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PubMed2006

Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway.

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway controls many cellular processes that are important for the formation and progression of cancer, including apoptosis, transcription, translation, metabolism, angiogenesis, and cell cycle progression. Genetic alterations and biochemical activation of the pathway are frequent events in preneoplastic lesions and advanced cancers and often portend a poor prognosis. Thus, inhibition of the PI3K/Akt/mTOR pathway is an attractive concept for cancer prevention and/or therapy. Inhibitors of individual components, such as PI3K, PDK-1, Akt, and mTOR, are being developed at a rapid pace and have promise for improving the care of cancer patients. Here, we review the published data on inhibitors of the pathway and discuss relevant issues, such as the complex regulation of the pathway, the design of clinical trials, and the likelihood of finding a therapeutic index when targeting such a critical signaling pathway.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2003

RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1.

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2004

Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression.

Hippocampal long-term depression (LTD) is a long-lasting decrease in synaptic strength that is most commonly studied at glutamatergic inputs to pyramidal cells in hippocampal area CA1. Activation of G-protein-coupled group I (including types 1 and 5) metabotropic glutamate receptors (mGluRs) by the pharmacological agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) elicits LTD in area CA1 of the hippocampus. Recent reports have shown that de novo protein synthesis is necessary for DHPG-induced LTD. However, relatively little is known about the signaling pathways that couple mGluRs to translation initiation. In this study, we investigated whether the activation of the phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which has been shown to regulate translation initiation, is necessary for mGluR-LTD induced by DHPG. We found that brief incubations of mouse hippocampal slices with DHPG resulted in increased phosphorylation of Akt and mTOR in hippocampal area CA1. Two structurally unrelated PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increases in phosphorylation of Akt and mTOR. Biochemical fractionation studies showed that the DHPG-induced increase in the phosphorylation of Akt and mTOR could be detected in synaptoneurosome preparations, and immunohistochemical analysis revealed that similar increases could be detected in both stratum pyramidale and stratum radiatum in area CA1. Finally, we observed that both PI3K inhibitors and rapamycin, an mTOR inhibitor, prevented mGluR-LTD induced by DHPG. Together, our findings indicate that activation of the PI3K-Akt-mTOR signaling cascade is required for mGluR-LTD and suggest that this pathway may couple group I mGluRs to translation initiation in hippocampal area CA1.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Evidence that SHIP-1 contributes to phosphatidylinositol 3,4,5-trisphosphate metabolism in T lymphocytes and can regulate novel phosphoinositide 3-kinase effectors.

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3); the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P(3) and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P(3) or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P(3) and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.

Animals↗
PubMed1996

Growth-promoting effects of Ca(2+)-mobilizing agents in hepatocytes: lack of correlation between the acute activation of phosphoinositide-specific phospholipase C and the stimulation of DNA synthesis by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2 alpha.

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide-specific phospholipase C (PI-PLC) and mobilize Ca2+, the role of PI-PLC in the growth-stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the beta-adrenoceptor blocker timolol) or PGF2 alpha, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5-trisphosphate (InsP3) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI-PLC, measured as the early rise (peak 15-60 s) in InsP3, was 8-10-fold with vasopressin or angiotensin II, 3-4-fold with norepinephrine, and approximately 2-fold with PGF2 alpha. For all the agonists, a rise in cytosolic free Ca2+ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP3. However, the growth-stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI-PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine approximately PGF2 alpha > angiotensin II > vasopressin. Also, norepinephrine, PGF2 alpha, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP3. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP3 responses were not. The results show that the extent of the initial activation of PI-PLC is not the determinant for the magnitude of the growth effects of Ca(2+)-mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI-PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI-PLC-independent, mechanisms are required.

Angiotensin II↗
PubMed2001

Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways.

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.

Androstadienes↗
PubMed2003

Epidermal growth factor up-regulates expression of transforming growth factor beta receptor type II in human dermal fibroblasts by phosphoinositide 3-kinase/Akt signaling pathway: Resistance to epidermal growth factor stimulation in scleroderma fibroblasts.

