Expression of p57 KIP2 Potently Blocks the Growth of Human Astrocytomas and Induces Cell Senescence

Abstract

Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57(KIP2) (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G(1) as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated beta-galactosidase marker within all astrocytoma cell lines such that approximately 75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells ( approximately 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural features characteristic of apoptosis. Examination of bax and poly-(ADP ribose) polymerase levels showed no change in bax, but decreased expression of poly-(ADP ribose) polymerase after p57 induction in all astrocytoma cell lines. These data demonstrate that the proliferative block imposed by p57 on human astrocytoma cells results in changes in the expression of a number of cell cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence.