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PubMed2002

RhoA-dependent murine prostate cancer cell proliferation and apoptosis: role of protein kinase Czeta.

We previously showed that RhoA played an important role in the proliferation of murine We prostate cancer (TRAMP) cells (P. M. Ghosh et al., Oncogene, 18: 4120-4130, 1999). Untransfected TRAMP cells as well as those expressing constitutively active RhoA (Q63L) mutant protein (Q63L cells) were highly proliferative. In contrast, TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein (T19N cells) were slow growing. In this study, we showed, in addition, that T19N cells displayed reduced rates of apoptotic cell death in response to serum deprivation, compared with TRAMP and Q63L cells, and we studied the mechanisms of the effects of RhoA on TRAMP cell proliferation and apoptosis. Both proliferation and apoptosis of TRAMP and Q63L cells were dependent on the activation of phosphatidylinositol 3-kinase (PI3K). The ubiquitous mitogen-activated Ser/Thr kinase, p70S6 kinase, a downstream effector of PI3K, was overexpressed in TRAMP and Q63L cells. Another PI3K effector, the cell survival protein Akt, displayed increased activity in T19N cells, which did not express active RhoA, compared with TRAMP and Q63L cells. The atypical protein kinase C (PKC) isoform PKCzeta, which is downstream of PI3K, was activated in cells expressing active RhoA. In addition, expression of constitutively activated PKCzeta in TRAMP cells enhanced proliferation and p70S6 kinase phosphorylation, whereas the inhibition of PKCzeta activation resulted in activation of Akt and enhanced cell survival. Thus, the effects of RhoA on TRAMP cell proliferation and apoptosis may be mediated by PKCzeta.

Animals↗
PubMed2002

Protein tyrosine kinase and protein phosphatase signaling pathways regulate volume-sensitive chloride currents in a nonpigmented ciliary epithelial cell line.

PURPOSE: To investigate whether signaling pathways that incorporate protein tyrosine kinases and phosphatases regulate PKC-sensitive, volume-sensitive Cl(-) currents (I(Cl,vol)) in cultured rabbit nonpigmented ciliary epithelial cells. METHODS: Activation of I(Cl,vol) in response to hyposmotic stimulation was recorded with whole-cell patch-clamp techniques in the presence of pharmacologic agents that activate or block kinases and phosphatases. RESULTS: I(Cl,vol) in rabbit nonpigmented ciliary epithelial cells was identified as a PKC-sensitive, volume-sensitive Cl(-) current, because current was downregulated during cell swelling by phorbol-12-dibutyrate, a PKC activator, and the PKC inhibitors, calphostin and chelerythrine, enhanced the current. Activation of c-Src tyrosine kinases, with an Src activator peptide (EPQ(pY)EEIPI), increased I(Cl,vol) after hyposmotic stimulation, whereas the protein tyrosine kinase inhibitor, genistein, but not its inactive analogue daidzein, inhibited the current. The phosphatidylinositol-3-kinase (PI3K) inhibitor, wortmannin, inhibited I(Cl,vol). Wortmannin did not further inhibit I(Cl,vol) in cells pretreated with the protein tyrosine kinase inhibitor, genistein, but blocked enhancement of I(Cl,vol) by PKC inhibitors. The serine-threonine protein phosphatase (PP) inhibitor, okadaic acid, blocked activation of I(Cl,vol), whereas insulin, which activates PI3K and PP-1, enhanced the current. The insulin-enhanced current was also blocked by okadaic acid. I(Cl,vol) was not activated under isosmotic conditions by the simultaneous inhibition of PKC with calphostin and activation of PP-1 by insulin. CONCLUSIONS: These data show that PKC-sensitive Cl(-) currents activated in response to cell swelling in nonpigmented ciliary epithelial cells are modulated by protein tyrosine kinase, PI3K, and PP signaling pathways. Activation of PP and PKC may involve the upstream intermediaries Src tyrosine kinase and PI3K.

