Selective Protein Tyrosine Kinase Inhibitor Research Articles

Therapeutic effects of dasatinib in mouse model of multiple sclerosis

Background: Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS). EAE is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. Dasatinib (Sprycel) is a selective protein tyrosine kinase inhibitor with immunomodulatory properties that abrogates multiple signal transduction pathways in immune cells. In the present research, our aim was to test the therapeutic efficacy of dasatinib in experimental model of MS.Methods: We performed EAE induction in female C57BL/6 mice by myelin oligodendrocyte glycoprotein35–55 (MOG35–55) in Complete Freund’s Adjuvant (CFA) emulsion, and used dasatinib for the treatment of EAE. During the course of study, clinical evaluation was assessed, and on day 21 post-immunization blood samples were taken from the heart of mice for tumor necrosis factor-alpha (TNF-α), nitric oxide (NO) and antioxidants capacity evaluation. The mice were sacrificed and brains and cerebellums of mice were removed for histological analysis. Also for in vitro analysis, we used C6 astrocytoma cell line to evaluate the inhibitory effects of dasatinib in cell proliferation and matrix metalloproteinase-2 (MMP-2) activity.Results: Our findings demonstrated that dasatinib had beneficial effects on EAE by lower incidence, attenuation in the severity and a delay in the onset of disease. The serum level of NO and TNF-α in dasatinib treated mice was significantly lower than control mice. In vitro, dasatinib inhibited cell proliferation and MMP-2 activity.Conclusion: Dasatinib with its potential therapeutic effects and immunomodulatory properties may be recommended, after additional necessary tests and trials, for the treatment of MS.

Imatinib Mesylate: An Innovation in Treatment of Autoimmune Diseases

Imatinib mesylate is a selective protein tyrosine kinase inhibitor, which can inhibit BCR/Abl, PDGF-R, c-KIT, c-fms, TCR/Abl, Lck, FLT-3 and MAPKs activities on various cell types. On immune system, imatinib has antiproliferative activity and immunomodulatory effects in lymphocytes, macrophages, mast cells and dendritic cells with abrogating multiple signal transduction pathways involved in pathogenesis of autoimmune diseases e.g. inhibiting IFN-γ, TNF-α, IL-1β and IL-17 pro-inflammatory cytokines and MMPs secretion. To date, the efficacy of imatinib in numerous animal model of autoimmune diseases (rheumatoid arthritis, multiple sclerosis, autoimmune diabetes and glomerulonephritis) has been demonstrated, but application of this drug in human autoimmune diseases should be tested in future clinical trials. This review provides an update on the use of tyrosine kinase inhibitor imatinib mesylate in treatment of autoimmune diseases and its related recent patents that could be developed as a novel and available therapy for the management of the autoimmunity improvement.

Is exon mutation analysis needed for adjuvant treatment of gastrointestinal stromal tumor?

Gastrointestinal stromal tumors (GISTs) are the most common soft tissue sarcoma of the gastrointestinal tract, resulting from an activating mutation of stem cell factor receptor (KIT), and an activating mutation of the homologous platelet-derived growth factor receptor alpha (PDGFRA) kinase. Most GISTs (90%-95%) are KIT-positive. About 5% of GISTs are truly negative for KIT expression. GISTs have been documented to resistant conventional chemotherapeutics. Due to the KIT activation that occurs in the majority of the cases, KIT inhibition is the primary treatment approach in the adjuvant treatment of metastatic GISTs. Imatinib mesylate is an oral agent that is a selective protein tyrosine kinase inhibitor of the KIT protein tyrosine kinase, and it has demonstrated clinical benefit and objective tumor responses in most GIST patients in phase II and III trials. The presence and the type of KIT or PDGFRA mutation are predictive of response to imatinib therapy in patients with advanced and metastatic disease. Molecular analysis in phase I-II trials revealed significant differences in objective response, progression-free survival, and overall survival between GISTs with different kinase mutations. The aim of this letter is to touch on the need for exon mutation analysis for adjuvant treatment with imatinib in GIST patients.

CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway

Abstract▪2447▪This icon denotes a clinically relevant abstractWe examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants.RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands.Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells.CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Fibroblasts Expressing PDGF-Receptor-Alpha Diminish During Alveolar Septal Thinning in Mice

In mice, secondary alveolar septal formation primarily occurs during a brief postnatal period and is accompanied by transient expansion of the interstitial lung fibroblast (LF) population. PDGF-A, which solely signals through PDGF-receptor-alpha (PDGF-Rα), is required for expansion, but the receptor's relevant downstream targets remain incompletely defined. We have evaluated the proliferation, apoptosis, and differential response to the selective protein tyrosine kinase inhibitor, imatinib, by pdgfrα-expressing LF (pdgfrα-LF) and compared them with their nonexpressing LF counterparts. Our objective was to determine whether diminished signaling through PDGF-Rα-mediated pathways regulates the decline in myofibroblasts, which accompanies septal thinning and ensures more efficient alveolar gas exchange. Using quantitative stereology and flow cytometry at postnatal d 12 and 14, we observed that imatinib caused a selective suppression of proliferation and an increase in apoptosis. The number of the alpha smooth muscle actin (αSMA) producing pdgfrα-LF was also reduced. Using cultures of neonatal mouse LF, we showed that imatinib did not suppress PDGF-Rα gene expression but reduced PDGF-A-mediated Akt phosphorylation, potentially explaining the increase in apoptosis. Our findings are relevant to bronchopulmonary dysplasia in which positive pressure ventilation interferes with myofibroblast depletion, septal thinning, and capillary maturation.

Masitinib in the treatment of active rheumatoid arthritis: results of a multicentre, open-label, dose-ranging, phase 2a study

IntroductionSince current treatment options for patients suffering from active rheumatoid arthritis (RA) remain inadequate, especially for those unresponsive to disease-modifying antirheumatic drugs (DMARDs), new and improved medication is needed. This study evaluates the safety and efficacy of masitinib (AB1010), a potent and selective protein tyrosine kinase inhibitor of c-KIT, in the monotherapy treatment of DMARD-refractory RA.MethodsThis was a multicentre, uncontrolled, open-label, randomised, dose-ranging, phase 2a trial. Masitinib was administered orally to 43 patients who had inadequate response to DMARDs, at initial randomised dosing levels of 3 and 6 mg/kg per day over a 12-week period. Dose adjustment was permitted based upon tolerability and response criteria. Efficacy was assessed via American College of Rheumatology 20%/50%/70% improvement criteria (ACR20/50/70) responses, disease activity score using 28 joint counts (DAS28), index of improvement in RA (ACRn) and C-reactive protein (CRP) improvement, relative to baseline at week 12.ResultsImprovement was observed in all efficacy endpoints, including ACR20/50/70 scores of 54%, 26% and 8%, respectively, and a reduction in CRP level by greater than 50% for approximately half the population. This improvement was sustainable throughout an extension phase (> 84 weeks) and was also independent of initial DMARD resistance (anti-tumour necrosis factor-alpha and/or methotrexate). A relatively high patient withdrawal rate (37%) required the use of last observation carried forward (LOCF) data imputation. Incidence of adverse events was high (95%), although the majority were of mild or moderate severity with a considerable decline in frequency observed after 12 weeks of treatment. Two nonfatal serious adverse events were reported. Dose-response analyses tentatively indicate that an initial dosing level of 6.0 mg/kg per day administered orally in two daily intakes is the most appropriate, based upon potency and tolerability trends.ConclusionsTreatment with masitinib improved DMARD-refractory active RA. Following an initial high incidence of mostly mild to moderate side effects during the first 12 weeks of treatment, masitinib appears to be generally well tolerated. This, together with evidence of a sustainable efficacy response, suggests that masitinib is suitable for long-term treatment regimens. Since this was the first study of masitinib in a nononcologic pathology, the relatively high patient withdrawal rate observed can be partly attributed to a highly cautious response to adverse events. There is sufficient compelling evidence to warrant further placebo-controlled investigation.Trial registrationClinicalTrials.gov NCT00831922.

