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Introduction: Nurses play a crucial role in global health promotion, and innovative teaching strategies are vital to addressing modern educational challenges. Service-learning, a credit-based approach, integrates community service with academic learning, enhancing students' understanding of course content while fostering civic values. Although established in the United States of America and growing in Europe, most studies on service-learning focus on student learning outcomes, often overlooking its impact on the communities served., Aims: The purpose of this study is to test whether the service-learning practice of nursing students teaching a pharmacology curriculum topic to school children promotes knowledge of the topic in both the nursing students and the school children., Design: This research involves two pretest-posttest quantitative analyses and two satisfaction surveys., Participants: Seventy-six nursing students participated during the 2022-2023 academic year, and 69 primary school pupils received the service-learning intervention., Results: Post-test scores of nursing students were significantly higher than pre-test scores (p = 0.0009). The percentage of correct answers post-intervention was significantly higher than pre-intervention (p < 0.0001). Additionally, 98.7 % of nursing students found the service-learning experience beneficial for learning, and 94.7 % reported increased social awareness. For the school children, the percentage of correct responses after the service-learning experience was statistically significantly higher than before the activity (p < 0.0001). The percentage of correct answers from pupils in each primary grade (fourth, fifth, and sixth) was statistically significantly higher after the application of the learning experience compared to before (p < 0.0001, p < 0.0001, and p < 0.001, respectively). The satisfaction survey indicated high acceptance of the methodology among both nursing students and school pupils., Conclusions: Service-learning enhances knowledge of the pharmacology topic in both nursing students and school children., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

GRB2-associated binder 1 (GAB1) is the inaugural member of the GAB/DOS family of pleckstrin homology (PH) domain-containing proteins. Upon receiving various stimuli, GAB1 transitions from the cytoplasm to the membrane where it is phosphorylated by a range of kinases. This event recruits SH2 domain-containing proteins like SHP2, PI3K's p85 subunit, CRK, and others, thereby activating distinct signaling pathways, including MAPK, PI3K/AKT, and JNK. GAB1-deficient embryos succumb in utero, presenting with developmental abnormalities in the heart, placenta, liver, skin, limb, and diaphragm myocytes. Oncogenic mutations have been identified in the context of cancer. GAB1 expression levels are disrupted in various tumors, and elevated levels in patients often portend a worse prognosis in multiple cancer types. This review focuses on GAB1's influence on cellular transformation particularly in proliferation, evasion of apoptosis, metastasis, and angiogenesis-each of these processes being a cancer hallmark. GAB1 also modulates the resistance/sensitivity to antitumor therapies, making it a promising target for future anticancer strategies.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice ( n = 165) generated by backcrossing were treated with doxorubicin and docetaxel. We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients ( n = 71) and women with breast cancer ( n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.

Metabolic changes that facilitate tumor growth are one of the hallmarks of cancer. These changes are not specific to tumors but also take place during the physiological growth of tissues. Indeed, the cellular and tissue mechanisms present in the tumor have their physiological counterpart in the repair of tissue lesions and wound healing. These molecular mechanisms have been acquired during metazoan evolution, first to eliminate the infection of the tissue injury, then to enter an effective regenerative phase. Cancer itself could be considered a phenomenon of antagonistic pleiotropy of the genes involved in effective tissue repair. Cancer and tissue repair are complex traits that share many intermediate phenotypes at the molecular, cellular, and tissue levels, and all of these are integrated within a Systems Biology structure. Complex traits are influenced by a multitude of common genes, each with a weak effect. This polygenic component of complex traits is mainly unknown and so makes up part of the missing heritability. Here, we try to integrate these different perspectives from the point of view of the metabolic changes observed in cancer.

Metabolic changes that facilitate tumor growth are one of the hallmarks of cancer. The triggers of these metabolic changes are located in the tumor parenchymal cells, where oncogenic mutations induce an imperative need to proliferate and cause tumor initiation and progression. Cancer cells undergo significant metabolic reorganization during disease progression that is tailored to their energy demands and fluctuating environmental conditions. Oxidative stress plays an essential role as a trigger under such conditions. These metabolic changes are the consequence of the interaction between tumor cells and stromal myofibroblasts. The metabolic changes in tumor cells include protein anabolism and the synthesis of cell membranes and nucleic acids, which all facilitate cell proliferation. They are linked to catabolism and autophagy in stromal myofibroblasts, causing the release of nutrients for the cells of the tumor parenchyma. Metabolic changes lead to an interstitium deficient in nutrients, such as glucose and amino acids, and acidification by lactic acid. Together with hypoxia, they produce functional changes in other cells of the tumor stroma, such as many immune subpopulations and endothelial cells, which lead to tumor growth. Thus, immune cells favor tissue growth through changes in immunosuppression. This review considers some of the metabolic changes described in breast cancer.

