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PubMed2005

Down-regulation of PTEN expression due to loss of promoter activity in human hepatocellular carcinoma cell lines.

AIM: To investigate the regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene expression in human hepatocellular carcinoma (HCC) cell lines. METHODS: The mRNA and protein levels of PTEN were detected by Northern blot and Western blot in HCC cell lines, respectively. Plasmids containing different fragments of PTEN promoter with Luciferase reporter were constructed and transiently transfected into HCC cell lines to study the promoter activity. DNA analysis and RT-PCR were performed to detect the mutation of PTEN promoter and PTEN cDNA. RESULTS: Either protein or mRNA levels of PTEN in L02 cells (as a control) were significantly higher than that in HCC cell lines. The profile of PTEN promoter activity in 8 cell lines was closely correlated with levels of PTEN mRNA and PTEN protein. Furthermore, the sequence analysis of 8 cells lines showed no mutation in the region of PTEN promoter and PTEN cDNA. CONCLUSION: PTEN expression is down-regulated in HCC cell lines probably due to loss of activity of PTEN promoter.

Carcinoma, Hepatocellular↗
PubMed2001

Mutation of the PTEN gene in advanced cervical cancer correlated with tumor progression and poor outcome after radiotherapy.

Improvement in management of advanced cervical cancer after radiotherapy requires a better understanding of its biological behavior. PTEN/MMAC/TEP(PTEN), a candidate tumor suppressor gene located at chromosome 10q23.3, was recently identified and found to be frequently mutated in several different types of human tumors. In contrast, rare mutations of the PTEN gene have been reported in cervical cancer. The aim of this study was to determine whether mutation of PTEN leads to increased genomic alteration in advanced cervical carcinoma, and to identify the correlation between mutation of PTEN and patient outcome after radiotherapy. We examined 50 primary advanced cervical carcinomas (37 patients of Stage IIIB, 13 patients of Stage IVA) treated with definitive radiotherapy using a PCR-based assay followed by SSCP and direct sequencing. The PTEN gene was mutated in 8 of the 50 (16%) patients (2 of Stage III, and 6 of Stage IV). There was a significant difference in Stage III versus IV between the wild-type PTEN patients and mutant PTEN patients (P=0.002). The tumor size was 6+/-2.1 cm in the wild-type PTEN tumors versus 8.5+/-2 cm in the mutant PTEN tumors (P=0.009). In addition, there was a significant difference in survival between the wild-type PTEN patients and mutant PTEN patients (P=0.009). The results of this study suggest that the PTEN gene mutation rate increases with tumor progression, and that the PTEN gene may play a role in both progression of cervical carcinoma and treatment outcome.

Adult↗
PubMed2004

The inositol 3-phosphatase PTEN negatively regulates Fc gamma receptor signaling, but supports Toll-like receptor 4 signaling in murine peritoneal macrophages.

Fc gamma R clustering in macrophages activates signaling events that result in phagocytosis. Phagocytosis is accompanied by the generation harmful byproducts such as reactive oxygen radicals and production of inflammatory cytokines, which mandate that the phagocytic process be subject to a tight regulation. The molecular mechanisms involved in this regulation are not fully understood. In this study, we have examined the role of the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in Fc gamma R-induced macrophage function. We demonstrate that in ex vivo murine peritoneal macrophages that are deficient in PTEN expression, Fc gamma R-induced Akt and extracellular signal-regulated kinase phosphorylation are enhanced. Notably, PTEN(-/-) macrophages showed constitutively high phosphorylation of Akt. However, PTEN did not seem to influence tyrosine phosphorylation events induced by Fc gamma R clustering. Furthermore, PTEN(-/-) macrophages displayed enhanced phagocytic ability. Likewise, Fc gamma R-induced production of TNF-alpha, IL-6, and IL-10 was significantly elevated in PTEN(-/-) macrophages. Surprisingly, LPS-induced TNF-alpha production was down-regulated in PTEN(-/-) macrophages. Analyzing the molecular events leading to PTEN influence on LPS/Toll-like receptor 4 (TLR4) signaling, we found that LPS-induced activation of mitogen-activated protein kinases is suppressed in PTEN(-/-) cells. Previous reports indicated that LPS-induced mitogen-activated protein kinase activation is down-regulated by phosphatidylinositol 3-kinase through the activation of Akt. Our observation that Akt activation is basally enhanced in PTEN(-/-) cells suggests that PTEN supports TLR4-induced inflammatory responses by suppressing the activation of Akt. Thus, we conclude that PTEN is a negative regulator of Fc gamma R signaling, but a positive regulator of TLR4 signaling. These findings are the first to demonstrate a role for PTEN in Fc gamma R- and TLR4-mediated macrophage inflammatory response.

