title: Evidence of a functional role for the cyclin-dependent kinase inhibitor p21CIP1 in leukemic cell (U937) differentiation induced by low concentrations of 1-beta-D-arabinofuranosylcytosine.
abstract: The functional role of the cyclin-dependent kinase inhibitor (CDKI) p21CIP1 in differentiation of human myelomonocytic leukemia cells (U937) exposed to low concentrations of the antimetabolite 1-beta-D-arabino-furanosylcytosine (ara-C) was examined utilizing a cell line stably expressing a p21CIP1 antisense construct. Continuous exposure to 50 nM ara-C led to marked induction of p21CIP1 at 48-72 h in empty-vector control cells but not in their antisense-expressing counterparts (p21AS/F4 and B8). Such treatment induced expression of the myelomonocytic differentiation marker CD11b in approximately 35% of control cells, but no evidence of maturation was noted in antisense-expressing lines. However, antisense-expressing cells exposed to low concentrations of ara-C exhibited a reciprocal increase in apoptosis, manifested by the appearance of cells with classic morphologic features and hypodiploid quantities of DNA, reduced mitochondrial membrane potential (deltapsim), an increase in cytochrome c release into the cytosol, cleavage/activation of procaspases-9 and -3, and degradation of PARP and p27Kip1. Whereas empty-vector control cells exposed to 50 nM ara-C exhibited a decline in Bcl-2 expression, dephosphorylation of pRb, and an initial accumulation in S-phase, antisense-expressing cells did not. However, c-Myc down-regulation induced by low concentrations of ara-C was, if anything, more complete in antisense-expressing cells. Exposure of control but not antisense-expressing 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 nM ara-C for 72 h resulted in detectable levels of cytoplasmic p21CIP1, a phenomenon associated with resistance to apoptosis, only in empty vector controls. Collectively, these findings demonstrate a functional role for p21CIP1 in leukemic cell maturation induced by low concentrations of ara-C. They also indicate that, as in the case of more conventional differentiation-inducers such as phorbol esters, disruption of the p21CIP1 response after exposure to low concentrations of the cytotoxic drug ara-C prevents leukemic cells from engaging a maturation program, but instead directs them along an apoptotic pathway.
authors.affiliation: Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Wang
authors.forename: P B
authors.initials: PB
authors.lastname: Fisher
authors.forename: P
authors.initials: P
authors.lastname: Dent
authors.forename: S
authors.initials: S
authors.lastname: Grant
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D003561:Cytarabine / Q000008:administration & dosage / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006651:Histocytochemistry; D006801:Humans; D007951:Leukemia, Myeloid / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D016177:Macrophage-1 Antigen / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D020298:U937 Cells
chemical_list: C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016177:Macrophage-1 Antigen; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D016271:Proto-Oncogene Proteins c-myc; D016160:Retinoblastoma Protein; D003561:Cytarabine; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases