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PubMed2000

Stem cell factor and erythropoietin inhibit apoptosis of human erythroid progenitor cells through different signalling pathways.

Erythropoietin (EPO) and stem cell factor (SCF) are two important factors in human erythropoiesis. We have recently demonstrated that SCF and EPO synergistically activate mitogen-activated protein (MAP) kinase, thereby promoting growth of human erythroid colony-forming cells (ECFCs). In the present study, we have examined the intracellular mechanisms by which SCF and EPO maintain survival of these cells. In the absence of SCF and EPO, human ECFCs underwent rapid apoptosis. The process was significantly inhibited by addition of a single factor and was totally prevented in the presence of both factors. Treatment of ECFCs with wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI3K), inhibited the antiapoptotic effect of SCF but had no effect on that of EPO, indicating that SCF but not EPO inhibits apoptosis through the PI3K pathway. In contrast, treatment of ECFCs with PD98059, a specific inhibitor of MAP kinase/ERK kinase (MEK), inhibited cell growth but had no effect on the antiapoptotic activity of either SCF or EPO, suggesting that SCF and EPO prevent apoptosis of human ECFCs independent of the extracellular signal-regulated kinase (ERK) pathway. Interestingly, both EPO and SCF induced activation of PI3K. However, through PI3K, SCF caused activation of protein kinase B (PKB), an anti-apoptosis signal, whereas EPO led to activation of ERKs. Furthermore, the SCF- and EPO-maintained expression of antiapoptotic protein Bcl-XL was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl-XL may not have a major role in preventing apoptosis of human ECFCs. Phosphorylated BAD was not affected by SCF, EPO or wortmannin. Taken together with our previous results, the present study indicates that SCF and EPO support survival and growth of human ECFCs through different signalling pathways and that they transduce distinctly different signals through activation of PI3K.

Androstadienes↗

PubMed2005

The neuroprotection of insulin on ischemic brain injury in rat hippocampus through negative regulation of JNK signaling pathway by PI3K/Akt activation.

Current studies demonstrated that cell survival is determined by a balance among signaling cascades, including those that recruit the Akt and JNK pathways. In our present work, the relationship between Akt1 and JNK1/2 was evaluated after cerebral ischemia-reperfusion in the hippocampus in a four-vessel occlusion model of Sprague-Dawley rats. This paper was based on our present and previous studies. Firstly, Akt1 had one active peak during reperfusion following 15 min ischemia. Secondly, two peaks of JNK1/2 activation occurred during reperfusion, respectively. Thirdly, the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 were detected. They only had one active peak, respectively, during reperfusion. To clarify the mechanism of Akt1 activation and further define whether JNK1/2 activation could be regulated by Akt1 through PI3K pathway, LY294002 and insulin were, respectively, administrated to the rats prior to ischemia. Our research indicated that LY294002, a PI3K inhibitor, significantly suppressed Akt1 activation. Furthermore, LY294002 significantly strengthened both peaks of JNK1/2 activation, c-Jun activation, Bcl-2 phosphorylation, and the activation of caspase-3 during reperfusion. In contrast, insulin, a PI3K agonist, not only obviously activated Akt1 during early and later reperfusion, but also inhibited phosphorylation of JNK1/2, c-Jun, and Bcl-2 and attenuated the activation of caspase-3. In addition, pretreatment of insulin significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Consequently, our results indicated that the cross-talk between Akt1 and JNK1/2 could be mediated by insulin receptor through PI3K in rat hippocampus during reperfusion. This signaling pathway might play a neuroprotective role against ischemic insults via inhibition of the JNK pathway, involving the death effector of caspase-3.

Analysis of Variance↗

PubMed2004

Role of Akt/protein kinase B in the activity of transcriptional coactivator p300.

Akt/protein kinase B is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway and plays a critical role in promotion of cell survival. The function of transcriptional coactivator p300 is required by many transcription factors to either activate or repress gene expression. Here, we show that induction of PI3K enhances the metabolic stability of endogenous p300 protein. On the other hand, repression of PI3K by LY294002 induces p300 degradation through the 26S proteasome pathway and impedes the transcriptional activity of the coactivator. In addition, Akt interacts with the coactivator and the activity of Akt is required to maintain the steady-state level of p300. Our study provides a new insight into the molecular mechanisms by which the critical concentration of p300 protein is regulated and suggests a role for Akt in control of various cellular activities through the transcriptional coactivator p300.

