The kinase inhibitor dasatinib induces apoptosis in chronic lymphocytic leukemia cells in vitro with preference for a subgroup of patients with unmutated IgVH genes. Veldurthy, Aditya, et al. Blood, 112: 1443-52 (2008)
2008
| | 18550857
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Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor signaling. Alexandre K Rouquette-Jazdanian,Claudette Pelassy,Jean-Philippe Breittmayer,Claude Aussel Cellular signalling
18
2006
Show Abstract
T lymphocytes contain two kinetic pools of cholesterol extractable with methyl-beta-cyclodextrin (m-beta-CD): a fast pool (31.5%, t1/2=17 s) and a slow pool (68.5%, t1/2=15 min). Purification of detergent-resistant membranes (DRMs) shows that the fast pool corresponds to buoyant cholesterol. Cholesterol extraction of the fast pool (i.e. cholesterol from rafts) still allows the buoyancy of signaling proteins and their phosphorylation under CD3 stimulation. Cholesterol depletion of the slow pool (i.e. cholesterol from membranes other than rafts) is accompanied by the extraction of the whole raft followed by the inhibition of CD3-induced tyrosine-phosphorylations. Cholesterol oxidase (COase) allows a specific oxidation of raft cholesterol into cholestenone. Cholestenone leaves the DRMs and accumulates as Triton X-100-soluble material. Specific cholesterol-rich raft disruption by COase does not inhibit the activation of either Jurkat cells or T CD4+ lymphocytes. Our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that a cholesterol-poor subtype of rafts is involved in signal transmission via the TCR. | | 15925486
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Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies. Banerji, U; O'Donnell, A; Scurr, M; Pacey, S; Stapleton, S; Asad, Y; Simmons, L; Maloney, A; Raynaud, F; Campbell, M; Walton, M; Lakhani, S; Kaye, S; Workman, P; Judson, I Journal of clinical oncology : official journal of the American Society of Clinical Oncology
23
4152-61
2005
Show Abstract
To study the toxicity and pharmacokinetic-pharmacodynamic profile of 17-allylamino, 17- demethoxygeldanamycin (17-AAG) and to recommend a dose for phase II trials.This was a phase I study examining a once-weekly dosing schedule of 17-AAG. Thirty patients with advanced malignancies were treated.The highest dose level reached was 450 mg/m(2)/week. The dose-limiting toxicities (DLTs) encountered were grade 3 diarrhea in three patients (one at 320 mg/m(2)/week and two at 450 mg/m(2)/week) and grade 3 to 4 hepatotoxicity (AST/ALT) in one patient at 450 mg/m(2)/week. Two of nine DLTs were at the highest dose level. Two patients with metastatic melanoma had stable disease and were treated for 15 and 41 months, respectively. The dose versus area under the curve-relationship for 17-AAG was linear (r(2) = .71) over the dose range 10 to 450 mg/m(2)/week, with peak plasma concentrations of 8,998 mug/L (standard deviation, 2,881) at the highest dose level. After the demonstration of pharmacodynamic changes in peripheral blood leukocytes, pre- and 24 hours post-treatment, tumor biopsies were performed and demonstrated target inhibition (c-RAF-1 inhibition in four of six patients, CDK4 depletion in eight of nine patients and HSP70 induction in eight of nine patients) at the dose levels 320 and 450 mg/m(2)/week. It was not possible to reproducibly demonstrate these changes in biopsies taken 5 days after treatment.It has been possible to demonstrate that 17-AAG exhibits a tolerable toxicity profile with therapeutic plasma concentrations and target inhibition for 24 hours after treatment and some indications of clinical activity at the dose level 450 mg/m(2)/week. We recommend this dose for phase II clinical trials. | | 15961763
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Pharmacokinetic-pharmacodynamic relationships for the heat shock protein 90 molecular chaperone inhibitor 17-allylamino, 17-demethoxygeldanamycin in human ovarian cancer xenograft models. Banerji, U; Walton, M; Raynaud, F; Grimshaw, R; Kelland, L; Valenti, M; Judson, I; Workman, P Clinical cancer research : an official journal of the American Association for Cancer Research