OBJECTIVE: Transforming growth factor beta receptors (TGFbetaRs) are known to be expressed at high levels in several fibrotic diseases, including systemic sclerosis. In the present study, we investigated the mechanism of up-regulation of TGFbetaR expression. METHODS: The levels of expression of TGFbetaR type II (TGFbetaRII) messenger RNA (mRNA), with or without stimulation by epidermal growth factor (EGF), were evaluated by Northern blot analysis, and the protein levels were determined by immunoblotting. The transcription activity of the TGFbetaRII gene was examined with luciferase assays using the -1670/+35 TGFbetaRII promoter luciferase construct. RESULTS: EGF up-regulates the expression of TGFbetaRII mRNA and protein in human dermal fibroblasts. Actinomycin D, an RNA synthesis inhibitor, significantly blocked the EGF-mediated up-regulation of TGFbetaRII mRNA expression, whereas cycloheximide, a protein synthesis inhibitor, did not block this up-regulation. In addition, EGF treatment did not significantly affect the TGFbetaRII mRNA half-life. EGF-mediated induction of TGFbetaRII expression was inhibited by treatment of fibroblasts with the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin or LY294002, and Akt inhibitor also blocked EGF-induced expression of TGFbetaRII. In addition, EGF induced TGFbetaRII promoter activity, and this induction was significantly blocked by wortmannin, LY294002, or Akt inhibitor. Cotransfection with a dominant-negative mutant of p85 (the regulatory component of PI 3-kinase) or Akt significantly reduced the induction of TGFbetaRII promoter activity by EGF. Moreover, a constitutive active form of p110 (a catalytic component of PI 3-kinase) induced TGFbetaRII promoter activity. In addition, scleroderma fibroblasts expressed increased levels of TGFbetaRII but did not show further up-regulation of TGFbetaRII expression by EGF. CONCLUSION: These results indicate that EGF-mediated induction of TGFbetaRII expression occurs at the transcription level, does not require de novo protein synthesis, and involves the PI 3-kinase/Akt signaling pathway, and that abnormal activation of EGF-mediated signaling pathways, including PI 3-kinase or Akt, might play a role in the up-regulation of TGFbetaRII in scleroderma fibroblasts.

Androstadienes↗
PubMed2003

Experimental researches on the role of phosphoinositide-specific phospholipase C in 4-hydroxynonenal induced exocytosis.

The action of 4-hydroxynonenal (HNE), a chemotactic aldehyde produced by lipid peroxidation, was analysed on exocytosis in parallel with its effects on phosphoinositide-specific phospholipase C (PLC) both in undifferentiated HL-60 cells and in cells induced to differentiate toward the granulocytic cell line by 1.25% DMSO. Exocytosis was evaluated by the secretion of beta-glucuronidase from cells incubated at 37 degrees C for 10 min in the presence of various aldehyde concentrations. HNE action was more pronounced in DMSO-differentiated cells, where concentrations between 10(-8) and 10(-6) m were able both to trigger exocytosis and to strongly activate PLC; in both processes maximal stimulation was given by 10(-7) m. HNE-induced exocytosis was completely prevented by pertussis toxin and by the PLC inhibitor U73122. The comparison between HNE and formyl-methionyl-leucyl-phenylalanine (fMLP), used as a positive control, showed that the tripeptide produced an higher stimulation of exocytosis than the aldehyde; by contrast HNE induced a stronger increase of PLC activity. Wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), strongly inhibited the exocytosis induced by fMLP, while it failed to induce a statistically significant inhibition of HNE action. We conclude that both compounds trigger exocytosis through a Ptx-sensitive G protein; the present data support the hypothesis that the lower ability of the aldehyde to trigger exocytosis as compared to fMLP might depend upon a low ability to activate PI3K, while PLC activation appears to play a key role in HNE-induced exocytosis.

Aldehydes↗
PubMed2003

Products of phosphoinositide specific phospholipase C can trigger dephosphorylation of cofilin in chemoattractant stimulated neutrophils.

