Tyrosine Kinase Signal Transduction Pathways Research Articles

Quantitative fret studies of EphA2-Lyn interactions.

The regulation of the receptor tyrosine kinase (RTK) signal transduction pathways is critical for proper cellular function. RTK activation occurs in response to ligand binding, which induces RTK dimerization or oligomerization to allow cross-phosphorylation of specific tyrosines in the kinase domains of the RTK. Downstream signals are then propagated upon the recruitment of soluble kinases or adaptor proteins to these phoshorylated tyrosines. The EphA2 receptor tyrosine kinase is a member of the largest RTK family and has been reported to initiate downstream signaling by recruiting Lyn, a member of the Src family of non-receptor tyrosine kinases. Contrary to expectations, quantitative FRET experiments demonstrate that ligand-induced EphA2 tyrosine phosphorylation does not lead to Lyn recruitment to EphA2. In control experiments, Lyn is efficiently recruited by a different RTK, EGFR, upon ligand stimulation. The differential interactions of Lyn with EphA2 and EGFR may be a means of signal diversification. Current work is focused on the mechanism behind the unexpected lack of interactions between EphA2 and Lyn upon EphA2 clustering and phosphorylation.

Trained innate immunity, long-lasting epigenetic modulation, and skewed myelopoiesis by heme

Trained immunity defines long-lasting adaptations of innate immunity based on transcriptional and epigenetic modifications of myeloid cells and their bone marrow progenitors [M. Divangahi et al., Nat. Immunol. 22, 2-6 (2021)]. Innate immune cells, however, do not exclusively differentiate between foreign and self but also react to host-derived molecules referred to as alarmins. Extracellular "labile" heme, released during infections, is a bona fide alarmin promoting myeloid cell activation [M. P. Soares, M. T. Bozza, Curr. Opin. Immunol. 38, 94-100 (2016)]. Here, we report that labile heme is a previously unrecognized inducer of trained immunity that confers long-term regulation of lineage specification of hematopoietic stem cells and progenitor cells. In contrast to previous reports on trained immunity, essentially mediated by pathogen-associated molecular patterns, heme training depends on spleen tyrosine kinase signal transduction pathway acting upstream of c-Jun N-terminal kinases. Heme training promotes resistance to sepsis, is associated with the expansion of self-renewing hematopoetic stem cells primed toward myelopoiesis and to the occurrence of a specific myeloid cell population. This is potentially evoked by sustained activity of Nfix, Runx1, and Nfe2l2 and dissociation of the transcriptional repressor Bach2. Previously reported trained immunity inducers are, however, infrequently present in the host, whereas heme abundantly occurs during noninfectious and infectious disease. This difference might explain the vanishing protection exerted by heme training in sepsis over time with sustained long-term myeloid adaptations. Hence, we propose that trained immunity is an integral component of innate immunity with distinct functional differences on infectious disease outcome depending on its induction by pathogenic or endogenous molecules.

Targeted therapy for small cell lung cancer

Targeted agents for small cell lung cancer (SCLC) include inhibitors of angiogenesis, tyrosine kinase inhibitors and signal transduction pathway inhibitors. Bevacizumab, a class of antiangiogenic agent, tends to have no effects on patients. The tyrosine kinase inhibitor, for example sunitinib, may be used as a monotherapy. Signaling inhibitors including Amuvatinib and LDE225 are undergoing phaseⅠand Ⅱ trials. Primary data show that SCLC is not sensitive to targeted therapy, needing more selection, so further studies are requested. Key words: Lung neoplasms; Carcinoma, small cell; Targeted therapy

Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

The interpretation of biological data sets is essential for generating hypotheses that guide research, yet modern methods of global analysis challenge our ability to discern meaningful patterns and then convey results in a way that can be easily appreciated. Proteomic data is especially challenging because mass spectrometry detectors often miss peptides in complex samples, resulting in sparsely populated data sets. Using the R programming language and techniques from the field of pattern recognition, we have devised methods to resolve and evaluate clusters of proteins related by their pattern of expression in different samples in proteomic data sets. We examined tyrosine phosphoproteomic data from lung cancer samples. We calculated dissimilarities between the proteins based on Pearson or Spearman correlations and on Euclidean distances, whilst dealing with large amounts of missing data. The dissimilarities were then used as feature vectors in clustering and visualization algorithms. The quality of the clusterings and visualizations were evaluated internally based on the primary data and externally based on gene ontology and protein interaction networks. The results show that t-distributed stochastic neighbor embedding (t-SNE) followed by minimum spanning tree methods groups sparse proteomic data into meaningful clusters more effectively than other methods such as k-means and classical multidimensional scaling. Furthermore, our results show that using a combination of Spearman correlation and Euclidean distance as a dissimilarity representation increases the resolution of clusters. Our analyses show that many clusters contain one or more tyrosine kinases and include known effectors as well as proteins with no known interactions. Visualizing these clusters as networks elucidated previously unknown tyrosine kinase signal transduction pathways that drive cancer. Our approach can be applied to other data types, and can be easily adopted because open source software packages are employed.

Can Activation Induced Cytidine Deaminase Lead to Genetic Instability in Bcr-Abl Negative Myeloproliferative Neoplasms?

AbstractAbstract 5056 Objective:Most patients with myeloproliferative neoplasms (MPN) have mutations that lead to constitutive activation of the tyrosine kinase signal transduction pathways. JAK2V617F mutation is present in about 97% of polycythemia vera (PV), and in 60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. JAK2-exon-12 mutations, MPL exon-10-mutations are also seen in MPNs. In addition to this mutations, some patients with MPN may acquire mutations of other genes, such as in genes involved in negative regulations of signaling pathways (LNK, CBL, SOCS1, SOCS2, SOCS3) and epigenetic regulations (TET2, ASXL1, DNMT3A, or EZH2). IKZF1 deletions and TP53 mutations are mainly found post-MPN acute myeloid leukemia (AML). Loss of function mutations in TET2, ASXL1 and EZH2 genes, may be early events preceding JAK2V617F but may also secondary genetic event. These mutations are more frequently observed in PMF than in PV and ET. They are also present in other myeloid malignancies such as myelodysplasia and AML. Moreover several cases have been described that are positive for BCR-ABL and JAK2V617F mutation. It is thought that the mutagenic phenotype existing inherently in patients with MPN can generate new mutations. With the contribution of these mutations, PV, ET and PMF might evolve into secondary myelofibrosis or leukemia at different ratios. Activation induced cytidine deaminase (AID) is required for a B cell terminal differentiation mechanism shared by somatic hypermutation and class-switch recombination. Aberrant expression of AID can induce imperfect mutations in non-immunoglobulin genes and was detected in human lymphomas, lung cancer and gastric or intestinal metaplasia. High AID expression is associated with poor prognosis in follicular lymphomas, chronic lymphocytic leukemia and promotes B lymphoid blast crisis in chronic myeloid leukemia. Current study aims to investigate AID expression levels in myeloproliferative neoplasms and their relationship to genetic instability in MPN patients. Material and methods:The patient group consisted of 120 patients with bcr-abl negative myeloproliferative neoplasms (PV: 32 patients, ET: 62 patients and PMF: 18 patients, post-ET or post-PV myelofibrosis: 5 patients, unclassified MPN: 1 patient, MPN-AML: 2 patients). The patients were admitted to Istanbul University, Istanbul Medical Faculty, Division of Hematology Outpatient Clinics. The control group consisted of 69 healthy persons. We evaluated AID expression levels of the patients and of the control group. RNA was extracted from peripheral blood samples of patients and controls, RNA was converted to complementary DNA (cDNA). Using the primers and probes of the target gene (AID) and reference gene HPRT (hypoxanthine phosphoribosyltransferase), cDNA was amplified by realtime polymerase chain reaction and expression levels of the genes were evaluated. Results:We found that AID expression levels in the patient group were significantly higher than in the control group (Target-AID/Reference-HPRT, mean value: 0. 1747 vs 0. 0417, respectively, p<0. 001). We found that the patients age, blood hemoglobin levels, history of thrombosis, bone marrow reticulin levels, serum LDH levels, organomegaly and having JAK2V617F mutation were not correlated with AID expression levels. Conclusion:We showed that AID expression levels in patient with myeloproliferative neoplasms were significantly higher than in healthy control group. The role and the biological significance of high AID expression levels in genetic instability in MPNs need to be elucidated. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract PR11: Dissecting signaling transduction network to infer master regulators of non small cell lung cancer

Abstract The dissection of signal transduction networks is lagging significantly with respect to their transcriptional and protein-complex counterparts. Innovative use of multiplex and microarray-based approaches, where multiple antibodies can be used to probe an ensemble of phosphoproteins, is starting to become sufficiently mature to allow characterization of small pathways. Yet, this is still far from providing an unbiased, genome-wide view of signal-transduction processes. Using data from the first genome-wide profiling of phosphopeptide abundance in normal and tumor-related lung tissue, we have designed a novel algorithm, pARACNe, to generate an unbiased, genome-wide network representing tyrosine kinase signal transduction pathways. Specifically, pARACNe was used to infer tyrosine kinase substrates in non-small cell lung cancer (NSCLC) from tandem mass-spec abundance profiling of phosphotyrosine-enriched peptides from ˜250 samples derived from primary tumors, cell lines, and normal lung tissue. SILAC validation of EGFR, MET and ALK substrates, confirmed &amp;gt;80% of the pARACNe-inferred interactions. Further analysis of large-scale NSCLC microarray expression profile data, using the MINDy algorithm, showed significant convergence between the signal transduction pathways inferred by proteomics and gene expression-based methods. The availability of an accurate NSCLC Tyrosine Kinase Interaction Network (TKIN) has significant implications for our ability to elucidate master regulators of oncogenesis and tumor progression and for the identification and prioritization of druggable targets. We used the MARINa algorithm and the TKIN to infer a complete repertoire of master regulator (MRs) kinases, both individual as well as synergistic, in 46 cell lines. Initial validation shows that the method could recapitulate both cancer-specific MRs, such as EGFR, as well as cell line-specific ones, such as ALK and PTK2 in H2228 cells. Follow-up experimental assays have shown that the PTK2, which is active in tumor-related but not in normal cells, is required for H2228 cell viability and may constitute a novel oncogene addiction in NSCLC and a candidate therapeutic target. We also validated synergistic addiction of EGFR and MET in several cell lines by showing no effect on colony formation after treating cell lines with inhibitors of individual genes but synergistic decrease in colony formation with dual treatment. The TKI suggests that current pathway models are incomplete and sets the stage for quantitative, network-based methods to identify key regulators of physiologic and pathologic cell phenotypes. This proffered talk is also presented as Poster A14. Citation Format: Mukesh Bansal, Michael Peyton, Manjunath Kustagi, Luc Girard, Klarisa Rikova, Michael Comb, Michael White, John Minna, Andrea Califano. Dissecting signaling transduction network to infer master regulators of non small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr PR11.

Focal adhesion kinase inhibitors are potent anti-angiogenic agents

Focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase and scaffold protein localized to focal adhesions, is uniquely positioned at the convergence point of integrin and receptor tyrosine kinase signal transduction pathways. FAK is overexpressed in many tumor cells, hence various inhibitors targeting its activity have been tested for anti-tumor activity. However, the direct effects of these pharmacologic agents on the endothelial cells of the vasculature have not been examined. Using primary human umbilical vein endothelial cells (HUVEC), we characterized the effects of two FAK inhibitors, PF-573,228 and FAK Inhibitor 14 on essential processes for angiogenesis, such as migration, proliferation, viability and endothelial cell tube formation. We observed that treatment with either FAK Inhibitor 14 or PF-573,228 resulted in reduced HUVEC viability, migration and tube formation in response to vascular endothelial growth factor (VEGF). Furthermore, we found that PF-573,228 had the added ability to induce apoptosis of endothelial cells within 36 h post-drug administration even in the continued presence of VEGF stimulation. FAK inhibitors also resulted in modification of the actin cytoskeleton within HUVEC, with observed increased stress fiber formation in the presence of drug. Given that endothelial cells were sensitive to FAK inhibitors at concentrations well below those reported to inhibit tumor cell migration, we confirmed their ability to inhibit endothelial-derived FAK autophosphorylation and FAK-mediated phosphorylation of recombinant paxillin at these doses. Taken together, our data indicate that small molecule inhibitors of FAK are potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting tumor angiogenesis.