11
7023-32
2005
Show Abstract
To establish the pharmacokinetic and pharmacodynamic profile of the heat shock protein 90 (HSP90) inhibitor 17-allylamino, 17-demethoxygeldanamycin (17-AAG) in ovarian cancer xenograft models.The effects of 17-AAG on growth inhibition and the expression of pharmacodynamic biomarkers c-RAF-1, CDK4, and HSP70 were studied in human ovarian cancer cell lines A2780 and CH1. Corresponding experiments were conducted with established tumor xenografts. The variability and specificity of pharmacodynamic markers in human peripheral blood lymphocytes (PBL) were studied.The IC50 values of 17-AAG in A2780 and CH1 cells were 18.3 nmol/L (SD, 2.3) and 410.1 nmol/L (SD, 9.4), respectively. Pharmacodynamic changes indicative of HSP90 inhibition were demonstrable at greater than or equal the IC50 concentration in both cell lines. Xenograft experiments confirmed tumor growth inhibition in vivo. Peak concentrations of 17-AAG achieved in A2780 and CH1 tumors were 15.6 and 16.5 micromol/L, respectively, and there was no significant difference between day 1 and 11 pharmacokinetic profiles. Reversible changes in pharmacodynamic biomarkers were shown in tumor and murine PBLs in both xenograft models. Expression of pharmacodynamic markers varied between human PBLs from different human volunteers but not within the same individual. Pharmacodynamic biomarker changes consistent with HSP90 inhibition were shown in human PBLs exposed ex vivo to 17-AAG but not to selected cytotoxic drugs.Pharmacokinetic-pharmacodynamic relationships were established for 17-AAG. This information formed the basis of a pharmacokinetic-pharmacodynamic-driven phase I trial. | | 16203796
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Volume expansion stimulates p72(syk) and p56(lyn) in skate erythrocytes. Musch, M W, et al. J. Biol. Chem., 274: 7923-8 (1999)
1999
Show Abstract
Hypotonic volume expansion of skate erythrocytes rapidly stimulates the tyrosine phosphorylation of band 3, the membrane protein thought to mediate the osmotically sensitive taurine efflux. Skate erythrocytes possess numerous tyrosine kinases including p59fyn, p56lyn, pp60(src), and p72(syk), demonstrated by immune complex assays measuring autocatalytic kinase activity. Inclusion of the cytoplasmic domain of band 3 in this assay showed that only Syk and Lyn can directly phosphorylate the cytoplasmic domain of band 3. Upon cell volume expansion, Syk activity was increased as assessed by three different assays (immune complex assay measuring autophosphorylation, assay of the level of phosphotyrosine of the immunoprecipitated kinase, and assay of level of 32P in the kinase immunoprecipitated from cells prelabeled with 32PO4 and then volume-expanded). The tyrosine kinase Lyn was also stimulated by volume expansion, most notably when analyzed by the latter two methods. Volume expansion stimulated a large increase in the ability of Syk to phosphorylate band 3 at times that coincide with the stimulation of taurine flux. The stilbene piceatannol inhibited Syk preferentially over Lyn and other tyrosine kinases and inhibited volume-stimulated taurine efflux in a concentration-dependent manner similar to that for the inhibition of Syk. Two major phosphorylation peaks were detected in tryptic digests of cdb3 separated by reverse phase HPLC. Edman degradation demonstrated a phosphotyrosine in a YXXL motif. In conclusion, p72(syk) appears to be a strong candidate as a pivotal signal-transducing step in the volume-activated taurine efflux in skate red cells. The level of band-3 phosphorylation may be regulated, in addition, by a protein-tyrosine phosphatase of the 1B variety. | Kinase Assay | 10075687
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Association of p59(fyn) with the T lymphocyte costimulatory receptor CD2. Binding of the Fyn Src homology (SH) 3 domain is regulated by the Fyn SH2 domain Lin, H., et al J Biol Chem, 273:19914-21 (1998)
1998
| Immunoprecipitation | 9677430
