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In normal cells in which DNA has been damaged, p53 induces the expression of p21(Waf1/Cip1); p21, in turn, binds to cyclin-dependent kinase 2 (cdk2) and inhibits its function. Inhibition of cdk2 results in cell cycle arrest in G(0)-G(1). Although p53 is transcriptionally active and induces p21 expression in neuroblastoma (NB) cells, the G(0)-G(1) checkpoint is attenuated. Here we report that the mechanism that mediates this defect in NB cells is the inability of p21 to bind to, or inhibit the activity of cdk2. However, when recombinant p21 protein was added to NB cell extracts in vitro, the protein inhibited the activity of cdk2. This finding suggests that endogenous p21 protein in NB cells is inactive and may be bound either to a protein complex or in a conformation that precludes its binding to cdk2. The dysfunction of p21 in NB cells represents a novel mechanism by which the G(0)-G(1) cell cycle checkpoint can be inactivated. This mechanism may be important in regulating the growth of NB and potentially other types of tumors. Cdk inhibitors currently being developed for clinical use may be useful therapy for tumors such as NB in which endogenous cdk inhibitors are defective.

p53 is a tumor suppressor protein important in the regulation of apoptosis. Because p53 functions as a transcription factor, cellular responses depend upon activity of p53 localized in the nucleus. Cytoplasmic sequestration of p53 has been proposed as a mechanism by which the function of this protein can be suppressed, particularly in tumor types such as neuroblastoma in which the frequency of mutations of p53 is low. Data presented here demonstrate that nuclear p53 protein is expressed in a panel of neuroblastoma cell lines, and after exposure to DNA damage, transcriptionally active p53 expression can be induced. After exposure to both equitoxic IC80 and 10-Gy doses of ionizing radiation, both p53 and p21 were induced, but G1 cell cycle arrest was attenuated. To investigate whether the DNA damage signaling pathway was incapable of inducing sufficient p53 in these cells, we expressed additional wild-type p53 after adenoviral vector transduction. This exogenous p53 expression also resulted in p21 induction but was unable to enhance the G1 arrest, suggesting that the pathway downstream from p53 is nonfunctional. Although p53-mediated G1 arrest is attenuated in neuroblastoma cells, the ability of p53 to induce apoptosis appears functional, consistent with its chemosensitive phenotype. This work demonstrates that p53 is expressed in the nucleus of neuroblastoma cells and can mediate induction of p21. However, this cell type appears to have an attenuated ability to mediate a DNA damage-induced G1 cell cycle arrest.

Cyclin-dependent kinases (Cdks) and their cyclin partners regulate mammalian cell proliferation and withdrawal from the cell cycle and, as such, control differentiation in many tissues. Studies were undertaken to examine the roles of cell cycle proteins in differentiating cytotrophoblasts. Cyclin E gene and protein expression was down-regulated after 24 h in cultured trophoblasts. Cdk2-associated kinase activity was decreased after 96 h in culture as was the amount of cyclin E in complexes with Cdk2; however, levels of the Cdk inhibitor, p27Kip1, were significantly increased. In freshly isolated trophoblasts and in 24-h cultures, the retinoblastoma gene product (pRb) was found in both the active and inactive forms, yet only hypophosphorylated, active pRb was present in syncytiotrophoblast. Thus, inactivation of Cdk2 through cyclin E down-regulation and increased p27Kip1 expression leads to an accumulation of active pRb in syncytiotrophoblast. Prevention of entry into S phase by hypophosphorylated pRb may allow trophoblasts to respond to signals that potentiate differentiation. Our studies suggest that regulation of G1-phase Cdk activity may be involved in the terminal differentiation process of cytotrophoblasts.