The relationship between mutated proteins and the cancer stem-cell population is unclear. Glioblastoma tumors frequently express EGFRvIII, an EGF receptor (EGFR) variant that arises via gene rearrangement and amplification. However, expression of EGFRvIII is restricted despite the prevalence of the alteration. Here, we show that EGFRvIII is highly coexpressed with CD133 and that EGFRvIII(+)/CD133(+) defines the population of cancer stem cells (CSC) with the highest degree of self-renewal and tumor-initiating ability. EGFRvIII(+) cells are associated with other stem/progenitor markers, whereas markers of differentiation are found in EGFRvIII(-) cells. EGFRvIII expression is lost in standard cell culture, but its expression is maintained in tumor sphere culture, and cultured cells also retain the EGFRvIII(+)/CD133(+) coexpression, self-renewal, and tumor initiating abilities. Elimination of the EGFRvIII(+)/CD133(+) population using a bispecific antibody reduced tumorigenicity of implanted tumor cells better than any reagent directed against a single epitope. This work demonstrates that a mutated oncogene can have CSC-specific expression and be used to specifically target this population., (©2013 AACR.)

Background: EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community., Results: In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 10⁷ M⁻¹ as determined by enzyme-linked immunosorbent assay (ELISA)., Conclusion: This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

c-Jun NH(2)-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and have been implicated in the formation of several human tumors, especially gliomas. We have previously shown that a 55 kDa JNK isoform is constitutively active in 86% of human brain tumors and then showed that it is specifically a JNK2 isoform and likely to be either JNK2alpha2 or JNK2beta2. Notably, we found that only JNK2 isoforms possess intrinsic autophosphorylation activity and that JNK2alpha2 has the strongest activity. In the present study, we have further explored the contribution of JNK2 isoforms to brain tumor formation. Analysis of mRNA expression by reverse transcription-PCR revealed that JNK2alpha2 is expressed in 91% (10 of 11) of glioblastoma tumors, whereas JNK2beta2 is found in only 27% (3 of 11) of tumors. Both JNK2alpha2 and JNK2beta2 mRNAs are expressed in normal brain (3 of 3). Using an antibody specific for JNK2alpha isoforms, we verified that JNK2alpha2 protein is expressed in 88.2% (15 of 17) of glioblastomas, but, interestingly, no JNK2alpha2 protein was found in six normal brain samples. To evaluate biological function, we transfected U87MG cells with green fluorescent protein-tagged versions of JNK1alpha1, JNK2alpha2, and JNK2alpha2APF (a dominant-negative mutant), and derived cell lines with stable expression. Each cell line was evaluated for various tumorigenic variables including cellular growth, soft agar colony formation, and tumor formation in athymic nude mice. In each assay, JNK2alpha2 was found to be the most effective in promoting that phenotype. To identify effectors specifically affected by JNK2alpha2, we analyzed gene expression. Gene profiling showed several genes whose expression was specifically up-regulated by JNK2alpha2 but down-regulated by JNK2alpha2APF, among which eukaryotic translation initiation factor 4E (eIF4E) shows the greatest change. Because AKT acts on eIF4E, we also examined AKT activation. Unexpectedly, we found that JNK2alpha2 could specifically activate AKT. Our data provides evidence that JNK2alpha2 is the major active JNK isoform and is involved in the promotion of proliferation and growth of human glioblastoma tumors through specific activation of AKT and overexpression of eIF4E.

c-Jun N-terminal kinases (JNKs) are a group of mitogen-activated protein kinase family members that are important in regulating cell growth, proliferation, and apoptosis. Activation of the JNK pathway has been implicated in the formation of several human tumors. We have previously demonstrated that a 55-kDa JNK isoform is constitutively activated in 86% of human brain tumors and more recently demonstrated that this isoform is either JNK2alpha2 or JNK2beta2. Importantly, we have also found that among the 10 known JNK isoforms, the JNK2 isoforms are unique in their ability to autophosphorylate in vitro and in vivo. This does not require the participation of any upstream kinases and also leads to substrate kinase activity in vitro and in vivo. To clarify the mechanism of JNK2alpha2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1alpha2, which does not have detectable autophosphorylation activity, with portions of JNK2alpha2, which has the strongest autophosphorylation activity. Through in vivo and in vitro kinase assays, we were able to define a domain ranging from amino acids 218 to 226 within JNK2alpha2 that is required for its autophosphorylation. Mutation of JNK2alpha2 to its counterpart of JNK1alpha2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1alpha2 to JNK2alpha2 at this 9-amino acid site enabled JNK1alpha2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2alpha2 activity. One site ranging from amino acids 363 to 382 of JNK2alpha2 is required for efficient c-Jun binding in vitro, and a site ranging from amino acids 383 to 424 enhances autophosphorylation intensity, although it is not required for triggering the autophosphorylation in vitro. These findings have uncovered the regions required for JNK2alpha2 autophosphorylation, and this information could be used as potential targets to block JNK2alpha2 activation.

