L-[1-11C]-tyrosine PET to evaluate response to hyperthermic isolated limb perfusion for locally advanced soft-tissue sarcoma and skin cancer.
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PET with L-[1-11C]-tyrosine (TYR) was investigated in patients undergoing hyperthermic isolated limb perfusion (HILP) with recombinant tumor necrosis factor alpha (rTNF-alpha) and melphalan for locally advanced soft-tissue sarcoma and skin cancer of the lower limb. METHODS: Seventeen patients (5 women, 12 men; age range 24-75 y; mean age 52 y) were studied. TYR PET studies were performed before HILP and 2 and 8 wk afterwards. The protein synthesis rates (PSRs) in nanomoles per milliliter per minute were calculated. After final PET studies, tumors were resected and pathologically examined. Patients with pathologically complete responses (pCR) showed no viable tumors after treatment. Those with pathologically partial responses (pPR) showed various amounts of viable tumors in the resected tumor specimens. RESULTS: Six patients (35%) showed a pCR and 11 patients (65%) showed a pPR. All tumors were depicted as hot spots on PET studies before HILP. The PSR in the pCR group at 2 and 8 wk after perfusion had decreased significantly (P < 0.05) in comparison to the PSR before HILP. A significant difference was found in PSR between the pCR and pPR groups at 2 and at 8 wk (P < 0.05). Median PSR in nonviable tumor tissue was 0.62 and ranged from 0.22 to 0.91. With a threshold PSR of 0.91, sensitivity and specificity of TYR PET were 82% and 100%, respectively. The predictive value of a PSR > 0.91 for having viable tumor after HILP was 100%, whereas the predictive value of a PSR < or = 0.91 for having nonviable tumor tissue after HILP was 75%. The 2 patients in the pPR groups with a PSR < 0.91 showed microscopic islets of tumor cells surrounded by extensive necrosis on pathological examination. CONCLUSION: Based on the calculated PSR after HILP, TYR PET gave a good indication of the pathological outcome. Inflammatory tissue after treatment did not interfere with viable tumor on the images, suggesting that it may be worthwhile to pursue TYR PET in other therapy evaluation settings. (+info)
An intramembrane modulator of the ErbB2 receptor tyrosine kinase that potentiates neuregulin signaling.
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The ErbB2 receptor tyrosine kinase plays a critical role in a variety of developmental processes, and its aberrant activation may contribute to the progression of some breast and ovarian tumors. ASGP2, a transmembrane glycoprotein found on the surface of the highly metastatic ascites 13762 rat mammary adenocarcinoma cell line, is constitutively associated with ErbB2 in these cells and in mammary tissue from pregnant rats. Expression studies indicate that ASGP2 interacts directly and specifically with ErbB2 through one of its epidermal growth factor-like domains and that the co-expression of the two proteins in the same cell dramatically facilitates their direct stable interaction. Ectopic expression of ASGP2 in human melanoma tumor cells potentiates the response of endogenous ErbB2 to the neuregulin-1 growth factor. These observations point to a novel intramembrane mechanism for the modulation of receptor tyrosine kinase activity. (+info)
Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Involvement of Jak2 in the stimulation of phosphatidylinositol 3-kinase.
(11/13395)
Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates many of the biological activities of human neutrophils. The signaling pathways via which these effects are mediated are not fully understood. We have shown previously that GM-CSF treatment of human neutrophils activates the Janus kinase/signal transducers and activators of transcription (Jak/STAT) pathway and, more specifically, Jak2, STAT3, and STAT5B in neutrophils. GM-CSF also stimulates the activity of the phosphatidylinositol 3-kinase (PI3-kinase) in a tyrosine kinase-dependent manner. Here we report that pretreating the cells with a Jak2 inhibitor (AG-490) abolishes tyrosine phosphorylation of the p85 subunit of PI3-kinase induced by GM-CSF. Furthermore, p85 was found to associate with Jak2, but not with Lyn, in stimulated cells in situ and with its autophosphorylated form in vitro; however, Jak2 did not bind to either of the two Src homology 2 (SH2) domains of the p85 subunit of PI3-kinase. Although STAT5B bound to the carboxyl-terminal SH2 domain of p85, it was absent from the complex containing PI3-kinase and Jak2. These results suggest that stimulation of the activity of PI3-kinase induced by GM-CSF is mediated by Jak2 and that the association between Jak2 and p85 depends on an adaptor protein yet to be identified. (+info)
gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization.
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We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis. (+info)
Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction.