Animals↗
PubMed2002

Inactivation of the antiapoptotic phosphatidylinositol 3-kinase-Akt pathway by the combined treatment of taxol and mitogen-activated protein kinase kinase inhibition.

Paclitaxel (Taxol) activates a number of signal transduction pathways that lead to apoptosis. In contrast, paclitaxel also activates cell survival pathways, such as the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway. Previously, we have shown that inhibition of MEK combined with paclitaxel treatment causes an impressive enhancement of apoptosis in various tumor cell lines. Here, we find that the combination of paclitaxel with a MEK inhibitor leads to a dramatic inactivation of the antiapoptotic Akt (protein kinase B) kinase. The decrease in Akt is not reflected at the protein or mRNA level but rather attributed to kinase inactivation. To confirm that inactivation of Akt is significant, a constitutively active Akt mutant was introduced and shown to reverse tumor cell apoptosis. Further analysis upstream of Akt shows that treatment with the combination of paclitaxel and MEK inhibitor down-regulates PI3K activity more than either agent alone. The direct pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) similarly enhances paclitaxel-induced tumor apoptosis in a dose-dependent manner. Our results suggest the combination of paclitaxel and MEK inhibitor leads to down-regulation of the PI3K-Akt signaling in addition to the proapoptotic effects of paclitaxel and MEK inhibitor alone. Overall, these findings render the combined use of paclitaxel with MEK inhibitors, or paclitaxel with PI3K inhibitors, as a promising new strategy for cancer chemotherapy.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase.

Transcription of the gene coding for the tumor-associated antigen MN/carbonic anhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1). Previous studies identified CAIX expression in areas adjacent to hypoxic regions in solid tumors and suggested supplementary/alternative modes of regulation. To better understand the mechanisms activating CAIX expression, we characterized the cell density-dependent induction of CAIX in HeLa cells. This process is anchorage and serum independent and is not mediated by a soluble factor, decreased pH, or lowered glucose concentration. Stabilization of HIF-1 alpha was not observed in dense cultures. In contrast to sparse cell culture conditions, phosphatidylinositol 3'-kinase (PI3K) activity was significantly increased in dense HeLa cultures. The PI3K inhibitors LY294002 and wortmannin inhibited CAIX expression in dense cultures in a dose-dependent manner, specifically targeting the CA9 promoter (-173/+31 region) that was transactivated by constitutively active p110 PI3K subunit. The mechanism controlling CAIX expression in dense cultures is, however, dependent on lowered O(2) tension because stirring abrogates induction of CAIX expression. Hypoxia- and cell density-induced CAIX expressions were mediated by two seemingly independent mechanisms, as documented by the additive effect of increased cell density and treatment with the hypoxia-mimic CoCl(2) on levels of CAIX expression. The minimal cell density-dependent region within the CA9 promoter consists of the juxtaposed protected region 1 and hypoxia-response elements. However cell density-dependent CAIX expression was abrogated in the HIF-1 alpha-deficient Kal3.5 cells, suggesting an important role of HIF-1 in the corresponding mechanism. Thus, induction of CAIX in high-density cultures requires separate but interdependent pathways of PI3K activation and a minimal level of HIF-1 alpha. These interdependent pathways function at a lowered O(2) concentration that is, however, above that necessary for HIF-1 alpha stabilization.

Antigens, Neoplasm↗
PubMed2002

Down-regulation of PIK3CG, a catalytic subunit of phosphatidylinositol 3-OH kinase, by CpG hypermethylation in human colorectal carcinoma.

