G-protein-gated K+ (KG) channels generate slow inhibitory postsynaptic potentials in the brain. Current opinion suggests that neuronal KG channels are heterotetramers of Kir3.1 and Kir3.2. In substantia nigra (SN), however, mRNA of Kir3.1 does not express, whereas that of Kir3.2 clearly does. Therefore, we have characterized the KG channels containing Kir3.2 subunits in SN using biochemical and immunological techniques. We found that they were composed of only Kir3.2 subunits and did not contain significant amounts of either Kir3.1 or Kir3.3. Furthermore, at least some of the KG channels in SN were assemblies of the splicing variants Kir3. 2a and Kir3.2c. The channels were localized specifically at the postsynaptic membrane on the dendrites of dopaminergic neurons. Kir3. 2c, but not Kir3.2a, could bind a PDZ domain-containing protein, PSD-95. The heterologously expressed KG channels composed of Kir3.2a plus Kir3.2c or Kir3.2a alone were activated by G-protein stimulation, but expression of Kir3.2c alone was not. This study reveals that the Kir3.2 splicing variants play distinct roles in the control of function and localization of some of the KG channels in dopaminergic neurons of SN.

Malignant transformation in melanoma is characterized by a phenotype ''switch'' from E-to N-cadherin, which is associated with increased motility and invasiveness of the tumor and altered signaling, leading to decreased apoptosis. We hypothesized that the novel pentapeptide (ADH-1), which disrupts N-cadherin adhesion, could sensitize melanoma tumors to the cytotoxic effects of chemotherapy. N-cadherinexpressing human melanoma-derived cell lines were used to generate xenografts in animal models to study isolated limb infusion with melphalan and systemic chemotherapy with temozolomide. We report here that melphalan in combination with ADH-1 significantly reduced tumor growth up to 30-fold over melphalan alone. ADH-1 enhancement of response to melphalan was associated with increased formation of DNA adducts, increased apoptosis, and intracellular signaling changes associated with focal adhesions and fibroblast growth factor receptors. Targeted therapy using an N-cadherin antagonist can dramatically augment the antitumor effects of chemotherapy and is a novel approach to optimizing treatment for melanoma. [Cancer Res 2008;68(10):3777-84]

Purpose: Despite objective response rates of only f13%, temozolomide remains one of the most effective single chemotherapy agents against metastatic melanoma, second only to dacarbazine, the current standard of care for systemic treatment of melanoma. The goal of this study was to identify molecular and/or genetic markers that correlate with, and could be used to predict, response to temozolomide-based treatment regimens and that reflect the intrinsic properties of a patient's tumor. Experimental Design: Using a panel of 26 human melanoma-derived cell lines, we determined in vitro temozolomide sensitivity, O 6 -methylguanine-DNA methyltransferase (MGMT) activity, MGMT expression and promoter methylation status, and mismatch repair proficiency, as well as the expression profile of 38,000 genes using an oligonucleotide-based microarray platform. Results: The results showed a broad spectrum of temozolomide sensitivity across the panel of cell lines, with IC 50 values ranging from 100 Amol/L to 1mmol/L. There was a significant correlation between measured temozolomide sensitivity and a gene expression signature^derived prediction of temozolomide sensitivity (P < 0.005). Notably, MGMT alone showed a significant correlation with temozolomide sensitivity (MGMT activity, P < 0.0001; MGMT expression, P V 0.0001). The promoter methylation status of the MGMT gene, however, was not consistent with MGMT gene expression or temozolomide sensitivity. Conclusions: These results show that melanoma resistance to temozolomide is conferred predominantly by MGMTactivity and suggest that MGMTexpression could potentially be a useful tool for predicting the response of melanoma patients to temozolomide therapy.Malignant melanoma is increasing at a rate faster than any other cancer, with an expected 62,000 new cases this year (1). Despite advances in our understanding of melanoma biology and the development of several targeted therapeutics, the overall response rates of malignant melanomas to therapy continue to be low.Currently, the drug of choice for the treatment of systemic melanoma is dacarbazine (DTIC). Although DTIC as a single agent has yielded response rates of f15% against melanoma, most of these are incomplete and last only a few months. Temozolomide is a second-generation alkylating agent with a mechanism of action similar to DTIC through the active metabolite 5-(3-methyltriazen-1-yl)imidazole-4-carboximide (MTIC; ref. 2). A randomized phase III trial comparing temozolomide with DTIC showed that temozolomide improved health-related quality of life, had greater systemic exposure to both the parent drug and active metabolite, and was associated with longer progression-free survival (3).

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