Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors.

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus.

The Mdm2 oncoprotein promotes cell survival and cell cycle progression by inhibiting the p53 tumor suppressor protein. To regulate p53, Mdm2 must gain nuclear entry, and the mechanism that induces this is now identified. Mitogen-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, the Akt/PKB serine-threonine kinase, results in phosphorylation of Mdm2 on serine 166 and serine 186. Phosphorylation on these sites is necessary for translocation of Mdm2 from the cytoplasm into the nucleus. Pharmacological blockade of PI3-kinase/Akt signaling or expression of dominant-negative PI3-kinase or Akt inhibits nuclear entry of Mdm2, increases cellular levels of p53, and augments p53 transcriptional activity. Expression of constitutively active Akt promotes nuclear entry of Mdm2, diminishes cellular levels of p53, and decreases p53 transcriptional activity. Mutation of the Akt phosphorylation sites in Mdm2 produces a mutant protein that is unable to enter the nucleus and increases p53 activity. The demonstration that PI3-kinase/Akt signaling affects Mdm2 localization provides insight into how this pathway, which is inappropriately activated in many malignancies, affects the function of p53.

The PTEN tumor suppressor protein inhibits tumor necrosis factor-induced nuclear factor kappa B activity.

Nuclear factor kappaB (NF-kappaB) transcriptionally activates genes that promote immunity and cell survival. Activation of NF-kappaB is induced by an IkappaB kinase (IKK) complex that phosphorylates and promotes dissociation of IkappaB from NF-kappaB, which then translocates into the nucleus. Activation of phosphatidylinositol (PI) 3-kinase/Akt signaling by tumor necrosis factor (TNF) activates IKK and NF-kappaB. The present study shows that PTEN, a tumor suppressor that inhibits PI 3-kinase function, impairs TNF activation of Akt and the IKK complex in 293 cells. Transient expression of PTEN suppressed IKK activation and TNF-induced NF-kappaB DNA binding and transactivation. Studies were conducted with PC-3 prostate cancer cells that do not express PTEN and DU145 prostate cancer cells that express PTEN. TNF activated Akt in PC-3 cells, but not in DU145 cells, and the ability of TNF to activate NF-kappaB was blocked by pharmacological inhibition of PI 3-kinase activity in PC-3 cells, but not in DU145 cells. Expression of PTEN in PC-3 cells to a level comparable with that endogenously present in DU145 cells inhibited TNF activation of NF-kappaB. The cell type-specific ability of PTEN to negatively regulate the PI 3-kinase/AKT/NF-kappaB pathway may be important to its tumor suppressor activity.

Vascular endothelial cell growth factor activates CRE-binding protein by signaling through the KDR receptor tyrosine kinase.

Vascular endothelial cell growth factor (VEGF) plays a crucial role in the development of the cardiovascular system and in promoting angiogenesis associated with physiological and pathological processes. Although a great deal is known of the cytoplasmic signaling pathways activated by VEGF, much less is known of the mechanisms through which VEGF communicates with the nucleus and alters the activity of transcription factors. Binding of VEGF to the KDR/Flk1 receptor tyrosine kinase induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. p38 MAPK/MSK-1 and protein kinase C/p90RSK pathways mediate CREB phosphorylation. Confocal microscopy shows that VEGF-induced phosphorylation of nuclear CREB is blocked by pharmacological inhibition of protein kinase C and p38 mitogen-activated protein kinase signaling. Thus, KDR/Flk1 uses multiple pathways to transmit signals into the nucleus where CREB becomes activated. These results suggest that CREB may play a role in alterations of gene expression important to angiogenesis.

A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1.

Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppressor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN.

Interferon alpha /beta promotes cell survival by activating nuclear factor kappa B through phosphatidylinositol 3-kinase and Akt.

