Factor Receptor Tyrosine Kinase Activity Research Articles

Human cytomegalovirus-inhibitory flavonoids: Studies on antiviral activity and mechanism of action

We report antiviral activity against human cytomegalovirus for certain dietary flavonoids and their likely biochemical mechanisms of action. Nine out of ten evaluated flavonoids blocked HCMV replication at concentrations that were significantly lower than those producing cytotoxicity against growing or stationary phase host cells. Baicalein was the most potent inhibitor in this series (IC 50 = 0.4–1.2 μM), including positive control ganciclovir. Baicalein and genistein were chosen as model compounds to study the antiviral mechanism(s) of action for this series. Both flavonoids significantly reduced the levels of HCMV early and late proteins, as well as viral DNA synthesis. Baicalein reduced the levels of HCMV immediate-early proteins to nearly background levels while genistein did not. The antiviral effects of genistein, but not baicalein, were fully reversible in cell culture. Pre-incubation of concentrated virus stocks with either flavonoid did not inhibit HCMV replication, suggesting that baicalein did not directly inactivate virus particles. Baicalein functionally blocked epidermal growth factor receptor tyrosine kinase activity and HCMV nuclear translocation, while genistein did not. At 24 h post infection HCMV-infected cells treated with genistein continued to express immediate-early proteins and efficiently phosphorylate IE1-72. However, HCMV induction of NF-κB and increases in the levels of cell cycle regulatory proteins—events that are associated with immediate-early protein functioning – were absent. The data suggested that the primary mechanism of action for baicalein may be to block HCMV infection at entry while the primary mechanism of action for genistein may be to block HCMV immediate-early protein functioning.

Current Knowledge and Future Directions of the Selective Epidermal Growth Factor Receptor Inhibitors Erlotinib (Tarceva®) and Gefitinib (Iressa®)

Gefitinib (Iressa); AstraZeneca Pharmaceuticals, Wilmington, DE, http://www.astrazeneca-us.com) and erlotinib (Tarceva); OSI Pharmaceuticals, Inc., Melville, NY, http://www.osip.com) are so-called small molecules that selectively inhibit epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both drugs received registration approval by the U.S. Food and Drug Administration (FDA) for the second- and third-line treatment of non-small cell lung cancer (NSCLC), but the failure of gefitinib to show a survival advantage over placebo has resulted in a discussion about the registration of gefitinib. Recently published results have revealed that mutations in the tyrosine kinase domain of EGFR are strongly associated with increased gefitinib and erlotinib sensitivity in patients with advanced NSCLC. Here, we present the current knowledge and the future directions of the EGFR tyrosine kinase inhibitors gefitinib and erlotinib.

Ethanol inhibits insulin expression and actions in the developing brain

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.

Epidermal Growth Factor Receptor Mutations, Small-Molecule Kinase Inhibitors, and Non–Small-Cell Lung Cancer: Current Knowledge and Future Directions

Gefitinib and erlotinib are small molecules that selectively inhibit epidermal growth factor receptor (EGFR) tyrosine kinase activity. When these drugs were introduced into the clinic, the specific targets affected in human tumors were unknown. In April 2004, two groups reported that mutations in the tyrosine kinase domain of EGFR are strongly associated with gefitinib sensitivity in patients with non-small-cell lung cancer (NSCLC). We subsequently extended these findings and showed that such mutations are also associated with sensitivity to erlotinib. Here, we present current knowledge about EGFR mutations in the context of clinical trials involving gefitinib and erlotinib in NSCLC. This article reviews the rationale for targeting EGFR, the development of gefitinib and erlotinib, the discovery of EGFR mutations, and subsequent studies to define the incidence, spectrum, and functions of EGFR mutations. The discovery of EGFR mutations promises to alter the ways in which we consider and treat NSCLC. This information can guide practitioners and help them inform their patients about EGFR mutations and their impact on the treatment of NSCLC.

VEGF and PDGF have distinct roles in development of experimental obliterative bronchiolitis—a therapeutic approach

Obliterative bronchiolitis (OB) is the leading cause of lung allograft loss but no efficient treatment for OB is exists. Inflammation and subsequent smooth muscle cell proliferation are central in OB development, leading to gradual occlusion of bronchioles. We examined the possibility to treat OB by selective inhibition of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinase activity in rat tracheal allografts with PTK787 and imatinib respectively.