Structural Characterization of Novel Adenine Dinucleotide Phosphate Conjugates of Imatinib in Incubations with Rat and Human Liver Microsomes

Imatinib, a potent and selective protein tyrosine kinase inhibitor, has been approved for the treatment of chronic myelogenous leukemia and metastatic and unresectable malignant gastrointestinal stromal tumors. In vitro metabolism of imatinib was investigated in rat and human liver microsomes. Besides several oxidative metabolites and an N-desmethyl metabolite, as previous reported, a novel metabolite with a mass addition of 621 atomic mass units to the parent was detected as the major metabolite in the incubations with rat liver microsomes, using NADPH as a cofactor. The analysis of MS(2) and MS(n) data revealed that this metabolite corresponded to adenine dinucleotide phosphate (ADP+) conjugate of imatinib. The ADP+ adduct was scaled up from rat liver microsomal incubations and isolated for NMR analysis. NMR data confirmed and conclusively showed the conjugation had occurred between the pyridine nitrogen of imatinib to the ribose ring of ADP+ moiety. The formation of this adduct was enzymatic and required NADP+ as a reactant. In addition, ADP+ adducts of imatinib N-oxide and desmethyl imatinib were also detected as minor metabolites in the incubations with rat liver microsomes. In contrast, only trace levels of ADP+ adducts of imatinib and desmethyl imatinib were detected in the incubations with human liver microsomes. Imatinib-ADP+ adducts have been observed only in in vitro studies to date. The physiological role of these adducts is not clear, nor is their in vivo relevance.

Modulation of the Brain Distribution of Imatinib and its Metabolites in Mice by Valspodar, Zosuquidar and Elacridar

The selective protein tyrosine kinase inhibitor, imatinib, inhibits the growth of glioma cells in preclinical models, but its poor brain distribution limits its efficacy in patients. P-glycoprotein (P-gp, rodent Mdr1a/1b or Abcb1a/1b) and Breast cancer resistance protein (rodent Bcrp1 or Abcg2) were suggested to restrict the delivery of imatinib to the brain. This study evaluates the effect of administering selective inhibitors of these transporters together with imatinib on the systemic and cerebral disposition of imatinib in mice. Wild-type, Mdr1a/1b(-/-) and Bcrp1(-/-) mice were given imatinib intravenously, either alone, or with valspodar, zosuquidar (P-gp inhibitors), or elacridar (a P-gp and Bcrp1 inhibitor). The blood and brain concentrations of [(14)C]imatinib and its radioactive metabolites were determined. The blockade of P-gp by valspodar or zosuquidar (>3 mg/kg) enhanced the brain uptake of imatinib ( approximately 4-fold) in wild-type mice, but not that of its metabolites. Blockade of both P-gp and Bcrp1 by elacridar (>3 mg/kg) produced significantly greater brain penetration of imatinib (9.3-fold) and its metabolites (2.8-fold). In contrast, only the lack of P-gp enhanced imatinib brain penetration (6.4-fold) in knockout mice. These results of brain uptake correlated reasonably well with those obtained previously by our group using in situ brain perfusion. Imatinib and its metabolites penetrate into the brain poorly and their penetration is limited by P-gp and (probably) Bcrp1. Administering imatinib together with P-gp (and Bcrp1) transporter inhibitors such as elacridar may improve the delivery of imatinib to the brain, making it potentially more effective against malignant gliomas.

