Endocrine disruptors of sex hormone activities.

Sex hormones, such as androgens, estrogens and progestins are naturally occurring compounds that tightly regulate endocrine systems in a variety of living organisms. Uncontrolled environmental exposure to these hormones or their biological and synthetic mimetics has been widely documented. Furthermore, water contaminants penetrate soil to affect flora, fauna and ultimately humans. Because endocrine systems evolved to respond to very small changes in hormone levels, the low levels found in the environment cannot be ignored. The combined actions of sex hormones with glucocorticoids and other nuclear receptors disruptors creates additional level of complexity including the newly described "dynamic assisted loading" mechanism. We reviewed the extensive literature pertaining to world-wide detection of these disruptors and created a detailed Table on the development and current status of methods used for their analysis.

Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer.

BACKGROUND: Side population (SP) fraction cells, identified by efflux of Hoechst dye, are present in virtually all normal and malignant tissues. The relationship between SP cells, drug resistance and cancer stem cells is poorly understood. Small-cell lung cancer (SCLC) is a highly aggressive human tumour with a 5-year survival rate of <10%. These features suggest enrichment in cancer stem cells. METHODS AND RESULTS: We examined several SCLC cell lines and found that they contain a consistent SP fraction that comprises <1% of the bulk population. Side population cells have higher proliferative capacity in vitro, efficient self-renewal and reduced cell surface expression of neuronal differentiation markers, CD56 and CD90, as compared with non-SP cells. Previous reports indicated that several thousand SP cells from non-small-cell lung cancer are required to form tumours in mice. In contrast, as few as 50 SP cells from H146 and H526 SCLC cell lines rapidly reconstituted tumours. Whereas non-SP cells formed fewer and slower-growing tumours, SP cells over-expressed many genes associated with cancer stem cell and drug resistance: ABCG2, FGF1, IGF1, MYC, SOX1/2, WNT1, as well as genes involved in angiogenesis, Notch and Hedgehog pathways. CONCLUSIONS: Side population cells from SCLC are highly enriched in tumourigenic cells and are characterised by a specific stem cell-associated gene expression signature. This gene signature may be used for development of targeted therapies for this rapidly fatal tumour.

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes.

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo.

Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)-DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA-DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.

Mitogenic signaling by cyclic adenosine monophosphate in chromaffin cells involves phosphatidylinositol 3-kinase activation.

Increase of intracellular cyclic adenosine monophosphate by the permeant cyclic adenosine monophosphate analog, 8-(4-chlorophenylthio)-adenosine 3':5'- cyclic monophosphate, is mitogenic for normal adult rat chromaffin cells. The mitogenic effect is blocked by the phosphatidylinositol 3-kinase inhibitor, LY294002, and is associated with accumulation of phosphorylated Akt and p70S6 kinase, suggesting that cyclic adenosine monophosphate activates Type l phosphatidylinositol 3-kinase. The mechanism of activation was examined in PC12 pheochromocytoma cells, which are neoplastic chromaffin cells that exhibit many of the biochemical characteristics of their normal counterparts. Incubation of PC12 cells with 8-(4-chlorophenylthio)-adenosine 3':5'- cyclic monophosphate led to a significant increase in total phosphatidylinositol 3-kinase activity that was sensitive to low concentrations of LY294002. The increase was maximal at 1 h and returned to basal levels within six hours. Immunoprecipitation studies showed no increase in phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immune complexes from PC12 cells stimulated by 8-(4-chlorophenylthio)-adenosine 3':5'- cyclic monophosphate, in contrast to cells stimulated by nerve growth factor. Instead, activity was demonstrated in association with p110gamma and p85. These findings suggest that cyclic adenosine monophosphate causes activation of Types IA and IB phosphatidylinositol 3-kinase by a novel mechanism in chromaffin and pheochromocytoma cells. That activation may contribute to chromaffin cell proliferation and to the development and progression of pheochromocytomas. J. Cell. Biochem. Suppl. 36: 89-98, 2001.

