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PubMed2004

Phosphatidylinositol 3-kinase offsets cAMP-mediated positive inotropic effect via inhibiting Ca2+ influx in cardiomyocytes.

Phosphoinositide 3-kinase (PI3K) has been implicated in beta2-adrenergic receptor (beta2-AR)/G(i)-mediated compartmentation of the concurrent G(s)-cAMP signaling, negating beta2-AR-induced phospholamban phosphorylation and the positive inotropic and lusitropic responses in cardiomyocytes. However, it is unclear whether PI3K crosstalks with the beta1-AR signal transduction, and even more generally, with the cAMP/PKA pathway. In this study, we show that selective beta1-AR stimulation markedly increases PI3K activity in adult rat cardiomyocytes. Inhibition of PI3K by LY294002 significantly enhances beta1-AR-induced increases in L-type Ca2+ currents, intracellular Ca2+ transients, and myocyte contractility, without altering the receptor-mediated phosphorylation of phospholamban. The LY294002 potentiating effects are completely prevented by betaARK-ct, a peptide inhibitor of beta-adrenergic receptor kinase-1 (betaARK1) as well as G(betagamma) signaling, but not by disrupting G(i) function with pertussis toxin. Moreover, forskolin, an adenylyl cyclase activator, also elevates PI3K activity and inhibition of PI3K enhances forskolin-induced contractile response in a betaARK-ct sensitive manner. In contrast, PI3K inhibition affects neither the basal contractility nor high extracellular Ca2+-induced increase in myocyte contraction. These results suggest that beta1-AR stimulation activates PI3K via a PKA-dependent mechanism, and that G(betagamma) and the subsequent activation of betaARK1 are critically involved in the PKA-induced PI3K signaling which, in turn, negates cAMP-induced positive inotropic effect via inhibiting sarcolemmal Ca2+ influx and the subsequent increase in intracellular Ca2+ transients, without altering the receptor-mediated phospholamban phosphorylation, in intact cardiomyocytes.

Androstadienes↗
PubMed2003

Angiotensin II regulation of the Na+ pump involves the phosphatidylinositol-3 kinase and p42/44 mitogen-activated protein kinase signaling pathways in vascular smooth muscle cells.

This investigation used primary cultured rat vascular smooth muscle cells to examine angiotensin II (Ang II) regulation of Na(+), K(+)-ATPase (Na(+) pump) activity, and Na(+) pump alpha(1)- and beta(1)-subunit gene transcription. This regulation was mediated through both phosphatidylinositol-3 kinase (PI3K) and p42/44 mitogen-activated protein kinase (p42/44(MAPK)) signaling pathways. Both acute (10 min) and prolonged (24 h) treatment with Ang II stimulated Na(+) pump activity. Also, prolonged exposure to Ang II (24 h) increased promoter transcription of the Na(+) pump alpha(1)- and beta(1)-subunits. Furthermore, PI3K activities because well because p42/44(MAPK) phosphorylation were increased within 10 min after Ang II treatment. To determine whether these stimulatory activities of Ang II are acting through Ang II receptors 1 and/or 2 (AT(1), AT(2)), cells were pretreated with either AT(1) receptor blocker losartan or the AT(2) receptor blocker PD 123,319. Indeed, these treatments prevented the stimulatory effect of Ang II on Na(+) pump activity at both acute and 24-h time points. Furthermore, the Ang II-stimulated alpha(1)-subunit promoter transcription was inhibited by losartan but not by the AT(2) receptor blocker. These results indicate that Ang II acts through both the AT(1) and AT(2) receptor to up-regulate Na(+) pump activity; however, Ang II regulates alpha(1)-gene transcription through AT(1) but not AT(2) receptors. It was also observed that the Ang II-stimulated beta(1)-subunit gene transcription is not mediated through either AT(1) or AT(2) receptors. To examine whether the Na(+)/H(+) exchanger is involved in Ang II-stimulated Na(+) pump activity, cells were pretreated with amiloride, a specific inhibitor of the Na(+)/H(+) exchanger. This pretreatment prevented 24 h, but not acute, Ang II-stimulated Na(+) pump activity. The 24-h Ang II-stimulated alpha(1)-subunit promoter transcription was also inhibited by amiloride. This suggests that the prolonged effect of Ang II on Na(+) pump activity is dependent on increased Na(+)/H(+) exchange. Because Ang II treatment for 10 min increased PI3K activity because well because p42/44(MAPK) phosphorylation, studies were performed to determine the involvement of PI3K and p42/44(MAPK) signaling pathways in both Ang II-stimulated Na(+) pump activity and alpha(1)- and beta(1)-gene transcription. Cells were pretreated with either the PI3K inhibitor wortmannin or the p42/44(MAPK) inhibitor PD 98059. Ang II-stimulated PI3K or p42/44(MAPK) activity was inhibited by these pretreatments. Furthermore, pretreatment of cells with the PI3K inhibitors wortmannin and LY29404 or the MAPK inhibitors U0126 and PD 98059 were all observed to inhibit Ang II-stimulated Na(+) pump activity. To more specifically determine the role of PI3K in Ang II-regulation of alpha(1)-and beta(1)-gene transcription, cells were cotransfected with a dominant-negative p85 construct. Cotransfection with dominant-negative p85 reduced Ang II-stimulated alpha(1)-but not beta(1)-gene transcription in vascular smooth muscle cells. These results indicate that Ang II acts through PI3K/p42/44(MAPK) signaling pathways to up-regulate Na(+) pump activity and alpha(1)-gene transcription and that Ang II-regulated beta(1)-gene transcription is not mediated through either PI3K or p42/44 (MAPK) signaling pathways.

Amiloride↗
PubMed2001

Growth factor-stimulated phosphorylation of Akt and p70(S6K) is differentially inhibited by LY294002 and Wortmannin.

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70(S6K). The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70(S6K), more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70(S6K) by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70(S6K); at concentrations which completely inhibit phosphorylation of p70(S6K), phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70(S6K) with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70(S6K)) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM in blocking PI3K-activation of Akt and p70(S6K) must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70(S6K) signaling pathways.

Androstadienes↗
PubMed2001

Role of phosphatidylinositol 3-kinase in anti-IgM- and anti-IgD-induced apoptosis in B cell lymphomas.

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.

Animals↗
PubMed2003

Phosphatidylinositol 3-kinase regulates the CD4/CD8 T cell differentiation ratio.

The signaling pathways that control T cell differentiation have only begun to be elucidated. Using T cell lines, it has been shown that class IA phosphatidylinositol 3-kinase (PI3K), a heterodimer composed of a p85 regulatory and a p110 catalytic subunit, is activated after TCR stimulation. Nonetheless, the contribution of p85/p110 PI3K isoforms in T cell development has not been described. Mice deficient in the other family of class I PI3K, p110gamma, which is regulated by G protein-coupled receptors, exhibit reduced thymus size. Here we examine T cell development in p110gamma-deficient mice and in mice expressing an activating mutation of the p85 regulatory subunit, p65(PI3K), in T cells. We show that p110gamma-deficient mice have a partial defect in pre-TCR-dependent differentiation, which is restored after expression of the p65(PI3K) activating mutation. Genetic alteration of both PI3K isoforms also affects positive selection; p110gamma deletion decreased and p65(PI3K) expression augmented the CD4(+)/CD8(+) differentiation ratio. Finally, data are presented showing that both PI3K isoforms influenced mature thymocyte migration to the periphery. These observations underscore the contribution of PI3K in T cell development, as well as its implication in determining the CD4(+)/CD8(+) T cell differentiation ratio in vivo.

Animals↗
PubMed2003

Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway.