Targeted therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade

The outcome for patients with lung cancer has not changed significantly for more than two decades. Several studies show that the overexpression of vascular endothelial growth factor (VEGF)/vascular permeability factor and epidermal growth factor (EGF) and their receptors correlates with the clinical outcome for lung cancer patients. However, clinical trials of agents that target either of these pathways alone have been disappointing. We hypothesize that targeting both the tumor and its vasculature by simultaneously blocking the VEGFR and EGFR pathways will improve the treatment of locoregional lung cancer. Human lung cancer specimens were first examined for the activation of VEGF receptor 2 (VEGFR2) and EGF receptor (EGFR) for tumor and tumor-associated endothelial cells, and both were found to be activated. The effects of ZD6474 (ZACTIMA), a small-molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were then studied in vitro using human lung cancer and microvascular endothelial cells. In vitro, ZD6474 inhibited EGFR, VEGFR2, mitogen-activated protein kinase and Akt phosphorylation, EGF- and VEGF-induced proliferation, and endothelial cell tube formation and also induced apoptosis. ZD6474 was further studied in vivo using an orthotopic mouse model of non-small cell lung cancer using NCI-H441 human lung adenocarcinoma cells. The inhibition of both VEGFR2 and EGFR signaling pathways by ZD6474 resulted in profound antiangiogenic, antivascular, and antitumor effects. These results provide a basis for the development of clinical strategies for the combination of selective protein tyrosine kinase inhibitors that block both EGFR and VEGFR signaling as part of the management of locally advanced lung cancer.

Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: evidence for dual regulation by Src and EGFR kinases.

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (ICl.vol) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO4 −3). ICl.vol evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC50 = 22.4 μM); 100 μM genistein stimulated ICl.vol by 122.4 ± 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 150 μM) and tamoxifen (20 μM), blockers of ICl.vol. Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl− current in 1T. In contrast to the stimulatory effects of genistein, 100 μM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited ICl.vol by 38.2 ± 4.9% and 40.9 ± 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter ICl.vol. In addition, the PTP inhibitor VO4 −3 (1 mM) reduced ICl.vol by 53.5 ± 4.5% (IC50 = 249.6 μM). Pretreatment with VO4 −3 antagonized genistein-induced augmentation and A23- or A25-induced suppression of ICl.vol. Furthermore, the selective Src-family PTK inhibitor PP2 (5 μM) stimulated ICl.vol, mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 μM) reduced ICl.vol, mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO4 −3. The results suggest that ICl.vol is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on ICl.vol, and multiple target proteins are likely to be involved.

EFFECT OF TYROSINE KINASE INHIBITORS ON TYROSINE PHOSPHORYLATION AND MOTILITY PARAMETERS IN HUMAN SPERM

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37°C in 5% CO 2, with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. Thisinhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

Involvement of spinal tyrosine kinase in inflammatory and N-methyl- d-aspartate-induced hyperalgesia in rats

Phosphorylation of a subunit of N-methyl- d-aspartate (NMDA) receptor by protein tyrosine kinase (PTK) Src or Trk is known to enhance its channel activity. We examined whether a spinally administered selective PTK inhibitor, lavendustin A, which has high affinity for Src and Trk tyrosine kinases, could influence the development and maintenance of inflammatory hyperalgesia or NMDA-induced hyperalgesia. Inflammation was induced by injection of a mixture of carrageenan and kaolin into the tail base of rats. In another group of rats, hyperalgesia was induced by intrathecal administration of NMDA. Intrathecal administration of lavendustin A (1.0 μg) or NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cycloheptane-5,10-iminemaleate, MK-801 (3.0 μg) before injection of a mixture of carrageenan and kaolin or after the development of inflammation inhibited carrageenan–kaolin-induced mechanical hyperalgesia. Intrathecal injection of 1.0 μg NMDA produced thermal and mechanical hyperalgesia. Co-administration of 1.0 μg lavendustin A with NMDA significantly reduced the duration of spontaneous pain behaviour and inhibited NMDA-induced hyperalgesia. Lavendustin A itself did not cause any sedation, motor impairment or analgesia. Our results suggest that inhibition of PTK could be therapeutically effective as an analgesic in some NMDA receptor-mediated hyperalgesic states.

Effect of tyrosine kinase inhibitors on tyrosine phosphorylation and motility parameters in human sperm.