PURPOSE: Premature death associated with colorectal adenocarcinoma occurs in PIK3CG(-/-) mice and overexpression of PIK3CG in colon cancer cells suppresses cell proliferation. We examined expression levels of PIK3CG, a catalytic subunit of phosphatidylinositide 3-OH kinase (PI3K), in colon cancer cells to investigate the hypothesis that PIK3CG might contribute to the growth and progression of colorectal cancers. EXPERIMENTAL DESIGN: The effects of LY294002, a PI3K inhibitor, on cell growth were examined to elucidate the role of the PI3K-Akt/protein kinase B (PKB) pathway in colon cancer. We used reverse transcription-PCR, Western blotting, and immunohistochemical analyses to examine PIK3CG mRNA and protein expression levels in colon cancer cells and primary colorectal cancers. To clarify the mechanism responsible for the silencing of this gene in colon cancers, we performed methylation-sensitive PCR analysis of DNA digested with HpaII and MspI and analyzed PI3KCG expression in DLD-1 and LoVo cells treated with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza). RESULTS: LY294002 suppressed growth and decreased expression of Akt (Ser(473)) expression in cancer cells. Three (60%) of 5 colon cancer cell lines did not express PIK3CG, but hypermethylation at CpG sites of the promoter regions of this gene was detected. However, 5-Aza-treated DLD-1 and LoVo cells did express PIK3CG. Reduction of PIK3CG expression was detected immunohistochemically in 85% of human colorectal cancers and was closely associated with invasion, metastasis, and poor differentiation. Down-regulation of PIK3CG expression and hypermethylation of promoter regions were also detected in primary colon cancers. CONCLUSIONS: Our findings suggest that the silencing of the PIK3CG gene plays an important role in inhibiting the PI3K-Akt/PKB signaling system responsible for tumorigenesis and the progression of colorectal cancers.

Adult↗
PubMed2002

Effects of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one on synaptic vesicle cycling at the frog neuromuscular junction.

Inositol phospholipids are thought to play an important regulatory role in synaptic membrane traffic. We investigated the effects of perturbing 3-phosphoinositide metabolism on neurotransmission at the frog neuromuscular junction. We used the reversible phosphoinositide-3 kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one [LY294002 (LY)] and we examined its effects by intracellular recording, fluorescence imaging with styryl dyes (FM 1-43 and FM 2-10), calcium imaging, and electron microscopy. LY treatment reversibly inhibited vesicle cycling; electron micrographs indicated a dramatic reduction in the number of vesicles, balanced by the appearance of numerous cisternas. LY wash-off reverted the phenotype; terminals were refilled with vesicles, and they resumed normal FM 1-43 uptake and release. Surprisingly, LY treatment also enhanced the frequency of spontaneous release up to 100-fold in a calcium-independent manner. LY evoked similar effects in normal frog Ringer's solution, Ca-free Ringer's solution, and BAPTA AM-pretreated preparations; imaging of nerve terminals loaded with the calcium-sensitive fluorescent dye fluo-3 showed no significant change in fluorescence intensity during LY treatment. FM 1-43 imaging data suggested that LY evoked the cycling of 70-90% of all vesicles. The LY-induced effect on spontaneous release was reproduced by the casein kinase 2 inhibitor 5,6-dichlorobenzimidazole riboside but not, however, by the PI3K inhibitor wortmannin. Because LY has been shown recently to potently inhibit casein kinase 2 as well as PI3K, we hypothesize that casein kinase 2 inhibition is responsible for the enhancement of spontaneous release, whereas PI3K inhibition induces the block of vesicle cycling.

Action Potentials↗
PubMed2003

PTEN mutation and epidermal growth factor receptor activation regulate vascular endothelial growth factor (VEGF) mRNA expression in human glioblastoma cells by transactivating the proximal VEGF promoter.

