Regulation of PTEN transcription by p53.

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.     

Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53‐dependent apoptosis and anticancer effect

This study investigated the efficacy of Toxoplasma GRA16, which binds to herpes virus‐associated ubiquitin‐specific protease (HAUSP), in anticancer treatment, and whether the expression of GRA16 in genetically modified hepatocellular carcinoma (HCC) cells (GRA16‐p53‐wild HepG2 and GRA16‐p53‐null Hep3B) regulates PTEN because alterations in phosphatase and tensin homologue (PTEN) and p53 are vital in liver carcinogenesis and the abnormal p53 gene appears in HCC. For this purpose, we established the GRA16 cell lines using the pBABE retrovirus system, assessed the detailed mechanism of PTEN regulation in vitro and established the anticancer effect in xenograft mice. Our study showed that cell proliferation, antiapoptotic factors, p‐AKT/AKT ratio, cell migration and invasive activity were decreased in GRA16‐stable HepG2 cells. Conversely, the apoptotic factors PTEN and p53 and apoptotic cells were elevated in GRA16‐stable HepG2 cells but not in Hep3B cells. The change in MDM2 was inconspicuous in both HepG2 and Hep3B; however, the PTEN level was remarkably elevated in HepG2 but not in Hep3B. HAUSP‐bound GRA16 preferentially increased p53 stabilization by the nuclear localization of PTEN rather than MDM2‐dependent mechanisms. These molecular changes appeared to correlate with the decreased tumour mass in GRA16‐stable‐HepG2 cell‐xenograft nude mice. This study establishes that GRA16 is a HAUSP inhibitor that targets the nuclear localization of PTEN and induces the anticancer effect in a p53‐dependent manner. The efficacy of GRA16 could be newly highlighted in HCC treatment in a p53‐dependent manner.     

调控p53蛋白的转录因子

肿瘤抑制因子p53被称为"分子警察",它在维持细胞正常生长及抑制恶性增殖过程中起重要作用。p53的表达水平受多种因素影响,其中转录水平的调控是基因发挥功能的一个重要步骤。因此,针对调控p53蛋白的转录因子这一环节阐明p53发挥功能的分子机理,有望为肿瘤治疗、预防和新药研发提供新的靶标。本文着重对调控p53蛋白的转录因子进行综述。     

Proteome profile changes that are differentially regulated by lipid and protein phosphatase activities of tumor suppressor PTEN in PTEN-expressing U-87 MG human glioblastoma cells

The phosphatase and tensin homolog tumor suppressor (PTEN) belongs to a class of "gatekeeper" tumor suppressors together with p53, retinoblastoma and adenomatous polyposis. It is considered one of the most important tumor suppressors in the post p53 era. Previously to identify the molecules involved in the signaling network regulated by PTEN using proteomic tools, we reported global proteome profiles at different time points using the PTEN inducible NIH3T3 cells (Kim, S.-y., Kim, Y. S., Bahk, Y. Y., Mol. Cells 2003, 15, 396-405). However, the system had a critical limitation that NIH3T3 cell has endogenous wild-type PTEN and, thus to be exact, the induced PTEN could not give the answer about the real physiological roles of this tumor suppressor. Here, to find out PTEN-related protein network we have established various PTEN (wild-type, an activity inert C124G, and a lipid phosphatase deficient G129E)-expressing cell clones in U-87 MG human glioblastoma cells lacking detectable PTEN as a result of genetic lesions. In this biological context, we compared their morphological and expression patterns, and proteome images of each PTEN-expressing cell clone by 2-DE followed by identification with MALDI-TOF MS. We obtained some pieces of evidence that morphological change by PTEN expression is mediated by its protein phosphatase activity and their growth rate by the lipid phosphatase activity. The proteomic approaches showed that 30 proteins possibly correlated with PTEN's protein phosphatase activity (13 down-regulated and 17 up-regulated) and 20 with the lipid phosphatase activity (14 down-regulated and 6 up-regulated) were identified. Taken together, we conclude that the comparative analysis of proteome from various PTEN-expressing cells has yielded interpretable data to elucidate the protein network directly and/or indirectly caused by individual phosphatase activities of PTEN in vivo.     

Inactivation of p53 signaling by p73 or PTEN ablation results in a transformed phenotype that remains susceptible to Nutlin-3 mediated apoptosis

The p53 signaling pathway is frequently disrupted in carcinogenesis. However, roughly 50% of all cancers express wild-type p53 and have alterations in accessory signaling components required for p53 activity. Using the well described E1A/RAS transformation model, in which p53 activity must be suppressed for transformation, we show here that p53 is inactive and unable to suppress transformation following ablation of p73 or PTEN. However, despite the transformed phenotype conferred by p53 inactivation following p73 or PTEN loss, p53 could be fully activated by Nutlin-3, resulting in efficient caspase-mediated apoptosis. Our novel and unexpected finding provides important information regarding the efficacy of Nutlin-3 and indicates that patients with tumors deficient in p53 function due to p73 or PTEN loss may benefit from Nutlin-3 treatment.     