Animals↗
PubMed2002

Expression and significance of a new tumor suppression gene PTEN in primary renal cell carcinoma.

BACKGROUND & OBJECTIVE: PTEN gene is a newly discovered tumor suppressor gene, that maps to chromosome 10q23, and of longstanding interest to those studying somatic mutations in human tumors. Mutation and deletion of PTEN gene probably resulted in a new signal transduction pathway related to human malignant tumors. It was reported that PTEN gene mutated and deleted in renal cell carcinoma, however, there was no report concerning its expression in renal cell carcinoma. This study was designed to investigate expression and significance of PTEN gene in primary renal cell carcinoma. METHODS: Immunohistochemical peroxidase-conjugated streptavidin (SP) method was used to detect expression of PTEN gene in 40 cases of primary renal cell carcinoma, 18 with renal tissues closely adjacent to carcinoma and 5 case of normal renal tissues. The relationship between expression of tumor suppressor gene of PTEN and the percentage of lymph node metastasis of renal cell carcinoma was analyzed. RESULT: It was showed that PTEN gene was expressed in all 5 case of normal renal tissues and 18 cases of renal tissues closely adjacent to carcinoma. The intensity was relatively strong and it was localized mainly in cytoplasm of epithelium cells of renal tubular. Expression of PTEN in 40 cases of renal cell carcinoma were different, 12.5% were negative, 17.5% were weak positive, and 70% were strong positive. PTEN protein was localized in cytoplasm of carcinoma cells. The percentage of lymph node metastasis of renal cell carcinoma negative or weak positive of PTEN protein was 80% and 51.74%, respectively. The percentage of lymph node metastasis of renal cell carcinoma positive of PTEN protein was 10.71%, comparing with those of renal cell carcinoma negative or weak positive of PTEN protein, the difference was significant (P < 0.05). CONCLUSION: This study suggested that PTEN gene was deleted or weakly expressed in primary renal significant cell carcinoma, which is probably related to tumorigenesis and development of renal cell carcinoma.

Aged↗
PubMed2003

[Expression of PTEN-encoding product in different stages of carcinogenesis and progression of gastric carcinoma].

OBJECTIVE: To illustrate the significance of expression of phosphatase and tensin homologue derived from chromosome ten (PTEN) encoding product in normal mucosa, intestinal metaplasia (IM), dysplasia and carcinoma of the stomach, and to evaluate its clinical implication in tumorigenesis and progression of gastric carcinoma. METHODS: Formalin-fixed and paraffin-embedded tissues from 184 cases of gastric carcinoma, its adjacent normal mucosa, IM and dysplasia were evaluated for the expression of PTEN by SABC immunohistochemistry. PTEN expression was assessed as to tumor stage, lymph node metastasis, Lauren's classification and WHO histological classification of gastric carcinoma. Expression of VEGF protein was also studied in 60 cases of gastric carcinoma, with its correlation with PTEN concerned. RESULTS: The positive rates of PTEN protein were 100% (102/102), 98.5% (65/66), 66.7% (4/6) and 47.8% (88/184) in normal mucosa, IM, dysplasia and carcinoma of stomach, respectively. The positive rates in the last two groups were lower than the first two (P < 0.01). PTEN was less expressed in advanced gastric carcinoma than in early ones (42.9% vs 67.6%, P < 0.01). The positive rate of PTEN protein was lower in gastric carcinoma with lymph node metastasis than without (40.3% vs 63.3%, P < 0.01). PTEN was less expressed in diffuse-type gastric carcinoma than in intestinal-type (41.5% vs 57.8%, P < 0.05). Signet ring cell carcinoma expressed PTEN stood the lowest (25.0%, 7/28), which was less than well and moderately differentiated ones (61.8%, 21/34) (P < 0.01). Expression of PTEN was inversely correlated with expression of VEGF though without any significance (P > 0.05). CONCLUSION: Loss or reduced expression of PTEN protein is common in carcinogenesis and progression of gastric cancer. Altered expression of PTEN may contribute to carcinogenesis and progression of gastric cancer by increasing angiogenesis, cellular adhesion and mobility and so on. PTEN may be an objective marker for pathologically biological behavior of gastric carcinoma.

