Role of basal calcium in the EGF activation of MAP kinases.
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The role of intracellular Ca2+ pools in the regulation of growth factor signal transduction pathways and mitogenesis is not well understood. We have examined the roles of basal and transiently mobilized Ca2+ in the regulation of MAP kinases by EGF. To assess the influence of Ca2+ transients we utilized Plcg1-/- and Plcg1+/+ mouse embryonic fibroblasts, while BAPTA/AM was employed to chelate total intracellular Ca2+ in the same cell lines. The MAP kinases erk-1, erk-2 and erk-5 exhibited similar patterns of activation in wild-type and Plcg1-/- cells treated with EGF. However, pretreatment with BAPTA/AM significantly increased and prolonged erk-1 and erk-2 activation in both cell types. In contrast, BAPTA/AM prevented the EGF activation of erk-5 in wild-type and Plcg1-/- cells. These data indicate that basal Ca2+, but not growth factor provoked Ca2+ transients, has a significant influence on the activation of these MAP kinases. AG1478, a specific EGF receptor kinase inhibitor, abolished the prolonged erk-1 and erk-2 activation produced by EGF in cells pretreated with BAPTA/AM. This indicates that the prolonged activation of erk-1 and erk-2 produced in the presence of BAPTA/AM requires continuous signaling from the EGF receptor kinase. (+info)
Carbachol-stimulated transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T(84) cells is mediated by intracellular ca(2+), PYK-2, and p60(src).
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Ca(2+)-dependent agonists, such as carbachol (CCh), stimulate epidermal growth factor receptor (EGFR) transactivation and mitogen-activated protein kinase activation in T(84) intestinal epithelial cells. This pathway constitutes an antisecretory mechanism by which CCh-stimulated chloride secretion is limited. Here, we investigated mechanisms underlying CCh-stimulated epidermal growth factor receptor (EGFR) transactivation. Thapsigargin (TG, 2 microM) stimulated EGFR and extracellular signal-regulated kinase (ERK) phosphorylation in T(84) cells. Inhibition of either EGFR or ERK activation, with tyrphostin AG1478 (1 microM) and PD 98059 (20 microM), respectively, potentiated chloride secretory responses to TG, as measured by changes in short-circuit current (I(sc)) across T(84) cells. CCh (100 microM) stimulated tyrosine phosphorylation and association of the Ca(2+)-dependent tyrosine kinase, PYK-2, with the EGFR, which was inhibited by the Ca(2+) chelator, BAPTA (20 microM). The calmodulin inhibitor, fluphenazine (50 microM) inhibited CCh-stimulated PYK-2 association with the EGFR and phosphorylation of EGFR and ERK. CCh also induced tyrosine phosphorylation of p60(src) and association of p60(src) with both PYK-2 and the EGFR. The Src family kinase inhibitor, PP2 (20 nM-20 microM) attenuated CCh-stimulated EGFR and ERK phosphorylation and potentiated chloride secretory responses to CCh. We conclude that CCh-stimulated transactivation of the EGFR is mediated by a pathway involving elevations in intracellular Ca(2+), calmodulin, PYK-2, and p60(src). This pathway represents a mechanism that limits CCh-stimulated chloride secretion across intestinal epithelia. (+info)
Clinical pharmacokinetic and in vitro combination studies of nolatrexed dihydrochloride (AG337, Thymitaq) and paclitaxel.
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A clinical study of nolatrexed dihydrochloride (AG337, Thymitaq) in combination with paclitaxel was performed. The aims were to optimize the schedule of administration and determine any pharmacokinetic (PK) interactions between the two drugs. In vitro combination studies were performed to assist with schedule optimization. Three patients were entered on each of three different schedules of administration of the two drugs: (1) paclitaxel 0-3 h, nolatrexed 24-144 h; (2) nolatrexed 0-120 h, paclitaxel 48-51 h; (3) nolatrexed 0-120 h, paclitaxel 126-129 h. Paclitaxel was administered at a dose of 80 mg m(-2) over 3 h and nolatrexed at a dose of 500 mg m(-2) day(-1) as a 120-h continuous intravenous infusion. Plasma concentrations of both drugs were determined by high performance liquid chromatography. In vitro growth inhibition studies using corresponding schedules were performed using two head and neck cancer cell lines. In both HNX14C and HNX22B cell lines, synergistic growth inhibition was observed on schedule 2, whereas schedules 1 and 3 demonstrated antagonistic effects. In the clinical study, there was no effect of schedule on the pharmacokinetics of nolatrexed. However, patients on schedules 1 and 3 had a higher clearance of paclitaxel (322-520 ml min(-1) m(-2)) than those on schedule 2 (165-238 ml min(-1) m(-2)). Peak plasma concentrations (1.66-1.93 vs. 0.86-1.32 microM) and areas under the curve (392-565 vs. 180-291 microM min(-1)) of paclitaxel were correspondingly higher on schedule 2. The pharmacokinetic interaction was confirmed by studies with human liver microsomes, nolatrexed being an inhibitor of the major routes of metabolism of paclitaxel. Toxicity was not schedule-dependent. Nolatrexed and paclitaxel may be safely given together when administered sequentially at the doses used in this study. Studies in vitro suggest some synergy, however, due to a pharmacokinetic interaction, paclitaxel doses should be reduced when administered during nolatrexed infusion. (+info)
Activation of epidermal growth factor receptor during corneal epithelial migration.