The signal transduction pathways that trigger dephosphorylation of cofilin in neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) were investigated with a phospho-specific antibody that recognized cofilin only when this protein was phosphorylated on ser-3. Unlike earlier studies that monitored changes in (32)P-labeled cofilin, this Ab allowed us to monitor changes in the total mass of phosphorylated cofilin during neutrophil stimulation. Neutrophils stimulated with fMLP (1.0 microM) for 1.0 min exhibited a massive loss (> 85%) of phosphate from cofilin, which was blocked by an antagonist of phosphoinositide-specific phospholipase C (PI-PLC) (1.0 microM U73122). Products of PI-PLC, sn-1,2-diglyceride and inositol (1,4,5)-trisphosphate, are known to activate protein kinase C (PKC) and increase intracellular Ca(2+), respectively. Treatment of neutrophils with agents that selectively activate PKC [4beta-phorbol 12-myristate 13-acetate (PMA) ] or cellular Ca(2+) (ionophore A23187) also triggered dephosphorylation of cofilin. Both a nonspecific (100 nM staurosporine) and a highly selective antagonist of PKC (200 nM bisindolylmaleimide I) blocked dephosphorylation of cofilin in neutrophils stimulated with PMA but not with fMLP or ionophore A23187. The calmodulin (CaM) antagonists trifluoperazine (15 microM) and W-7 (50 microM) blocked dephosphorylation of cofilin in stimulated neutrophils whereas inactive/less-active analogs of these inhibitors (15 microM promethazine, 50 microM W-5) were substantially less effective. Calyculin A (40 nM), an antagonist of type 1 and 2A protein phosphatases, also triggered a massive dephosphorylation of cofilin in unstimulated neutrophils through a pathway that was insensitive to inhibitors of type 2B phosphatases. These data suggest that both PKC-dependent and independent pathways can trigger dephosphorylation of cofilin in neutrophils with the latter pathway predominating in fMLP-stimulated cells. These pathways may also contain CaM and a type 2C and/or novel phosphatase (e.g., slingshot).

Actin Depolymerizing Factors↗
PubMed2004

Frontline: The p85alpha isoform of phosphoinositide 3-kinase is essential for a subset of B cell receptor-initiated signaling responses.

Phosphoinositide 3-kinase (PI3K) is a ubiquitously expressed signaling enzyme that plays an integral role in development and activation of B cells. B cell receptor (BCR)-driven proliferation is completely blocked either in cells lacking the p85alpha regulatory isoform of PI3K or in wild-type cells treated with pharmacological PI3K inhibitors. However, the contribution of p85alpha to early signaling events has not been fully investigated. Here we show that B cells lacking p85alpha have signaling impairments that are both quantitatively and qualitatively different from those in cells treated with PI3K inhibitors. Loss of p85alpha results in partial reductions in Ca2+ mobilization and IkappaB phosphorylation, whereas ERK phosphorylation is not diminished. Moreover, although Akt phosphorylation is partially reduced, phosphorylation of several proteins downstream of Akt is preserved. These partial impairments suggest that there are other routes to PI3K activation in B cells apart from p85alpha-associated catalytic subunits. Notably, addition of phorbol ester restores BCR-mediated proliferation in p85alpha-deficient cells but not wild-type cells treated with PI3K inhibitors. These findings suggest that the primary BCR signaling defect in B cells lacking p85alpha is a failure to activate diacylglycerol-regulated signaling enzymes, most likely protein kinase C.

Animals↗
PubMed2005

Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-kappa B activation and IFN-beta synthesis downstream of Toll-like receptor 3 and 4.

Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)-mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF)-dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappaB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IkappaB kinase (IKK)-alpha/beta phosphorylation and IkappaB-alpha degradation with a concomitant increase in NF-kappaB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappaB-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappaB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappaB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappaB activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.

Adaptor Proteins, Vesicular Transport↗
PubMedDate not supplied

Inhibitors of phosphoinositide 3-kinase, LY294002 and wortmannin, affect sperm capacitation and associated phosphorylation of proteins differently: Ca2+-dependent divergences.