Adult↗
PubMed2006

PTEN gene transfer suppresses the invasive potential of human malignant gliomas by regulating cell invasion-related molecules.

Loss of function of the tumor suppressor gene PTEN is more frequently encountered in high-grade malignant gliomas than in low-grade gliomas. High-grade gliomas are characterized by their extremely invasive behavior, suggesting that PTEN is one of the important regulators of cell motility and that alterations of its coding gene contribute to a much more invasive tumor cell phenotype. In order to clarify a role of PTEN in glioma invasion, we introduced the wild-type PTEN gene into human malignant glioma cell lines and investigated their motile and invasive activity in a brain slice model that presents circumstances analogous to normal brain conditions in vivo. In addition, we analyzed biochemical and molecular changes resulting from the transfer of PTEN in the glioma cells. Infection of recombinant replication-defective adenovirus vector containing the wild-type PTEN cDNA (Ad5CMV-PTEN) significantly inhibited the cell migration and invasion activities of PTEN-mutated glioma cell lines in in vitro migration and chemoinvasion assays. In an organotypic brain slice model, co-culture of glioma spheroids and rat brain slices demonstrated that Ad5CMV-PTEN transfected cells failed to invade surrounding normal brain tissues. Ad5CMV-PTEN transfer into the glioma cell lines lacking the wild-type gene product decreased the levels of matrix metalloproteinase (MMP)-2 mRNA and inhibited the enzymatic activities of MMP-2 and MMP-9. In contrast, mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-2 was upregulated by the PTEN gene transfer. Introduction of PTEN gene in glioma cell lines markedly reduced the levels of Rac-GTP and Cdc42-GTP, activated forms of these small GTP-binding proteins, and decreased the phosphorylation levels of focal adhesion kinase. These results suggest that PTEN inhibits glioma cell invasion in two ways: suppressing proteolysis of the extracellular matrix by MMPs and modulating the migratory activity of glioma cells to a less motile nature by inactivating two Rho-family GTP-binding proteins, Rac and Cdc42.

Animals↗
PubMed2006

[Adenoviral-mediated expression of PTEN cDNA induces apoptosis in endometrial carcinoma cells].

OBJECTIVE: To investigate whether the human endometrial carcinoma cells, RL95-2 infected with recombinant Ad-PTEN can steadily produce PTEN protein and enter apoptosis. METHODS: The recombinant adenovirus containing PTEN cDNA was constructed using the method of homologous recombination in bacteria. The viral titer was examined by plaque assay and the expression of PTEN protein was detected by western blot assay. The apoptosis of RL95-2 cells was evaluated as following: flipping of membrane phosphatidylserine (PS) and identification of activating caspase-3 positive cells was determined by flow cytometer (FCM), and furthermore genomic DNA fragmentation was detected by agarose electrophoresis. RESULTS: The recombinant adenovirus encoding PTEN cDNA was successfully constructed, and viral titers of Ad-PTEN were 5 x 10(9) pfu/ml. After infected by Ad-PTEN, the expression of PTEN protein was steady in human RL95-2 cells. After infected by Ad-PTEN for 24, 48, 72 and 96 h, the relative cell number of membrane PS flipping were (6.09 +/- 1.01)%, (9.98 +/- 2.17)%, (11.74 +/- 2.65)%, (27.69 +/- 8.67)%, which significantly increased than control group (P < 0.05), the relative cell number of activated caspase-3 positive were (2.6 +/- 0.5)%, (18.0 +/- 4.4)%, (21.8 +/- 5.1)%, (33.7 +/- 9.9)%, respectively, which significantly increased than control group (P < 0.05), and genomic DNA fragmentation was verified also. CONCLUSIONS: The recombinant Ad-PTEN vector is constructed successfully and the expression of specific PTEN is steady in RL95-2 cell line. The expression of PTEN induces RL95-2 cells to apoptosis. PTEN gene may be a novel therapeutic target in endometrial carcinoma.

Adenoviridae↗
PubMed2003

Mechanisms underlying PTEN regulation of vascular endothelial growth factor and angiogenesis.