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PURPOSE: Epidermal growth factor (EGF) and related growth factors: transforming growth factor (TGF)-alpha, heprin-ding (HB)-EGF, and amphiregulin (AR), have been shown to stimulate events associated with epithelial wound repair. These growth factors function by binding to a common EGF receptor (EGFR), tyrosine kinase. We have used in vivo and organ culture wound-healing models to examine the kinetics and extent of EGFR activation during corneal epithelial wound repair and whether the epithelium itself produces EGFR ligands capable of stimulating the healing process. METHODS: In the in vivo model, 3-mm debridement wounds were made in rat corneas and allowed to heal in situ. Activation of EGFR was analyzed by 1) indirect immunofluorescence microscopy, 2) immunoprecipitation using anti-EGFR and anti-phosphotyrosine (anti-PT), and 3) binding-site localization using EGF-fluorescein isothiocyanate (FITC). Relative levels of mRNA for EGF, TGF-alpha, HB-EGF, and AR were determined using reverse transcription-polymerase chain reaction. To determine whether inhibiting EGFR activation slows epithelial migration, wounded corneas were allowed to heal in organ culture in the presence of tyrphostin AG1478 (0-50 microM), a specific inhibitor of EGFR kinase activity. RESULTS: In unwounded corneas, EGFR was localized in basal cells and appeared to be membranous. Within 1 hour after wounding, EGFR was no longer immunolocalized in the membranes of cells migrating into the wound area. EGF-FITC-binding assays indicated that EGFR ligands could penetrate all the way to the limbus. Immunoprecipitation showed that EGFR was phosphorylated on tyrosine residues within 30 minutes after wounding and that phosphorylation levels increased after wounding. Levels of mRNA for TGF-alpha, HBEGF, and AR all appeared to increase after wounding. In organ culture experiments, tyrphostin AG1478 inhibited migration rates in a dose-dependent manner. CONCLUSIONS: These data indicate that EGFR was activated during corneal epithelial wound healing in vivo. Furthermore, this activation appears to be a necessary component of the process, because inhibition of the EGFR signaling cascade significantly slowed migration rates. (+info)
Murine cytomegalovirus stimulates cellular thymidylate synthase gene expression in quiescent cells and requires the enzyme for replication.
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Herpesviruses accomplish DNA replication either by expressing their own deoxyribonucleotide biosynthetic genes or by stimulating the expression of the corresponding cellular genes. Cytomegalovirus (CMV) has adopted the latter strategy to allow efficient replication in quiescent cells. In the present report, we show that murine CMV (MCMV) infection of quiescent fibroblasts induces both mRNA and protein corresponding to the cellular thymidylate synthase (TS) gene, which encodes the enzyme that catalyzes the de novo synthesis of thymidylic acid. The increase in TS gene expression was due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse TS promoter was induced following MCMV infection. Mutagenesis of the potential E2F-responsive element immediately upstream from the TS essential promoter region abolished the virus-mediated stimulation of the TS promoter, suggesting that the transactivating activity of MCMV infection was E2F dependent. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in TS promoter activity. Finally, MCMV replication and viral DNA synthesis were found to be inhibited by ZD1694, a quinazoline-based folate analog that inhibits TS activity. These results demonstrate that upregulation of cellular TS expression is required for efficient MCMV replication in quiescent cells. (+info)
Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-selective tyrosine kinase inhibitor.