PTEN and p53 are required for hypoxia induced expression of maspin in glioblastoma cells

In response to genotoxic stress, p53 induces the tumor suppressors maspin and PTEN. Here we demonstrate that in response to limited oxygen conditions PTEN and p53 work in tandem to induce maspin in glioblastoma cells. In response to hypoxia a portion of PTEN migrates to the nucleus and complexes with p53, while cytoplasmic PTEN prevents Mdm2 nuclear localization by attenuating Akt signaling. Subcellular distribution of PTEN in the cytoplasm or nucleus protects p53 from inac-tivation and degradation. The presence of nuclear PTEN and p53 coordinates the induction of maspin and p21 (both p53 gene targets) in response to hypoxia. Altering the expression of PTEN and/or p53 attenuated maspin gene induction under hypoxic conditions. Furthermore, implanting U87 (PTEN null) and PTEN reconstituted U87 cells (U87PTEN) in mice we observed by immuno-histochemistry and western blot that Maspin was only detectable in cells with PTEN. The integra-tion of PTEN and p53 into a common pathway for the induction of another tumor suppressor, Maspin, constitutes a tumor suppressor network of PTEN/p53/Mapsin that is operational under limited oxygen conditions.     

p53 Down Regulates PDGF-Induced Formation of Circular Dorsal Ruffles in Rat Aortic Smooth Muscle Cells

The tumor suppressor, p53, negatively regulates cell migration and invasion in addition to its role in apoptosis, cell cycle regulation and senescence. Here, we study the roles of p53 in PDGF-induced circular dorsal ruffle (CDR) formation in rat aortic smooth muscle (RASM) cells. In primary and immortalized RASM cells, up-regulation of p53 expression or increase in activity with doxorubicin inhibits CDR formation. In contrast, shRNA-knockdown of p53 or inhibition of its activity with pifithrin α promotes CDR formation. p53 acts by up-regulating PTEN expression, which antagonizes Rac and Cdc42 activation. Both lipid and protein phosphatase activities of PTEN are required for maximal suppression of CDR, but the lipid activity clearly plays the dominant role. N-WASP, the downstream effector of Cdc42, is the major positive contributor to CDR formation in RASM, and is an indirect target of p53. The Rac effector, WAVE2, appears to also play a minor role, while WAVE1 has no significant effect in CDR formation. In sum, we propose that p53 suppresses PDGF-induced CDR formation in RASM cells by upregulating PTEN leading mainly to the inhibition of the Cdc42-N-WASP pathway.     

Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified

The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.     

PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy.

The PTEN tumor suppressor protein inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signaling that promotes translocation of Mdm2 into the nucleus. When restricted to the cytoplasm, Mdm2 is degraded. The ability of PTEN to inhibit the nuclear entry of Mdm2 increases the cellular content and transactivation of the p53 tumor suppressor protein. Retroviral transduction of PTEN into U87MG (PTEN null) glioblastoma cells increases p53 activity and expression of p53 target genes and induces cell cycle arrest. U87MG/PTEN glioblastoma cells are more sensitive than U87MG/PTEN null cells to death induced by etoposide, a chemotherapeutic agent that induces DNA damage. Previously, tumor suppressor proteins have been supposed to act individually to suppress cancers. Our results establish a direct connection between the activities of two major tumor suppressors and show that they act together to respond to stresses and malignancies. PTEN protects p53 from survival signals, permitting p53 to function as a guardian of the genome. By virtue of its capacity to protect p53, PTEN can sensitize tumor cells to chemotherapy that relies on p53 activity. p53 induces PTEN gene expression, and here it is shown that PTEN protects p53, indicating that a positive feedback loop may amplify the cellular response to stress, damage, and cancer.     

Promoter analysis of tumor suppressor gene PTEN: identification of minimum promoter region

Mutations of PTEN, a tumor suppressor gene located on chromosome 10, which encodes a protein-tyrosine and lipid-phosphatase, are prevalent in various human cancers, including glioblastoma. Despite extensive characterization of PTEN mutations in human cancers and a relatively good understanding of the molecular roles of PTEN in the control of cellular processes, little is known about modes of PTEN regulation. To understand the regulation of expression of the tumor suppressor gene PTEN, we isolated a 2212 bp fragment from the human BAC clone 46B12 DNA. The 3' end of this fragment starts at the Not I site of -745 relative to the first translation codon ATG (+1) and ends at the Sal I site of -2957 at the 5' end. Using classical 5'RACE and primer extension techniques, nine start sites were observed between -817 and -984 upstream of the ATG start site. We located a 137 bp fragment (-958/-821) as the minimum promoter region using promoter deletion and luciferase assays. A 704 bp fragment (-33/-737) downstream of the 2212 bp fragment was also cloned. As indicated by luciferase assays, the data show that this region possesses no promoter function. Interestingly, a p53 binding sequence is located within the 599 bp fragment (-1344/-745), although p53 expression had a minimal effect on PTEN, demonstrating its insignificant role in PTEN gene expression.     

