Expression of p57(KIP2) potently blocks the growth of human astrocytomas and induces cell senescence

Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression, Inhibition of kinase activ ity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-d ependent kinase inhibitors is an important mechanism by which the effects o f growth signals are down-regulated. We undertook the present study to dete rmine the role of p57(KIP2) (p57) in human astrocytomas, We demonstrate her e that whereas p57 is expressed in fetal brain tissue, specimens of astrocy tomas 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-character ized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using th e tetracycline repressor system leads to a potent proliferative block in G( 1) as determined by growth curve and flow cytometric analyses. After the in duction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, an d retinoblastoma protein is shifted to a hypophosphorylated form. Morpholog ically, p57-induced astrocytoma cells became large and hat with an expanded cytoplasm, The inducible expression of p57 leads to the accumulation of se nescence-associated beta-galactosidase marker within all astrocytoma cell l ines such that similar to 75% of cells were positive at 1 week after induct ion. Induction of p57 in U373 astrocytoma cells generated a small populatio n of cells (similar to 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural feat ures characteristic of apoptosis, Examination of bar and poly-(ADP ribose) polymerase levels showed no change in bar, 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 huma n astrocytoma cells results in changes in the expression of a number of cel l cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence.