Specific PI 3-kinase Inhibitor Research Articles

Regulation of cytosolic phospholipase A2 activity in macrophages stimulated with receptor-recognized forms of alpha 2-macroglobulin: role in mitogenesis and cell proliferation.

Macrophages exposed to receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) demonstrate increased DNA synthesis and cell division. In the current study, we have probed the role of cytosolic phospholipase A(2) (cPLA(2)) activity in the cellular response to alpha(2)M*. Ligation of the alpha(2)M* signaling receptor by alpha(2)M*, or its receptor binding fragment, increased cPLA(2) activity 2-3-fold in a concentration and time-dependent manner. This activation required a pertussis toxin-insensitive G protein. Cellular binding of alpha(2)M* also induced transient translocation of cPLA(2) activity to nuclei and membrane fractions. Inhibition of protein kinase C activity or chelation of Ca(2+) inhibited alpha(2)M*-induced increased cPLA(2) activity. Binding of alpha(2)M* to macrophages, moreover, increased phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK, and JNK. Incubation of macrophages with inhibitors of MEK 1/2 or p38 MAPK before stimulation with alpha(2)M* profoundly decreased phosphorylation of MAPKs, blocking cPLA(2) activation. alpha(2)M*-induced increase in [(3)H]thymidine uptake and cell proliferation was completely abolished if activation of cPLA(2) was prevented. The response of macrophages to alpha(2)M* requires transcription factors nuclear factor kappaB, and cAMP-responsive element-binding protein as well as expression of the proto-oncogenes c-fos and c-myc. These studies indicate that the activation of cPLA(2) plays a crucial role in alpha(2)M*-induced mitogenesis and cell proliferation.

Phosphatidylinositol 3-OH kinase-Akt/protein kinase B pathway mediates Gas6 induction of scavenger receptor a in immortalized human vascular smooth muscle cell line.

The growth arrest-specific gene 6 encodes a secreted protein, Gas6, which was originally identified as the ligand of a receptor, Axl, with tyrosine kinase activity. The class A scavenger receptor (SRA) mediates lipid uptake into cells, leading to the formation of foam cells, an important step in atherogenesis. Although Gas6 induces SRA expression, the underlying mechanism is not clear. In this report, we show that the Gas6-induced expression of SRA was mediated by the phosphatidylinositol 3-OH kinase (PI3-kinase)-serine/threonine kinase (Akt/protein kinase B [PKB]) pathway involving Akt phosphorylation. This pathway was activated by exposure to Gas6. Furthermore, the effect of Gas6 was abrogated by wortmannin, a specific inhibitor of PI3-kinase. We also demonstrated that the constitutively active form of Akt enhanced activity of the SRA promoter but that the dominant-negative mutant of Akt completely abolished the expression of SRA after treatment with Gas6. These results show that the PI3-kinase-Akt/PKB pathway participates in Gas6-induced SRA expression and suggests that the activation of Akt/PKB plays an important role in Gas6-induced atherosclerosis and foam cell formation in human vascular smooth muscle cells.

Arginine-specific ADP-ribosyltransferase on the surface of gizzard smooth muscle cells and the involvement of phosphatidylinositol 3-kinase in maintaining the activity of this transferase.

An arginine-specific ADP-ribosyltransferase activity was detected in chicken gizzard smooth muscle, and the specific activity is highest in the membrane fraction. This transferase is released from the membrane fraction by phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that it is a glycosylphosphatidylinositol (GPI)-anchored protein. When primary cultured gizzard smooth muscle cells (SMCs) were incubated with [adenylate-(32)P]NAD, several proteins were labeled. The labeling was inhibited by preincubation of the cells with PI-PLC, or by the addition of L-arginine to the reaction, and was sensitive to hydroxylamine treatment. The activity of the transferase was maintained in differentiated SMCs cultured with insulin, but was dramatically decreased concomitantly with cell dedifferentiation induced by serum or a specific PI3-kinase inhibitor, LY294002. These results indicate that the GPI-anchored arginine-specific ADP-ribosyltransferase is expressed on the surface of differentiated SMCs and can modify several cell surface proteins. Our results also suggest that PI3-kinase is involved in the regulation of transferase activity during differentiation.

Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-kappa B activation in primary B cells.

The NF-kappaB/Rel transcription factors play an important role in the expression of genes involved in B cell development, differentiation and function. Nuclear NF-kappaB is induced in B cells by engagement of either the BCR or CD40 or by stimulation with lipopolysaccharide (LPS). Despite the importance of NF-kappaB to B cell function, little is known about the signaling pathways leading to NF-kappaB activation. In this report we address the role of phosphoinositide 3'-kinase (PI 3-kinase) in BCR- and LPS-induced NF-kappaB activation using populations of primary murine resting B cells. Using the specific pharmacological inhibitors of PI 3-kinase, Wortmannin and LY294002, we demonstrate that PI 3-kinase activity is vital for BCR-induced NF-kappaB DNA-binding activity. Furthermore, we show that this is achieved via protein kinase C-dependent degradation of IkappaBalpha. Similar analyses reveal that PI 3-kinase is also critical in triggering NF-kappaB DNA-binding activity and IkappaBalpha degradation following LPS stimulation. Interestingly, a PKC inhibitor which blocked the BCR-induced IkappaBalpha degradation had no effect on the degradation of IkappaBalpha after LPS stimulation. Taken together, our results indicate the involvement of PI 3-kinase in at least two distinct signaling pathways leading to activation of NF-kappaB in B cells.

Roles of phosphatidylinositol 3-kinase and phospholipase D in temporal activation of superoxide production in FMLP-stimulated human neutrophils.

To determine the temporal roles of phosphatidylinositol 3-kinase (PI3-kinase) and phospholipase D (PLD) during human neutrophil activation stimulated by a chemotactic peptide, we examined the kinetics of these enzymes and related them to a neutrophil function (superoxide production). Both wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), potent and specific inhibitors of PI3-kinase, inhibit PI3-kinase activity in human neutrophils and significantly inhibit superoxide production from the early phase. Ethanol has no effect on PI3-kinase and markedly inhibits superoxide production at the late phase. Although these agents are inhibitory to different degrees, when neutrophils are simultaneously treated with ethanol and PI3-kinase inhibitors, superoxide is not produced. These results suggest that PI3-kinase and PLD play a pivotal role in the signal transduction pathway of the chemo-attractant-receptor involved neutrophil activation. These enzymes produce second messengers which are required for subsequent superoxide production in human neutrophils. NADPH oxidase is activated in a PI3-kinase-dependent manner at the early phase, and PLD activity follows it and is related to superoxide production at the late phase in human neutrophils by stimulation with FMLP.

The flightless I protein colocalizes with actin- and microtubule-based structures in motile Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small GTPases.

The flightless I protein contains an actin-binding domain with homology to the gelsolin family and is likely to be involved in actin cytoskeletal rearrangements. It has been suggested that this protein is involved in linking the cytoskeletal network with signal transduction pathways. We have developed antibodies directed toward the leucine rich repeat and gelsolin-like domains of the human and mouse homologues of flightless I that specifically recognize expressed and endogenous forms of the protein. We have also constructed a flightless I-enhanced green fluorescent fusion vector and used this to examine the localization of the expressed protein in Swiss 3T3 fibroblasts. The flightless I protein localizes predominantly to the nucleus and translocates to the cytoplasm following serum stimulation. In cells stimulated to migrate, the flightless I protein colocalizes with beta-tubulin- and actin-based structures. Members of the small GTPase family, also implicated in cytoskeletal control, were found to colocalize with flightless I in migrating Swiss 3T3 fibroblasts. LY294002, a specific inhibitor of PI 3-kinase, inhibits the translocation of flightless I to actin-based structures. Our results suggest that PI 3-kinase and the small GTPases, Ras, RhoA and Cdc42 may be part of a common functional pathway involved in Fliih-mediated cytoskeletal regulation. Functionally, we suggest that flightless I may act to prepare actin filaments or provide factors required for cytoskeletal rearrangements necessary for cell migration and/or adhesion.

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways

Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.

Nucleophosmin–anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway

More than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-γ as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK–expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK–transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK–transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL.

Nucleophosmin–anaplastic lymphoma kinase associated with anaplastic large-cell lymphoma activates the phosphatidylinositol 3-kinase/Akt antiapoptotic signaling pathway

AbstractMore than half of anaplastic large-cell lymphomas (ALCLs) have a chromosomal translocation t(2;5) that leads to the expression of a hybrid protein composed of the nucleolar phosphoprotein nucleophosmin (NPM) and the anaplastic lymphoma kinase (ALK) that exhibits an unregulated tyrosine kinase activity. We have previously identified PLC-γ as a crucial downstream signaling molecule of NPM-ALK that contributes to its mitogenic potential. Here, we show that NPM-ALK recruits the C-terminal SH2 domain of the phosphatidylinositol 3-kinase (PI 3kinase) p85 subunit. PI 3-kinase assays revealed that the kinase is activated by NPM-ALK in vivo, in turn activating PKB/Akt in NPM-ALK–expressing cells. The use of 2 specific PI 3-kinase inhibitors, wortmannin and LY294002, demonstrated the requirement of PI 3-kinase for the growth of NPM-ALK–transformed cell lines, as well as a cell line established from a patient with ALCL. Primary murine bone marrow retrovirally transduced with NPM-ALK showed a transformed phenotype that was reversible on treatment with PI 3-kinase inhibitors. Flow cytometric analysis revealed that wortmannin-treated NPM-ALK–transformed cell lines underwent apoptosis. Furthermore, apoptosis induced by overexpression of the proapoptotic molecule Bad could be partially blocked by the overexpression of NPM-ALK. Thus, NPM-ALK activates the antiapoptotic PI 3-kinase/Akt pathway, which likely contributes to the molecular pathogenesis of ALCL.

Rapid Activation of Protein Kinase B/Akt Has a Key Role in Antiapoptotic Signaling during Liver Regeneration

Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones, and cytokines. Here we report that protein kinase B (PKB)/Akt, part of a key cell survival signaling pathway, is markedly activated after partial hepatectomy (PHX). The antiapoptotic protein Bad, a downstream target of PKB/Akt, is also phosphorylated. This cascade can be activated by various factors in primary hepatocytes, with the strongest activation by insulin and the α1-adrenergic agonist phenylephrine (PE), followed by IL-6, epidermal growth factor (EGF), and hepatocyte growth factor (HGF). Pretreatment of cells with the specific PI3 kinase inhibitor LY294002 abolished insulin- or PE-activation of PKB/Akt, suggesting that activation of PKB/Akt is mediated by a PI3 kinase-dependent mechanism. In vivo administration of PE, insulin, IL-6, HGF, or EGF to mice markedly stimulated PKB/Akt in the liver, with the strongest stimulation induced by insulin and PE. Moreover, HGF and insulin were able to attenuate transforming growth factor β-induced apoptosis in hepatic cells, and these effects were antagonized by LY294002. Taken together, these findings suggest that rapid activation of PKB/Akt is a key antiapoptotic signaling pathway involved in liver regeneration.

Glucose deprivation induces Akt-dependent synthesis and incorporation of GLUT1, but not of GLUT4, into the plasma membrane of 3T3-L1adipocytes

Reduction of the glucose concentration in the culture medium of 3T3-L1 adipose cells below 1.25 mM produces a 4-8-fold stimulation of 2-deoxyglucose uptake which starts after a lag phase of 2 h and is maximal after 10-16 h. In the present study, we employed the 'membrane sheet assay' in order to re-assess the contribution of the transporter isoforms GLUT1 and GLUT4 to this effect. Immunochemical assay of glucose transporters in membranes prepared with the 'sheet assay' revealed that the effect reflected a marked increase of GLUT1 in the plasma membrane with no effect on GLUT4. Glucose deprivation increased the total cellular GLUT1 protein in parallel with the transport activity, whereas GLUT4 was unaltered. The specific PI 3-kinase inhibitor wortmannin inhibited the effect of glucose deprivation on transport activity and also on GLUT1 synthesis. Glucose deprivation produced a moderate, biphasic increase in the activity of the protein kinase Akt/PKB that was inhibitable by wortmannin. When wortmannin was added after stimulation of cells in order to assess the internalization rate of transporters, the effect of insulin was reversed considerably faster (T1/2 = 18 min) than that of glucose deprivation (T1/2 > 60 min). These data are consistent with the conclusion that the effect of glucose deprivation reflects a specific, Akt-dependent de-novo synthesis of GLUT1, and not of GLUT4, and its insertion into a plasma membrane compartment which is distinct from that of the insulin-sensitive GLUT1.

Activation of Akt Is Induced by Heat Shock and Involved in Suppression of Heat-Shock-Induced Apoptosis of NIH3T3 Cells

Heat shock exposure to NIH3T3 cells for 15 min at 45°C activated Akt, which is mediated by PI3-kinase, as evidenced by the significant inhibition of heat-shock-induced phosphorylation by specific inhibitors of PI3-kinase. The phosphorylated Akt was gradually decreased to the basal level within 9 h after heat shock. This resulted in growth arrest, but cell growth could be recovered within 24 h accompanied with a high rate of proliferation. However, heat shock for 60 min failed to activate Akt, resulting in apoptosis. The recovery of cell growth after heat-shock-inducing activation of Akt was completely blocked by wortmannin. Moreover, overexpression of a dominant-negative Akt mutant significantly inhibited the apoptosis-suppressive effect of heat shock, indicating the direct involvement of heat-shock-induced Akt activation in the apoptosis suppression. The results indicate that a signal transduction pathway, namely, PI3-kinase/Akt, may contribute to an apoptosis-suppressive function after heat shock in NIH3T3 cells.

Mitogenic and Anti-Apoptotic Actions of Hepatocyte Growth Factor Through Erk, Stat3 and Akt in Human Aortic Endothelial Cells

P83 Background: Hepatocyte growth factor (HGF), an angiogenic growth factor, may play a pivotal role in the regulation of endothelial cells (EC), since HGF demonstrated the mitogenic and anti-apoptotic actions in endothelial cells. However, it is still unclear how HGF demonstrated those actions. Therefore, in this study, we have focused on the signal transduction systems. Methods: Human aortic EC death was induced by serum-deprived condition. Cell growth was assessed by WST assay, and thymidine incorporation and cell death was measured by LDH release and caspase 3 activity. Phosphorylation of ERK, STAT and Akt was measured by western blotting as assessed by the specific phospho-specific antibody. Results: Treatment of EC with recombinant HGF (rHGF) resulted in a significant increase in cell growth (WST: control; 0.267±0.02, rHGF 100ng/ml; 0.385±0.003, p≤0.01) and also attenuated the cell death (LDH release: control; 0.94±0.04, rHGF 100ng/ml; 0.43±0.024, p≤0.01). ERK and STAT3 (Ser727) was phosphorylated by rHGF (100 ng/ml) as assessed by the specific phospho-specific antibody, while PD98059, the inhibitor of MEK, attenuated endothelial cell growth (HGF; 0.385±0.003, HGF+PD 30uM; 0.203±0.003, p≤0.01) and the phosphorylation of STAT3 (Ser727). On the other hand, STAT3 (Tyr705), which was usually stimulated by JAK, was not phosphorylated by rHGF. In addition, Akt on the down stream of PI3 kinase was also phosphorylated by rHGF, while the specific inhibitor of PI3kinase, wortomanin and LY294002, markedly attenuated endothelial cell death assessed by LDH release and caspase3 activity. Conclusions: Overall, this study demonstrated that HGF stimulated cell growth through ERK cascade including STAT3(Ser727) activation, not JAK-STAT3(Tyr705) pathway, and inhibited endothelial cell death through PI3 kinase-Akt pathway. HGF may stimulate angiogenesis through both mitogenic and anti-cell death function in endothelial cells.

Mitogenic and Anti-Apoptotic Actions of Hepatocyte Growth Factor Through Erk, Stat3 and Akt in Human Aortic Endothelial Cells

P83 Background: Hepatocyte growth factor (HGF), an angiogenic growth factor, may play a pivotal role in the regulation of endothelial cells (EC), since HGF demonstrated the mitogenic and anti-apoptotic actions in endothelial cells. However, it is still unclear how HGF demonstrated those actions. Therefore, in this study, we have focused on the signal transduction systems. Methods: Human aortic EC death was induced by serum-deprived condition. Cell growth was assessed by WST assay, and thymidine incorporation and cell death was measured by LDH release and caspase 3 activity. Phosphorylation of ERK, STAT and Akt was measured by western blotting as assessed by the specific phospho-specific antibody. Results: Treatment of EC with recombinant HGF (rHGF) resulted in a significant increase in cell growth (WST: control; 0.267±0.02, rHGF 100ng/ml; 0.385±0.003, p≤0.01) and also attenuated the cell death (LDH release: control; 0.94±0.04, rHGF 100ng/ml; 0.43±0.024, p≤0.01). ERK and STAT3 (Ser727) was phosphorylated by rHGF (100 ng/ml) as assessed by the specific phospho-specific antibody, while PD98059, the inhibitor of MEK, attenuated endothelial cell growth (HGF; 0.385±0.003, HGF+PD 30uM; 0.203±0.003, p≤0.01) and the phosphorylation of STAT3 (Ser727). On the other hand, STAT3 (Tyr705), which was usually stimulated by JAK, was not phosphorylated by rHGF. In addition, Akt on the down stream of PI3 kinase was also phosphorylated by rHGF, while the specific inhibitor of PI3kinase, wortomanin and LY294002, markedly attenuated endothelial cell death assessed by LDH release and caspase3 activity. Conclusions: Overall, this study demonstrated that HGF stimulated cell growth through ERK cascade including STAT3(Ser727) activation, not JAK-STAT3(Tyr705) pathway, and inhibited endothelial cell death through PI3 kinase-Akt pathway. HGF may stimulate angiogenesis through both mitogenic and anti-cell death function in endothelial cells.

Activation of NF-κB Binding in HT-29 Colon Cancer Cells by Inhibition of Phosphatidylinositol 3-Kinase

The ubiquitous transcription factor NF-κB, which is activated in cells by diverse stimuli including phosphatidylinositol 3-kinase (PI3-kinase), is a critical factor for cell survival and growth. Inhibition of PI3-kinase enhances enterocyte-like differentiation of the human colon cancer cell line HT-29. The purpose of our study was to determine whether PI3-kinase alters NF-κB in HT-29 cells. Wortmannin, a specific PI3-kinase inhibitor, stimulated NF-κB binding activity in HT-29 cells by 4 h after treatment. Activation of NF-κB occurred without degradation of IκBα, a protein that sequesters NF-κB in the cytosol. In addition to increasing NF-κB binding, either wortmannin or cotransfection with a dominant negative mutant of the p85 regulatory subunit of PI3-kinase (Δp85) induced NF-κB transactivation. Taken together, these results suggest that inhibition of PI3-kinase in HT-29 cells results in induction of NF-κB binding activity and transactivation which is independent of IκBα degradation.

Effects of wortmannin on the kinetics of gvbd and the activities of the maturation-promoting factor and mitogen-activated protein kinase during bovine oocyte maturation in vitro

The present study was conducted with the objective of examining the effect of wortmanin, a specific PI 3-kinase inhibitor, on the kinetic of GVBD, and on the activities of the maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase during bovine oocyte maturation. The time sequence for GVBD was not different between oocytes cultured with or without wortmannin. Most of the cultured oocytes were at the filamentous bivalents stage after 4 h of culture. Six hours after the start of culture, most of the oocytes possessed germinal vesicles with condensed bivalent, and by 10 h of culture nearly all of the cultured oocytes underwent GVBD. A gradual increase in MPF activity until 12 h of culture was observed in the presence and absence of wortmannin. A sharp decrease in MPF activity in oocytes cultured without wortmannin treatment was recorded at 14 h of culture. Thereafter, MPF regained activity, reaching a maximum level at 20 to 24 h of culture. For oocytes cultured with wortmannin, no decline in the activity of MPF was observed during the interval from 12 to 24 h of culture. For these oocytes the MPF activity remained nearly stable during this transition until the end of incubation. The presence of wortmannin in the maturation medium did not alter MAP kinase activity. Taken together, these observations indicate that inhibition of PI 3-kinase does not modulate the time sequence of GVBD or the pattern of MAP kinase activity in bovine oocytes. However, PI 3-kinase might be one of the molecules that regulate the sharp reduction in the activity of MPF during the MItransition.

PIK3CA as an oncogene in cervical cancer.

Amplification of chromosome arm 3q is the most consistent aberration in cervical cancer, and is implicated in the progression of dysplastic uterine cervical cells into invasive cancer. The present study employed the 'positional candidate gene' strategy to determine the contribution of PIK3CA, which is located in 3q26.3, in cervical tumorigenesis. PIK3CA is known to be involved in the PI 3-kinase/AKT signaling pathway, which plays an important role in regulating cell growth and apoptosis. The results of comparative genomic hybridization show that the 3q26.3 amplification was the most consistent chromosomal aberration in primary tissues of cervical carcinoma, and a positive correlation between an increased copy number of PIK3CA (detected by competitive PCR) and 3q26.3 amplification was found in tumor tissues and in cervical cancer cell lines. In cervical cancer cell lines harboring amplified PIK3CA, the expression of gene product (p110alpha) of PIK3CA was increased, and was subsequently associated with high kinase activity. In addition, transformation phenotypes in these lines, including increased cell growth and decreased apoptosis, were found to be significantly affected by the treatment of specific PI 3-kinase inhibitor, suggesting that increased expression of PIK3CA in cervical cancer may result in promoting cell proliferation and reducing apoptosis. These evidences support that PIK3CA is an oncogene in cervical cancer and PIK3CA amplification may be linked to cervical tumorigenesis. Oncogene (2000).

