The Cyclin-Dependent Kinase Inhibitor p57KIP2 Functions as a Tumor Suppressor Gene in Human Leukemia.

Abstract

p57KIP2 is a cyclin-dependent kinase inhibitor with a role in the regulation of cell cycle progression, proliferation, differentiation, and development. Disruption any of these biological processes could result in malignant transformation. We have previously reported that epigenetic inactivation of p57KIP2 by aberrant DNA methylation is associated with a worse prognosis in patients with adult acute lymphocytic leukemia (Blood 2003; 101:4131–6 and JCO 2005; 23:3932–9). To further study the role of p57KIP2 in human leukemia we have developed several knock-in and knock-down cellular models of p57KIP2 in human leukemia cell lines. First, we established the methylation/expression patterns of p57KIP2 in 21 leukemia cell lines of both myeloid and lymphoid origin and of different phenotypes. We found that p57KIP2 was methylated and silenced in all except 6 (28%) cell lines. Methylation of the p57KIP2 promoter region correlated with gene silencing, and lack of protein expression, and treatment of methylated/silenced cell lines with a 5-aza-2′-deoxycytidine (DAC), a demethylating agent, resulted in p57KIP2 gene and protein re-expression. In unmethylated p57KIP2 cell lines, p57KIP2 mRNA was rapidly up-regulated by stimulation by growth-inhibitory signals such as TNF alpha, LPS and TGF beta, and when cell-cycle progression was arrested. In contrast, treatment with dexamethasone, DAC or the histone deacetylase inhibitor SAHA did not have such an effect. The reverse was observed in methylated cell lines. Transient transfection of p57KIP2 in silenced cell lines resulted in cell-cycle arrest at the G0/G1 phase. Stable transfection of p57KIP2 using a lentivirus system in Raji cells, in which p57kip2 is methylated and silenced, resulted in the induction of apoptosis and the suppression of cell growth in soft agar. Stable transfection of p57KIP2 in Jurkat cells, in which p57KIP2 is unmethylated and expressed, resulted in partial inhibition of cell growth, and increased susceptibility to the induction of apoptosis by staurosporine. Knock-down of p57KIP2 using an shRNA lentivirus in K562 cells, in which p57kip2 is not methylated and is expressed, promoted cell growth and proliferation in soft agar. These studies provide evidence that p57KIP2 has the properties of a bona fide tumor suppressor gene in human leukemia and provide further weight to the notion that p57KIP2 has an important role in human leukomogenesis.