Sperm capacitation is regulated by multiple pathways that also control sperm motility and tyrosine (Tyr) phosphorylation of several sperm proteins. Among the reported pathways, phosphoinositide 3-kinase (PI3K) signaling and its role in modulating sperm postejaculatory changes and motility remain elusive. It was shown that wortmannin, a selective inhibitor of PI3K, prevents human sperm acrosome reaction. Using LY294002 (2-(4-morphlinyl)-8-phenyl-4H-1-benzopyran-4-one), another chemically different inhibitor of PI3K, it was suggested that this enzyme inhibits human sperm motility. In this study, we used the 2 known inhibitors of PI3K to investigate their effect on sperm capacitation and associated protein phosphorylation events. Our data show that sperm incubated with LY294002 undergo capacitation and increased Tyr phosphorylation of specific sperm proteins in a manner similar to that promoted by the capacitation inducer fetal cord serum ultrafiltrate (FCSu), as well as double phosphorylation of the threonine (Thr)-glutamine (Glu)-Tyr motif. Under similar conditions, wortmannin did not affect these sperm functions on its own, although it did prevent the effect induced by FCSu. Consistently, wortmannin decreased the phospho (P)-Tyr content of sperm proteins and prevented the phosphorylation of their Thr-Glu-Tyr motif. We also show by means of immunoblotting and cell fractionation experiments the presence of PI3K and its downstream effector Akt (protein kinase B) at the membrane level, as well as sperm heads and flagella. Our data show that human spermatozoa contain a consensus motif usually phosphorylated by Akt and that its P-serine (Ser)/Thr content is increased by both LY294002 and FCSu, while it is decreased by wortmannin. In addition, the 2 inhibitors differently affected the intracellular calcium concentration, [Ca(2+)](i). While LY294002 increased [Ca(2+)](i), wortmannin did not affect its content and did not prevent the LY294002 effect. Thus, we propose that the LY294002-promoted increase in [Ca(2+)](i) operates independently of PI3K. In conclusion, we suggest that special care be taken when using LY294002 to investigate the role that PI3K plays in a cellular phenomenon.

Androstadienes↗
PubMed2004

Involvement of phosphoinositide 3-kinase signaling pathway in chondrocytic differentiation of ATDC5 cells: application of a gene-trap mutagenesis.

Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. Here, we applied this method to murine mesenchymal ATDC5 cells, which differentiate into mature chondrocytes in the presence of insulin. As the trap vector we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene as a reporter and selection marker driven by the promoter of the trapped gene. After pPT1-geo was introduced into ATDC5 cells by electroporation, the neomycin-resistant clones were screened for beta-galactosidase activity. The selected clones were cultured in differentiation medium to evaluate the chondrogenic phenotype. The clones no. 6-30 and 6-175, which exhibited impaired and accelerated mineralization, respectively, were subjected to further analysis. In clone no. 6-30 in which the gene coding for the p85alpha subunit of phosphoinositide 3-kinase (PI3K) was trapped, the expression of marker genes of early chondrocytes including collagen type II, aggrecan, and PTH/PTHrP receptor was delayed. The insulin-induced stimulation of growth was reduced in clone no. 6-30 compared with the parental ATDC5 cells. Moreover, treatment of parental ATDC5 cells with a specific inhibitor of PI3K, LY294002, phenocopied clone no. 6-30, suggesting the involvement of PI3K signaling in the chondrogenic differentiation of ATDC5 cells. Clone no. 6-175 with accelerated mineralization was revealed to have a gene homologous to human KIAA0312 trapped, whose function remains unclear. Taken together, the gene-trap in ATDC5 cells might be useful to identify the molecules involved in chondrogenic differentiation.

Animals↗
PubMed2007

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes.

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.

Animals↗
PubMed2001

Role of phosphoinositide 3OH-kinase in autocrine transformation by PDGF-BB.

Phosphoinositide 3OH-kinases (PI3K) are a family of lipid kinases that activates signalling pathways important for migration, cytoskeletal rearrangements, and cell survival. These processes are important hallmarks in transformation. We have evaluated the functional role of PI3K for development of a transformed morphology and migratory responses of murine fibroblasts (NIH/sis and COL1A1/NIH3T3 cell lines) stimulated in an autocrine fashion by constitutive expression of platelet-derived growth factor-BB (PDGF-BB). We show that prolonged treatment with the specific PI3K inhibitor LY294002, induced a reversion of the transformed morphology, and prevented density-independent growth and focus formation. Functional PI3K was also required for development of the transformed morphology of NIH/sis and COL1A1/NIH3T3. Furthermore, treatment with LY294002 completely perturbed random migration of the cells. In addition our data show that, in the signalling pathways downstream of PI3K, activation of the small GTPase Rac was a prerequisite for the transformation signal. Our data also indicate the presence of a suramin-insensitive PI3K activity. Most likely this was due to the presence of a suramin-insensitive intracellular PDGFR pool that allowed activation of PI3K located in intracellular compartments. In conclusion these data show that intact PI3K activity was required for the morphological alterations and the enhanced migratory response that are hallmarks for PDGF induced autocrine transformation.

Animals↗
PubMed2005

Phosphoinositide 3-kinase/Akt involvement in arsenic trioxide resistance of human leukemia cells.

The purpose of this study was to evaluate the possible involvement of the phosphoinositide 3-kinase (PI3K)/Akt survival pathway in determining resistance to arsenic trioxide (As2O3)-induced apoptosis. We employed a HL60 cell clone (HL60AR) with a constitutively active PI3K/Akt survival pathway, as well as U937 and K562 cells. In addition, we used parental (PT) HL60 cells overexpressing a constitutively active Akt. Selective pharmacological inhibitors of the PI3K/Akt axis (LY294002, wortmannin) were employed to influence the sensitivity to As2O3. While HL60PT cells were sensitive to 2.5 microM As2O3 and died of apoptosis, HL60AR cells were resistant up to 5 microM As2O3. Treatment with either LY294002 or wortmannin lowered resistance of HL60AR cells to As2O3. Also in U937 and K562 cells, inhibitors of the PI3K/Akt axis caused a decrease in As2O3 resistance. Overexpression of constitutively active Akt in HL60PT cells caused the induction of resistance to 2.5 microM As2O3. Conversely, forced expression of a dominant negative Akt in HL60AR cells resulted in a decrease in As2O3 resistance. Moreover, HL60 cell resistance to 2.5 microM As2O3 could be significantly reduced by incubation with SN50, a peptide inhibitor selective for the NF-kappaB transcription factor. Taken together our findings suggest that a constitutive activation of the PI3K/Akt pathway, which is increasingly detected in some types of acute myeloid leukemia, may contribute to As2O3 resistance, most likely through NF-kappaB activation. Selective pharmacological inhibitors of this survival pathway, as well as of NF-kappaB, might be usefully employed in the future to reverse resistance to this treatment.

Androstadienes↗
PubMed1998

Fas or ceramide induce apoptosis by Ras-regulated phosphoinositide-3-kinase activation.

We demonstrate a rapid and transient activation of phosphoinositide-3-kinase (PI-3-K) by Fas receptor triggering or cellular treatment with synthetic C6-ceramide. The stimulation of PI-3-K is critical for Fas or C6-ceramide-induced programmed cell death because transfection with a transdominant inhibitory PI-3-K construct or pre-treatment with the PI-3-K inhibitor wortmannin almost completely prevented Fas or C6-ceramide-mediated apoptosis. Treatment with the caspase inhibitor Ac-YVAD-cmk or cellular transfection with transdominant inhibitory N17Ras prevented PI-3-K stimulation by Fas, suggesting that Fas activates PI-3-K via caspases and Ras. N17Ras expression also prevented C6-ceramide-initiated PI-3-K stimulation. The notion of a PI-3-K regulation by Ras upon Fas receptor ligation or ceramide treatment is supported by co-immunoprecipitation experiments revealing an activation-dependent association of PI-3-K and Ras.

Androstadienes↗
PubMed1998

Phosphoinositide-specific phospholipase C interacts with phosphatidylinositol kinase homolog TOR2.

The Saccharomyces cerevisiae PLC1 gene encodes a homolog of the delta isoform of mammalian phosphoinositide-specific phospholipase C. Cells deleted for PLC1 gene (plc1Delta) are viable but display several phenotypes, including temperature and osmotic sensitivity and defects in utilization of carbon sources other than glucose. We have used the two hybrid screen to identify Plc1p-interacting proteins. One of the identified proteins was Tor2p, a putative phosphatidylinositol (PtdIns) kinase involved in regulation of protein synthesis, cell cycle progression and organization of the actin cytoskeleton. This interaction was confirmed biochemically by coprecipitation of Plc1p and Tor2p. The results suggest that Tor2p, as a PtdIns kinase, produces phosphorylated PtdIns, which is then hydrolyzed by the associated Plc1p. The proximity of Tor2p to Plc1p may therefore result in a regulated spatial and temporal coupling of synthesis and hydrolysis of phosphorylated forms of PtdIns.

Animals↗