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O(2)(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O(2)(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O(2)(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I(A) PI3K, but not translocation of the p110gamma subunit of class I(B) PI3K, to the neutrophil membrane. Inhibition of protein kinase Czeta (PKCzeta) inhibited MBP-stimulated O(2)(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta (Thr(505)) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I(A) PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I(A) PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O(2)(-) production in response to MBP stimulation.

Adult↗
PubMed2004

Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus.

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca(2+) flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca(2+) flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca(2+) flux has a PI3K-independent component, while Ca(2+) flux following mIg/CD19 coligation is totally PI3K dependent. The p85alpha and p110delta subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca(2+) flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110alpha becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.

Animals↗
PubMed2005

Heparin-binding epidermal growth factor-like growth factor inhibits cytokine-induced NF-kappa B activation and nitric oxide production via activation of the phosphatidylinositol 3-kinase pathway.

NO produced by inducible NO synthase (iNOS) has been implicated in various pathophysiological processes including inflammation. Therefore, inhibitors of NO synthesis or iNOS gene expression have been considered as potential anti-inflammatory agents. We have previously demonstrated that heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) decreases proinflammatory cytokine IL-8 and NO production in cytokine-stimulated intestinal epithelial cells by interfering with the NF-kappaB signaling pathway. However, the upstream signaling mechanisms involved in these responses have not yet been defined. In this report, we show that in intestinal epithelial cells, HB-EGF triggered PI3K-dependent phosphorylation of Akt. Inhibition of PI3K reversed the ability of HB-EGF to block NF-kappaB activation, expression of iNOS, and NO production. Small interfering RNA of PI3K also reversed the inhibitory effect of HB-EGF on iNOS expression. Alternatively, transient expression of constitutively active PI3K decreased NO production by approximately 2-fold more than treatment with HB-EGF alone. This PI3K effect was HB-EGF dependent. Thus, activation of PI3K is essential but not sufficient for decreased NO synthesis. PI3K and HB-EGF act synergistically to decrease NO synthesis. Neither overexpression or inhibition of MEK, Ras, or Akt affected HB-EGF-mediated inhibition of NF-kappaB activation. These data demonstrate that HB-EGF decreases proinflammatory cytokine-stimulated NF-kappaB activation and NO production via activation of the PI3K signaling pathway. These results also suggest that inhibition of NF-kappaB and activation of the PI3K-dependent signaling cascade by HB-EGF may represent key signals responsible for the anti-inflammatory effects of HB-EGF.

Active Transport, Cell Nucleus↗
PubMed2001

[Role of phosphatidylinositol signalling path in developing hormonal resistance in tumor cells].

Phosphatidylinositol 3-kinase (PI3K) is a key regulatory protein which is responsible for anti-apoptotic signal transduction regulating cell survival during exposure to damaging factors. The report deals with the role of the PI3K signaling pathway in regulating cellular response to hormones and, particularly, in development of resistance as a result of long-term exposure of cells to steroid cytostatic hormones. In our study, even a short-term exposure of transformed fibroblasts of hamster (line 2PK) resulted in an activation of main PI3K effectors (MAP-kinase and protein kinase B (PKB)) which appeared against the background of hormone-induced inhibition of cellular growth. A long-term (3 months) cell culturing with dexamethasone was followed by formation of subpopulations of cells which were refractory to the growth inhibition by hormone and were characterized by high levels of activity of PI3K, MAP-kinases and PKB. Activation of PI3K and PI3K-dependent enzymes correlated with enhancement of synthesis of c-jun, a component of the AP-1 transcriptional factor, was observed both in short- and long-term application of dexamethasone. We believe that, during long-term exposure of cells to cytostatic hormones, continuous activation of PI3K and PI3K-dependent transcriptional factors may result in a significant restructuring of intracellular signal pathway, and, finally, constitutive PI3K-signal pathway and partial overcoming the proliferative block by cells.

Cell Division↗
PubMed2002

Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction.