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37 degrees C in 5% CO(2), with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. This inhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

Extracellular matrix regulation of drug resistance in small-cell lung cancer

Tumour recurrence following chemotherapy remains a major obstacle to the cure of many cancers. This is exemplified by small-cell lung cancer (SCLC). Host-tumour interactions are central to tumour survival and proliferation. We hypothesized that a factor(s) within the local environment of SCLC cells could provide a survival signal or block a death signal, thereby accounting for the protection of SCLC cells from chemotherapy-induced apoptosis. Here we review recent work undertaken in our laboratory addressing this issue. We have shown that, in vivo, SCLC cells are surrounded by an extensive stroma of extracellular matrix (ECM) at both primary and metastatic sites which contains, among other proteins, fibronectin, laminin and collagen IV. Furthermore, adhesion of SCLC cells to fibronectin, laminin and collagen IV through beta1 integrins enhances tumorigenicity and confers resistance to apoptosis induced by standard chemotherapeutic agents, including etoposide, cis-platinum and adriamycin. Adhesion to ECM proteins stimulated protein tyrosine kinase (PTK) activity in both untreated and etoposide-treated cells. This effect could be completely blocked by a selective PTK inhibitor or by a function-blocking beta1 integrin antibody. PTK activation was found to block chemotherapy-induced activation of the death protease caspase-3 and, hence, apoptosis. Adhesion to ECM or treatment with a PTK inhibitor did not affect etoposide inhibition of topoisomerase II. Thus adhesion to ECM through beta1 integrins protects SCLC cells from chemotherapy-induced caspase-3 activation and apoptosis by activating PTK signalling downstream of DNA damage. Survival of tumour cells attached to ECM within this microenvironment could explain the local recurrence of SCLC and other tumours that is often seen clinically after chemotherapy.

PROTEIN TYROSINE KINASE‐DEPENDENT REGULATION OF ADENYLATE CYCLASE AND PHOSPHATIDYLINOSITOL 3‐KINASE ACTIVATES THE EXPRESSION OF GLIAL FIBRILLARY ACIDIC PROTEIN UPON INDUCTION OF DIFFERENTIATION IN RAT C6 GLIOMA

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

Biomolecular Basis for Cold-Induced Contraction of Coronary Arteries in the Newborn Lamb: Implication in Myocardial Dysfunction after Hypothermic Cardiopulmonary Bypass ♦ 98

Introduction: Despite improved overall outcome after open heart surgery, early myocardial dysfunction remains a problem in the infant and the neonate in the post hypothermic cardiopulmonary bypass recovery period. We hypothesize that a vasoconstriction of the conductance coronary arteries (CA) occurs when exposed to cold, and that this cold induced contraction involves protein tyrosine kinase (PTK)-/protein tyrosine phosphatase (PTP)-dependent signal transduction pathways. Methods: Newborn lamb 3-mm CA ring segments (1mm diameter) were studied in tissue bath (Krebs buffer, 21%O2, 5%CO2) for isometric contraction during a 2-hour exposure to hypothermia (7° & 17°C). In parallel, 8-mm CA ring segments were evaluated for changes in protein tyrosine phosphorylation using standard electrophoretic and immunoblotting techniques (monoclonal anti-phosphotyrosine antibody & chemiluminescence detection). Na-orthovanadate (SOV), a potent PTP inhibitor, and genestein (Gen), a non selective PTK inhibitor, were used separately and in combination to evaluate their effect on contractile behavior and tyrosine phosphorylation of CA during cooling (7° or 17°C) vs. 37°C. Results: Tissue bath cooling to 17°C resulted in sustained contraction (25% KCl, n=4), reversible upon rewarming. CA segments subjected to 7°C cooling demonstrated a transitory contraction at≈17°C, more prominent during rewarming (5-15% KCl). Cold-induced contraction was abolished in the presence of Gen and significantly enhanced(2-fold) in the presence of SOV, the latter being partially inhibited in the presence of Gen. There was increased tyrosine phosphorylation of 25, 30-33 and 100-110 kDa proteins detected at 17°C compared to 37°C (n=4), similarly enhanced by SOV and abolished by Gen.

Herbimycin A accelerates the induction of apoptosis following etoposide treatment or gamma-irradiation of bcr/abl-positive leukaemia cells.