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.

Grb2-associated binder 1 (Gab1) is a docking protein that is tyrosine phosphorylated following the activation of multiple cytokine receptors and receptor tyrosine kinases. Its function then is to recruit and activate multiple signaling molecules. In our previous work, we showed that Gab1 enhances cell growth and induces the transformed phenotype in NIH3T3 cells downstream of the epidermal growth factor (EGF) receptor. In this report, we analyze how it produces these effects. Because SHP-2 is the major binding partner of Gab1, we mutated its binding site in the Gab1 cDNA (Gab1/DeltaSHP-2). This construct was stably overexpressed in NIH3T3 cells (3T3-Gab1/DeltaSHP-2) and in the wild-type Gab1 cDNA (3T3-Gab1) or an empty expression vector (3T3-CTR). Our findings show that after EGF stimulation, Gab1/DeltaSHP-2 has a higher level of tyrosine phosphorylation at early time points than Gab1. Gab1/DeltaSHP-2 recruits more phosphatidylinositol 3'-kinase than Gab1 after EGF triggering, which accounts for a higher and more sustained AKT activation in 3T3-Gab1/DeltaSHP-2 cells relative to 3T3-Gab1 fibroblasts. Moreover, 3T3-Gab1/DeltaSHP-2 cells demonstrate a higher level of extracellular-regulated kinase 1 activation at early time points of EGF stimulation. However, there was an unexpected decrease in c-fos promoter induction in 3T3-Gab1/DeltaSHP-2 cells when compared with 3T3-Gab1 cells. Additionally, the 3T3-Gab1/DeltaSHP-2 cells show a reversion of the transformed phenotype, including fewer morphologic changes, an increase in stress fiber cytoskeletal organization, and a decrease in cell proliferation and anchorage independent growth. These results reveal that the Gab1/SHP-2 interaction is essential for cell growth and transformation but that this must occur through a novel pathway that is independent of extracellular-regulated kinase or AKT. On the basis of its role in growth and transformation, the Gab1/SHP-2 interaction may become an attractive target for the pharmacologic intervention of malignant cell growth.

Upon the addition of different growth factors and cytokines, the Gab1 docking protein is tyrosine phosphorylated and in turn activates different signaling pathways. On the basis of the large body of evidence concerning cross talk between the signaling pathways activated by growth factors and oxidative stress, we decided to investigate the role of Gab1 in oxidative injury. We stimulated wild-type mouse embryo fibroblasts (MEF) or MEF with a homozygous deletion of the Gab1 gene (-/- MEF) with H(2)O(2). Our results show that Gab1 is phosphorylated in a dose- and time-dependent manner after H(2)O(2) triggering. Gab1 then recruits molecules such as SHP2, phosphatidylinositol 3-kinase (PI3K), and Shc. Gab1 phosphorylation is sensitive to the Src family kinase inhibitor PP2. Furthermore, we demonstrate that Gab1 is required for H(2)O(2)-induced c-Jun N-terminal kinase (JNK) activation but not for ERK2 or p38 activation. Reconstitution of Gab1 in -/- MEF rescues JNK activation, and we find that this is dependent on the SHP2 binding site in Gab1. Cell viability assays reveal that Gab1 has a dual role in cell survival: a positive one through its interaction with PI3K and a negative one through its interaction with SHP2. This is the first report identifying Gab1 as a component in oxidative stress signaling and one that is required for JNK activation.