Levels of Cyclooxygenase-2 Are Increased in the Oral Mucosa of Smokers: Evidence for the Role of Epidermal Growth Factor Receptor and Its Ligands

Cyclooxygenase-2 (COX-2) is a promising pharmacologic target for preventing aerodigestive malignancies. In this study, we investigated the effects of tobacco smoke on the expression of COX-2 in oral mucosa. An approximately 4-fold increase in amount of COX-2 mRNA was observed in the oral mucosa of active smokers versus never smokers. Thus, a series of in vitro studies were carried out to elucidate the mechanism by which tobacco smoke induced COX-2. Treatment of a nontumorigenic oral epithelial cell line (MSK-Leuk1) with a saline extract of tobacco smoke (TS) stimulated COX-2 transcription, resulting in increased amounts of COX-2 mRNA, COX-2 protein, and prostaglandin E(2) (PGE(2)) synthesis. Exposure of cells to TS also caused an increase in epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both an inhibitor of EGFR tyrosine kinase activity and a neutralizing anti-EGFR antibody blocked TS-mediated induction of COX-2. To define the mechanism by which TS activated EGFR, the release of amphiregulin and transforming growth factor alpha, two ligands of the EGFR, was measured. Exposure to TS caused a rapid increase in the release of both ligands. TS also markedly induced the expression of mRNAs for amphiregulin and transforming growth factor alpha. Importantly, increased expression of both ligands was also detected in the oral mucosa of active smokers. Taken together, these results suggest that activation of EGFR signaling contributes to the elevated levels of COX-2 found in the oral mucosa of smokers. Moreover, these findings strengthen the rationale for determining whether inhibitors of COX-2 or EGFR tyrosine kinase activity can reduce the risk of tobacco smoke-related malignancies of the aerodigestive tract.

Implication de la voie PI3K/Akt dans le cancer de la prostate. Nouvelles stratégies pour concevoir des thérapies ciblées

Because of the unavailability of effective therapies to block or reverse the progression of androgen-independent prostate cancer, it seems obvious to target growth signaling pathways for which frequently recurring mutations have been identified. Acquired mutations of the PTEN gene have been reported in several tumor types, including up to 30% - 60% of prostate cancer tumors. This results in constitutive activation of the PI3K/Akt pathway which then represents a major target to prevent dysfunctions in cell growth, survival and motility. Our experience and, therefore, our own tools allow us to design new inhibitors of growth factor receptor tyrosine kinase, PDK-1 and farnesyltransferase activities. These original compounds could selectively switch off one or several steps of the multifunctional pathway and constitute lead compounds in the design of new classes of potent drugs.

New directions in oncology nursing care: focus on gefitinib in patients with lung cancer.

Treatment of solid tumors with chemotherapy regimens commonly is associated with debilitating or life-threatening side effects. Careful patient management, appropriate and prompt management of side effects, and interruption of therapy frequently are required for patients receiving chemotherapy. Furthermore, the systemic toxicity associated with chemotherapy may result in irreversible and incapacitating side effects, such as peripheral neuropathy, that lead to poor quality of life in patients. Gefitinib (Iressa, ZD1839, AstraZeneca Pharmaceuticals LP, Wilmington, DE) is a biologically based, molecular targeted therapy with a novel mechanism of action: selective inhibition of the epidermal growth factor receptortyrosine kinase (EGFR-TK) activity. Once-daily oral treatment with gefitinib is well tolerated. In clinical trials, treatment with gefitinib resulted in durable tumor responses and improvement in lung cancer-related symptoms in patients with advanced non-small cell lung cancer who had received prior chemotherapy. Trials are under way to explore the full potential of gefitinib and additional EGFR-TK inhibitors for other solid tumors and in other treatment settings, including prevention. Biologically based, molecular-targeted therapies such as gefitinib are providing new treatment options for patients and adding a new dimension to clinical practice for oncology nurses.