Acetyltransferases↗

PubMed2003

Insulin activation of phosphatidylinositol 3-kinase in the hypothalamic arcuate nucleus: a key mediator of insulin-induced anorexia.

In peripheral tissues, insulin signaling involves activation of the insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI3K) enzyme system. In the hypothalamus, insulin functions with leptin as an afferent adiposity signal important for the regulation of body fat stores and hepatic glucose metabolism. To test the hypothesis that hypothalamic insulin action involves intracellular PI3K signaling, we used histochemical and biochemical methods to determine the effect of insulin on hypothalamic IRS-PI3K activity. Here, we report that insulin induces tyrosine phosphorylation of the insulin receptor and IRS-1 and -2, increases binding of activated IRS-1 and -2 to the regulatory subunit of PI3K, and activates protein kinase B/Akt, a downstream target of PI3K. Using an immunohistochemical technique to detect PI 3,4,5-triphosphate, the main product of PI3K activity, we further demonstrate that in the arcuate nucleus, insulin-induced PI3K activity occurs preferentially within cells that contain IRS-2. Finally, we show that the food intake- lowering effects of insulin are reversed by intracerebroventricular infusion of either of two PI3K inhibitors at doses that have no independent feeding effects. These findings support the hypothesis that the IRS-PI3K pathway is a mediator of insulin action in the arcuate nucleus and, combined with recent evidence that leptin activates PI3K signaling in the hypothalamus, provide a plausible mechanism for neuronal cross-talk between insulin and leptin signaling.

Animals↗

PubMed2006

Vitronectin and collagen I differentially regulate osteogenesis in mesenchymal stem cells.

The roles of various soluble factors in promoting the osteogenic differentiation of adult mesenchymal stem cells (MSCs) have been widely studied, but little is known about how the extracellular matrix (ECM) instructs the phenotypic transition between growth and differentiation. To investigate this question, we cultured MSCs on purified vitronectin or type-I collagen, motivated by our earlier tissue engineering work demonstrating that MSC adhesion to polymer scaffolds is primarily mediated by the passive adsorption of these two ECM ligands from serum. Using alkaline phosphatase activity and matrix mineralization as indicators of the early and late stages of osteogenesis, respectively, we report here that both substrates supported differentiation, but the mechanism was substrate dependent. Specifically, osteogenesis on vitronectin correlated with enhanced focal adhesion formation, the activation of focal adhesion kinase (FAK) and paxillin, and the diminished activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol-3 kinase (PI3K) pathways. By contrast, MSCs on type-I collagen exhibited reduced focal adhesion formation, reduced activation of FAK and paxillin, and increased activation of ERK and PI3K. Inhibition of ERK and FAK blocked mineral deposition on both substrates, suggesting that the observed differences in signaling pathways ultimately converge to the same cell fate. Understanding these mechanistic differences is essential to predictably control the osteogenic differentiation of MSCs and widen their use in regenerative medicine.

Adsorption↗

PubMed2002

Effects of the Pit1 mutation on the insulin signaling pathway: implications on the longevity of the long-lived Snell dwarf mouse.

Mutations in Caenorhabditis elegans and mice have identified candidate genes that increase their lifespan via hormonal signal transduction, i.e. the insulin/IGF-1-like pathway. In this study we propose that longevity of the Snell dwarf (Pit1(dw)/Pit1(dw)) mouse is associated with a decrease of the insulin/IGF-1 signaling pathway caused by the Pit1 mutation. We recently demonstrated that the growth hormone deficiency of the dwarf mouse alters circulating insulin levels, thereby resulting in a decreased activity of the insulin/IGF-1 signaling pathway, which is a determining factor in the increased nematode lifespan. The decreased activity of the insulin/IGF-1 signaling pathway is indicated by decrease of (a) IRS-two pool levels; (b) docking of p85 alpha to IRS-2; (c) docking of p 85 alpha to p110 alpha or p110 beta, and (d) IRS-2-associated PI3K activity. In this study we present data suggesting that the InR beta-IRS-1-PI3K pathway is attenuated in the Snell dwarf mouse liver. Our data show that the PI3K activity associated with IRS-1, the docking of IRS-1 to InR beta and the docking of p85 alpha to IRS-1 are attenuated in the aged Snell dwarf. Our studies suggest that the Pit1 mutation results in a decreased activity of the insulin/IGF-1 pathway; that this plays a key role in the longevity of the Snell dwarf mouse and conforms to the nematode longevity paradigm.