PURPOSE: Locoregional recurrence is the dominant form of treatment failure in head and neck (H&N) cancer. The epidermal growth factor receptor (EGFR) is frequently amplified in this disease (<or=80%) and can lead to activation of phosphatidylinositol-3-kinase (PI3K), both directly and indirectly through Ras. We have shown previously that radioresistance could be conferred via the Ras-PI3K pathway. Here we investigate the contribution of EGFR to this pathway and its impact on treatment outcome. EXPERIMENTAL DESIGN: In a series of 38 H&N cancer patients, overexpression of EGFR by immunohistochemical staining was assessed. PI3K signaling was evaluated by staining for phosphorylated Akt (P-Akt), a downstream target of PI3K. Both EGFR and P-Akt were then related to outcome. Radiation survival was determined in the SQ20B cell line, a radioresistant squamous cell line derived from a recurrent laryngeal cancer, after pharmacological blockade of EGFR with Iressa, of Ras by the FTI L744,832, or of PI3K by LY294002. RESULTS: A significant association was found between P-Akt staining and local control in the patient series. Two-year local control was 100% for patients staining 0-1+ for P-Akt as compared with 70.6% for patients staining 2-3+ (P = 0.04). In our series of 38 H&N cancers, 30 (78.9%) of the specimens were strongly (3+) positive for EGFR, whereas 25 (65.8%) were moderately to strongly (2-3+) positive for P-Akt. Pharmacologically inhibiting EGFR, Ras, and PI3K led to radiosensitization of SQ20B cells. CONCLUSIONS: Evaluation of PI3K activation by Akt phosphorylation might be a prognostic marker for response to therapy, and PI3K could be a useful target for therapy. These results also suggest that signaling from EGFR to PI3K can lead to radioresistance.

Adult↗
PubMed2005

Overexpression of the N-terminal end of the p55gamma regulatory subunit of phosphatidylinositol 3-kinase blocks cell cycle progression in gastric carcinoma cells.

pRb and its family members p130 and p107 regulate cell cycle progression and direct G1/S transition in mammalian cells through interaction with the transcription factor E2F. Phosphatidylinositol 3-kinase (PI3K) is an essential component of growth factor-regulated pathways and plays a crucial role in the regulation of cellular proliferation and differentiation. It has been demonstrated that PI3K can regulate cell cycle progression via Akt-mediated pathway. However, the possible interactions between PI3K and Rb pathways remain to be defined. It was reported that the unique 24-amino-acid N-terminal end of the p55 regulatory subunits of PI3K is an Rb-binding domain and affects Rb action or Rb-E2F interaction. The 24 N-terminal amino acids of p55gamma encoded by a cDNA construct could compete with the endogenous p55gamma for binding to Rb, which influences Rb-mediated signaling and blocks cell cycle progression. In the current study, we investigated the effects of this 24-peptide on cell proliferation in human gastric carcinoma MKN-28 cells by means of cell cycle analysis, BrdU incorporation, and determining the levels of cell cycle regulatory molecule expression. Our results showed that p55gammaPI3K and the Rb family members p130 and p107 exist in MKN-28 cells, while the p110PI3K was not detected. Moreover, p55gammaPI3K was found binding to p130/p107 in these cells. We demonstrated that the introduction of the plasmid N24p55-GFP (harboring the cDNA for the 24 N-terminal amino acids of p55gamma) into MKN-28 cells caused cell cycle arrest at G1. Furthermore, we showed that the over-expression of the 24-peptide in MKN-28 cells decreased the population of cells incorporating BrdU and reduced the levels of cyclin D1 and cyclin A. These observations suggest that PI3K can regulate cell cycle progression and cell proliferation in human gastric tumor cells via Rb-mediated pathway, and that this effect of PI3K is mediated through a direct association with Rb via the N-terminal end of its p55 kDa regulatory subunits and modulating Rb-E2F interactions. Taken together with previous studies, our data provide a new therapeutic target in human stomach cancer. Strategies targeting PI3K signal transduction or the association of PI3K with Rb, or regulating PI3K-Rb interactions could be employed for gene therapy or chemotherapy of gastric cancer and other tumors.