Inactivation of the tumor suppressor gene PTEN and overexpression of VEGF are two of the most common events observed in high-grade malignant gliomas. The purpose of this study was to determine whether PTEN controls VEGF expression in gliomas under normoxic conditions. Transfer of PTEN to human glioma cells resulted in the transduction of a functional PTEN protein as evidenced by the upregulation of p27 and modification of the phosphorylation status of Akt. Under normoxic conditions, enzyme-linked immunosorbent assay and Northern blot analyses showed downregulation of VEGF in PTEN-treated cells. Moreover, conditioned media from PTEN-treated glioma cells significantly diminished the ability of endothelial cells to grow and migrate. Western blot assays demonstrated that, in a normoxic environment, PTEN downregulates HIF-1 alpha. Finally, promoter activity assays showed that the VEGF promoter region containing the HIF-1alpha binding site is necessary and sufficient for PTEN-mediated downregulation of VEGF. Experiments with PI3-K inhibitors and kinase assays suggested that PI3-K is mediating the effect of PTEN on VEGF, and not the p42/p48 or p38 MAP kinases. These results indicate that restoration of PTEN function in gliomas may induce therapeutic effect by downregulating VEGF. Furthermore, this close functional relationship between PTEN and VEGF suggests that a better understanding of the transduction signal regulated by PTEN might enhance the knowledge of the cause and physiology of vascular and inflammatory diseases.

Cell Division↗
PubMed2002

PTEN expression is associated with prognosis for patients with advanced endometrial carcinoma undergoing postoperative chemotherapy.

The prognostic significance of PTEN expression in endometrial carcinoma has not been clear. We conducted the present study to clarify the relationship between PTEN expression and prognosis in advanced endometrial carcinoma. Of 784 patients with endometrial carcinoma who underwent primary treatment between 1985 and 2000 at 5 institutions, 98 pure endometrioid carcinomas with retroperitoneal lymph node metastasis were provided for our study. PTEN expression was determined by immunohistochemic staining. Negative or mixed PTEN staining was observed in 64 (65.3%) patients. The survival rate for PTEN-positive patients was significantly higher than that for PTEN-negative or -mixed patients. PTEN-staining status was not associated with patient age, International Federation of Gynecology and Obstetrics (FIGO) stage, myometrial invasion or histologic grade. Of the 98 patients, 87 received radiation therapy (n = 25) or chemotherapy (n = 62) after surgery. PTEN expression did not relate to survival for patients receiving radiation therapy. In contrast, the survival rate for PTEN-positive cases was significantly higher than that for PTEN-negative or -mixed cases when patients underwent chemotherapy (62.4% vs. 11.8%). Subsequent multivariate analysis revealed that PTEN staining was an independent prognostic factor for patients undergoing chemotherapy. PTEN-positive staining was a significant prognostic indicator of favorable survival for patients with advanced endometrial carcinoma who underwent postoperative chemotherapy.

Adult↗
PubMed2003

Frequent monoallelic deletion of PTEN and its reciprocal associatioin with PIK3CA amplification in gastric carcinoma.

Mutational alterations of PTEN and PIK3CA, which negatively and positively regulate PI3-kinase activity, respectively, have been observed in many types of human cancer. To explore the implication of PTEN and PIK3CA mutations in gastric tumorigenesis, we characterized the expression and mutation status of the genes in 126 gastric tissues and 15 cell lines. Expression of PTEN transcript was abnormally low in 5 of 15 (33%) cell lines and 20 of 55 (36%) primary carcinomas, whereas 0 of 71 noncancerous tissues including 16 benign tumors showed altered expression. Allelotyping analysis using an intragenic polymorphism (IVS4+109) revealed that 14 of 30 (47%) informative cases carried LOH of the gene, which is closely linked to low expression. The LOH rate was significantly higher in advanced tumors [12 of 19 (63%)] compared to early-stage tumors [2 of 11 (18%)] and more frequent in poorly differentiated tumors [9 of 13 (69%)] than well- or moderately differentiated tumors [5 of 17 (29%)]. Interestingly, however, none of the LOH tumors carried mutational disruption of the remaining allele, suggesting haploinsufficiency of PTEN in gastric tumorigenesis. Methylation studies revealed that PTEN pseudogene, but not PTEN, is methylated in cell lines and primary tumors, indicating that PTEN is not a target of epigenetic silencing in gastric cancers and that the pseudogene should be considered more carefully in methylation analysis of the PTEN promoter. Genomic amplification of PIK3CA was found in 9 of 15 (60%) cell lines and 20 of 55 (36.4%) primary tumors but in no noncancerous tissues. Furthermore, PIK3CA amplification was predominantly detected in tumors with no PTEN alterations, suggesting that mutations of PTEN and PIK3CA are mutually exclusive events in gastric tumorigenesis. Amplification of PIK3CA was strongly associated with increased expression of PIK3CA transcript and elevated levels of phospho-AKT. Collectively, our data reveal that 13 of 15 (87%) gastric cell lines and 31 of 55 (56%) primary carcinomas harbored either amplification of PIK3CA or abnormal reduction of PTEN. Mutually exclusive alterations of PTEN and PIK3CA also suggest that mutations of either gene could activate the PI3-kinase/AKT signaling pathway, which is directly linked to the malignant progression of gastric tumor cells.