Philadelphia chromosome (Ph)-positive leukaemia cells express the chimeric bcr/abl oncoprotein, whose deregulated protein tyrosine kinase (PTK) activity antagonizes the induction of apoptosis by DNA damaging agents. Treatment of Ph-positive K562, TOM 1 and KCL-22 cells with etoposide for 2d induced cytosolic vacuolation, but not nuclear condensation or DNA fragmentation. The bcr/abl kinase-selective inhibitor herbimycin A increased the induction of nuclear apoptosis by etoposide or gamma-radiation. The concentration of herbimycin required to synergize with etoposide was similar to that required to decrease the level of tyrosine phosphorylated proteins or of the protein tyrosine kinase activity of anti-abl immune complexes in K562 cells. The ability of herbimycin A to sensitize K562, TOM 1 or KCL-22 cells to apoptosis induction correlated with its ability to decrease the cellular content of phosphotyrosyl proteins in these Philadelphia-positive lines. Enhancement of nuclear apoptosis by herbimycin was not attributable to downregulation of the bcl-2 or bcl-XL anti-apoptotic proteins. In contrast, herbimycin protected Philadelphia-negative HL60 cells from apoptosis induction by etoposide and did not affect killing of NC37 and CEM cells. The data suggest that the induction of apoptosis is blocked in cells expressing the bcr/abl oncoprotein and that herbimycin A increases induction of programmed cell death following DNA damage. Selective PTK inhibitors may therefore be of value in securing the genetic death of Ph-positive leukaemia cells.

Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation. However, co-administration of these two peptides to a single sponge together caused a significant increase in the rate of 133Xe clearance and angiogenesis similar to that seen with the high dose of VEGF165 (250 ng) acting alone. This VEGF/bFGF neovascular response was also blocked by daily co-administration of lavendustin A (10 jig),suramin (3 mg) or a monoclonal anti-bFGF antibody (DG2, I jig), but not lavendustin B (10 g).6 These results suggest that selective inhibition of PTK could have therapeutic potential in angiogenic diseases where VEGF plays a dominant role. Furthermore, blockade of the angiogenic activity of VEGF and VEGF,/bFGF by suramin reveals an alternative strategy in angio suppression.

Effect of tyrphostin on cell growth and tyrosine kinase activity of epidermal growth factor receptor in human gliomas

The effects of tyrphostin, a selective protein tyrosine kinase inhibitor, on epidermal growth factor (EGF)-stimulated cell growth and EGF-receptor tyrosine kinase activity were studied in four human glioma cell lines. Stimulation by EGF induced variable enhancements of cell growth as well as tyrosine phosphorylation of EGF receptor and intracellular target proteins in all glioma cell lines. The level of immunoreactive EGF receptor detected with antibodies against extra- and intracellular domains was moderate in all four glioma cell lines, but markedly decreased with the latter antibody in two glioma cell lines. This variation was associated with considerable reduction of the EGF-stimulated tyrosine autophosphorylation level. Tyrphostin inhibited dose-dependently the EGF-stimulated cell growth and tyrosine autophosphorylation in all glioma cell lines, and the optimum time for the maximum inhibitory effect on tyrosine autophosphorylation was 12 to 18 hours after treatment with tyrphostin. The antiproliferative activity of tyrphostin nearly correlated quantitatively with its potency as an inhibitor of the EGF-stimulated EGF receptor tyrosine kinase activity. Tyrphostin had no significant effect on the immunoreactive EGF receptor levels, on the affinity constants and numbers of EGF receptor, or on the down-regulation and specific internalization of EGF receptor in any glioma cell line, suggesting that the effects of tyrphostin are not likely to be the results of reduction in EGF receptor and EGF binding capacity. In addition, the serum-stimulated cell growth was also inhibited dose-dependently by higher concentrations of tyrphostin in all glioma cell lines. It might be suggested, therefore, that tyrphostin inhibits EGF-stimulated cell growth by a specific suppression of EGF receptor tyrosine kinase activity, and at higher concentrations there appears to be some degree of either nonspecific inhibition or inhibition of serum-stimulated protein tyrosine kinase activity to induce the cell growth inhibition of gliomas.