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Changes in expression of signal transduction proteins in T lymphocytes of patients with leprosy Zea, A. H., et al Infect Immun, 66:499-504 (1998)
1998
| Immunoblotting (Western) | 9453602
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Zinc is essential for binding of p56(lck) to CD4 and CD8alpha. Lin, R S, et al. J. Biol. Chem., 273: 32878-82 (1998)
1998
Show Abstract
Binding of the protein tyrosine kinase p56(lck) to T-cell co-receptors CD4 and CD8alpha is necessary for T-lymphocyte development and activation. Association of p56(lck) with CD4 requires two conserved cysteine residues in the cytosolic domain of CD4 and two in the amino terminus of p56(lck), consistent with the notion that these four residues coordinate a single metal atom (1-5). Here we demonstrate that Zn2+ is essential for complex formation. In an in vitro binding reaction, Zn2+ mediates p56(lck) association with a glutathione S-transferase (GST) fusion protein containing the cytosolic domains of CD4 or CD8alpha; no other metals tested support binding. Treatment of preformed GST-CD4.p56(lck) dimers with the Zn2+ chelators 1,10-O-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid results in dissociation of GST-CD4 from p56(lck), consistent with the finding of Huse et al. (5) that Zn2+ is contained within similar complexes. Furthermore, we show that, within live cells, CD4.p56(lck) and CD8alpha.p56(lck) interactions occur in a zinc-dependent fashion. Specifically, pretreatment of the human Jurkat T-cell line with membrane permeable zinc chelators disrupts CD4.p56(lck) complexes, and treatment of COS cells co-expressing CD8alpha and p56(lck) with such chelators likewise leads to dissociation of CD8alpha.p56(lck) complexes. CD4. p56(lck) and CD8alpha.p56(lck) represent the first examples of intracellular proteins that require zinc as a bridge for heterodimerization. | Immunoprecipitation, Immunoblotting (Western) | 9830036
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Signaling through intercellular adhesion molecule 1 (ICAM-1) in a B cell lymphoma line. The activation of Lyn tyrosine kinase and the mitogen-activated protein kinase pathway Holland, J. and Owens, T. J Biol Chem, 272:9108-12 (1997)
1997
| Immunoprecipitation | 9083038
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Pattern of expression of five alternative transcripts of the lck gene in different hematopoietic malignancies: correlation of the level of lck messenger RNA I B with the immature phenotype of the malignancy. Rouer, E, et al. Cell Growth Differ., 5: 659-66 (1994)
1994
Show Abstract
The lck gene encodes a tyrosine protein kinase of the src family which is highly expressed in T-lymphocytes. Two widely separated promoters govern expression of the lck gene. We report in this study that alternative splicing between cryptic donor and acceptor sites in the 5' untranslated region of the transcripts initiated at the type I promoter give rise to three different type I lck mRNAs, I A, I B, and I C. Altogether with the types II A and II B reported previously, the lck transcription is thus characterized by five alternative transcripts. We further used the complementary DNA-polymerase chain reaction assay to describe the pattern of expression of these five lck transcripts in different hematopoietic cell lines and in blood or bone marrow samples from healthy donors and leukemic patients. We report that the transcript II A is by far the major transcript present both in human samples and in T-cell lines. The low lck expression in B-cell lines is characterized by the quite exclusive presence of the transcript I B. We show that hematopoietic diseases characterized by the presence of immature cells [acute myeloid leukemia (AML-0 and AML-1) and T- and B-cell acute lymphoid leukemia] exhibit a marked increase of the transcript I B. No significant difference from the normal pattern is observed in AML according to the differentiation stage (AML-2 to AML-5). A normal pattern of lck expression is restored in AML-0 and AML-1 patients at complete remission. | | 8086341
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