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.

Abstract LB-27: Dissecting signaling transduction network to infer master regulators of non small cell lung cancer

Abstract Abstract The dissection of signal transduction networks is lagging significantly with respect to their transcriptional and protein-complex counterparts. Innovative use of multiplex and microarray-based approaches, where multiple antibodies can be used to probe an ensemble of phosphoproteins, is starting to become sufficiently mature to allow characterization of small pathways. Yet, this is still far from providing an unbiased, genome-wide view of signal-transduction processes. Using data from the first genome-wide profiling of phosphopeptide abundance in normal and tumor related lung tissue1, we have designed a novel algorithm, pARACNe, to generate an unbiased, genome-wide network representing tyrosine kinase signal transduction pathways. Specifically, pARACNe was used to infer tyrosine kinase substrates in non-small cell lung cancer (NSCLC) from tandem mass-spec abundance profiling of phosphotyrosine enriched peptides from ∼250 samples derived from primary tumors, cell lines, and normal lung tissue. SILAC validation of EGFR, MET and ALK substrates, confirmed &amp;gt;80% of the pARACNe inferred interactions. Further analysis of large-scale NSCLC microarray expression profile data, using the MINDy algorithm2, showed significant convergence between the signal transduction pathways inferred by proteomics and gene expression based methods. The availability of an accurate NSCLC Tyrosine Kinase Interaction Network (TKIN) has significant implications for our ability to elucidate master regulators of oncogenesis and tumor progression and for the identification and prioritization of druggable targets. We used the MARINa algorithm and the TKIN to infer a complete repertoire of master regulator (MRs) kinases of 46 cell lines. Initial validation shows that the method could recapitulate both cancer specific MRs, such as EGFR, as well as cell line specific ones, such as ALK and PTK2 in H2228 cells. Follow up experimental assays have shown that the PTK2, which is active in tumor-related but not in normal cells, is required for H2228 cell viability and may constitute a novel oncogene addiction in NSCLC and a candidate therapeutic target. The TKI suggests that current pathway models are incomplete and sets the stage for quantitative, network-based methods to identify key regulators of physiologic and pathologic cell phenotypes.

P2X7 receptor-Pannexin1 complex: pharmacology and signaling

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 microM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.

Implications of HER family inhibition in targeted therapy of ovarian cancer

13120 Background: The human epidermal receptor (HER) family couples binding of extracellular ligands to intracellular tyrosine kinase (TK) signal transduction pathways, contributing to cellular proliferation. Overexpression of HER family members has been associated with poor prognosis in ovarian cancer. There is growing evidence to support that none of the individual HER members can be considered as the stand-alone target in ovarian cancer and that cooperation between them influences therapeutic response. To evaluate this receptor cooperation in ovarian cancer our goal was to determine HER family expression and to quantify inhibition of HER1 alone compared to inhibition of HER1/HER2 combined. Methods: HER1, HER2, HER3 and HER4 expression was determined by Western blot in 9 ovarian cancer cell lines. Ovarian cancer cell proliferation was determined after inhibition of HER1 using the monoclonal antibody cetuximab, the TK inhibitor gefitinib, or combination of the two. To quantify the combined inhibition of HER1/HER2, 3 cell lines were selected with variable HER1 and HER2 expression. Cell proliferation was then determined in these 3 cell lines after treatment with cetuximab combined with the monoclonal HER2 antibody, trastuzumab. IC50 values were calculated for all treatment arms and compared. A lung cancer cell line (HCC827), with a HER1 TK mutation and sensitivity to gefitinib and cetuximab, was used as a positive control. Results: HER1 was overexpressed in 3/9 ovarian cancer cell lines, HER2 in 1/9, HER3 in 2/9, and HER4 in 4/9. Minimal to no growth inhibition was seen in the 9 cell lines after blocking HER1 with cetuximab, gefitinib, or the combination of both. In the 3 cell lines selected for HER1 and HER2 expression, there was no growth inhibition achieved despite combining cetuximab with trastuzumab. However, this treatment combination increased resistance in 1 of the 3 cell lines (HCC60), noted to overexpress HER1, 2, and 4. Conclusions: Ovarian cancer has variable HER family expression. No correlation was found between HER1 and HER2 overexpression and response to their targeted inhibition. Our findings support the concept the entire HER family plays a role in ovarian cancer growth and suggest an equilibrium shift of HER heterodimerization may play an important role in maintaining cell signaling. No significant financial relationships to disclose.