The c-Jun NH(2)-terminal kinases (JNKs) have a role both in promoting apoptosis and tumorigenesis. The JNKs are encoded by three separate genes (JNK1, 2, and 3), which are spliced alternatively to create 10 JNK isoforms that are either M(r) 55,000 or 46,000 in size. However, the functional significance and distinct role for each splice variant remains unclear. We have noted previously that 86% of primary human glial tumors show activation of almost exclusively the M(r) 55,000 isoforms of JNK. To further study which isoforms are involved, we constructed glutathione S-transferase fusion proteins for all 10 JNK isoforms and examined kinase activity with or without the activating upstream kinase. Surprisingly, five JNK isoforms demonstrate autophosphorylation activity, and in addition, all four JNK2 isoforms (either M(r) 55,000 or 46,000) show a high basal level of substrate kinase activity in the absence of the upstream kinase, especially a M(r) 55,000 JNK2 isoform. Examination revealed autophosphorylation activity at the T-P-Y motif, which is critical for JNK activation, because a mutant lacking the dual phosphorylation sites did not show autophosphorylation or basal kinase activity. Using green fluorescence protein-JNK expression vectors, transient transfection into U87MG cells demonstrates that although the JNK1 isoforms localize predominantly to the cytoplasm, the JNK2 isoforms localize to the nucleus and are phosphorylated, confirming the constitutive activation seen in vitro. We then examined which JNK isoforms are active in glial tumors by performing two-dimensional electrophoresis. This revealed that the M(r) 55,000 isoforms of JNK2 are the principal active JNK isoforms present in tumors. Collectively, these results suggest that these constitutively active JNK isoforms play a significant role in glial tumors. Aside from epidermal growth factor receptor vIII, this is the only other kinase that has been shown to be basally active in glioma. The presence of constitutively active JNK isoforms may have implications for the design of inhibitors of the JNK pathway.

The ERK pathway is typically associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. An analogous signaling module, the JNK pathway, has not been shown to be consistently activated by the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines. The function of the JNK pathway in primary tumors is unclear as it has been implicated in both promoting apoptosis and cell growth in vitro, which may be a reflection of the cell lines chosen. Primary human brain tumors frequently show overexpression of the EGF receptor. To clarify the role of JNK in tumorigenesis, we have investigated the role of JNK in a large panel of primary human brain tumors and tumor derived cell lines. Here we present evidence that JNK has a major role in promoting tumorigenesis both in vivo and in vitro. Western blot analysis demonstrated that 86% (18 of 21) of primary brain tumors showed evidence of JNK activation but only 38% (8 of 21) showed evidence of ERK activation. Kinase assays revealed that 77% of brain tumor cell lines activated JNK in response to EGF (7 of 13) or had high levels of basal activity (3 of 13), whereas none of six normal cell lines analysed, including astrocytes, had these properties. Of several growth factors examined, EGF produced the highest level of JNK induction in tumor cell lines and the duration of activation was greater than that seen for ERK. Expression of a dominant-negative (dn) form of JNK potently inhibited EGF mediated anchorage independent growth and protection from cell death in two glial tumor cell lines. These findings demonstrate that enhanced JNK activation is frequently found in primary brain tumors and that this activation contributes to phenotypes related to transformation.

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.

The Grb2-associated binder-1 (Gab1) is one of the major adapter molecules downstream of growth factor receptor signaling. Even though insulin causes tyrosine phosphorylation of Gab1, its role in insulin signaling has not been identified yet. We have demonstrated that insulin increased expression of early growth response gene-1 (egr-1), which is one of the most important transcription factors involved in cell proliferation and differentiation. In the present study, the possible role of Gab1 in insulin-induced egr-1 expression was studied using Rat1 fibroblasts expressing human insulin receptors and wildtype Gab1 (HIRc/Gab1(WT)), Gab1 with three tyrosines in the phosphatidylinositol (PI) 3'-kinase binding domain mutated to phenylalanine (HIRc/Gab1(DeltaPI3K)), or histidinol resistance only (HIRc/HIS). Insulin-induced egr-1 expression in HIRc/Gab1(DeltaPI3K) cells was much lower than in the other cells, as determined by Northern blot analysis. These results suggest that Gab1 is involved in the signaling pathway for insulin-induced egr-1 expression through increasing PI3'-kinase activity. The MAP kinase activity increased less with insulin treatment in HIRc/Gab1(DeltaPI3K) cells than in other cells. Inhibition of MAP kinase by the MEK inhibitor completely abolished insulin-induced egr-1 expression. These results suggest that Gab1 increases MAP kinase activity through its PI3'-kinase binding site, which then leads to egr-1 expression. Our results indicate that Gab1 is involved in the control of egr-1 expression regulated by insulin.

Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cgamma, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

Loss of all or part of one copy of chromosome 17p is very common in ovarian and breast tumors. OVCA1 is a candidate tumor suppressor gene mapping to a highly conserved region on chromosome 17p13.3 that shows frequent loss of heterozygosity in breast and ovarian carcinomas. Western blot analysis of extracts prepared from breast and ovarian carcinomas revealed reduced expression of OVCA1 compared with extracts from normal epithelial cells from these tissues. Subcellular localization studies indicate that OVCA1 is localized to punctate bodies scattered throughout the cell but is primarily clustered around the nucleus. Attempts to create cell lines that stably expressed OVCA1 from the cytomegalovirus promoter were generally unsuccessful in a variety of different cell lines. This reduction of colony formation was quantified in the ovarian cancer cell line A2780, where it was demonstrated that cells transfected with plasmids expressing OVCA1 had a 50-60% reduction in colony number as compared with appropriate controls, and only a few of these clones expressed OVCA1, albeit at low levels. The clones that expressed exogenous OVCA1 were found to have dramatically reduced rates of proliferation. Reduced growth rates correlated with an increased proportion of the cells in the G1 fraction of the cell cycle compared with the parental cell line and decreased levels of cyclin D1. The low levels of cyclin D1 appeared to be caused by an accelerated rate of cyclin D1 degradation. Overexpression of cyclin D1 was able to override OVCA1's suppression of clonal outgrowth. These results suggest that slight alterations in the level of OVCA1, such as would occur after reduction of chromosome 17p13.13 to hemizygosity, may result in cell cycle deregulation and promote tumorigenesis.

Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.

The newly identified insulin receptor (IR) substrate, Gab1 [growth factor receptor bound 2 (Grb2)-associated binder-1] is rapidly phosphorylated on several tyrosine residues by the activated IR. Phosphorylated Gab1 acts as a docking protein for Src homology-2 (SH2) domain-containing proteins. These include the regulatory subunit p85 of phosphatidylinositol 3-kinase and phosphotyrosine phosphatase, SHP-2. In this report, using a modified version of the yeast two-hybrid system, we localized which Gab1 phospho-tyrosine residues are required for its interaction with phosphatidylinositol 3-kinase and with SHP-2. Our results demonstrate that to interact with p85 or SHP-2 SH2 domains, Gab1 must be tyrosine phosphorylated by IR. Further, we found that Gab1 tyrosine 472 is the major site for association with p85, while tyrosines 447 and 589 are participating in this process. Concerning Gab1/SHP-2 interaction, only mutation of tyrosine 627 prevents binding of Gab1 to SHP-2 SH2 domains, suggesting the occurrence of a monovalent binding event. Finally, we examined the role of Gab1 PH (Pleckstrin homology) domain in Gab1/IR interaction and in Gab1 tyrosine phosphorylation by IR. Using the modified two-hybrid system and in vitro experiments, we found that the Gab1 PH domain is not important for IR/ Gab1 interaction and for Gab1 tyrosine phosphorylation. In contrast, in intact mammalian cells, Gab1 PH domain appears to be crucial for its tyrosine phosphorylation and association with SHP-2 after insulin stimulation.

Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF.

The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.

The epidermal growth factor receptor has received much interest as a target for various antineoplastic agents, but a complication is that many normal tissues also express this receptor. We have previously identified in human glial tumors an 801-bp in-frame deletion within the epidermal growth factor receptor gene that created a novel epitope at the junction. By using Western blot assays with a mutant-specific antibody as a rapid and sensitive means for detecting this alteration in primary human tumors, it was found that 57% (26 of 46) of high-grade and 86% (6 of 7) of low-grade glial tumors, but not normal brain, express this protein. This altered receptor was also present in 66% (4 of 6) of pediatric gliomas and 86% (6 of 7) of medulloblastomas, 78% (21 of 27) of breast carcinomas, and 73% (24 of 32) of ovarian carcinomas. The fact that this receptor is frequently found in tumors but not in normal tissue makes it an attractive candidate for various antitumor strategies.

During the last few years, the scientific field has focused its attention on the pathogenic role of free radicals in the process of ischemia-revascularization. It is a well-known fact that xanthine oxidase is an important source of tissular free radicals. Bearing this in mind, we designed an experimental protocol to analyse the effect of allopurinol (a xanthine oxidase inhibitor) in the survival of rats after the occlusion of the superior mesenteric artery during a period of 90 minutes and its action on the superoxide radical liberation. The concentration of oxipurinol and allopurinol in the ischemic area (intestine), liver and blood were measured. We concluded that the administration of allopurinol increased the survival rate, which is correlated to higher concentrations of allopurinol and oxipurinol in the inner part of the intestinal cells. A correlation between the survival rate and superoxide radicals was not found.