Mechanistic insights into maintenance of high p53 acetylation by PTEN

Earlier studies have shown that PTEN regulated p53 protein stability both in a phosphatase-dependent manner through antagonizing Akt-Mdm2 pathway and in a phosphatase-independent manner through interacting with p53. In this study, we report that PTEN forms a complex with p300 in the nucleus and plays a role in maintenance of high p53 acetylation in response to DNA damage. Furthermore, p300 is required for nuclear PTEN-regulated cell cycle arrest. Interestingly, however, p53 acetylation was found to promote PTEN-p53 interaction. To investigate the molecular mechanisms, we show that acetylation promotes p53 tetramerization, which, in turn, is required for the PTEN-p53 interaction and subsequent maintenance of high p53 acetylation. Taken together, our results suggest a physiological role for the PTEN tumor suppressor in the nucleus and provide a molecular explanation for our previous observation that PTEN controls p53 protein levels independent of its phosphatase activity.     

The Pharmacodynamics of the p53-Mdm2 Targeting Drug Nutlin: The Role of Gene-Switching Noise

In this work we investigate, by means of a computational stochastic model, how tumor cells with wild-type p53 gene respond to the drug Nutlin, an agent that interferes with the Mdm2-mediated p53 regulation. In particular, we show how the stochastic gene-switching controlled by p53 can explain experimental dose-response curves, i.e., the observed inter-cell variability of the cell viability under Nutlin action. The proposed model describes in some detail the regulation network of p53, including the negative feedback loop mediated by Mdm2 and the positive loop mediated by PTEN, as well as the reversible inhibition of Mdm2 caused by Nutlin binding. The fate of the individual cell is assumed to be decided by the rising of nuclear-phosphorylated p53 over a certain threshold. We also performed in silico experiments to evaluate the dose-response curve after a single drug dose delivered in mice, or after its fractionated administration. Our results suggest that dose-splitting may be ineffective at low doses and effective at high doses. This complex behavior can be due to the interplay among the existence of a threshold on the p53 level for its cell activity, the nonlinearity of the relationship between the bolus dose and the peak of active p53, and the relatively fast elimination of the drug.     

Cell Surface Area and Membrane Folding in Glioblastoma Cell Lines Differing in PTEN and p53 Status

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo−/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C _m = 1.9 µF/cm². In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C _m values of 3.7–4.0 µF/cm², which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types.     

Temporal PTEN inactivation causes proliferation of saphenous vein smooth muscle cells of human CABG conduits

Internal mammary artery (IMA) coronary artery bypass grafts (CABG) are remarkably resistant to intimal hyperplasia (IH) as compared to saphenous vein (SV) grafts following aorto-coronary anastomosis. The reason behind this puzzling difference still remains an enigma. In this study, we examined the effects of IGF-1 stimulation on the PI3K-AKT/PKB pathway mediating proliferation of smooth muscle cells (SMCs) of IMA and SV origin and the specific contribution of phosphatase and tensin homologue (PTEN) in regulating the IGF-1-PI3K-AKT/PKB axis under these conditions. Mitogenic activation with IGF-1, time-dependently stimulated the phosphorylation of PI3K and AKT/PKB in the SV SMCs to a much greater extent than the IMA. Conversely, PTEN was found to be significantly more active in IMA SMCs. Transient overexpression of PTEN in SMCs of SV and IMA inhibited AKT/PKB activity and upstream of AKT/PKB, caused a reduction of IGF-1 receptors. Downstream, PTEN overexpression in SV SMCs induced the transactivation of tumour suppressor protein p53 by down-regulating the expression of its inhibitor MDM2. However, PTEN overexpression had no significant effect on MDM2 and p53 expression in IMA SMCs. PTEN overexpression inhibited IGF-1-induced SMC proliferation in both SV and IMA. PTEN suppression, induced by siRNA transfection of IMA SMCs diminished the negative regulation of PI3K-PKB signalling leading to greater proliferative response induced by IGF-1 stimulation. Thus, we show for the first time that early inactivation of PTEN in SV SMCs leads to temporally increased activity of the pro-hyperplasia PI3K-AKT/PKB pathway leading to IH-induced vein graft occlusion. Therefore, modulation of the PI3K-AKT/PKB pathway via PTEN might be a novel and effective strategy in combating SV graft failure following CABG.