Targeted molecular therapy of malignant gliomas

Malignant gliomas are the most common form of primary brain tumors in adults. Despite advances in diagnosis and standard therapies such as surgery, radiation, and chemotherapy, the prognosis remains poor. Recent scientific advances have enhanced our understanding of the biology of gliomas and the role of tyrosine kinase receptors and signal transduction pathways in tumor initiation and maintenance, such as the epidermal growth factor receptors, platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and the Ras/Raf/mitogen-activated protein (MAP)-kinase and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways. Novel targeted drugs such as small molecular inhibitors of these receptors and signaling pathways are showing some activity in initial studies. As we learn more about these drugs and how to optimize their use as single agents and in combination with radiation, chemotherapy, and other targeted molecular agents, they will likely play an increasing role in the management of this devastating disease. This review summarizes the current results with targeted molecular agents in malignant gliomas and strategies under evaluation to increase their effectiveness.

Targeted molecular therapy of malignant gliomas

Malignant gliomas are the most common form of primary brain tumors in adults. Despite advances in diagnosis and standard therapies such as surgery, radiation, and chemotherapy, the prognosis remains poor. Recent scientific advances have enhanced our understanding of the biology of gliomas and the role of tyrosine kinase receptors and signal transduction pathways in tumor initiation and maintenance, such as the epidermal growth factor receptors, platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and the Ras/Raf/mitogen-activated protein (MAP)-kinase and phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways. Novel targeted drugs such as small molecular inhibitors of these receptors and signaling pathways are showing some activity in initial studies. As we learn more about these drugs and how to optimize their use as single agents and in combination with radiation, chemotherapy, and other targeted molecular agents, they will likely play an increasing role in the management of this devastating disease. This review summarizes the current results with targeted molecular agents in malignant gliomas and strategies under evaluation to increase their effectiveness.

In vitro formation and activity-dependent plasticity of synapses between Helix neurons involved in the neural control of feeding and withdrawal behaviors

Short-term activity-dependent synaptic plasticity has a fundamental role in short-term memory and information processing in the nervous system. Although the neuronal circuitry controlling different behaviors of land snails of the genus Helix has been characterized in some detail, little is known about the activity-dependent plasticity of synapses between identified neurons regulating specific behavioral acts. In order to study homosynaptic activity-dependent plasticity of behaviorally relevant Helix synapses independently of heterosynaptic influences, we sought to reconstruct them in cell culture. To this aim, we first investigated in culture the factors regulating synapse formation between Helix neurons, and then we studied the short-term plasticity of in vitro-reconstructed monosynaptic connections involved in the neural control of salivary secretion and whole-body withdrawal. We found that independently of extrinsic factors, cell–cell interactions are seemingly sufficient to trigger the formation of electrical and chemical synapses, although mostly inappropriate—in their type or association—with respect to the in vivo synaptic connectivity. The presence of ganglia-derived factors in the culture medium was required for the in vitro reestablishment of the appropriate in vivo-like connectivity, by reducing the occurrence of electrical connections and promoting the formation of chemical excitatory synapses, while apparently not influencing the formation of inhibitory connections. These heat-labile factors modulated electrical and chemical synaptogenesis through distinct protein tyrosine kinase signal transduction pathways. Taking advantage of in vitro-reconstructed synapses, we have found that feeding interneuron–efferent neuron synapses and mechanosensory neuron–withdrawal interneuron synapses display multiple forms of short-term enhancement-like facilitation, augmentation and posttetanic potentiation as well as homosynaptic depression. These forms of plasticity are thought to be relevant in the regulation of Helix feeding and withdrawal behaviors by inducing dramatic activity-dependent changes in the strength of input and output synapses of high-order interneurons with a crucial role in the control of Helix behavioral hierarchy.

