Glutamate, the major excitatory neurotransmitter in the central nervous system, gates three types of ionotropic receptors: NMDA, 1 AMPA, and kainate (3). Five kainate receptor subunits in two homology groups have been identified: KA1, KA2, and GluR5, GluR6, and GluR7 (4, 5). Expression of individual GluR5-7 subunits in heterologous systems results in homomeric receptors that respond to glutamate or kainic acid with a rapidly desensitizing current (5, 6). KA1 and KA2, on the other hand, are functional only when coexpressed with GluR5, -6, or -7 (5, 6).PSD-95, also known as synapse-associated protein 90, is a scaffold protein that contains three PDZ domains, a SH3 domain, and a guanylate kinase domain (7). The PDZ domains have been shown to bind to the C terminus of NMDA receptor NR2 and kainate receptor GluR6 subunits, and these interactions are important for the clustering of NMDA or kainate receptors in the postsynaptic membrane (7-9). In addition, PSD-95 also binds to cytoskeletal linker proteins and cytoplasmic signaling proteins such as neuronal nitric oxide synthase and the Src family protein tyrosine kinase Fyn (10, 11). PSD-95 appears to link NMDA or kainate receptors to a variety of cellular signaling cascades. In transgenic mice lacking PSD-95, although the localization of NMDA receptors at post-synaptic density remains unaltered, the frequency dependence of NMDAdependent long-term potentiation and long-term depression is shifted, and spatial learning is severely impaired (12). Suppression of PSD-95 expression inhibits NMDA receptor-mediated activation of nitric-oxide synthase and neuronal excitotoxicity (13), which suggests that PSD-95 is critical in coupling glutamate receptors to cellular signaling networks and plays an important role in their biological function within the central nervous system. JNK is a major stress-activated kinase in mammalian systems that is implicated in mediating neuronal death induced by various detrimental stimuli and by glutamate-mediated excitotoxicity (14 -16). In the absence of JNK3, a neuronal form of JNK, kainic acid-induced seizure activity and neuronal degeneration are significantly attenuated (1). This phenotype is strikingly similar to that observed in GluR6-deficient mice (2), which suggests that JNK activation may be involved in GluR6-mediated excitotoxicity.Both MLK2 and MLK3 are members of the mixed lineage kinase family typified by a N-terminal SH3 domain, a middle kinase domain, and a C-terminal proline-rich region that may bind to SH3 domain-containing proteins. MLK2/3 can directly bind and activate MKK4 and MKK7, which in turn phosphorylate and activate JNKs (17-18). Studies from our group and others show that expression of MLK2 induces JNK activation and apoptotic cell death (18 -19).The current study was undertaken to investigate the molec-* This work is supported by United States Army Medical Research and Materiel Command under cooperative agreement DAMD17-00-2-0012 (to Y. F. L.). The costs of publication of this article were defrayed in part by the pay...

Using the polymerase chain reaction to study mRNA expressed in human epithelial tumor cells, a member of a new family of protein kinases was identified. The catalytic domain of this kinase has amino‐acid‐sequence similarity to both the Tyr‐specific and the Ser/Thr‐specific kinase classes. Clones representing two members of this new family have been isolated from a human colonic epithelial cDNA library and sequenced. The predicted amino‐acid sequences of these clones reveal that, in addition to the unusual nature of their kinase catalytic domains, they contain two Leu/Ilezipper motifs and a basic sequence, near their C‐termini. As they possess domains associated with proteins from two distinct functional groups, these kinases have been named mixed‐lineage kinases (MLK) 1 and 2. mRNA from MLK1 has been found to be expressed in epithelial tumor cell lines of colonic, breast and esophageal origin. The MLK1 gene has been mapped to human chromosome 14q24.3–31.

In vivo models that recapitulate oncogene-dependent tumorigenesis will greatly facilitate development of molecularly targeted anticancer therapies. We have developed a model based on activating mutations in c-KIT in gastrointestinal stromal tumors (GISTs). This model comprises murine tumors of FDC-P1 cell lines expressing c-KIT mutations that render the tumors either responsive (V560G) or resistant (D816V) to the small-molecule c-KIT inhibitor, imatinib. Clinically, GIST response to imatinib is associated with rapid reduction in fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET), preceding changes in conventional response criteria by several weeks. Using the FDC-P1 model in small animal PET, FDG uptake into tumors expressing the c-KIT V560G mutation was significantly reduced as early as 4 hours after imatinib treatment. In contrast, no change in FDG uptake was observed in resistant c-KIT D816V-expressing tumors after 48 hours of imatinib treatment. Consistent with the PET results, expression of the glucose transporter, GLUT1, was significantly reduced in V560G tumors at 4 hours, preceding changes in markers of proliferation by several hours. In vitro, imatinib treatment of V560G cells resulted in a reduction of glucose transporter numbers at the cell surface and decreased glucose uptake well before changes in cell viability. Notably, decreased ambient glucose concentrations enhanced the cytotoxic effect of imatinib. Taken together, these data account for the rapidity and significance of the PET response to imatinib and suggest that metabolic effects may contribute to imatinib cytotoxicity. Further, the FDC-P1 model represents a very useful paradigm for molecularly targeted drug development. (Cancer Res 2005; 65(21): 9633-6)

The aim of this study was to evaluate the novel probe 18 F-6-fluoro-N-[2-(diethylamino)ethyl] pyridine-3-carboxamide ( 18 F-MEL050) for the imaging of primary and metastatic melanoma. Methods: PET using 18 F-MEL050 was performed in murine models of melanoma. The specificity of 18 F-MEL050 was studied by comparing its accumulation in pigmented B16-F0 allograft tumors with that of human amelanotic A375 xenografts using PET and high-resolution autoradiography. 18 F-MEL050 PET results were compared with 18 F-FDG PET, the current standard in melanoma molecular imaging. To test the ability of 18 F-MEL050 to assess the metastatic spread of melanoma, a murine model of lung metastasis was imaged by PET/CT, and results correlated with physical assessment of tumor burden in the lungs. Results: In pigmented B16-F0 grafts, 18 F-MEL050 PET yielded a tumor-to-background ratio of approximately 20:1 at 1 h and greater than 50:1 at 2 and 3 h. In the B16-F0 melanoma allograft model, tumor-to-background ratio was more than 9-fold higher for 18 F-MEL050 than for 18 F-FDG (50.9 6 6.9 vs. 5.8 6 0.5). No uptake was observed in the amelanotic melanoma xenografts. Intense uptake of 18 F-MEL050 was evident in metastatic lesions in the lungs of B16-BL6 tumor-bearing mice on PET at 2 h after tracer injection, with high concordance between 18 F-MEL050 accumulation on PET/CT and tumor burden determined at necroscopy. Conclusion: 18 F-MEL050 has a rapid tumor uptake and high retention with specificity for melanin, suggesting great potential for noninvasive clinical evaluation of suspected metastatic melanoma.

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