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Transforming growth factor alpha (TGF-alpha) is an autocrine growth factor for human cancer. Overexpression of TGF-alpha and its specific receptor, the epidermal growth factor receptor (EGFR), is associated with aggressive disease and poor prognosis. The EGFR has been proposed as a target for anticancer therapy. Compounds that block ligand-induced EGFR activation have been developed. ZD-1839 (Iressa) is a p.o.-active, quinazoline derivative that selectively inhibits the EGFR tyrosine kinase and is under clinical development in cancer patients. The antiproliferative activity of ZD-1839 alone or in combination with cytotoxic drugs differing in mechanism(s) of action, such as cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, doxorubicin, etoposide, topotecan, and raltitrexed, was evaluated in human ovarian (OVCAR-3), breast (ZR-75-1, MCF-10A ras), and colon cancer (GEO) cells that coexpress EGFR and TGF-alpha. ZD-1839 inhibited colony formation in soft agar in a dose-dependent manner in all cancer cell lines. The antiproliferative effect was mainly cytostatic. However, treatment with higher doses resulted in a 2-4-fold increase in apoptosis. A dose-dependent supra-additive increase in growth inhibition was observed when cancer cells were treated with each cytotoxic drug and ZD-1839. The combined treatment markedly enhanced apoptotic cell death induced by single-agent treatment. ZD-1839 treatment of nude mice bearing established human GEO colon cancer xenografts revealed a reversible dose-dependent inhibition of tumor growth because GEO tumors resumed the growth rate of controls at the end of the treatment. In contrast, the combined treatment with a cytotoxic agent, such as topotecan, raltitrexed, or paclitaxel, and ZD-1839 produced tumor growth arrest in all mice. Tumors grew slowly for approximately 4-8 weeks after the end of treatment, when they finally resumed a growth rate similar to controls. GEO tumors reached a size not compatible with normal life in all control mice within 4-6 weeks and in all single agent-treated mice within 6-8 weeks after GEO cell injection. In contrast, 50% of mice treated with ZD-1839 plus topotecan, raltitrexed, or paclitaxel were still alive 10, 12, and 15 weeks after cancer cell injection, respectively. These results demonstrate the antitumor effect of this EGFR-selective tyrosine kinase inhibitor and provide a rationale for its clinical evaluation in combination with cytotoxic drugs. (+info)
Phase I, dose-finding, and pharmacokinetic study of raltitrexed combined with oxaliplatin in patients with advanced cancer.
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PURPOSE: To determine the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of the raltitrexed plus oxaliplatin combination regimen, to explore its safety and pharmacokinetics, and to assess its antitumor activity in patients with advanced solid tumors. PATIENTS AND METHODS: Forty-eight patients received the combination of raltitrexed plus oxaliplatin. Raltitrexed was administered as a 15-minute infusion followed by oxaliplatin as a 2-hour infusion 1 hour later, repeated every 3 weeks. Seven dose levels were explored, ranging from 2 to 3.75 mg/m(2) and from 85 to 130 mg/m(2) for raltitrexed and oxaliplatin, respectively. The pharmacokinetics of both raltitrexed and oxaliplatin was assessed at the last three dose levels. RESULTS: Forty-six patients were assessable for toxicity. Severe toxicities usually occurred from dose level V (raltitrexed 3 mg/m(2) and oxaliplatin 130 mg/m(2)). This combination was not myelosuppressive, eliciting only sporadic grades 3 and 4 neutropenia and/or thrombocytopenia without complications. There was no alopecia. DLTs were asthenia and nausea/vomiting, despite systematic antiemetic prophylaxis. Dose level VI (raltitrexed 3.5 mg/m(2) and oxaliplatin 130 mg/m(2)) was deemed to be the MTD. Eight confirmed partial responses were observed: six patients with malignant mesothelioma (both pretreated and nonpretreated), one with fluorouracil-refractory pancreatic carcinoma, and one with renal carcinoma. Evaluation of the pharmacokinetics of both drugs did not suggest any drug interaction. CONCLUSION: The combination of raltitrexed and oxaliplatin given as consecutive short infusions every 3 weeks seems to be an acceptable regimen that allows a dose-intensity as high as the sum of the recommended doses of each agent given alone. The dose recommended for further phase II studies is raltitrexed 3 mg/m(2) and oxaliplatin 130 mg/m(2) every 3 weeks. Promising antitumor activity has been observed in patients with malignant mesothelioma. (+info)
The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism.
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The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated, and by which ATP hydrolysis is coupled to drug transport, are not known. Based on equilibrium binding experiments, photoaffinity labeling and drug transport assays, we conclude that homodimeric LmrA mediates drug transport by an alternating two-site transport (two-cylinder engine) mechanism. The transporter possesses two drug-binding sites: a transport-competent site on the inner membrane surface and a drug-release site on the outer membrane surface. The interconversion of these two sites, driven by the hydrolysis of ATP, occurs via a catalytic transition state intermediate in which the drug transport site is occluded. The mechanism proposed for LmrA may also be relevant for P-glycoprotein and other ABC transporters. (+info)