Adenocarcinoma↗
PubMed2006

Increased PTEN expression due to transcriptional activation of PPARgamma by Lovastatin and Rosiglitazone.

Germline mutations in the tumor suppressor gene PTEN (protein phosphatase and tensin homolog located on chromosome ten) predispose to heritable breast cancer. The transcription factor PPARgamma has also been implicated as a tumor suppressor pertinent to a range of neoplasias, including breast cancer. A putative PPARgamma binding site in the PTEN promoter indicates that PPARgamma may regulate PTEN expression. We show here that the PPARgamma agonist Rosiglitazone, along with Lovastatin, induce PTEN in a dose- and time-dependent manner. Lovastatin- or Rosiglitazone-induced PTEN expression was accompanied by a decrease in phosphorylated-AKT and phosphorylated-MAPK and an increase in G1 arrest. We demonstrate that the mechanism of Lovastatin- and Rosiglitazone-associated PTEN expression was a result of an increase in PTEN mRNA, suggesting that this increase was transcriptionally-mediated. Compound-66, an inactive form of Rosiglitazone, which is incapable of activating PPARgamma, was unable to elicit the same response as Rosiglitazone, signifying that the Rosiglitazone response is PPARgamma-mediated. To support this, we show, using reporter assays including dominant-negative constructs of PPARgamma, that both Lovastatin and Rosiglitazone specifically mediate PPARgamma activation. Additionally, we demonstrated that cells lacking PTEN or PPARgamma were unable to induce PTEN mediated cellular events in the presence of Lovastatin or Rosiglitazone. These data are the first to demonstrate that Lovastatin can signal through PPARgamma and directly demonstrate that PPARgamma can upregulate PTEN at the transcriptional level. Since PTEN is constitutively active, our data indicates it may be worthwhile to examine Rosiglitazone and Lovastatin stimulation as mechanisms to increase PTEN expression for therapeutic and preventative strategies including cancer, diabetes mellitus and cardiovascular disease.

Antineoplastic Agents↗
PubMed2003

Expression of PTEN in renal cell carcinoma and its relation to tumor behavior and growth.

BACKGROUND AND OBJECTIVES: PTEN is a candidate tumor suppressor gene in a variety of malignant tumors, including renal cell carcinoma (RCC). PTEN regulates cell cycle progression and cell survival in vivo. However, the role of PTEN alterations and their association with tumor growth and behavior in patients with RCC has not been well established. The aim of our study was to evaluate PTEN expression in RCC and its correlation with clinicopathologic features, cell proliferation, and apoptosis. METHODS: Sixty-seven RCC specimens were examined immunohistochemically with anti-PTEN antibody. The apoptotic cells were visualized by terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) and proliferative cells were visualized by staining with Ki-67 antibody. RESULTS: Twenty-one (31.3%) of the 67 RCCs showed reduced PTEN expression. The apoptotic index (AI) varied from 0.2 to 25.5%, and the Ki-67 index (KI) ranged from 1.6 to 69.8%. Reduced PTEN expression correlated with TNM stage (P < 0.05) and nuclear grade (P < 0.05). Tumors with reduced PTEN expression had a significantly higher KI than those with normal PTEN expression (P < 0.01). By univariate analysis, nuclear grade (P = 0.0005), TNM stage (P < 0.0001), AI (P = 0.0220), KI (P = 0.0002), and reduced PTEN expression (P < 0.0001) were associated with shortened survival. However, TNM stage was the only independent prognostic factors by multivariate analysis (P = 0.0007). CONCLUSIONS: Our results suggest that PTEN expression is frequently reduced in advanced RCC. The PTEN gene seems to be important for the growth suppression of RCC, by inhibiting cell proliferation.