Our previous work showed that, compared with parental U87MG human glioblastoma cells, vascular endothelial growth factor (VEGF) mRNA levels are decreased in U87/T691, a derivative line in which epidermal growth factor receptor (EGFR) signaling is inhibited by introduction of a truncated p185(Neu) protein (A. Maity et al., Cancer Res., 60: 5879-5886, 2000). The effect of EGFR activation on VEGF was mediated at the level of transcription via a phosphatidylinositol 3'-kinase (PI3K)-dependent pathway. In the current study we investigated the effect of PTEN, a negative regulator of PI3K signaling commonly mutated in glioblastoma cells, on VEGF expression. Several glioblastoma cell lines containing mutant PTEN, including U87MG, U87/T691, and U251MG, were infected with adenovirus expressing wild-type PTEN. This led to a decrease in the levels of both VEGF mRNA and phosphorylated Akt, a marker for PI3K activation. Treatment of U87MG cells with LY294002, a PI3K inhibitor, or cotransfection with a vector expressing wild-type PTEN decreased VEGF promoter activity using reporters containing either 1.5 kb of the promoter or a fragment extending from -88 to +54 bp. Activity of the -88/+54 VEGF promoter was down-regulated by dominant negative Akt and up-regulated by constitutively active myristoylated Akt. Introduction of wild-type PTEN and pharmacological inhibition of EGFR decreased VEGF mRNA expression and VEGF promoter activity in U87MG cells to a greater extent that did either manipulation by itself. Therefore, in human glioblastoma cells, PTEN mutation can cooperate with EGFR activation to increase VEGF mRNA levels by transcriptionally up-regulating the proximal VEGF promoter via the PI3K/Akt pathway.

Endothelial Growth Factors↗
PubMed2003

The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells.

Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.

Antineoplastic Agents↗
PubMed2003

Cytokine activation of phosphoinositide 3-kinase sensitizes hematopoietic cells to cisplatin-induced death.

Cytokine growth factors regulate the normal proliferation of hematopoietic cells but can also override irradiation-induced growth arrest checkpoints through activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. In the present study, we assessed the effect that erythropoietin and interleukin-3 have on cisplatin-treated hematopoietic cells. When cultured in the presence of cytokine, cisplatin-treated 32D cells transiently accumulated in a G(2)-M phase arrest and ultimately died by a nonapoptotic mechanism. By comparison, reduction of cytokine-induced PI3K activity, either through cytokine receptor mutation or direct inhibition with LY294002, caused cisplatin-treated cells to enter a biphasic G(1) and G(2)-M arrest. The arrest of these cells coincided with an absence of cyclin-dependent kinase (Cdk)1 and Cdk2 activity and significantly reduced cell death during cisplatin treatment. Indeed, LY294002 treatment during cisplatin exposure allowed the recovery of a viable, proliferating cell population after removal of cisplatin. In contrast, Cdks remained active in the G(2)-M-arrested population of cisplatin-treated cells with continuous cytokine activation of PI3K, and even transient exposure to cisplatin resulted in death of the entire population. These data suggest that cytokine activation of PI3K signaling pathways overrides cisplatin-induced growth arrest checkpoints, thereby sensitizing hematopoietic cells to DNA damage-induced death.

Animals↗
PubMed2003

The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent process.

The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human leukemia cells to pharmacological cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic leukemia cells to minimally toxic concentrations of flavopiridol (FP), roscovitine, or CGP74514A for 3 h in conjunction with the PI3K inhibitor LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g., cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis (IAP)-binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release], caspase activation, and apoptosis. Similar interactions were observed in a variety of other leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited protein kinase (PK) A (H89), PKC (GFX), mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1/2; U0126), p38 MAP kinase (MAPK; SB202190), m-target of rapamycin (TOR; rapamycin), or ataxia-telangiectasia mutation (ATM; caffeine), whereas the PI3K inhibitor wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of glycogen synthase kinase (GSK)-3, forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites, retinoblastoma protein cleavage, and down-regulation of cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in hematological malignancies.

2-Aminopurine↗
PubMed2003

Phosphatidylinositol 3'-kinase is required for growth of mast cells expressing the kit catalytic domain mutant.

