The Cyclin-Dependent Kinase Inhibitor p21^WAF1 Is Required for Survival of Differentiating Neuroblastoma Cells

Abstract

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21 WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21 WAF1 , even after removal of aphidicolin from the culture medium. The p21 WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21 WAF1 mRNA partially blocks expression of p21 WAF1 and promotes programmed cell death. These data indicate that p21 WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.We are using the SH-SY5Y neuroblastoma cell line as a model for neuronal terminal differentiation (33, 48). These cells express low levels of both the low-affinity nerve growth factor (NGF) receptor and the trkA NGF receptor (2, 48). SH-SY5Y cells treated with NGF and aphidicolin, a specific and reversible inhibitor of DNA polymerases ␣ and ␦, cease proliferation and extend long neurites (25, 33). The differentiated cells require NGF for survival and, in the presence of NGF, are stable for 4 to 6 weeks. These cells express neuronal markers and cytologically resemble sympathetic neurons. In contrast, NGF alone does not stop cell proliferation and induces only slight neurite extension (8, 55). Treatment with aphidicolin does not induce neurite extension, and the cells resume proliferation following removal of aphidicolin.Several mechanisms have been proposed for cessation of neuronal proliferation. One way by which growth arrest might occur is by down-regulating the expression of proteins required for progression of the cell cycle through the G 1 -S and G 2 -M transitions (24). cdc2 is the p34 cdc2 kinase, which in conjunction with cyclin B triggers mitosis (40). The kinases cdk2 and cdk4, in association with cyclins A, D1, D2, D3, and E, are required for the G 1 -S transition. Neuronal precursor cells, but not neurons in the adult brain, express cdc2, cdk2, and cyclins A and D2 (24,31,45,58,59). In contrast, cyclins D1 and E are expressed in both the developing and the adult brain (37, 58). Sympathetic neurons do not express cdc2, cdk2, or cyclin A but do express cdk4 and cyclins B, D1, D2, D3, and E (18). PC12 pheochromocytoma cells differentiated with NGF express decreased levels of cdc2 and cdk2 but continue to express cyclins A and E (6). Hence, although the details vary, down-regulation of cell cycle-associated proteins occurs in a variety of neuronal systems.A second potential mechanism for growth arrest involves induction of p21 WAF1, which binds to and inhibits cdc2, cdk2, and cdk4 (16,23,43). Transcription of p21 WAF1 is indu...

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line

“…This argues in favor of a repressor role for p21 in inhibiting paclitaxel-induced apoptosis mediated by p34 cdc2 . Several studies have shown that p21 acts as a survival factor (Poluha et al, 1996;Polyak et al, 1996;Waldman et al, 1996;Wang and Walsh, 1996). We found that inhibition of p21 expression using a p21 antisense oligonucleotide led to a signi®cant increase in paclitaxel cytotoxicity in MCF-7 cells.…”

p21WAF1 induces permanent growth arrest and enhances differentiation, but does not alter apoptosis in PC12 cells

“…It was initially shown to increase during DNA damage induced cell death (El-Deiry et al, 1994). More recently, it has been shown that inducing p21 can decrease apoptosis in di erentiating muscle cell lines (Wang and Walsh, 1996) and prostaglandin treated colon carcinoma cell lines (Gorospe et al, 1996), while decreasing expression during di erentiation of a neuronal line increases apoptosis (Poluha et al, 1996). p21 has also been demonstrated to inhibit two members of the stress activated protein kinase family (SAPK), JNK and p38 (Shim et al, 1996), proteins thought to be involved in the signalling pathways leading to cell death in response to stress (Davis, 1994;.…”

Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation

“…Since p27 has been shown to confer resistance against anticancer agents to tumor cells (Croix et al, 1996), it appeared likely that p27 has antiapoptotic activity similar to other cdk inhibitors such as p16 and p21 (Wang and Walsh, 1996;Poluha et al, 1996). We therefore hypothesized that c-Myc may mediate apoptosis through the down-regulation of p27, and that Pim-1 might facilitate this process.…”