The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G 1 and G 2 ͞M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.

The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G 1 and G 2 /M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. The cdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p53 22/23 . An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since the cdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at ؊76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNAprotein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulates cdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.Inactivation of p53 tumor suppressor gene occurs in over half of all human tumors, implying that loss of this gene represents a fundamentally important step in genomic instability and susceptibility to malignant transformation (1, 2). The underlying mechanism of tumor suppressor activity of p53 resides in part in its ability to bind DNA in a sequence-specific manner to activate gene transcription (2). It has been reported that a substantial number of genes containing the p53-binding site(s) are activated by p53. These include mdm2 (3, 4), p21/WAF-1 (5), Gadd45 (6), cyclin G (7), bax (8), and an insulin-like growth factor-binding protein (IGF-BP3) (9). p21 and Gadd45 were implicated in the p53-mediated cell cycle regulation (10, 11), while bax and IGF-BP3 were involved in the induction of apoptosis (8,9).In addition to playing a role as a DNA-binding dependent transcription activator, p53 has also been reported to negatively regulate the transcription of a number of genes. These genes include presenilin 1 (12), topoisomerase II␣ (13, 14), map4 (15), O 6 -methylguanine-DNA methyltransferase (16), insulin receptor (17), mdr-1 (18), hsp70 1 (19), interleukin-6 (20), bcl2 (21), c-fos (22), and other viral and cellular promoters (23). In contrast to the transcription activation by p53, no consensus sequence has been found in the promoters that are repressed by p53. It was initially reported that only the promoters containing a TATA box, but not those containing an initiator element, are repressed by p53 (24). This finding...

Nitric oxide (NO) during primary culture of articular chondrocytes causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation, and differentiation status. In this study, we characterized the molecular mechanism by which p38 kinase induces apoptosis through activation of p53. We report here that NO-induced activation of p38 kinase leads to activation of NFB, which in turn induces transcription of the p53 gene. Activated p38 kinase also physically associates and phosphorylates the serine 15 residue of p53, which results in accumulation of p53 protein during NO-induced apoptosis. Ectopic expression of wild-type p53 enhanced NO-induced apoptosis, whereas expression of a dominant negative p53 blocked it, indicating that p53 plays an essential role in NO-induced apoptosis of chondrocytes. The increased accumulation of p53 caused expression of Bax, a pro-apoptotic member of the Bcl-2 family that is known to cause apoptosis via release of cytochrome c and caspase activation. These results suggest that NO-activated p38 kinase activates p53 function in two different ways, transcriptional activation by NFB and direct phosphorylation of p53 protein, leading to apoptosis of articular chondrocytes.

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in many glioma cell lines. However, treatment with TRAIL in combination with subtoxic doses of roscovitine, a specific inhibitor of Cdc2 and Cdk2, induced rapid apoptosis in TRAIL-resistant glioma cells. Roscovitine could sensitize Bcl-2-or Bcl-xL-overexpressing glioma cells, but not human astrocytes, to TRAIL-induced apoptosis, offering an attractive strategy for safely treating resistant gliomas. Treatment with roscovitine significantly inhibited Cdc2 activity, and expression of a dominant-negative Cdc2 mutant sensitized glioma cells to TRAIL-induced apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in U87MG and T98 glioma cells, treatment with roscovitine recovered TRAIL-induced activation of caspases very efficiently in these cells. We found that treatment with roscovitine or expression of a dominant-negative Cdc2 mutant downregulated the protein levels of survivin and XIAP, two major caspase inhibitors. Overexpression of survivin or XIAP attenuated the apoptosis induced by roscovitine and TRAIL. Taken together, these results suggest that downregulation of survivin and XIAP by subtoxic doses of roscovitine contributes to the amplification of caspase cascades, thereby overcoming glioma cell resistance to TRAILmediated apoptosis.