Novel Anticancer Targets and Drug Discovery in Post Genomic Age

Cancer is a serious disease with a complex pathogenesis, which threats human life greatly. Currently, great efforts have been put to the identification of novel anticancer targets and the discovery of anticancer drugs following the progress of chemogenomics, which will be reviewed briefly in this article. Furthermore, during the past 5 years, the global effort of sequencing human genome has provided us with an enormous number of potential targets associated with cancer therapy. As a result, the New Drug Discovery (NDD) is undergoing a transition "from gene to drug". Accordingly, the targets for anticancer drugs studies now are focused on some biological macromolecular targets associated with cancer and several interactive mechanisms involved in the growth and metastasis of cancer cells as well as tumor angiogenesis, such as Matrix Metalloproteinases (MMPs), Aminopeptidase N (APN), Tyrosine Kinase (TK), Farnesyltransferase (FTase) and cell Signal Transduction Pathway and so forth. Among these targets the MMP-2, -9 and APN are the most extensively studied enzymes in our laboratory. The peptidomimetics Matrix Metalloproteinase Inhibitors (MMPIs) and APN inhibitors (APNIs) with the molecular scaffold of pyrrolidine, 3-amino-2-hydroxy-4-phenyl butyric acid (AHPA) and glutamylide, which have been designed and synthesized in our laboratory, will be described in the review, among which the pyrrolidine scaffold is patented with the IC(50) ranging from 1 nM to 300 nM against MMP-2, and MMP-9.

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts.

In recent years, new strategies in cancer therapy have been developed targeting key signaling molecules in the receptor tyrosine kinase signal transduction pathway. In contrast, most therapeutical concepts to manipulate G protein-coupled receptors (GPCR)-mediated disorders are still limited to the use of receptor-specific agonists or antagonists. Visible progress in the understanding of GPCR signaling complexity, especially the detection of several families of highly target- and cell-specific regulator proteins of GPCRs, G proteins, and effector components may open new horizons to develop novel therapeutical concepts targeting GPCR signaling elements. Thus, this review will focus on different molecular levels that may be of particular interest in terms of new drug development such as: (i) GPCR subtypes, allosteric binding sites, dimerization and constitutive activity, the use of RAMPs (receptor-activity-modifying proteins) and RASSLs (receptor activated solely by synthetic ligands); (ii) AGS (activators of G protein signaling) and RGS (regulators of G protein signaling) proteins which modify G protein activity; (iii) the high diversity of isozymes involved in the generation, signal transmission, and degradation of second messenger molecules.

Reactive oxygen species as mediators of signal transduction in ischemic preconditioning.

Ischemic preconditioning (IPC) is a most powerful endogenous mechanism for myocardial protection against ischemia/reperfusion injury. It is now apparent that reactive oxygen species (ROS) generated in the mitochondrial respiratory chain act as a trigger of IPC. ROS mediate signal transduction in the early phase of IPC through the posttranslational modification of redox-sensitive proteins. ROS-mediated activation of Src tyrosine kinases serves a scaffold for interaction of proteins recruited by G protein-coupled receptors and growth factor receptors that is necessary for amplification of cardioprotective signal transduction. Protein kinase C (PKC) plays a central role in this signaling cascade. A crucial target of PKC is the mitochondrial ATP-sensitive potassium channel, which acts as a trigger and a mediator of IPC. Mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase, p38 MAP kinase, and c-Jun NH(2)-terminal kinase) are thought to exist downstream of the Src-PKC signaling module, although the role of MAP kinases in IPC remains undetermined. The late phase of IPC is mediated by cardioprotective gene expression. This mechanism involves redox-sensitive activation of transcription factors through PKC and tyrosine kinase signal transduction pathways that are in common with the early phase of IPC. The effector proteins then act against myocardial necrosis and stunning presumably through alleviation of oxidative stress and Ca(2+) overload. Elucidation of IPC-mediated complex signaling processes will help in the development of more effective pharmacological approaches for prevention of myocardial ischemia/reperfusion injury.