Adult↗
PubMed2006

PTEN: a crucial mediator of mitochondria-dependent apoptosis.

The highly frequent mutation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in various cancers has attracted much attention to study its role in tumorigenesis. As an important tumor suppressor, the pro-apoptotic function of PTEN has been linked to its capacity antagonizing the PI3K/Akt signaling pathway. However, less data are available concerning its role in neurodegeneration in which apoptotic processes are also involved. In the present study, we attempted to study the role and the underlying mechanism of PTEN in neuronal apoptosis. Using primary rat hippocampal cultures, staurosporine (STS, 100 nM) induced a time-dependent apoptosis, accompanied by a marked production of reactive oxygen species (ROS), release of cytochrome c and activation of caspase 9 and 3. However, the expression of PTEN, and the levels of phospho-PTEN and phospho-Akt were not changed at all time points tested (0.5-24 h) after STS stimulation, suggesting that the protein level as well as the phosphorylation status of PTEN were not related to the procession of apoptosis. Interestingly, immunostaining revealed a punctate intracellular distribution of PTEN from 2 to 8 h after adding STS. Double labeling and Western blotting of mitochondrial fraction demonstrated a mitochondrial location and accumulation of PTEN, respectively, after challenging with STS. Furthermore, we provide evidence for the first time that PTEN was associated with Bax in the absence and the presence of STS. Of note, the STS-induced marked increase in the cellular ROS level, release of cytochrome c and activation of caspase 3 were inhibited in cultured hippocampal cells when PTEN was knocked down by a specific antisense. Moreover, knockdown of PTEN significantly protected hippocampal cells from apoptotic damage. These findings demonstrated that PTEN is a crucial mediator of mitochondria-dependent apoptosis, and thus could become a molecular target for interfering with neurodegenerative diseases.

Animals↗
PubMed2005

Transient strong reduction of PTEN expression by specific RNAi induces loss of adhesion of the cells.

The tumor suppressor gene pten encodes a lipid phosphatase that dephosphorylates D3 of phosphatidylinositol(3,4,5)trisphosphate, producing phosphatidylinositol(4,5)bisphosphate. Although PTEN has been implicated in cell adhesion and migration, the underlying molecular mechanism is unknown. To investigate the role of PTEN in cell adhesion, we designed three different siRNAs (siRNA PTEN-a, siRNA PTEN-b, and siRNA PTEN-c) and transfected into 293T cells. Two days later, only the cells transfected with siRNA PTEN-b became round and detached from the culture dishes, whereas cells transfected with a control siRNA against GFP or the two other siRNAs against PTEN did not. Evaluation of the RNAi effect revealed that siRNA PTEN-b inhibited >95% of PTEN expression, the most effective among the three siRNAs. To check for non-specific effects such as interferon response and inhibition of off-target genes, we then used quantitative PCR analysis and DNA microarray analysis. None was detected, indicating that the RNAi system was highly specific. Immunofluorescence studies using PTEN-knockdown HeLa cells revealed that the loss of adhesion was accompanied by a reduction in the number of focal adhesion plaques and disorganization of the actin cytoskeleton. Transient and near-complete loss of PTEN expression induces loss of adhesion of the cells.

Apoptosis↗
PubMed2005

Physiological levels of PTEN control the size of the cellular Ins(1,3,4,5,6)P(5) pool.

To understand how a signaling molecule's activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30-40% (p<0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P(5) levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p<0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to D/L-Ins(1,2,4,5,6)P5, levels of which increased 60% (p<0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein.

Catalysis↗
PubMed2006

Regulation of protein kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines.