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.

Probing fibroblast growth factor dimerization and role of heparin-like glycosaminoglycans in modulating dimerization and signaling.

For a number of growth factors and cytokines, ligand dimerization is believed to be central to the formation of an active signaling complex. In the case of fibroblast growth factor-2 (FGF2) signaling, heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) are involved through interaction with both FGF2 and its receptors (FGFRs) in assembling a tertiary complex and modulating FGF2 activity. Biochemical data have suggested different modes of HLGAG-induced FGF2 dimerization involving specific protein-protein contacts. In addition, several recent x-ray crystallography studies of FGF.FGFR and FGF.FGFR.HLGAG complexes have revealed other modes of molecular assemblage, with no FGF-FGF contacts. All these different biochemical and structural findings have clarified less and in fact raised more questions as to which mode of FGF2 dimerization, if any, is essential for signaling. In this study, we address the issue of FGF2 dimerization in signaling using a combination of biochemical, biophysical, and site-directed mutagenesis approaches. Our findings presented here provide direct evidence of FGF2 dimerization in mediating FGF2 signaling.

The src homology 2 domain of Bcr/Abl is required for efficient induction of chronic myeloid leukemia-like disease in mice but not for lymphoid leukemogenesis or activation of phosphatidylinositol 3-kinase.

The effect of mutations in the Src homology 2 (SH2) domain of the BCR/ABL oncogene on leukemogenesis was tested in a quantitative murine bone marrow transduction/transplantation assay that accurately models human Philadelphia-positive B-lymphoid leukemia and chronic myeloid leukemia (CML). The SH2 domain was not required for induction of B-lymphoid leukemia in mice by BCR/ABL. Under conditions where the p190 and p210 forms of BCR/ABL induce fatal CML-like myeloproliferative disease within 4 weeks, p210 SH2 mutants induced CML-like disease in some mice only after a significant delay, with other recipients succumbing to B-lymphoid leukemia instead. In contrast, p190 BCR/ABL SH2 point and deletion mutants rapidly induced CML-like disease. These results provide the first direct evidence of significant differences in cell signaling by the Bcr/Abl tyrosine kinase between these distinct leukemias. Contrary to previous observations, high levels of phosphatidylinositol 3-kinase (PI 3-kinase) activity in primary malignant lymphoblasts and myeloid cells from recipients of marrow transduced with the BCR/ABL SH2 mutants were found. Hence, the decreased induction of CML-like disease by the p210 BCR/ABL SH2 mutants is not due to impaired activation of PI 3-kinase.

Inactivation of wild-type BCR/ABL tyrosine kinase in hematopoietic cells by mild hyperthermia.

Temperature-sensitive mutants of BCR/ABL tyrosine kinase have been extensively used to study the mechanisms of cell transformation and signal transduction. However, little is known about the effect of temperature on the activity of wild-type BCR/ABL gene product. In this study, we demonstrate that in vivo tyrosine kinase activity of p210, p190 BCR/ABL and v-abl are temperature-sensitive when expressed in hematopoietic cells and decline when temperature is raised 2 degrees C above normal range. In vitro tyrosine kinase activities of purified recombinant Abl and immunoprecipitated p210 BCR/ABL were also sensitive to increased temperature. Tyrosine phosphorylation of cellular proteins was markedly reduced in BCR/ABL transformed cells after 16 h at 39 degrees C, whereas the expression of BCR/ABL was unchanged. Temperature-induced downregulation of BCR/ABL kinase activity was reversible when cells were shifted back to 37 degrees C. The downregulation of Abl tyrosine kinase activity was not influenced by mutation or deletion of SH2 or SH3 domains or mutation of the GRB2 binding site. No increase in functional activity or expression of protein-tyrosine phosphatases, PTP-1B, SH-PTP1 or SH-PTP2 was detected in cells grown at 39 degrees C. Temperature-induced downregulation in tyrosine kinase activity correlated with decline in phosphotyrosine-associated PI 3-kinase whereas there was no change in growth factor independence of transformed hematopoietic cells. In conclusion, Abl tyrosine kinase has intrinsic sensitivity to temperature and BCR/ABL expressed in hematopoietic cells is downregulated by increasing temperature 2 degrees C. These observations provide a unique opportunity to identify cellular factor(s) which regulate BCR/ABL kinase in vivo and suggests possible novel treatment of CML by a mild hyperthermia.

Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells.

Angiopoietin-1 is a unique growth factor which induces Tie2 receptor autophosphorylation and interaction with signal transduction molecules, GRB2 and p85 subunit of PI 3-kinase, but no detectable mitogenic response. Here we show that PI 3-kinase-dependent activation of Akt and attachment to extracellular matrix are required for angiopoietin-1-mediated endothelial cell survival. Apoptosis of growth factor-deprived cells grown in monolayer was decreased by angiopoietin-1 and correlated with Akt activation. In contrast, angiopoietin-1, bFGF or VEGF failed to protect cells in suspension culture. Ceramide, an intermediate of several apoptotic pathways, interferes with growth factor-mediated Akt activation. Ceramide induced endothelial cell death and abolished angiopoietin-1-mediated activation of Akt and the effect on cell survival. In addition, we found that PI 3-kinase activity is necessary for migration of endothelial cells in response to Angiopoietin-1. A transient activation of MAPK/ERKs was also detected within 10 min after stimulation with angiopoietin-1. In contrast to VEGF-mediated biological effects, inhibition of MAPK/ERKs by PD98059 in endothelial cells did not affect angiopoietin-1 mediated survival or migration. These findings indicate significant differences in intracellular signaling between VEGF and angiopoietin-1 and that PI 3-kinase lipid products are key mediators of the biological effects of angiopoietin-1.

Expression of tie1 and tie2 proteins during reendothelialization in balloon-injured rat carotid artery.

The novel endothelial cell tyrosine kinase receptors, Tie1 and Tie2, are essential for vascular development and remodeling in the embryo but little is known regarding the regulation of their expression and their role in the maintenance and repair of the adult vascular system. We examined the expression of Tie1 and Tie2 in normal vessels and during reendothelialization following balloon injury of the adult rat carotid artery. Tie proteins were detected in quiescent endothelial cells of the adult rat carotid artery. Tie1 and Tie2 proteins were also detected in human and rat platelets. A weak expression of Tie1 and Tie2 proteins was detected in young endothelial cells which sparsely repopulated the denuded surface by day 14. Protein levels increased in the confluent layer of endothelial cells by day 28. Based on these observations, we tested whether Tie1 and Tie2 mRNA and protein levels are regulated by cell density. Tie1 and Tie2 expression significantly increased with higher density in cultured human endothelial cells, and this upregulation required cell-cell interaction. These data suggest that Tie1 and Tie2 may play a role in the maintenance and repair of the adult vascular system and that the expression of these proteins is regulated by cell density.

Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles.