Influence of chelidonine, an inhibitor of tubulin polymerisation on tyrosine kinase activity in normal, transformed and malignant cell lines

Chelidonine, a tertiary hexahydro-benzophenanthridine alkaloid is an inhibitor of tubulin polymerisation and has been revealed to arrest cells in G2/M. Since enhanced tyrosine kinase (TK) activity is linked to the transition from normal to the immortal malignant phenotype, the effect of 10 mM chelidonine was evaluated on TK activity in normal, transformed and malignant cell lines after 2 hours of exposure. Chelidonine caused a stimulation of TK activity in two normal cell lines (human foreskin fibroblast (Hs27) and normal monkey kidney (NMK)). In contrast, an inhibition of TK activity was observed in transformed human embryonic kidney (Graham 293) and transformed African green monkey kidney (Vero), as well as in human cervical carcinoma (HeLa) and squamous oesophageal carcinoma (WHCO5) cells. Hs27 cells exposed to chelidonine, revealed an increase in TK activity of 1.27-fold (P < 0.05). NMK cells showed a 1.15-fold increase in TK activity. A decrease in TK activity was observed in Graham 293 (0.91-fold) and Vero (0.45-fold) (P < 0.005) cells. In both HeLa and WHCO5 cells, the TK activity was reduced to 0.68-fold (P < 0.05) and 0.56-fold (P < 0.005) respectively. These data, including results from our previous studies, suggest a potential cross talk between the SAPK/JNK and TK signal transduction pathways and a possible differential effect of chelidonine on the phosphorylation status of role players involved in determining the length of G2 arrest in normal versus transformed and malignant cells.

Tyrosine phosphorylation-mediated signal transduction in MCP-1-induced macrophage activation: role for receptor dimerization, focal adhesion protein complex and JAK/STAT pathway

Monocyte chemoattractant protein-1 (MCP-1) plays a crucial role in the recruitment of monocytes/macrophages associated with several inflammatory diseases and malignancies. The early signal transduction mechanism of macrophage activation in response to in vitro MCP-1 treatment was investigated. The treatment of murine peritoneal macrophages with MCP-1 resulted in a significant enhancement in the tyrosine phosphorylation of cellular proteins, which peaked within 2.5–5 min of MCP-1 treatment. The MCP-1-induced tyrosine phosphorylation of cellular proteins involved the phosphorylation of non-receptor tyrosine kinases Lyn, JAK2, cytoskeletal binding protein paxillin and downstream transcription factors STAT3 and STAT5. Immunoflourescence microscopical studies on MCP-1-treated macrophages showed the cellular localization of the tyrosine-phosphorylated proteins and bundling of actin filaments at the focal adhesion points. MCP-1-induced association of focal adhesion proteins Lyn/phospho-paxillin with CCR2 was also observed by co-precipitation. Inhibitor studies with genistein on MCP-1-induced macrophage TNF and IL-1 production additionally supported the role of protein tyrosine phosphorylation in the process of macrophage activation with MCP-1. Present investigations suggest that the early events in the tyrosine kinase signal transduction pathway for macrophage activation in response to MCP-1 probably involve (1) CCR2 receptor dimerization, (2) enhanced tyrosine phosphorylation and assembly of focal adhesion complex, and (3) the activation of JAK/STAT pathway in the murine peritoneal macrophages.

Tumor Cells Express FcγRl Which Contributes to Tumor Cell Growth and a Metastatic Phenotype

High levels of circulating immune complexes containing tumor-associated antigens are associated with a poor prognosis for individuals with cancer. The ability of B cells, previously exposed to tumor-associated antigens, to promote both in vitro and in vivo tumor growth formed the rationale to evaluate the mechanism by which immune complexes may promote tumor growth. In elucidating this mechanism, FcγRl expression by tumor cells was characterized by flow cytometry, polymerase chain reaction, and sequence analysis. Immune complexes containing shed tumor antigen and anti-shed tumor antigen Ab cross-linked FcγRl-expressing tumor cells, which resulted in an induction of tumor cell proliferation and of shed tumor antigen production. Use of selective tyrosine kinase inhibitors demonstrated that tumor cell proliferation induced by immune complex cross-linking of FcγRl is dependent on the tyrosine kinase signal transduction pathway. A selective inhibitor of phosphatidylinositol-3 kinase also inhibited this induction of tumor cell proliferation. These findings support a role for immune complexes and FcγRl expression by tumor cells in augmentation of tumor growth and a metastatic phenotype.