Interferons (IFNs) play critical roles in host defense by modulating gene expression via activation of signal transducer and activator of transcription (STAT) factors. IFN-alpha/beta also activates another transcription factor, nuclear factor kappaB (NF-kappaB), which protects cells against apoptotic stimuli. NF-kappaB activation requires the IFN-dependent association of STAT3 with the IFNAR1 chain of the IFN receptor. IFN-dependent NF-kappaB activation involves the sequential activation of a serine kinase cascade involving phosphatidylinositol 3-kinase (PI-3K) and Akt. Whereas constitutively active PI-3K and Akt induce NF-kappaB activation, Ly294002 (a PI-3K inhibitor), dominant-negative PI-3K, and kinase-dead Akt block IFN-dependent NF-kappaB activation. Moreover, dominant-negative PI-3K blocks IFN-promoted degradation of kappaBox alpha. Ly294002, a dominant-negative PI-3K construct, and kinase-dead Akt block IFN-promoted cell survival, enhancing apoptotic cell death. Therefore, STAT3, PI-3K, and Akt are components of an IFN signaling pathway that promotes cell survival through NF-kappaB activation.

Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF.

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation.

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.

VRAP is an adaptor protein that binds KDR, a receptor for vascular endothelial cell growth factor.

A protein that binds the intracellular domain of KDR (KDR-IC), a receptor for vascular endothelial cell growth factor (VEGF), was identified by two-hybrid screening. Two-hybrid mapping showed that the VEGF receptor-associated protein (VRAP) interacted with tyrosine 951 in the kinase insert domain of KDR. Northern blot analysis identified multiple VRAP transcripts in peripheral leukocytes, spleen, thymus, heart, lung, and human umbilical vein endothelial cells (HUVEC). The predominant VRAP mRNA encodes a 389-amino acid protein that contains an SH2 domain and a C-terminal proline-rich motif. In HUVEC, VEGF promotes association of VRAP with KDR. Phospholipase C gamma and phosphatidylinositol 3-kinase, effector proteins that are downstream of KDR and important to VEGF-induced endothelial cell survival and proliferative responses, associate constitutively with VRAP. These observations identify VRAP as an adaptor that recruits cytoplasmic signaling proteins to KDR, which plays an important role in normal and pathological angiogenesis.

The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties.

The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.

Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation.

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.

Mouse receptor interacting protein 3 does not contain a caspase-recruiting or a death domain but induces apoptosis and activates NF-kappaB.

The death domain-containing receptor superfamily and their respective downstream mediators control whether or not cells initiate apoptosis or activate NF-kappaB, events critical for proper immune system function. A screen for upstream activators of NF-kappaB identified a novel serine-threonine kinase capable of activating NF-kappaB and inducing apoptosis. Based upon domain organization and sequence similarity, this novel kinase, named mRIP3 (mouse receptor interacting protein 3), appears to be a new RIP family member. RIP, RIP2, and mRIP3 contain an N-terminal kinase domain that share 30 to 40% homology. In contrast to the C-terminal death domain found in RIP or the C-terminal caspase-recruiting domain found in RIP2, the C-terminal tail of mRIP3 contains neither motif and is unique. Despite this feature, overexpression of the mRIP3 C terminus is sufficient to induce apoptosis, suggesting that mRIP3 uses a novel mechanism to induce death. mRIP3 also induced NF-kappaB activity which was inhibited by overexpression of either dominant-negative NIK or dominant-negative TRAF2. In vitro kinase assays demonstrate that mRIP3 is catalytically active and has autophosphorylation site(s) in the C-terminal domain, but the mRIP3 catalytic activity is not required for mRIP3 induced apoptosis and NF-kappaB activation. Unlike RIP and RIP2, mRIP3 mRNA is expressed in a subset of adult tissues and is thus likely to be a tissue-specific regulator of apoptosis and NF-kappaB activity. While the lack of a dominant-negative mutant precludes linking mRIP3 to a known upstream regulator, characterizing the expression pattern and the in vitro functions of mRIP3 provides insight into the mechanism(s) by which cells modulate the balance between survival and death in a cell-type-specific manner.

NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase.

Activation of the nuclear transcription factor NF-kappaB by inflammatory cytokines requires the successive action of NF-kappaB-inducing kinase (NIK) and an IKB-kinase (IKK) complex composed of IKKalpha and IKKbeta. Here we show that the Akt serine-threonine kinase is involved in the activation of NF-kappaB by tumour necrosis factor (TNF). TNF activates phosphatidylinositol-3-OH kinase (PI(3)K) and its downstream target Akt (protein kinase B). Wortmannin (a PI(3)K inhibitor), dominant-negative PI(3)K or kinase-dead Akt inhibits TNF-mediated NF-kappaB activation. Constitutively active Akt induces NF-kappaB activity and this effect is blocked by dominant-negative NIK. Conversely, NIK activates NF-kappaB and this is blocked by kinase-dead Akt. Thus, both Akt and NIK are necessary for TNF activation of NF-kappaB. Akt mediates IKKalpha phosphorylation at threonine 23. Mutation of this amino acid blocks phosphorylation by Akt or TNF and activation of NF-kappaB. These findings indicate that Akt is part of a signalling pathway that is necessary for inducing key immune and inflammatory responses.

The proteasome regulates caspase-dependent and caspase-independent protease cascades during apoptosis of MO7e hematopoietic progenitor cells.

Withdrawal of trophic support from growth factor-dependent MO7e human myeloid progenitor cells induces apoptosis characterized by DNA fragmentation and degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Inhibitors of caspase (ICE) protease family members did not inhibit apoptosis or DNA fragmentation induced by factor withdrawal, but blocked degradation of DNA-PKcs. Thus, caspase activity accounts for only a component of the apoptotic program in MO7e hematopoietic cells. The protease inhibitor TPCK, but not other protease inhibitors, blocked DNA fragmentation, but not degradation of DNA-PKcs during apoptosis of MO7e cells. Thus, caspase-independent and caspase-dependent protease cascades mediate distinct features of MO7e cell apoptosis. The proteasome inhibitors calpain inhibitor I and lactacystin promoted DNA fragmentation, degradation of DNA-PKcs and apoptosis of MO7e cells. The ability of lactacystin to promote DNA fragmentation was abrogated by TPCK, but not by caspase inhibitors, whereas the ability of lactacystin to promote degradation of DNA-PKcs was blocked by caspase inhibitors, but not by TPCK. Thus, caspase-dependent and caspase-independent protease cascades are downstream of and regulated by the proteasome, which plays a central role in regulating the multiple protease cascades that induce apoptosis.

Modulation of tumor necrosis factor and interleukin-1-dependent NF-kappaB activity by mPLK/IRAK.

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-kappaB-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, the mouse Pelle-like kinase/human interleukin-1 receptor-associated kinase (mPLK/IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-kappaB activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-kappaB-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-kappaB activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD- and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-kappaB-dependent gene expression.

Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.

Homology PCR has been used to identify receptor tyrosine kinases (RTKs) expressed during activation of rat hepatic stellate cells, the key fibrogenic mesenchymal element in the liver. Partial cDNAs encoding several RTKs were cloned from stellate cells activated in vivo, including those of Flt-1, Flk-1, c-met, PDGFR, and Tyro10/DDR2. RNAse protection from cells activated in vivo demonstrated biphasic induction of flt-1 and flk-1 mRNAs, receptors for vascular endothelial growth factor (VEGF). Culture-activation of stellate cells was associated with increased [125I]VEGF binding and Flt-1 and Flk-1 receptor protein. Induction of VEGF binding sites correlated with an 2.5-fold increase in DNA synthesis in response to VEGF, but only if cells were activated by growth on collagen 1, whereas cells maintained in a quiescent state on a basement membrane-like substratum (EHS matrix) were nonproliferative. In both stellate and endothelial cells VEGF-induced mitogenesis was augmented by co-incubation with basic fibroblast growth factor (bFGF), a cytokine with known synergy with VEGF. These findings suggest that the cellular targets of VEGF in liver may not be confined to sinusoidal endothelial cells, and that VEGF responses reflect combined effects on both hepatic stellate cells and sinusoidal endothelium.