Aging↗

PubMed2001

Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF-1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2.

In the present study we investigated the mechanisms responsible for and the biological consequences of the constitutive activation of the insulin-like growth factor-1 receptor (IGF-1R) in the MIA PaCa-2 cells. An aberrant increase in the expression and activation of the IGF-1R was observed during the transition of growth states from exponential to quiescent. The increase in IGF-1R expression is preceded by an increase in IGF-1R mRNA transcript and is associated with an increase in the IGF-1R promoter activity. Inhibition of de novo transcription by actinomycin D increased the stability of IGF-1R mRNA in exponentially growing cells, thereby increasing the expression of IGF-1R to a level similar to that seen in quiescent cells. Increased IGF-1R signaling mediated the growth factor independence of quiescent MIA PaCa-2 cells through the constitutive activation of mitogen-activated protein kinase (MAPK). Exogenous IGF-1 increased cell proliferation and activated MAPK and AKT signaling pathways. The resistance of cells to apoptosis by IGF-1R signaling was mediated through MAPK and phosphatidylinositol 3-kinase (PI3K) pathways and a yet unidentified pathway(s). Thus, aberrant regulation of IGF-1R signaling is required for resistance to apoptosis and growth factor independence of MIA PaCa-2 cells. This likely protects cells from unfavorable conditions and allows cells to rapidly re-enter the cell cycle when conditions are favorable.

Apoptosis↗

PubMed2004

Platelet-derived growth factor stimulates membrane lipid synthesis through activation of phosphatidylinositol 3-kinase and sterol regulatory element-binding proteins.

We analyzed the transcriptional program elicited by stimulation of normal human fibroblasts with platelet-derived growth factor (PDGF) using cDNA microarrays. 103 significantly regulated transcripts that had not been previously linked to PDGF signaling were identified. Among them, a cluster of genes involved in fatty acid and cholesterol biosynthesis, including stearoyl-CoA desaturase (SCD), fatty acid synthase, and hydroxymethylglutaryl-CoA synthase (HMGCS), was up-regulated by PDGF after 24 h of treatment, and their expression correlated with increased membrane lipid production. These genes are known to be controlled by sterol regulatory element-binding proteins (SREBP). PDGF increased the amount of mature SREBP-1 and regulated the promoters of SCD and HMGCS in an SREBP-dependent manner. In line with these results, blocking SREBP processing by addition of 25-hydroxycholesterol blunted the effects of PDGF on lipogenic enzymes. SREBP activation was dependent on the phosphatidylinositol 3-kinase (PI3K) pathway, as judged from the effects of the inhibitor LY294002 and mutation of the PDGFbeta receptor tyrosines that bind the PI3K adaptor subunit p85. Fibroblast growth factors (FGF-2 and FGF-4) and other growth factors mimicked the effects of PDGF on NIH3T3 and human fibroblasts. In conclusion, our results suggest that growth factors induce membrane lipid synthesis via the activation SREBP and PI3K.

CCAAT-Enhancer-Binding Proteins↗

PubMed2002

Signaling pathways involved in IL-8-dependent activation of adhesion through Mac-1.

In human neutrophils, IL-8 induces chemotaxis, the respiratory burst, and granule release, and enhances cellular adhesion, a beta(2) integrin-dependent event. IL-8 stimulates neutrophil adhesion to purified fibrinogen in a Mac-1-dependent manner. Mitogen-activated protein kinase (MAPK) activation was detected in human neutrophil lysates after treatment with IL-8 and PMA, but not the activating mAb CBR LFA 1/2. IL-8-stimulated neutrophil adhesion to fibrinogen was blocked 50% by the MAPK/extracellular signal-related kinase-activating enzyme inhibitor PD098059. Adhesion was blocked approximately 75% by inhibition of the phosphatidylinositol-3 kinase (PI3K) pathway with LY294002, supporting that activation of both MAPK and PI3K may play a role in IL-8-dependent inside-out signals that activate Mac-1. Activation of MAPK was inhibited in IL-8-stimulated cells in the presence of PI3K inhibitors LY294002 or wortmannin, supporting a model in which PI3K is upstream of MAPK. IL-8-stimulated neutrophil adhesion was inhibited 50% by bisindolylmaleimide-I, implicating protein kinase C (PKC) in the intracellular signaling from the IL-8R to Mac-1. A 74-kDa molecular mass species was detected by an activation-specific Ab to PKC when cells were stimulated with PMA or IL-8, but not a beta(2)-activating Ab. Inhibition of either MAPK or PKC resulted in partial inhibition of IL-8-stimulated polymorphonuclear neutrophil adhesion, and treatment with both inhibitors simultaneously completely abolished IL-8-stimulated adhesion to ligand. Inhibition of PI3K blocked MAPK activation, but not PKC activation, suggesting a branch point that precedes PI3K activation. These data suggest that both MAPK and PKC are activated in response to IL-8 stimulation, and that these may represent independent pathways for beta(2) integrin activation in neutrophils.