DUAL ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN EXPERIMENTAL OBLITERATIVE BRONCHIOLITIS

P964 Aims: We investigated the role of vascular endothelial growth factor (VEGF) in the development of obliterative bronchiolitis (OB) in rat tracheal allografts. Methods: Tracheal allografts were transplanted heterotopically from DA- to WF-rats into the greater omentum. Syngeneic controls were performed DA to DA rats. Expression of VEGF ligand and receptor protein and mRNA was analysed from normal, syngeneic, and allogeneic tracheal transplants. Adenovirally mediated VEGF164 (AdVEGF) gene transfer was used to over-express VEGF while AdLacZ-treated allografts served as controls. PTK787, an inhibitor of VEGF receptor tyrosine kinase activity, was used for blocking VEGF action. Imatinib, an inhibitor of platelet-derived growth factor receptor (PDGFR) tyrosine kinase activity, was used to inhibit PDGF activity. Cyclosporine A (CyA) was used for background immunosuppression. Tracheal allografts were removed 10 and 30 days after transplantation for in situ hybridization, immunohistochemistry, and histology. Results: An increase in VEGF mRNA and protein expression was demonstrated in smooth muscle cells and mononuclear inflammatory cells of allograft airway wall compared to normal trachea and syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene decreased early epithelial necrosis but increased microvascular remodeling and luminal occlusion by more than 50% compared to AdLacZ-treated control allografts. Inhibition of VEGF receptor tyrosine kinase activity with PTK787 negated the effect of VEGF overexpression. Although VEGFR inhibition with PTK787 increased early epithelial necrosis in uninfected allografts, it significantly reduced microvascular remodeling, intragraft infiltration by CD4+ and CD8+ T cells and the degree of luminal occlusion. Simultaneous adenoviral VEGF overexpression and treatment with a kinase inhibitor incorporating selectivity for PDGFR (imatinib) totally prevented luminal occlusion (Table 1). Table 1. Effect of the different treatment regimens on allograft histologyFigureConclusions: In conclusion, our findings indicate that VEGF has a dual role in transplant obliterative airway disease. VEGF may protect epithelial integrity by reducing the early ischemic injury. On the other hand, VEGF may increase luminal occlusion by enhancing inflammatory response and growth factor production. Thus, multiple treatment strategies may be needed to prevent transplant obliterative airway disease.

Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 μM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future.

Inhibition of Platelet-Derived Growth Factor Promotes Pericyte Loss and Angiogenesis in Ischemic Retinopathy

We investigated whether inhibition of platelet-derived growth factor (PDGF) receptor tyrosine kinase activity would affect pericyte viability, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR-2) expression and angiogenesis in a model of retinopathy of prematurity (ROP). ROP was induced in Sprague Dawley rats by exposure to 80% oxygen from postnatal (P) days 0 to 11 (with 3 hours/day in room air), and then room air from P12-18 (angiogenesis period). Shams were neonatal rats in room air from P0-18. STI571, a potent inhibitor of PDGF receptor tyrosine kinase, was administered from P12-18 at 50 or 100 mg/kg/day intraperitoneal (i.p.). Electron microscopy revealed that pericytes in the inner retina of both sham and ROP rats appeared normal; however STI571 induced a selective pericyte and vascular smooth muscle degeneration. Immunolabeling for caspase-3 and alpha-smooth muscle cell actin in consecutive paraffin sections of retinas confirmed that these degenerating cells were apoptotic pericytes. In all groups, VEGF and VEGFR-2 gene expression was located in ganglion cells, the inner nuclear layer, and retinal pigment epithelium. ROP was associated with an increase in both VEGF and VEGFR-2 gene expression and blood vessel profiles in the inner retina compared to sham rats. STI571 at both doses increased VEGF and VEGFR-2 mRNA and exacerbated angiogenesis in ROP rats, and in sham rats at 100 mg/kg/day. In conclusion, PDGF is required for pericyte viability and the subsequent prevention of VEGF/VEGFR-2 overexpression and angiogenesis in ROP.

Epidermal growth factor receptor tyrosine kinase inhibitors: evolving role in the treatment of solid tumors.