The Kit receptor tyrosine kinase is critical for the growth and development of hematopoietic cells, germ cells, and the interstitial cells of Cajal. Gain-of-function mutations in codon 816 of the catalytic domain of human Kit [codon 814 of murine Kit (mKit)] are found in patients with mastocytosis, leukemia, and germ cell tumors. There are no drugs that inhibit the activity of Kit catalytic domain mutants to a greater extent than wild-type Kit. The objective of this study was to understand the biochemical mechanisms mediating mast cell transformation by this Kit mutant to identify molecular targets for pharmacological intervention. To this end, we examined signaling pathways activated in the murine mast cell line IC2 infected with either wild-type (IC2-mKit) or mutant mKit (IC2-mKit(D814Y)). In this study, we show that mKit(D814Y) is constitutively phosphorylated on tyrosine 719, and this likely results in constitutive association with activated phosphatidylinositol 3'-kinase (PI3K). In vitro growth of IC2-mKit(D814Y) cells is more sensitive to inhibition of PI3K than SCF-induced growth of IC2-mKit cells. s.c. injection of IC2-mKit(D814Y) in syngeneic mice results in mast cell tumors. To determine whether inhibition of PI3K could reduce mKit(D814Y)-mediated tumorigenicity, mice were treated with 1.5 mg/kg wortmannin three times a week. Five weeks after injection of tumor cells, a 75% reduction in tumor weight was observed when wortmannin treatments were initiated 2 days after inoculation with tumor cells. A 66% reduction occurred when treatment was initiated 2 weeks after inoculation. Treatment with wortmannin increased necrosis in the tumors, and this was associated with apoptosis. Interestingly, there was no effect on tumor vasculature. Thus, PI3K is required for survival and growth of the IC2-mKit(D814Y) mast cell line both in vitro and in vivo. These findings may provide insight into designing strategies for treatment of mastocytosis and other diseases associated with mutations in the Kit catalytic domain.

Androstadienes↗
PubMed2005

GSK-3beta reactivation with LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis.

Constitutive activation of phosphatidylinositol 3-kinase (PI3K) confers resistance to apoptotic stimuli induced by chemotherapeutic agents in a variety of cancer cells. Therefore, the comprehension of mechanisms whereby PI3K downregulation interferes with chemotherapy is of major clinical interest for the elaboration of combined anticancer treatment modalities. Here, we examined the molecular mechanisms whereby the PI3K inhibitor LY294002 sensitized p53- and Fas-deficient hepatoma cells to etoposide and camptothecin. LY294002 increased Hep3B cell susceptibility to chemotherapy-induced apoptosis by enhancing the expression of DR4 and DR5 and the activation of caspase-8 and -3. Moreover, LY294002-mediated sensitization to chemotherapy involved mitochondrial Bax translocation and cytosolic cytochrome c accumulation. In Hep3B cells, LY294002 led to the reactivation of glycogen synthase kinase-3beta (GSK-3beta) by promoting its dephosphorylation on the serine 9 residue independently from Akt inhibition. The transient transfection of a constitutively active and non-phosphorylable S9AGSK-3beta mutant sensitized cells to etoposide cytotoxic effects while cell treatment with the small GSK-3beta inhibitor SB-415286 repressed the sensitizing effect of LY294002 on chemotherapy-induced apoptosis and caspase-8 activation. Altogether, our results show that LY294002 sensitizes hepatoma cells to chemotherapy-induced apoptosis via death receptor and mitochondria signalling pathways and that GSK-3beta reactivation is involved in this process. Therefore, PI3K-mediated GSK-3beta inhibition could be a mechanism by which cancer cells escape from chemotherapy-induced apoptosis.

Antineoplastic Agents↗
PubMed2006

[PI3 kinases and the control of autophagia].

Macroautophagy or autophagy is a degradative pathway terminating in the lysosomal compartment after the formation of a cytoplasmic vacuole that engulfs macromolecules and organelles. The recent discovery of the molecular controls of autophagy that are common to eukaryotic cells from yeast to human suggests that the role of autophagy in cell functioning is far beyond its nonselective degradative capacity. The downregulation of autophagy observed in cancer cells is associated with tumor progression. The regulation of autophagy by signalling pathways overlaps with the control of cell growth, proliferation, cell survival and death. Two of these pathways play an important role in control of autophagy, the class I and III PI3K pathways. Several tumor suppressor genes (PTEN, TSC1 and 2, p53) involved in the class I PI3K mTOR signalling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network (Ras, class I PI3K and Akt) have the opposite effect. These findings, together with the discovery that Beclin 1, which forms a complex with the class III PI3K to initiate autophagy, is a tumor suppressor gene product give credibility of the idea that autophagy is a tumor suppressor mechanism. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli, suggesting that they can also exploit autophagy for their own benefit.