Protein Kinase B (PKB/Akt) is a key regulator of cell proliferation, motility and survival. The activation status of PKB is regulated by phosphatidylinositol 3-kinase (PI3K) via the synthesis of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3, PIP3). PTEN antagonises PI3K by degrading PIP3 to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Deregulation of PKB through loss of functional PTEN has frequently been implicated in the progression of tumours, including prostate cancer, and the PTEN-negative prostate cancer cell lines LNCaP and PC3 have been widely used as models for this mechanism of constitutive PKB activation. However, other enzymes in addition to PTEN can antagonise PI3K, including SHIP2, which degrades PIP3 to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). We investigated the role of PTEN and SHIP2 in the regulation of PKB phosphorylation in a panel of human prostate-derived epithelial cell lines. In the PTEN-positive prostate-derived cell lines PNT2, PNT1a and P4E6, PI3K inhibition by LY294002 caused rapid dephosphorylation of PKB at ser473 (T(1/2)<2 min), leading to its inactivation. In the PTEN-null line LNCaP, LY294002-induced PKB dephosphorylation was much slower (T(1/2)>20 min), but in PC3 cells (also PTEN-null) it was only slightly slower than in PTEN-positive cells (T(1/2)=3 min). PKB dephosphorylation paralleled loss of plasma membrane PIP3. PNT1a, P4E6 and PC3, but not PNT2 or LNCaP, expressed SHIP2. SiRNA-mediated knockdown of SHIP2 expression markedly slowed PKB inactivation in response to LY294002 in PC3 but not in other SHIP2-positive cells, whereas knockdown of PTEN expression in PNT2, PNT1a and P4E6 resulted in higher steady-state levels of PKB phosphorylation and slowed, but did not prevent, LY294002-induced PKB inactivation. Thus SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells but not other prostate cell lines, where PTEN may share this role with further PIP3-degrading mechanisms.

Cell Line↗
PubMed2003

Profiling of genes expressed by PTEN haploinsufficient neural precursor cells.

PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- and PTEN +/+ mice to prepare subventricular zone (SVZ) precursor cells. Using DNA microarrays, we compared the expression profiles of PTEN +/+ and PTEN +/- cells and identified 91 differentially expressed genes in PTEN +/- precursor cells. Many of the PTEN-regulated genes are involved with signaling, cytoskeleton, extracellular matrix, metabolism, and transcription factors. Some of these changes are likely mediated by the transcription factor, HIF-1. We confirmed a subset of these changes by real-time PCR. In addition, we examined protein levels for two of the PTEN-up-regulated genes, vascular endothelial growth factor (VEGF) and doublecortin (DCX). PTEN haploinsufficiency increases immunostaining for VEGF for both cultured precursor cells and sections of the SVZ. PTEN haploinsufficiency shifted most of the DCX-positive cells from the SVZ to the olfactory bulb. These observations indicate that even a small decrease in PTEN levels results in substantial changes in gene expression and precursor cell function.

Animals↗
PubMed2006

PTEN is recruited to specific microdomains of the plasma membrane during lactacystin-induced neuronal apoptosis.

The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) is a novel phosphatase displaying both protein and lipid phosphatase activities. In the central nervous system, PTEN plays an important role in the regulation of cell growth, differentiation and death. The tumor suppressor function of PTEN is attributed to its phospholipid phosphatase activity that dephosphorylates the plasma membrane phosphatidylinositol-(3,4,5)-triphosphate (PtdIns(3,4,5)P(3)). Since PTEN is normally localized in the cytosol, it needs to be targeted to the plasma membrane to dephosphorylate PtdIns(3,4,5)P(3). We previously demonstrated that lactacystin-induced apoptosis of culture cortical neuron is associated with: (i) cleavage of PTEN (55 kDa) to a 50 kDa truncated form and (ii) accumulation of PTEN and all the truncated PTEN in a detergent-insoluble membrane fraction of the neuronal cells. Herein we demonstrate that a caspase-3 inhibitor suppresses cleavage of PTEN to the 50kDa truncated form in culture cortical neurons in response to lactacystin treatment. Using immunogold transmission electron microscopy, we examined the distribution of PTEN in plasma membrane sheets stripped from cultured cortical neurons with and without treatment of lactacystin. Our results demonstrate that lactacystin treatment induces accumulation of PTEN to the inner surface of the plasma membrane sheets of the neuronal cells. Taken together, our data suggest that caspase-3-like proteases are involved in the conversion of PTEN to a 50-kDa truncated form and that PTEN and its truncated form accumulate at specific microdomains of the plasma membrane in neuronal cells undergoing apoptosis.

Acetylcysteine↗