Hepatocyte growth factor protects cultured rat cerebellar granule neurons from apoptosis via the phosphatidylinositol-3 kinase/Akt pathway.

Recent studies suggest that hepatocyte growth factor (HGF) functions as a neurotrophic factor in the central nervous system. In this study, we investigated the neuroprotective effect of HGF and its mechanism of action. We used cultured cerebellar granule neurons that underwent apoptosis when the culture medium was changed from that containing serum with 25 mM K(+) to serum-free medium containing 5 mM K(+), and HGF prevented apoptotic cell death. HGF stimulated both mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI3)-kinase activity in cerebellar granule neurons. Two specific inhibitors of PI3-kinase, wortmannin and LY294002, efficiently blocked this neuroprotective effect of HGF. In contrast, PD98059, a selective inhibitor of MAP kinase kinase (MEK), did not affect the anti-apoptotic effect of HGF. The downstream signal of PI3-kinase in this protection was further investigated. HGF-induced phosphorylation of Akt and pretreatment of the cells with wortmannin completely impaired Akt activation. These results suggest that HGF prevents apoptosis in cerebellar granule neurons via the PI3-kinase/Akt pathway.

Meiotic Maturation of Bovine Oocytes Cultured In Vitro with EGF and Wortmannin.

This study was designed to examine the effect of EGF on meiotic maturation of bovine oocytes and to evaluate the ability of wortmannin, a specific PI 3-kinase inhibitor, to modulate EGF-induced oocyte maturation. EGF significantly improved meiotic maturation of both cumulus-intact and denuded oocytes. Wortmannin effectively and irreversibly blocked oocytes from progressing to the MII stage. When oocytes were transferred to wortmannin-treated medium after being cultured for 2 h in wortmannin-free medium, 36% of them progressed to the MII stage. By delaying the time of transfer to 4 h, 64% of the oocytes developed to MII, and by further delaying to 6 h, the rate of MII formation was almost similar to that of oocytes cultured without wortmannin supplementation. Culturing of the oocytes with EGF for 2 h did not improve their meiotic maturation. Increasing the incubation period with EGF for 4 or 6 h resulted in a maturation rate similar to that of those cultured with EGF for the total period of incubation. Data suggest that EGF is capable of stimulating bovine oocyte meiotic maturation regardless of the presence of cumulus cells, and that PI 3-kinase is involved in this process. Results also show that inhibition of PI 3-kinase from 6 h after the onset of culture does not block meiotic progression, and that EGF induces its positive maturation signal during the first 4 h of culture period.

Insulin-like Growth Factor (IGF)-I Regulates IGF-binding Protein-5 Gene Expression through the Phosphatidylinositol 3-Kinase, Protein Kinase B/Akt, and p70 S6 Kinase Signaling Pathway

Expression of the insulin-like growth factor-binding protein 5 (IGFBP-5) gene in vascular smooth muscle cells is up-regulated by IGF-I through an IGF-I receptor-mediated mechanism. In this study, we studied the possible involvement of the mitogen-activated protein kinase (MAPK) and PI 3-kinase signaling pathways in mediating IGF-I-regulated IGFBP-5 gene expression. The addition of Des(1-3)IGF-I, an IGF analog with reduced affinity to IGFBPs, resulted in a transient activation of p44 and p42 MAPK. Inhibition of the MAPK activation by PD98059, however, did not affect IGF-I-stimulated IGFBP-5 expression. Des(1-3)IGF-I treatment also strongly activated PI 3-kinase. This activation was probably mediated through IRS-1, because IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI 3-kinase activity. This activation occurred within 5 min and was sustained at high levels for over 6 h. Likewise, Des(1-3)IGF-I caused a long lasting activation of PKB/Akt and p70(s6k). When LY294002 and wortmannin, two specific inhibitors of PI 3-kinase, were added with Des(1-3)IGF-I, the IGF-I-regulated IGFBP-5 expression was negated. The addition of rapamycin, which inhibits IGF-I-induced p70(s6k) activation, significantly inhibited IGF-I-regulated IGFBP-5 gene expression. These results suggest that the action of IGF-I on IGFBP-5 gene expression requires the activation of the PI 3-kinase-PKB/Akt-p70(s6k) pathway but not the MAPK pathway in vascular smooth muscle cells.