Animals↗
PubMed2006

[Study of biological behavior of stromal cell-derived factor-1 on multiple myeloma cell migration and adhesion].

OBJECTIVE: To investigate the biological behavior of stromal cell-derived factor-1 (SDF-1) on multiple myeloma (MM) cell migration and adhesion and it related signaling pathways. METHODS: Expression of adhesion molecules on MM cells of RPMI8226, XG-1 and XG-7 cells was analysed by flow cytometry, the influence of SDF-1 on CD29 and CD49e distribution by immunofluorescence, the effect of SDF-1 on chemotaxis of MM cells by transwell assay. Activation of phosphoinositide-3 kinase (PI3K) in MM cells treated with SDF-1 and by immunoblotting. RESULTS: 3 strains of MM cell line expressed many adhesion molecule. RPMI8226, XG-7 cells were all high level of expression of CD29 (> 70%). XG-1, XG-7 cells were all high level of expression of CD44 (> 80%), and XG-7 cells was of CD49d (> 90%). In all of 3 strains, the levels of expression of CD49e were low (< 30%). SDF-1 could not upregulate their expression, but could trigger the establishment of polarized morphology of MM cells and the redistribution of CD29 and CD49e. SDF-1 promoted MM cells adhesion to endothelial cells, stimulated phosphorylation of P85 subunit of PI3K in MM cells and induced MM cells migration, which were inhibited by G protein inhibitor PTX and PI3K inhibitor wortmannin. CONCLUSION: SDF-1 can promote MM cell adhesion to endothelial cells, trigger establishment of a polarized morphology of MM cells and redistribution of adhesion molecules and induce MM cells migration via PI3K signaling pathway.

Blotting, Western↗
PubMed2006

Expression of interleukin (IL)-11 and IL-11 receptor in human colorectal adenocarcinoma: IL-11 up-regulation of the invasive and proliferative activity of human colorectal carcinoma cells.

Previous investigations have shown that interleukin (IL)-11/IL-11 receptor alpha-chain (IL-11Ralpha), a member of the PI3K, MAPK and JAK-STAT activating family of cytokines/receptors, correlates with the regulation of tumor progression. In this study, we established the IL-11/IL-11Ralpha expression profile in human colorectal adenocarcinoma (CRC) and clarified its signaling pathway and role in the invasion activity of CRC cell lines. To elucidate the role of IL-11/IL-11Ralpha, we examined 103 cases of CRC and 24 cases of colorectal adenoma by immunohistochemistry. In addition, we investigated the invasive activity of cell signaling pathway of CRC cell lines. The IL-11Ralpha expression was correlated with tumor invasion and lymphatic infiltration (p<0.01, respectively). Recombinant human IL-11 (rhIL-11) promoted the migration and proliferation of HT-29 cells and activated the PI3K and p44/p42 MAPK pathways. Wortmannin, a PI3K inhibitor, and PD98059, a p44/p42 MAPK inhibitor, significantly reduced the promotion of invasion and proliferation activity by rhIL-11, respectively. In summary, the IL-11Ralpha expression was correlated with clinicopathological features and IL-11 promoted the invasion via the PI3K and up-regulated the proliferation via the p44/p42 MAPK in CRC cells. These findings suggested that the IL-11/IL-11R pathway plays an important role in the progression of CRC.

Adenocarcinoma↗
PubMed1994

Insulin and insulin-like growth factor signaling are defective in the MDA MB-468 human breast cancer cell line.