Inhibitors of epidermal growth factor receptor tyrosine kinase (EGFR-TK) activity have shown promise as novel anticancer agents in a variety of common solid tumors. In preclinical studies and phase I trials, tumor responses to EGFR-TK inhibitors (EGFR-TKIs), such as gefitinib (Iressa, AstraZeneca Pharmaceuticals LP, Wilmington, DE) and erlotinib (Tarceva, OSI Pharmaceuticals, Melville, NY, and Genentech, Inc., South San Francisco, CA) were observed in heavily pretreated patients with advanced non-small cell lung cancer (NSCLC), head and neck cancer, breast cancer, colorectal cancer, and other solid tumors. Subsequent phase II studies resulted in tumor responses, disease stabilization, symptom improvement, and improved quality of life in patients with advanced NSCLC who had received prior platinum-based chemotherapy or platinum and docetaxel chemotherapies. Side effects related to treatment with EGFR-TKIs were generally mild, reversible, and noncumulative. Severity and frequency of drug-related adverse events were related directly to dose. The potential role of EGFR-TKIs in treating other solid tumors currently is being studied. Furthermore, research is being conducted to explore the potential use of EGFR-TKIs in novel combinations with chemotherapy, radiation therapy, endocrine therapy, and other molecular targeted therapies.

HER1/EGFR targeting: refining the strategy.

Human epidermal growth factor receptor (EGFR), HER, targeting has formed the basis of extensive and growing drug development programs in various companies. However, receptor biology is often poorly explained and confusing. The HER family of four naturally occurring receptors and one tumor-specific mutant can activate signaling via a complex and sophisticated range of mechanisms, which we are only beginning to understand. HER1/EGFR downstream signaling can lead to tumor growth and development via a host of processes, including enhanced cellular proliferation, survival, and metastasis. A range of potential therapeutic targets exists within the HER signaling system, both inside and outside the cell. Monoclonal antibodies and tyrosine kinase inhibitors, acting extracellularly and intracellularly, respectively, comprise two classes of agents most advanced in clinical development or already available for use. Despite promising single-agent activity in chemotherapy-resistant patients with non-small cell lung cancer (NSCLC), disappointing results from two phase III trials of the tyrosine kinase inhibitor gefitinib in NSCLC have been of concern to some. However, many factors may have contributed to this outcome, and it is not necessarily predictive of the future usefulness of these agents. Patient characteristics, lack of patient selection, dosing schedule, and trial design may all have played roles. It is important to remember that intracellular targeting of HER is a relatively novel approach, and our knowledge of how best to optimize such treatment is still unfolding. More clinical experience is needed.

Primary and Secondary Prevention of Non–Small-Cell Lung Cancer: The SPORE Trials of Lung Cancer Prevention

These chemotherapy trials have been closed prior to completion.</p><p>The aims of chemoprevention in lung cancer are to prevent the appearance of disease (primary prevention) and to stop or reverse the progression of premalignant lesions (secondary prevention). Until recently, there was little hope that these goals could be attained. However, the results achieved with tamoxifen in the prevention of breast cancer, and the emergence of new therapies specifically targeted to molecules involved in the pathogenesis of lung cancer have set the stage for investigation of these agents for chemoprevention of lung cancer. Two of these new molecular targeted agents are gefitinib, an inhibitor of epidermal growth factor receptor–tyrosine kinase activity, and tipifarnib (R115777, Zarnestra®), an inhibitor of the farnesyltransferase enzyme, which is required for the proper localization and function of the ras oncogene. Tumor responses and disease stabilization have been achieved with both agents in clinical trials. In the Iressa Dose Evaluation in Advanced Lung Cancer (IDEAL)–1 and IDEAL-2 phase II trials, gefitinib was demonstrated to be effective for disease control in patients with advanced non–small-cell lung cancer. The SPORE (Specialized Program of Research Excellence) Trials of Lung Cancer Prevention (STOP) are 2 parallel studies that will investigate the potential effectiveness of gefitinib and tipifarnib in preventing the appearance and progression of premalignant lesions in former or current smokers with a history of smoking-related cancer. These trials should provide information not only about the potential role of gefitinib and tipifarnib in lung cancer chemoprevention, but also about the molecular changes that underlie tumorigenesis and that may serve as markers of disease progression. The STOP trial objectives are to evaluate the effect of gefitinib and tipifarnib on histologic and biologic parameters in patients with evidence of sputum atypia, to evaluate various parameters as potential predictors of the effectiveness of these agents, and to evaluate the tolerability of these agents over a 6-month course of treatment. Histologic response, defined as prevention of appearance or progression of premalignant lesions, is the primary endpoint of these trials. New targeted molecular therapies such as gefitinib and tipifarnib may offer the opportunity to make chemoprevention a viable treatment modality in lung cancer as well as in other human solid tumors.