Carcinoma↗
PubMed2006

[Expression and significance of PTEN/PI3K signal transduction-related proteins in non-small cell lung cancer].

BACKGROUND & OBJECTIVE: PTEN/PI3K signal transduction pathway regulates cell proliferation and survival, and is closely associated with the development and progress of various tumors. However, the relationship between the function of this pathway with lung carcinoma has not been completely elucidated. This study was to investigate the expression and clinical significance of PTEN/PI3K signal transduction-related proteins, such as insulin-like growth factor-1 receptor (IGF-1R), PTEN and phosphatidylinositol 3-kinase (PI3K), in non-small cell lung carcinoma (NSCLC). METHODS: The expression of IGF-1R, PTEN, and PI3K in 59 specimens of primary NSCLC, 19 specimens of metastatic lymph nodes, 16 specimens of para-cancerous hyperplastic lung tissues, and 7 specimens of normal lung tissues were detected by SP immunohistochemistry. The difference of positive rates of IGF-1R,PTEN and PI3K was evaluated by Chi-square test and Fisher's exact test. RESULTS: The positive rates of IGF-1R, PTEN, and PI3K in NSCLC were 72.88%, 27.12%, and 84.75%, respectively. The positive rates of IGF-1R and PI3K were not associated with the clinicopathologic features of NSCLC (P>0.05). The positive rate of PTEN was highly associated with lymph node metastasis of NSCLC (P=0.009), but was not associated with pathologic type and differentiation grade of NSCLC (P>0.05). The positive rate of PTEN was significantly lower and that of PI3K was significantly higher in NSCLC and metastatic lymph node tissues than in para-cancerous hyperplastic and normal lung tissues (P<0.05). The expression of IGF-1R in NSCLC was positively correlated to that of PI3K (r=0.432, P=0.001); while the expression of PTEN was negatively correlated to that of PI3K (r=0.505, P<0.001). CONCLUSION: Overexpression of IGF-1R and PI3K, and low expression of PTEN are closely correlated to the development, invasion and metastasis of NSCLC.

Adult↗
PubMed1998

Phosphatidylinositol 3-kinase is required for CD28 but not CD3 regulation of the TEC family tyrosine kinase EMT/ITK/TSK: functional and physical interaction of EMT with phosphatidylinositol 3-kinase.

Ligation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase, EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears to play downstream of CD28 and the TCR.

Animals↗
PubMed1999

Contribution of phosphatidylinositol 3-kinase to radiation resistance in human melanoma cells.

The activity of phosphatidylinositol 3-kinase (PI3K), a key component of multiple signal transduction pathways, was investigated in early- and late-stage melanoma cells that have varying degrees of radiation resistance. Analysis of PI3K biproducts (PI-3,4-P2 and PI-3,4,5-triphosphate) revealed a direct correlation between radiation resistance and levels of PI3K activity. Treating melanoma cells with wortmanin or LY294002, two different PI3K inhibitors, decreased PI3K activity and caused a dose-dependent decrease in resistance to ultraviolet radiation. Lower resistance to radiation elicited by LY294002 coincided with increased apoptosis. To further establish the role of PI3K in radiation resistance, we transfected early-stage melanoma cells with the cDNA of p85, the regulatory subunit of PI3K. Clones that constitutively overexpressed p85 exhibited a higher degree of PI-3,4-P2 synthesis and a corresponding increase in their resistance to ultraviolet radiation. The results of this study point to the role of PI3K and its biproducts in radiation resistance of human melanoma cells.

Androstadienes↗
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↗
PubMed2002

A Role for phosphoinositide 3-kinase in the control of cell division and survival during retinal development.

