PTEN tumor suppressor is frequently mutated in human cancers and is a negative regulator of PI3'K/PKB/Akt-dependent cellular survival. Investigation of the human genomic PTEN locus revealed a p53 binding element directly upstream of the PTEN gene. Deletion and mutation analyses showed that this element is necessary for inducible transactivation of PTEN by p53. A p53-independent element controlling constitutive expression of PTEN was also identified. In contrast to p53 mutant cell lines, induction of p53 in primary and tumor cell lines with wild-type p53 increased PTEN mRNA levels. PTEN was required for p53-mediated apoptosis in immortalized mouse embryonic fibroblasts. Our results reveal a unique role for p53 in regulation of cellular survival and an interesting connection in tumor suppressor signaling.

The p53 tumor suppressor exerts anti-proliferative effects in response to various types of stress including DNA damage and abnormal proliferative signals. Tight regulation of p53 is essential for maintaining normal cell growth and this occurs primarily through posttranslational modifications of p53. Here, we describe Pirh2, a gene regulated by p53 that encodes a RING-H2 domain-containing protein with intrinsic ubiquitin-protein ligase activity. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of Mdm2. Expression of Pirh2 decreases the level of p53 protein and abrogation of endogenous Pirh2 expression increases the level of p53. Furthermore, Pirh2 represses p53 functions including p53-dependent transactivation and growth inhibition. We propose that Pirh2 is involved in the negative regulation of p53 function through physical interaction and ubiquitin-mediated proteolysis. Hence, Pirh2, like Mdm2, participates in an autoregulatory feedback loop that controls p53 function.

The p53 tumour suppressor promotes cell-cycle arrest or apoptosis in response to cellular stress, such as DNA damage and oncogenesis. This role of p53 is important for its tumour-suppression function and depends, at least in part, on its ability to bind to specific DNA sequences and activate the transcription of target genes. The pathway through which p53 promotes apoptosis is not fully understood. Here we describe a new gene regulated by p53 that encodes a predicted protein of 915 amino acids in mice (910 amino acids in humans), which we have named Pidd. The mouse Pidd cDNA contains a p53 consensus DNA binding sequence upstream of the Pidd-coding region. This sequence element bound to p53 and conferred p53-dependent inducibility on a heterologous reporter gene. Moreover, Pidd RNA was induced by ionizing radiation in a p53-dependent manner and the basal level of Pidd RNA was dependent on Trp53 status. Overexpression of Pidd inhibited cell growth in a p53-like manner by inducing apoptosis. Antisense inhibition of Pidd expression attenuated p53-mediated apoptosis. Our data suggest that Pidd is an effector of p53-dependent apoptosis.

The p53 protein is activated by stress signals and exhibits both protective and death-promoting functions that are considered important for its tumor suppressor function. Emerging evidence points toward an additional role for p53 in metabolism. Here, we identify Lpin1 as a p53-responsive gene that is induced in response to DNA damage and glucose deprivation. Lpin1 is essential for adipocyte development and fat metabolism, and mutation in this gene is responsible for the lypodystrophy phenotype in fld mice. We show that p53 and Lpin1 regulate fatty acid oxidation in mouse C2C12 myoblasts. p53 phosphorylation on Ser18 in response to low glucose is ROS and ATM dependent. Lpin1 expression in response to nutritional stress is controlled through the ROS-ATM-p53 pathway and is conserved in human cells. Lpin1 provides a critical link between p53 and metabolism that may be an important component in mediating the tumor suppressor function of p53.

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Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperaturesensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37؇C and as a wild-type polypeptide at 32؇C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G 1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32؇C by undergoing G 1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G 1 . The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G 1 arrest. These observations indicate that apoptosis and G 1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G 1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32؇C by undergoing G 1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.

Various cytokines have been shown to protect cells from p53-dependent apoptosis. To investigate the mechanism underlying cytokine-mediated survival, we used a Friend virus-transformed erythroleukemia cell line that expresses a temperaturesensitive p53 allele. These cells express the spleen focus-forming virus-encoded envelope glycoprotein gp55 that allows the cells to proliferate in the absence of erythropoietin (EPO). These cells respond to p53 activation at 32°C by undergoing G 1 cell cycle arrest and apoptosis.In the presence of EPO, p53 activation leads only to prolonged but viable G 1 arrest. These findings indicate that EPO functions as a survival factor and that gp55/EPO receptor signaling is distinct from EPO/EPO receptor signaling. We demonstrate that p53-dependent apoptosis results in mitochondrial damage as shown by loss of mitochondrial membrane potential, increase in intracellular calcium, and release of mitochondrial cytochrome c into the cytosol. EPO prevented all of these changes including the subsequent activation of caspases. We identify an intrinsic phosphatidylinositol-3-OH kinase/protein kinase B (PI3K/ PKB)-dependent survival pathway that is constitutively active in these cells. This survival pathway limits p53-dependent apoptosis. We propose that EPO promotes survival through a distinct pathway that is dependent on JAK2 but independent of STAT5 and PI3K. IntroductionThe p53 tumor suppressor gene plays a fundamental role in promoting apoptosis in response to abnormal proliferative signals and stress including DNA damage. 1 Loss of p53-mediated apoptosis results in the survival of oncogene-expressing cells undergoing inappropriate cell growth and in the survival of cells carrying mutations and carcinogenic lesions. Failure to eliminate such cells has been shown to accelerate tumorigenesis. 2 The evasion of apoptosis through genetic or epigenetic mechanisms that target death and survival pathways is considered to be a hallmark of cancer cells. 3 The cellular decision to undergo apoptosis is governed by the integration of survival and death signals. Growth factors can inhibit apoptosis through increased expression of prosurvival genes and through posttranslational modification and inactivation of proapoptotic proteins. One of the most widely studied cytokines capable of promoting survival in hematopoietic cells is erythropoietin (EPO). The binding of EPO to its receptor (EPO-R) activates multiple signaling pathways that ultimately control the survival, proliferation, and development of immature erythroid cells (for a review, see Wojchowski et al 4 ). EPO-dependent tyrosine phosphorylation of the receptor, which lacks intrinsic kinase activity, and many of its associated signaling molecules is mediated by the cytoplasmic tyrosine kinase JAK2. EPO signaling has been shown to rescue committed erythroid progenitors from undergoing apoptosis during normal erythropoiesis. 5,6 The function of EPO as a survival factor has been confirmed using EPO-dependent erythroid cells in which EPO deprivation res...

The p53 tumor suppressor gene encodes a transcription factor that is commonly mutated in cancer. Tumors arise when premalignant cells are unable to undergo p53-dependent apoptosis, cell cycle arrest or DNA repair. The p53-signaling pathway affects not only tumor development, but also the response of tumors to chemotherapeutic drugs. In this study, we use cell penetrating peptide conjugates of phosphorodiamidate morpholino oligomers (PPMOs) to inhibit p53 expression. We examine the functional properties of endogenous p53 isoforms that are produced upon PPMO-mediated inhibition of p53 translation and splicing, and report that loss of N-terminal or C-terminal sequences interferes with the transcriptional activity of p53. Importantly, we report that PPMOmediated inhibition of p53 expression sensitizes human cancer cells with wild-type p53 to chemotherapeutic drugs.