Resuscitative hypothermia after cardiac arrest in adults

Resuscitative hypothermia after cardiac arrest in adults

Extracellular signal-regulated kinase 1/2 activation by myometrial oxytocin receptor involves Galpha(q)Gbetagamma and epidermal growth factor receptor tyrosine kinase activation.

The mechanisms by which oxytocin (OT) stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) are only partially understood. OT receptor (OTR) signals predominantly through Galpha(q), but ERK1/2 phosphorylation (ERK1/2-P) in PHM1 myometrial cells was not eliminated by inhibition of downstream effectors such as phospholipase C or protein kinase C. Inconsistent with a Galpha(i)-coupled response, pertussis toxin inhibition of OT-induced ERK1/2-P was reversed by the protein kinase A inhibitors Rp-cAMPS and KT5720. Consistent with an inhibitory role for protein kinase A, pertussis toxin pretreatment raised cellular cAMP and 8-(4-chlorophenylthio)-cAMP inhibited OT-induced ERK1/2-P. Attenuation of the OT response by the Gbetagamma scavenger carboxyl terminus of the beta-adrenergic receptor kinase implicated a Gbetagamma-mediated pathway. In both COSM6 cells overexpressing OTR (OTR-COSM6) and in PHM1 cells, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 markedly reduced OT-induced ERK1/2-P, whereas the platelet-derived growth factor receptor tyrosine kinase inhibitor AG1296 had no effect. Furthermore, OT increased EGFR tyrosine phosphorylation in OTR-COSM6 cells, which was inhibited by AG1478 or EGTA plus thapsigargin pretreatment. AG1478 did not affect inositol 1,4,5-triphosphate production by OT or protein kinase C-stimulated ERK1/2-P but completely blocked ionomycin-induced ERK1/2-P and EGFR tyrosine phosphorylation. In both OTR-COSM6 and PHM1 cells, EGTA reduced OT-stimulated ERK1/2-P; no ERK1/2-P was observed when intracellular calcium increases were blocked by pretreatment with thapsigargin plus EGTA. These data are consistent with activation of a Gbetagamma-mediated pathway as a consequence of Galpha(q) activation in myometrium and OTR-COSM6 cells that results in increased ERK1/2-P. This pathway involves both EGFR activation and an influence of calcium.

PKC-zeta is required for angiotensin II-induced activation of ERK and synthesis of C-FOS in MCF-7 cells.

We examined the signalling pathways responsible for the Ang II induction of growth in MCF-7 human breast cancer cells. Ang II in MCF-7 cells induced: (a) the translocation from the cytosol to membrane and nucleus of atypical protein kinase C-zeta (PKC-zeta) but not of PKC-alpha, -delta, - epsilon and -eta; (b) the expression of c-fos mRNA and protein; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). All these effects were due to the activation of the Ang II type I receptor (AT1) since they were blocked by the AT1 antagonist losartan. The Ang II-stimulated ERK1/2 phosphorylation was blocked by (a) high doses of staurosporine, inhibitor of PKC-zeta, and by a synthetic myristoylated peptide with sequences based on the endogenous PKC-zeta pseudosubstrate region (zeta-PS); (b) PD098059, a mitogen-activated protein kinase kinase inhibitor (MAPKK/MEK); and, moreover, (c) the inhibitors of phosphoinositide 3-kinases (PI3K), LY294002 and wortmannin, thus indicating that PI3K may act upstream of ERK1/2. The Ang II-evoked c-fos induction was blocked only by high doses of staurosporine and by zeta-PS whilst PD098059, LY294002 and wortmannin were ineffective, thus indicating that c-fos induction is not due to ERK1/2 activity. When the epidermal growth factor-receptor (EGFR) tyrosine kinase activity was inhibited by the use of its inhibitor AG1478, Ang II was still able to induce ERK1/2 phosphorylation and c-fos expression, therefore proving that the transactivation of EGFR was not required for these Ang II effects in MCF-7 cells. The previously reported proliferation of MCF-7 cells induced by Ang II was blocked by PD098059 and by wortmannin in a dose-dependent manner, thereby indicating that in MCF-7 cells the PI3K and ERK pathways mediate the mitogenic signalling of AT1. Our results suggest that in MCF-7 cells Ang II activates multiple signalling pathways involving PKC-zeta, PI3K and MAPK; of these pathways only PKC-zeta appears responsible for the induction of c-fos.

