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.

Figure 1.

Western analysis of inducible expression of p57 in human astrocytoma cell lines. Before transfection with a human p57 cDNA, U343, U87, and U373 astrocytoma cell lines did not express p57. With tetracycline in the medium (+), p57-transfected astrocytoma cell clones did not express p57. However, after the removal of tetracycline from the medium (−), clones were identified with strong expression of p57 in the three astrocytoma cell lines. A specimen of nonneoplastic human brain does not express p57.

Figure 2.

Temporal analysis of p57 expression in astrocytoma cell clones in the uninduced and induced states. Without induction, there is no expression of p57 in U343 astrocytoma cells (uninduced). After induction, p57 is strongly expressed in U343^C9 and U373^C3 astrocytoma cell clones on day 1, and in U87^C3 cells on day 3. High expression levels of p57 for all cell clones could be maintained for periods longer than 9 days after induction. A specimen of human fetal brain is shown to express p57.

Figure 3.

Growth inhibitory effects of p57 expression on human astrocytoma cell clones. A: Induction of p57 causes a potent proliferative block in all astrocytoma cell clones as demonstrated by growth curve analysis. Open rectangles, uninduced astrocytoma cells; filled circles, p57-induced astrocytoma cells. Error bars show the SD of three separate counts for each data point. B: Flow cytometric analysis demonstrates that induction of p57 causes astrocytoma cells to accumulate in G₁ phase of the cell cycle. Open rectangles, uninduced astrocytoma cells; filled circles, p57-induced astrocytoma cells. Error bars show the SD of the results of three separate FACS analyses for each astrocytoma cell clone.

Figure 4.

Morphological alterations in human astrocytoma cell clones after the induction of p57, day 5. Uninduced U343^C9 cells are characterized by their tightly packed, cobblestone appearance. The cells are cuboidal with a high nuclear:cytoplasmic ratio. After p57 expression, these cells become large, round, and flat with abundant cytoplasmic-containing perinuclear vacuoles. Uninduced U87^C2 cells are bipolar in configuration. After p57 expression, these cells become large and frequently triangular in shape with a markedly expanded cytoplasm. U373^C3 cells are characterized by being round and flat with a ruffled peripheral plasma membrane before p57 induction. After p57 expression, they also developed an expanded cytoplasm, but maintained their overall round, flat shape. Phase microscopy for all panels, ×350.

Figure 5.

Expression of pRB- and E2F-family proteins in uninduced (right) and p57-induced (left) U87^C2 human astrocytoma cells. Without induction of p57, expression levels of pRB- and E2F-family proteins are not significantly altered throughout a 5-day time interval. However, after induction of p57, decreased levels of pRB, p107, and E2F-1 are observed. pRB is shifted to a faster migrating, hypophosphorylated form. Levels of p130 and E2F-4 are unchanged in this analysis throughout the 5-day time interval.

Figure 6.

Induction of SA-β-gal-positive cells after p57 induction. Uninduced astrocytoma cell clones (left) demonstrated rare cells positive for the SA-β-gal marker. After induction of p57 (day 5), the majority of astrocytoma cells are seen to be positive for SA-β-gal (right). Phase microscopy for U87^C2 and U373^C3 p57-induced cell panels, ×250; phase microscopy for all other panels, ×125.

Figure 7.

Generation of SA-β-gal-positive cells over time after p57 induction. Uninduced astrocytoma cells have rare SA-β-gal-positive cells. After p57 induction, there is an increase in the number of SA-β-gal-positive astrocytoma cells throughout time such that by day 7 ∼75% of p57-induced cells express the marker.

Figure 8.

Viability of human astrocytoma cells after p57 induction. After p57 induction, U343^C9 and U373^C3 astrocytoma cells responded by having diminished numbers of viable cells as determined by trypan blue dye exclusion when compared to uninduced cells. U87^C2 astrocytoma cells showed no loss in cell viability after p57 induction.

Figure 9.

Hoechst 33258 staining of p57-induced human astrocytoma cell clones, day 5. Nuclear integrity was unaltered in U343^C9 and U373^C2 astrocytoma cells. However, U373^C3 cells were characterized by exhibiting fragmentation of the nucleus in ∼15% of cells. Fluorescence microscopy, ×200.

Figure 10.

Number of cells demonstrating micronuclear fragmentation by Hoechst 33258 staining. Unlike U343^C9 and U87^C2 astrocytoma cell clones which showed no increase in numbers of cells with nuclear fragmentation after p57 induction, ∼15% of U373^C3 astrocytoma cells demonstrated micronuclear fragmentation.

Figure 11.

Ultrastructural features of p57-induced human astrocytoma cells. A: Typical nucleus (N) from U343C9 cells. B: Typical nucleus (N) from U87C2 cells. C: Apoptotic nucleus (N) from U373C3 cells. Approximately 15% of these cells were found to contain nuclei similar to this with dense peripheral chromatin and bizarre shapes. Scale bars, 1 μm.

Figure 12.

Western analysis for PARP and Bax in uninduced and p57-induced astrocytoma cell clones. There is no change in Bax expression with p57 induction. After p57 induction, PARP levels are observed to decrease. This is most marked for U373^C3 astrocytoma cells. No cleavage products for PARP were observed.