Bile acid transport and secretion in hepatocytes require phosphatidylinositol (PI) 3-kinase-dependent recruitment of ATP-dependent transporters to the bile canalicular membrane and are accompanied by increased canalicular PI 3-kinase activity. We report here that the lipid products of PI 3-kinase also regulate ATP-dependent transport of taurocholate and dinitrophenyl-glutathione directly in canalicular membranes. ATP-dependent transport of taurocholate and dinitrophenyl-glutathione in isolated canalicular vesicles from rat liver was reduced 50-70% by PI 3-kinase inhibitors, wortmannin, and LY294002, at concentrations that are specific for Type I PI 3-kinase. Inhibition was reversed by addition of lipid products of PI 3-kinase (PI 3,4-bisphosphate and, to a lesser extent, PI 3-phosphate and PI 3,4,5-trisphosphate) but not by PI 4, 5-bisphosphate. A membrane-permeant synthetic 10-mer peptide that binds polyphosphoinositides and leads to activation of PI 3-kinase in macrophages doubled PI 3-kinase activity in canalicular membrane vesicles and enhanced taurocholate and dinitrophenyl-glutathione transport in canalicular membrane vesicles above maximal ATP-dependent transport. The effect of the peptide was blocked by wortmannin and LY294002. PI 3-kinase activity was also necessary for function of the transporters in vivo. ATP-dependent transport of taurocholate and PI 3-kinase activity were reduced in canalicular membrane vesicles isolated from rat liver that had been perfused with taurocholate and wortmannin. PI 3,4-bisphosphate enhanced ATP-dependent transport of taurocholate in these vesicles above control levels. Our results indicate that PI 3-kinase lipid products are necessary in vivo and in vitro for maximal ATP-dependent transport of bile acid and nonbile acid organic anions across the canalicular membrane. Our results demonstrate regulation of membrane ATP binding cassette transporters by PI 3-kinase lipid products.

The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver.

Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking. We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATP-dependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions. Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system. Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs. These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.

Effect of a xanthine analog on human hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice.

Hepatocellular carcinoma (HCC) frequently overexpresses the MDR1 gene and is resistant to drugs transported by the multidrug-resistance efflux pump. A xanthine analog, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (CT-2584,CTI), is cytotoxic to many tumors in culture and was four times more effective than verapamil in inhibiting Rhodamine 123 secretion in MDR1-overexpressing Chinese hamster ovary cells. However, studies using PRF/PLC/5 (Alexander) cells revealed that CT-2584 is cytotoxic by another mechanism not involving inhibition of MDR1 function. Alexander cells have integrated the hepatitis B surface antigen (HBsAg) gene and quantitatively secrete HBsAg. The parent cell line, Alex 0, has low MDR1 expression and is drug-sensitive, whereas a derived line, Alex 0.5, is drug-resistant and overexpresses MDR1 100 times. Both cell lines were similarly killed within 24 or 48 hours by CT-2584. Freshly isolated rat and human hepatocytes were considerably more resistant to killing by CT-2584. In vivo, CT-2584 significantly reduced tumor growth in SCID mice bearing Alex 0 or 0.5 xenografts as determined by serial measurements of HBsAg. Hepatic parenchyma was normal, whereas apoptosis and cellular loss were observed in xenografts. The xenograft model is useful for testing pharmacological therapy of HCC.

Tyrosine phosphorylation of the CD3-epsilon subunit of the T cell antigen receptor mediates enhanced association with phosphatidylinositol 3-kinase in Jurkat T cells.

T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta chain (zetaA-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon, respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon, p85alpha, and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon-ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon-ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.

Tyrosine phosphorylation of p120cbl in BCR/abl transformed hematopoietic cells mediates enhanced association with phosphatidylinositol 3-kinase.