Cell Adhesion↗

PubMed2001

Ultraviolet irradiation- and dimethyl sulfoxide-induced telomerase activity in ovarian epithelial cell lines.

Information about telomerase regulation is incomplete, especially since various studies suggest complexity in telomerase regulation. Given the important association between telomerase and cancer, it is imperative to design and develop a model system in which telomerase activity can be regulated and studied. We employed ultraviolet (UV) radiation or dimethyl sulfoxide (DMSO) to transiently induce telomerase activity in a telomerase-positive cell line and, most importantly, in a telomerase-negative cell line. UV- or DMSO-induced telomerase activity was associated with increased hTRT, but not hTR, mRNA transcription in the telomerase-negative cells. However, no changes in hTRT or hTR mRNA transcription were noted with UV- or DMSO-induced telomerase activity in the telomerase-positive cells. Inhibition of protein synthesis or the phosphotidyl inositol 3-kinase (PI3K) pathway suppressed telomerase induction and/or activity in all cell lines examined, suggesting telomerase activity was dependent on protein synthesis and PI3K-mediated phosphorylation. Furthermore, enhanced telomerase activity was limited to UV and DMSO, since a variety of chemotherapeutic agents failed to induce telomerase activity. Therefore, our data provide a useful culture model system to study telomerase regulation in telomerase-negative and -positive cell lines and from which to obtain information about telomerase as a target for cancer intervention.

Dimethyl Sulfoxide↗

PubMed2001

Airway wall remodelling and hyperresponsiveness: modelling remodelling in vitro and in vivo.

Airway wall remodelling contributes to the airway hyperresponsiveness that characterizes asthma. An increase in airway smooth muscle (ASM) volume is quantitatively important in the overall remodelling response and may be considered as a target for new therapeutic approaches to chronic asthma. ASM volume increases result from both hypertrophic as well as hyperplastic growth, the latter having been more extensively investigated. There are relatively few in vivo models available for analysis of the underlying mechanism(s) or their regulation by drugs. Human ASM in culture has been used to investigate potential stimuli for ASM proliferation and the signal transduction pathways that subserve these responses. The mitogen-activated protein kinase (MAPK) family members, ERK 1/2 and the phosphoinositol-3-kinase (PI3K) pathways each contribute to the signalling of G1 progression/S-phase entry in ASM. Glucocorticoids and beta(2)-adrenoceptor agonists attenuate, but do not prevent proliferative responses of ASM. Thus, there is scope for improved pharmacological control of this chronic and progressive aspect of asthma pathogenesis.

Adrenergic beta-Agonists↗

PubMed2006

Chronic inflammation and pathogenesis of GI and pancreatic cancers.

The pathogenesis of cancer represents a complex and multifactorial process requiring a number of acquired and genetic defects. It is becoming increasingly apparent that many cancers originate from a chronic inflammatory process. The topic of this review is the inflammatory response and development of gastrointestinal (GI) and pancreatic cancers. Here, we describe the development of various gastric colorectal and pancreatic cancers through an inflammatory process. The tumor microenvironment which predisposes to tissue destruction, subsequent attempts at healing and accumulation of cellular damage with loss of cell cycle control mechanisms is discussed. Components of the tumor microenvironment that are important in the final common pathway leading to cancer include the tumor stroma, tumor-associated macrophages, cytokines and chemokines and reactive oxygen and nitrogen species. Common signaling pathways that link inflammation with cancer are described and include the COX-2, NF-kappaB and phosphatidyl inositol 3-kinase (PI3K) pathways. Finally, therapies that can be directed to the inflammatory process as either treatment or prevention of these cancers will be discussed including novel inhibitors of signaling pathways which are currently in development.