Neurogenesis in the retina requires the concerted action of three different cellular processes: proliferation, differentiation, and apoptosis. Class IA phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit. p110alpha has been shown to regulate cell division and survival. Little is known of its function in development, however, as p110alpha knockout mice exhibit CNS defects, but death at early embryonic stages impairs further study. Here, we examine the role of PI3K in mouse retina development by expressing an activating form of PI3K regulatory subunit, p65(PI3K), as a transgene in the retina. Mice expressing p65(PI3K) showed severely disrupted retina morphogenesis, with ectopic cell masses in the neuroepithelium that evolved into infoldings of adult retinal cell layers. These changes correlated with an altered cell proliferation/cell death balance at early developmental stages. Nonetheless, the most affected cell layer in adult retina was that of photoreceptors, which correlated with selectively increased survival of these cells at developmental stages at which cell division has ceased. These results demonstrate the relevance of accurate PI3K regulation for normal retinal development, supporting class IA PI3K involvement in induction of cell division at early stages of neurogenesis. These data also show that, even after cell division decline, PI3K activation mediates survival of differentiated neurons in vivo.

Animals↗
PubMed2001

Overexpression of phosphatidylinositol 3-kinase in human lung cancer.

BACKGROUND: This study was conducted to investigate the expression of phosphatidylinositol 3-kinase (PI3K) and AKT2, a downstream effector, in primary human lung carcinomas of different histological type. METHODS: Specimens from 105 human lung carcinomas and their corresponding lymph nodes and liver metastases were examined using immunohistochemistry and Northern-blot assays to study the PI3K p85 and p110 subunits and the AKT2-expression patterns. RESULTS: The p85 and p110 subunits of PI3K were overexpressed at the protein level in 77% and 59% of 80 primary lung carcinomas, respectively, irrespective of the histological type. PI3K overexpression was correlated with tumor grading. In contrast, no overexpression of PI3K subunits was observed in normal lung tissue and benign lung tumors. Consistent with these findings, upregulation of p110 mRNA transcripts was restricted to primary lung carcinomas. Overexpression of AKT2 was observed in 10% of the investigated lung tumor specimens, but in none of the healthy lung sections. A profound increase of PI3K expression was uniformly observed in lung tissue specimens and in corresponding extra-pulmonary lymph-node and liver metastases with low-differentiation grades. CONCLUSION: PI3K appears to be associated with the process of tumor-cell transformation and proliferation. One of its major downstream effector molecules, AKT2, which contributes to apoptotic cell death, was not upregulated by PI3K overexpression in primary lung carcinomas.

Adult↗
PubMed2004

Phosphoinositide 3-kinase regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling cPKC/PKCdelta but not Akt.

Superoxide production by NADPH oxidase is essential for the bactericidal properties of phagocytes. Phosphorylation of p47(phox), one of the cytosolic components of NADPH oxidase, is a crucial step of the oxidase activation. Some evidences suggest that phosphoinositide 3-kinase (PI3K) is involved in p47(phox) phosphorylation, but it has not been fully understood how PI3K regulates it. The aim of this study was to examine the mechanism underlying the PI3K regulation of p47(phox) phosphorylation. Pharmacological inhibition of PI3K attenuated both fMLP-stimulated p47(phox) phosphorylation and NADPH oxidase activity in HL-60 cells differentiated to a neutrophil-like phenotype. Although fMLP elicited Akt activation in a PI3K-dependent manner, an Akt inhibitor had no effect on the oxidase activity triggered by fMLP. In vitro kinase assay revealed that Akt was unable to catalyze p47(phox) phosphorylation. Interestingly, the activation of cPKC and PKCdelta after fMLP stimulation was dependent on PI3K. Furthermore, PI3K inhibitors reduced the activation of phospholipase Cgamma2 without affecting tyrosine phosphorylation on it. These results suggest that PI3K regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling diacylglycerol-dependent PKCs but not Akt.

Androstadienes↗