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Fibronectin (FN) is comprised of multiple isoforms arising from alternative splicing of a single gene transcript. One of the alternatively spliced segments, EDA, is expressed prominently in embryonic development, malignant transformation, and wound healing. We showed previously that EDA+ FN was more potent than EDA- FN in promoting cell spreading and cell migration because of its enhanced binding affinity to integrin alpha5beta1 (Manabe, R., Oh-e, N., Maeda, T., Fukuda, T., and Sekiguchi, K. (1997) J. Cell Biol. 139, 295-307). In this study, we compared the cell cycle progression and its associated signal transduction events induced by FN isoforms with or without the EDA segment to examine whether the EDA segment modulates the cell proliferative potential of FN. We found that EDA+ FN was more potent than EDA- FN in inducing G1-S phase transition. Inclusion of the EDA segment potentiated the ability of FN to induce expression of cyclin D1, hyperphosphorylation of pRb, and activation of mitogen-activated protein kinase extracellular signal regulated kinase 2 (ERK2). EDA+ FN was also more potent than EDA- FN in promoting FN-mediated tyrosine phosphorylation of p130(Cas), but not focal adhesion kinase, which occurred in parallel with the activation of ERK2, suggesting that p130(Cas) may be involved in activation of ERK2. These results indicated that alternative splicing at the EDA region is a novel mechanism that promotes FN-induced cell cycle progression through up-regulation of integrin-mediated mitogenic signal transduction. (+info)
Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets.
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Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets. (+info)
Control of ketogenesis from amino acids. IV. Tissue specificity in oxidation of leucine, tyrosine, and lysine.
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In vitro and in vivo studies were made on the tissue specificity of oxidation of the ketogenic amino acids, leucine, tyrosine, and lysine. In in vitro studies the abilities of slices of various tissues of rats to form 14CO2 from 14C-amino acids were examined. With liver, but not kidney slices, addition of alpha-ketoglutarate was required for the maximum activities with these amino acids. Among the various tissues tested, kidney had the highest activity for lysine oxidation, followed by liver; other tissues showed very low activity. Kidney also had the highest activity for leucine oxidation, followed by diaphragm; liver and adipose tissue had lower activities. Liver had the highest activity for tyrosine oxidation, but kidney also showed considerable activity; other tissues had negligible activity. In in vivo studies the blood flow through the liver or kidney was stopped by ligation of the blood vessels. Then labeled amino acids were injected and recovery of radioactivity in respiratory 14CO2 was measured. In contrast to results with slices, no difference was found in the respiratory 14CO2 when the renal blood vessels were or were not ligated. On the contrary ligation of the hepatic vessels suppressed the oxidations of lysine and tyrosine completely and that of leucine partially. Thus in vivo, lysine and tyrosine seem to be metabolized mainly in the liver, whereas leucine is metabolized mostly in extrahepatic tissues and partly in liver. Use of tissue slices seems to be of only limited value in elucidating the metabolisms of these amino acids. (+info)
Changes in protein tyrosine phosphorylation in the rat brain after cerebral ischemia in a model of ischemic tolerance.
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A brief period of sublethal cerebral ischemia, followed by several days of recovery, renders the brain resistant to a subsequent lethal ischemic insult, a phenomenon termed ischemic preconditioning or tolerance. Ischemic tolerance was established in the rat two-vessel occlusion model of ischemia, induced by occlusion of both carotid arteries in combination with hypotension. Ischemic preconditioning (3 minutes) provided maximal neuroprotection when induced 2 days prior to a lethal ischemic insult of 9-minute duration. Neuroprotection persisted for at least 8 weeks. Since neurotransmission has been implicated in ischemic cell death, the effect of ischemic preconditioning on tyrosine phosphorylation of proteins and on the levels of glutamate receptor subunits in hippocampus and neocortex was studied. Regional levels of tyrosine phosphorylation of proteins in general and the N-methyl-D-aspartate receptor subunit NR2 in particular are markedly enhanced after ischemia in nonconditioned brains, in both the synaptosomal fraction and the whole-tissue homogenate of rat neocortex and hippocampus, but recover to control levels only in the preconditioned brain. Ischemic preconditioning selectively induces a decrease in the levels of the NR2A and NR2B subunits and a modest decrease in the levels of NR1 subunit proteins in the synaptosomal fraction of the neocortex but not hippocampus after the second lethal ischemia. It was concluded that ischemic preconditioning prevents a persistent change in cell signaling as evidenced by the tyrosine phosphorylation of proteins after the second lethal ischemic insult, which may abrogate the activation of detrimental cellular processes leading to cell death. (+info)