Increased tyrosine kinase activity of abl oncogene in Philadelphia chromosome positive-leukemic cells leads to activation of p21ras and phosphatidylinositol 3'-kinase (PI 3-Kinase). The mechanism of activation of these signaling pathways is not understood, but numerous studies have focused on the identification and characterization of downstream substrates of BCR/abl tyrosine kinase as potential mediators of oncogenic signaling. It was recently found that the 120 kDa protein product of the c-cbl proto-oncogene is highly tyrosine phosphorylated and associates with BCR/abl in transformed hematopoietic cells. We have characterized further cbl's involvement in BCR/abl mediated tumorigenesis using growth factor independent BCR/abl transformed BaF3 cells. Our experiments show that, in contrast to other cell types, the in vivo interaction of cbl with GRB2 and p85 is significantly enhanced in BCR/abl transformed BaF3 cells and that tyrosine phosphorylation of cbl leads to a direct interaction with GRB2, p85 and abl SH2 domains. A 14-fold increase in cbl associated PI 3-kinase activity in BCR/abl transformed cells suggests that the binding of p85 SH2 domains to tyrosine phosphorylated cbl may contribute to PI 3-kinase activation. Domain analysis studies indicate that both SH3 domains of GRB2 bind to the proline rich region of cbl in quiescent BaF3 cells, whereas GRB2 SH2 domain interacts with a non-contiguous sequence of cbl in transformed cells. Although the interaction of cbl with GRB2 in transformed cells was facilitated by binding of GRB2 to BCR/abl, phosphorylation of cbl and its interaction with p190 BCR/abl remained unaltered in BaF3 cells transformed by p190Y177F BCR/abl mutant which is unable to bind GRB2. The current information and the data presented here suggest that, although cbl lacks src homology domains, it represents a novel intermediate protein which, by interaction with key SH-containing adaptor proteins, may participate in regulation of the Ras and PI 3-kinase pathways in BCR/abl transformed hematopoietic cells.

Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling.

Platelet-derived growth factor (PDGF) and serum, but not epidermal growth factor (EGF), stimulated sphingosine kinase activity in Swiss 3T3 fibroblasts and increased intracellular concentrations of sphingosine 1-phosphate (SPP), a sphingolipid second messenger (Olivera, A., and Spiegel, S. (1993) Nature 365, 557-560). We report herein that DL-threo-dihydrosphingosine (DHS), a competitive inhibitor of sphingosine kinase that prevents PDGF-induced SPP formation, specifically inhibited the activation of two cyclin-dependent kinases (p34(cdc2) kinase and Cdk2 kinase) induced by PDGF, but not by EGF. SPP reversed the inhibitory effects of DHS on PDGF-stimulated cyclin-dependent kinases and DNA synthesis, demonstrating that the DHS effects were mediated via inhibition of sphingosine kinase. DHS also markedly reduced PDGF-stimulated but not EGF-stimulated mitogen-activated protein kinase activity and DNA binding activity of activator protein-1. Examination of the early signaling events of PDGF action revealed that DHS did not affect PDGF-induced autophosphorylation of the growth factor receptor or phosphorylation of the SH2/SH3 adaptor protein Shc and its association with Grb2. This sphingosine kinase inhibitor did not abrogate activation of phosphatidylinositol 3-kinase by PDGF. In agreement, treatment with SPP had no effect on these responses but did, however, potently stimulate phosphorylation of Crk, another SH2/SH3 adaptor protein. Moreover, DHS inhibited PDGF-stimulated, but not EGF-stimulated, Crk phosphorylation. Thus, regulation of sphingosine kinase activity defines divergence in signal transduction pathways of PDGF and EGF receptors leading to mitogen-activated protein kinase activation.

Evidence for phosphatidylinositol 3-kinase-dependent T cell antigen receptor (TCR) signal transduction.

Recent evidence implicates PI 3-kinase in TCR signal transduction. The fungal metabolite wortmannin is a specific inhibitor of PI 3-kinase both in vitro and in vivo when used at nanomolar concentrations. Therefore, we examined the effect of wortmannin on stimulation of primary T cells and T cell lines. Wortmannin had a dose-dependent inhibitory effect on TCR-dependent primary T cell proliferation with IC50 in the nanomolar range. Furthermore, activation of T cell lines independently of antigen presenting cells and, therefore of any CD28 co-stimulatory signaling, was also sensitive to wortmannin. As expected, phorbol ester stimulation bypassed PI 3-kinase signal transduction. Importantly, the effect of wortmannin correlated with inhibition of activation of PI 3-kinase in stimulated T cells. The earliest step in T cell activation, tyrosine kinase activation, was not significantly affected by wortmannin. We conclude that a wortmannin-sensitive enzyme, probably PI 3-kinase, acting downstream of tyrosine kinases, but independently of the phorbol ester activated pathway, is necessary for stimulation of T cells via the TCR, and that this requirement is independent of any role of PI 3-kinase in co-stimulation via CD28 coreceptor. PI 3-kinase is most probably involved in generation of 3-phosphorylated lipid products, and is not merely an adaptor.

