RM
authors.lastname: Tombes
authors.forename: H
authors.initials: H
authors.lastname: Jiang
authors.forename: S P
authors.initials: SP
authors.lastname: Chellappan
authors.forename: P B
authors.initials: PB
authors.lastname: Fisher
authors.forename: S
authors.initials: S
authors.lastname: Grant
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D018845:CD11 Antigens / Q000032:analysis; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D003561:Cytarabine / Q000494:pharmacology; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D005822:Genetic Vectors; D018922:HL-60 Cells; D006801:Humans; D056465:Retinoblastoma-Binding Protein 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
chemical_list: D018845:CD11 Antigens; C493956:CDKN1A protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D003561:Cytarabine; D013755:Tetradecanoylphorbol Acetate