Polypeptide growth factors including the insulin-like growth factors (IGFs), insulin, and transforming growth factor-alpha are mitogens for many breast cancer cell lines and may act as regulators of cancer cell growth. In a human breast cancer cell line MCF-7, which expresses IGF-I receptor (IGF-IR), stimulation with insulin or IGFs resulted in autophosphorylation of the IGF-IR in an increased proportion of ras bound to GTP and in the association of phosphatidylinositol 3'-kinase (PI3K) activity and of p85-PI3K with M(r) 185,000 phosphotyrosinylated proteins corresponding in size to insulin/IGF-IR substrates. These events were associated with enhanced proliferation. MDA MB-468 is a human breast cancer cell line which expresses insulin receptor and high levels of epidermal growth factor/transforming growth factor alpha receptor but low levels of IGF-IR. In this cell line, insulin stimulated autophosphorylation of IR at physiological concentrations and promoted the association of PI3K activity and of p85 with phosphotyrosine-containing proteins. Insulin did not, however, induce increased ras.GTP, and the cells exhibited minimal proliferation in response to insulin. Unlike insulin treatment, epidermal growth factor stimulation of MDA MB-468 cells is mitogenic and resulted in increased ras.GTP content, suggesting that the failure of insulin to induce these changes is not due to alterations in these signaling molecules. We conclude that there is a postreceptor defect in insulin signaling in MDA MB-468 which prevents the activation of ras and the induction of mitogenesis. Activation of PI3K by insulin is not sufficient to mediate mitogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Oncostatin M activates phosphatidylinositol-3-kinase in Kaposi's sarcoma cells.

Oncostatin M (OM) is a polypeptide cytokine that induces autocrine and paracrine effects on AIDS-Kaposi's sarcoma (KS) cells (Nair et al., Science, 255, 1430-1432, 1992; Miles et al., Science, 255, 1432-1434, 1992). The signalling pathways underlying this activation are largely unknown. We have found that OM binding to KS cell lines in vitro identifies a higher affinity binding site (Kd 10-20 pM) with a lower affinity (Kd 1.5 nM), high capacity binding site. The binding of OM to its receptor at the KS cell surface stimulates a rapid tyrosine phosphorylation of multiple proteins, including the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K). In addition OM can stimulate the in vivo activation of PI3K and increases the PI3K activity in anti-phosphotyrosine and anti-src kinase family antibody directed immunoprecipitates. Genistein, an inhibitor of tyrosine kinases, inhibits the synthesis of phosphatidylinositol 3,4-biphosphate and the growth of KS cells. Finally, OM enhances tyrosine kinase activity in immune complex kinase assay performed with antibody anti-src kinase family. These data suggest that in KS cells OM can stimulate formation of tyrosine kinase co-ordinate signalling complexes, containing at least src kinase family and PI3K, which can drive the accumulation of the putative second-messengers D3-phosphorylated phosphoinositides.

Antineoplastic Agents↗
PubMed1996

The protein product of the proto-oncogene c-cbl forms a complex with phosphatidylinositol 3-kinase p85 and CD19 in anti-IgM-stimulated human B-lymphoma cells.

Multiple signal transduction cascades, consisting of multiple interacting proteins, are activated following stimulation through most cell surface receptors, including the immunoglobulin receptor of B lymphocytes. In this report, we investigated the multimolecular complexes formed following anti-Ig stimulation of a human B-lymphoma cell line, resulting in activation of phosphatidylinositol 3-kinase (PI3K). PI3K is a lipid kinase that consists of an 85-kD regulatory subunit, bound to a 110-kD catalytic subunit. CD19 is a 95-kD B-cell surface marker that contains a consensus binding motif for PI3Kp85 in the cytoplasmic domain and recruits PI3K activity in activated B cells. The protein product of the c-cbl protooncogene is a 120-kD protein that is expressed in early B-lineage cells and in myeloid cells and is phosphorylated on tyrosine following receptor-mediated signaling in T and B lymphocytes. We demonstrate here that phosphorylated c-cbl complexes with CD19 and with PI3Kp85 via its C-terminal SH2 domain, and that both c-cbl and CD19 are associated with active PI3K in anti-Ig-stimulated cells. Although we cannot differentiate between a three-component, c-cbl/CD19/p85 complex and individual two-component complexes, these studies suggest that c-cbl may function as a docking protein, possibly linking distinct signal transduction pathways.

Antigens, CD19↗