Anti-Inflammatory Agents↗

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↗

PubMed2005

Activation of PI3K-Akt-GSK3beta pathway mediates hepatocyte growth factor inhibition of RANTES expression in renal tubular epithelial cells.

Hepatocyte growth factor (HGF) was recently reported to ameliorate renal inflammation in a rat model of chronic renal failure. HGF exerted its action through suppression of RANTES expression in renal tubules. In the present study, we utilized an in vitro model of human kidney proximal tubule epithelial cells (HKC) to elucidate the mechanisms of RANTES suppression by HGF. HGF significantly suppressed basal and TNF-alpha-induced mRNA and protein expression of RANTES in a time and dose dependent fashion. HGF elicited PI3K-Akt activation and inhibited GSK3, a downstream transducer of PI3K-Akt, by inhibitory phosphorylation at Ser-9. When the PI3K-Akt pathway was blocked by wortmannin, HGF inhibition of RANTES was abrogated, demonstrating that the PI3K-Akt pathway is necessary for HGF action. In addition, specific inhibition of GSK3 activity by lithium ion suppressed basal and TNF-alpha-induced RANTES expression, reminiscent of the action of HGF. To further investigate the role of GSK3 in modulating RANTES expression, we examined the effect of forced expression of wild type GSK3beta or an uninhibitable mutant GSK3beta, in which the regulatory Ser-9 residue is changed to alanine (S9A-GSK3beta) in HKC. Overexpression of wild type GSK3beta did not alter the inhibitory action of HGF on RANTES. In contrast, expression of S9A-GSK3beta abolished HGF inhibition of basal and TNF-alpha stimulated RANTES expression. These findings suggest that PI3K-Akt activation and subsequent inhibitory phosphorylation of GSK3beta are required for HGF-induced suppression of RANTES in HKC.

Cell Line↗

PubMed2005

The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFkappaB pathway in osteosarcoma cells.

1 Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFkappaB pathway, the major cell survival pathway. 2 The PI3K-Akt-NFkappaB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-alpha or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor kappaB (IkappaB) kinase (IKK) and finally phosphorylation of IkappaB and its subsequent degradation. 3 Pretreatment with alendronate at 100 microM for 24 h prior to the stimulation with tumor necrosis factor-alpha or insulin partially inhibited the IkappaB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IkappaB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P(3). 4 The inhibitory effect of alendronate on IkappaB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5 The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFkappaB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.

Alendronate↗

PubMed2002

The role of phosphatidylinositol 3-kinase, rho family GTPases, and STAT3 in Ros-induced cell transformation.

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Ros-induced cell transformation. Inhibition of the mitogen-activated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, Deltap85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros- and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.

Animals↗

PubMed2004

Regulation of anoikis by Cdc42 and Rac1.

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.

Actins↗

PubMed2005

LY294002 and LY303511 sensitize tumor cells to drug-induced apoptosis via intracellular hydrogen peroxide production independent of the phosphoinositide 3-kinase-Akt pathway.

The phosphoinositide 3-kinase (PI3K)-Akt pathway is constitutively active in many tumors, and inhibitors of this prosurvival network, such as LY294002, have been shown to sensitize tumor cells to death stimuli. Here, we report a novel, PI3K-independent mechanism of LY-mediated sensitization of LNCaP prostate carcinoma cells to drug-induced apoptosis. Preincubation of tumor cells to LY294002 or its inactive analogue LY303511 resulted in a significant increase in intracellular hydrogen peroxide (H2O2) production and enhanced sensitivity to non-apoptotic concentrations of the chemotherapeutic agent vincristine. The critical role of intracellular H2O2 in LY-induced death sensitization is corroborated by transient transfection of cells with a vector containing human catalase gene. Indeed, overexpression of catalase significantly blocked the amplifying effect of LY pretreatment on caspase-2 and caspase-3 activation and cell death triggered by vincristine. Furthermore, the inability of wortmannin, another inhibitor of PI3K, to induce an increase in H2O2 production at doses that effectively blocked Akt phosphorylation provides strong evidence to unlink inhibition of PI3K from intracellular H2O2 production. These data strongly support death-sensitizing effect of LY compounds independent of the PI3K pathway and underscore the critical role of H2O2 in creating a permissive intracellular milieu for efficient drug-induced execution of tumor cells.

Apoptosis↗