Induction of Jak/STAT signaling by activation of the type 1 TNF receptor.

Cellular responses to TNF are initiated by either of two cell surface receptors, the type 1 TNF receptor (TNFR1) and the type 2 TNF receptor (TNFR2). Although neither receptor contains an intrinsic protein tyrosine kinase, such activity has been implicated in TNF action. In this study, we show that murine TNF induces the tyrosine phosphorylation and activation of the intracellular Janus tyrosine kinases Jak1, Jak2, and Tyk2 in murine 3T3-L1 adipocytes. Activation of Jak kinases by TNF was associated with tyrosine phosphorylation of STAT1, STAT3, STAT5, and STAT6, but not STAT2 or STAT4, showing that TNF acts on a specific subset of these latent cytoplasmic transcription factors in 3T3-L1 adipocytes. Agonist antiserum to TNFR1 induced Jak kinase and STAT protein phosphorylation. Phosphorylation of Jak proteins was also induced by human TNF, which selectively binds to TNFR1 on murine cells. 35S-labeled Jak kinases were precipitated from a cell-free system and from lysates of 3T3-L1 adipocytes by a glutathione S-transferase fusion protein containing the cytoplasmic domain of TNFR1. These results suggest that the cytoplasmic domain of TNFR1 can directly interact with and form signaling complexes with Jak kinases. Jak2 was precipitated from HeLa cells by antiserum to TNFR1, directly demonstrating their association in vivo. Thus, TNF activates a Jak/STAT signal-transduction cascade by acting through TNFR1.

Two-hybrid cloning of a gene encoding TNF receptor-associated protein 2, a protein that interacts with the intracellular domain of the type 1 TNF receptor: identity with subunit 2 of the 26S protease.

A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.

Inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine.

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

Induction of vascular endothelial growth factor by insulin-like growth factor 1 in colorectal carcinoma.

Vascular endothelial growth factor (VEGF) is an angiogenic hormone that is produced by and supports the growth of many types of malignancies. The present study shows that insulin-like growth factor 1 (IGF-I), a mitogen that promotes the propagation of cancers through autocrine and paracrine mechanisms, increases the expression of mRNA for VEGF and production of VEGF protein by COLO 205 colon carcinoma cells. IGF-I also induces expression of VEGF mRNA in SW620, LSLiM6, and HCT15 colon carcinoma cells showing that this is a common response to IGF-I. Whereas IGF-I induced VEGF mRNA in each cell line examined (2.3-12-fold), it induced proliferation only in COLO 205 and LSLiM6 cells. Thus, the proliferative response induced by IGF-I and its ability to induce VEGF occur through distinguishable mechanisms. IGF-I increases the cellular content of VEGF mRNA by increasing the rate of transcription (5-fold after 4 h) and also by increasing the half-life of VEGF mRNA (0.6 +/- 0.07 h in control cells to 2.0 +/- 0.37 h in IGF-I-treated cells). Monoclonal antibody (alphaIR3) directed against the type 1 IGF receptor significantly attenuated the ability of IGF-I to promote expression of VEGF mRNA. Interestingly, by itself alphaIR3 acted as a weak agonist and induced a modest increase in the cellular content of VEGF mRNA. alphaIR3 also promoted tyrosine phosphorylation of the beta subunit of the IGF-I receptor, and the magnitude of this response was comparable with that induced by IGF-I. These observations point to a nonlinear relationship between activation of the IGF-I receptor and induction of VEGF mRNA. Thus, in addition to its direct, growth stimulatory effect on transformed cells, IGF-I induces the expression of VEGF which can promote the progression of cancer by regulating the development of new blood vessels.