The activity of c-Src protein-tyrosine kinase is upregulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.

Background: Shc is the adaptor protein that exists in three isoforms, P46, P52 and P66, and acts as a bridge between activated cell surface receptors and downstream signalling molecules which act in extracellular signal-regulated cell events such as cell cycle progression. In our previous studies, Shc was shown to be a substrate of the tyrosine kinase c-Src in vitro and in vivo.

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This study aimed at quantitatively evaluating hippocampal central-type benzodiazepine receptors (BZRs) in the kainate model of temporal lobe epilepsy (TLE) by in vitro autoradiography (ARG) using [(125)I] Iomazenil (IMZ) specific ligand for central-type BZRs. Kainate (1 microg/0.5 microl) was injected into the left amygdala to induce limbic status epilepticus. One, three, or six months after injection, in vitro ARG with [(125)I] IMZ and cell counts were performed in the hippocampal CA1-4 regions and dentate gyrus ipsilateral to the kainate injection site, and were compared with the vehicle-injected control group. In all kainate-treated rats, clear pyramidal neuron loss was observed in left hippocampal areas CA1-4. Compared with the control group, progressive reduction of [(125)I] IMZ binding was also observed. This resulted in a marked binding decrease paralleling pyramidal neuron loss in hippocampal areas CA1 (down to 83% of control), CA2 (76%), CA3 (75%), and CA4 (90%) at 6 months after kainate administration. Conversely, [(125)I] IMZ binding significantly increased in the dentate gyrus (up to 106% of control) at 1 month, but returned to nearly normal at 3-6 months. These results suggest that central-type BZR neuroimaging is useful in detecting hippocampal sclerosis in the mesial TLE, though central BZR alterations differ depending on hippocampal subfields and post-seizure time-courses.

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According to Fearon and Vogelstein, the process of malignant transformation in intestinal cells is caused by stepwise gene mutations with phenotypic adenoma-carcinoma-sequence. COX inhibitors show tumorsuppressive effects both in vitro and in vivo. Since intestinal tumors of humans, rats and mice show overexpression of the COX-2 isoenzyme, the specific inhibition of COX-2 is considered to be a basic mechanism of tumor prevention. Protein kinase C (PKC) family plays an important role in signal transduction. PKCS isoenzyme is reported to be involved in cell differentiation and apoptosis onset. To investigate changes in PKCS expression and distribution under APC gene defect and COX-2 inhibition, we used the MIN-mouse model. Mice were treated with three different COX-inhibitors of various specificity over 60 days against control. Piroxicam, Sulindac, and Meloxicam were administered with drinking water in adequate doses. Formalin-fixed samples of the small intestine were investigated by indirect immunohistochemistry with antibodies against PKCS. Both in untreated and in Balb C mice, rare positive signals of PKCS were found mainly at the luminal surface of the villi. The cellular distribution was ubiquitous in the cytoplasm. In contrast, a distinct nuclear immunoreactivity was found under COX inhibition. PKCS immunoreactivity seemed to increase with COX-2 inhibitor specificity. These results corresponded with increased apoptotic activity (TUNEL, KLENOW) in the intestinal tissue after treatment with COX inhibitors. Considering that the translocation of PKCG from the cytosol to membrane structures and nucleus is a sign of its activation, a role for this isoenzyme in the regulation of the anticarcinogenic effects of COX-2 inhibition is to be concluded.