Previous studies indicated that genetic deletion of c-fos prevents malignant conversion of benign skin tumors. Conversely, overexpression of c-fos accelerates conversion, and AP-1 transcriptional activity increases as skin tumors progress from benign to malignant. To assess the contribution of AP-1 activity on tumor development, a dominant-negative AP-1 construct has been targeted to the epidermis of transgenic mice using a tetracycline regulated promoter system. When the transgene is continuously expressed postnatally, aging mice develop alopecia and skin erosions. The incidence of benign tumors increases in the transgenic mice during initiation-promotion protocols, but all of the tumors are sebaceous adenomas as contrasted to squamous papillomas in control groups. When the transgene is suppressed, squamous tumors develop but these undergo sebaceous metaplasia upon activation of the transgene. Under conditions of transgene expression, malignant conversion is rare while control groups have a high conversion frequency. Gene profiling analysis of phorbol ester treated skin and skin tumors is revealing a trove of AP-1 regulated genes associated with epidermal differentiation, inflammation and retinoid metabolism. The p53 regulated pro-apoptotic chloride channel protein, mtCLIC, is localized to human chromosome 1p36, a site of abnormalities in many human tumors. MtCLIC RNA expression is reduced in many human tumors, most commonly in breast, renal and ovarian sites. MtCLIC protein is also reduced in mouse skin carcinomas. Analysis of human tumor tissue arrays reveals a spectrum of changes in mtCLIC expression and intracellular localization which we are currently comparing to clinical course. MtCLIC translocates from the cytoplasm to the nucleus after DNA damage, induction of differentiation or senescence, and treatment with TNF-alpha. Deletion mutant studies indicate that the lysine rich nuclear localization sequence (NLS) is required for translocation, and the NH2 terminus of mtCLIC masks the NLS in resting cells. MtCLIC interacts with Ran, Importin-alpha and NTF2 when induced to translocate to the nucleus. Targeting mtCLIC to the nucleus with adenovirus encoding full-length mtCLIC modified with an enhanced NLS accelerates apoptosis and changes nuclear chloride content and pH. TNF-alpha also upregulates mtCLIC. Suppressing NFkappaB further upregulates mtCLIC in the presence of TNF-alpha and increases TNF-alpha mediated apoptosis. However, preventing the increase in mtCLIC by an antisense construct does not prevent apoptosis by TNF-alpha and NFkappaB suppression. Paradoxically expression of mtCLIC antisense in TNF-alpha treated cells enhances the apoptotic response. Likewise, induction of antisense mtCLIC in grafted tumor cells markedly inhibits tumor growth.
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