Topical N-Acetyl Cysteine and Genistein Prevent Ultraviolet-Light-Induced Signaling That Leads to Photoaging in Human Skin in vivo

Human skin is exposed to solar ultraviolet radiation. Ultraviolet radiation damages human skin and results in an old and wrinkled appearance, called photoaging. We have previously reported that molecular mechanisms by which ultraviolet light causes photoaging involve activation of growth factor and cytokine receptors in keratinocytes and dermal cells. They lead to downstream signal transduction through activation of mitogen-activated protein kinase (extracellular signal-regulated kinase, c-jun N-terminal protein kinase, and p38) pathways. These signaling pathways converge in the nucleus of cells to form an activated complex of transcription factor activator protein 1 (cFos/cJun), which induces matrix metalloproteinases that degrade skin connective tissue. In addition to cell surface receptor activation, generation of reactive oxygen species by ultraviolet radiation is believed to be critical in triggering mitogen-activated protein kinase pathways. We investigated the ability of (i) ultraviolet irradiation to generate reactive oxygen species in human skin in vivo; and (ii) genistein, which possesses both tyrosine kinase inhibitory and antioxidant activities, and n-acetyl cysteine, which can be converted into the endogenous antioxidant glutathione, to impair responses to ultraviolet light that eventuate in photoaging in human skin in vivo. Ultraviolet irradiation caused a rapid and significant increase in hydrogen peroxide levels in human skin in vivo. Pretreatment of human skin with genistein inhibited ultraviolet-induced epidermal growth factor receptor tyrosine kinase activity, whereas n-acetyl cysteine did not. Genistein inhibited ultraviolet induction of both extracellular signal-regulated kinase and cJun N-terminal protein kinase activities. n-Acetyl cysteine inhibited extracellular signal-regulated kinase but not cJun N-terminal protein kinase activation. Both genistein and n-acetyl cysteine prevented ultraviolet induction of cJun protein. Consistent with this, genistein and n-acetyl cysteine blocked ultraviolet induction of cJun-driven enzyme, collagenase. Neither genistein nor n-acetyl cysteine acted as sunscreens as they had no effect on ultraviolet-induced erythema. These data indicate that compounds similar to genistein and n-acetyl cysteine, which possess tyrosine kinase inhibitory and/or antioxidant activities, may prevent photoaging.

53 The blockade of epidermal growth factor receptor tyrosine kinase activity reduces the emergence of hepatocellular carcinoma within cirrhotic rat liver

53 The blockade of epidermal growth factor receptor tyrosine kinase activity reduces the emergence of hepatocellular carcinoma within cirrhotic rat liver

Monocytic activation of protein tyrosine kinase, protein kinase A and protein kinase C induced by porins isolated from Salmonella enterica serovar Typhimurium.

In the present study a monocytic cell line, U937, was used to investigate the possible involvement of protein tyrosine kinases (NT-PTKs), protein kinase A (PKA) and protein kinase C (PKC) in cell signaling pathways following Salmonella enterica serovar Typhimurium porin stimulation. Different concentrations of porins and lipopolysaccharide (LPS) were analysed to evaluate changes in PTK activity by a non radioactive tyrosine kinase assay and in PKA and PKC phosphorylation by Western blotting analysis. The inhibitors of PTK, PKA and PKC activation used, were: 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase activity; dihychloride (H-89), a selective inhibitor of PKA which is useful to discriminate between the effects of PKC and PKA; diacylglycerol kinase inhibitor II (R59949), which is useful for elucidating roles of PKC; calphostin C, a specific inhibitor of PKC. Porins of the outer membrane of the ST were isolated to be used as a stimulus in the performed experiments. Following porin treatment, a dose-dependent increase in PTK, PKA and PKC activation was observed. U937 monocytes pretreated with inhibitors induced an evident decrease in PTK activity and PKA and PKC phosphorylation pattern in porin stimulated monocytes. Our data support the important role played by NT-PTK, PKA and PKC in transducing the activating signal in macrophages stimulated with porins through the activation of the mitogen-activated protein kinase (MAPK) pathway that participate in the regulation of gene expression.