BCR/abl leads to the constitutive activation of Stat proteins, and shares an epitope with tyrosine phosphorylated Stats.

The mechanism by which BCR/abl leads to the transformation of hematopoietic cells is not understood. The introduction of BCR/abl into BaF3 cells, an IL-3-dependent pro-lymphocytic cell line, abrogates the requirement of IL-3 for growth. Given that IL-3 leads to the phosphorylation of Stat proteins, we tested the hypothesis that BCR/abl transformation of hematopoietic cells induces the phosphorylation of Stats. We found that BaF3 cells transformed by either the p190 or p210 forms of BCR/abl possess constitutively phosphorylated Stat1 and Stat5. Phosphorylation of Stat proteins was greater in cells transformed by p190 BCR/abl than in cells transformed by p210 BCR/abl, suggesting that the magnitude of phosphorylation of Stat proteins may play a role in the biological effects of BCR/abl. Expression of BCR/abl containing a mutation (Y177F) that prevents its interaction with GRB2 led to a decrease in the phosphorylation of Stat1 and Stat5. This suggested that GRB2, or its binding site on BCR/abl, may participate in the phosphorylation of Stat proteins. We also observed that the anti-phospho-Stat antibody directly recognized both the p190 and p210 forms of BCR/abl. This indicated that a tyrosine residue that becomes phosphorylated in BCR/abl may share homology with the tyrosine phosphorylation site of Stat1 and Stat5. These findings may have implications for the mechanisms by which BCR/abl interacts with signaling pathways to confer growth factor independence and induce transformation of hematopoietic cells.

PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl.

BCR/abl is a chimeric oncogene implicated in the pathogenesis of human chronic myelogenous leukemia. Expression of the BCR/abl gene induces hematologic malignancies in transgenic mice and transformation of interleukin-3-dependent hematopoietic cells. The mechanism of BCR/abl-mediated transformation of hematopoietic cells is poorly understood and involves activation of at least two signaling pathways, p21ras and PI 3-kinase. Here we report that PI 3,4-P2 and PI 3,4,5-P3, the enzymatic products of PI 3-kinase, accumulate in metabolically labeled transformed hematopoietic cells, in contrast to our previous report on the lack of accumulation of PI 3-kinase products in nontransformed NIH 3T3 fibroblasts that express p210 BCR/abl. Transformed cells also have increased PI 3-kinase activity in total cell extracts and membrane fractions. Activation of PI 3-kinase occurs by occupancy of SH2 domains of PI 3-kinase regulatory subunit, p85, by phosphorylated YXXM motifs. Therefore, we investigated whether BCR/abl binds to p85 and whether this binding is mediated by interaction of p85 SH2 domains with YXXM motif of BCR/abl. Association of p210 BCR/abl with p85 in immune complexes and with p85 SH2 domains was evident in hematopoietic cells that express the wt p210 BCR/abl. However, the binding of BCR/abl to p85 SH2 domains was abolished in cells expressing mutant, temperature-sensitive (ts) p210 BCR/abl in which the tyrosine in the YXXM motif of p210 BCR/abl was replaced by histidine. Despite lack of direct interaction with p85 SH2 domains, expression of ts p210 BCR/abl resulted in rapid, time-dependent activation of total and membrane-associated PI 3-kinase and increased PI 3-kinase activity in anti-P-tyr and anti-abl immunoprecipitates. These data suggest that BCR/abl-induced activation of PI 3-kinase in hematopoietic cells does not require binding of p85 SH2 domains to BCR/abl gene product and involves interaction with other tyrosine phosphorylated intermediate proteins.