We have previously reported that human IFN-beta suppresses the growth of transplanted ovarian tumor xenografls in athymic nude mice (1), and that IFN-beta induces apoptosis in these cells (2). To identify the genes responsible for cell death, we employed an antisense technical knockout approach (3). In this approach, death regulatory genes are identified by their ability, when expressed in an antisense orientation, to confer resistance to death inducers. Using this technique, we have identified several genes, the Regulators of Interferon-induced Death (RIDs), that enhance IFN-beta activated death in ovarian carcinoma cells. In this study we have characterized one of these genes, RID-2, and identified it as human inositol hexakisphosphate kinase 2 (IP6K2) (4, 5). IP6K2 catalyses the synthesis of diphosphoinositol pentakisphosphate (PP-IP5, contains 7 phosphates) using inositol hexaphosphate (IP6, contains 6 phosphates) as a substrate in the presence of ATP. Cellular IP6K2 levels are post-transcriptionally enhanced by IFN- beta. Ovarian carcinoma cells undergoing IFN-beta-induced apoptosis display elevated TRAIL, elevated IP6K enzymatic activity and elevated PP-IP5 levels. Overexpression of IP6K2 enhances both growth suppressive and apoptotic activities of IFN-beta. Overexpression of IP6K2 also sensitizes cells to ionizing radiation (101). A dominant negative mutant carrying a disrupted inositol phosphate binding domain (IPBD) causes resistance to both the antiproliferative and anti-apoptotic functions of IFN-beta IP6K2 binds TRAF2, an inducer of NFKB activity. We hypothesize that IP6K2 inactivates TRAF2, inhibiting NFKappaB activation. Thus, our studies ascribe a novel function for IP6K2 in cell growth control via apoptosis. Based on our preliminary studies we propose that IP6K2 is a novel regulator of cell death pathways induced by IFN-beta, and we propose to examine the mechanism of its antitumor action.
SPECIFIC AIMS :1. Define the functional relationship between IP6K2 and TRAIL - By TRAIL inactivation, confirm itsfunctional role as a mediator of IFN-beta-induced apoptosis2. Identify specific domains required for IP6K2 action - Identify TRAF-binding domains3. Determine relative importance of IP6K1, IP6K2, and IP6K3 as mediators of IFN-beta- induced apoptosis - Determine if IP6K1 and IP6K3 inhibit NFKB activation4. Determine the relevance of IP6K2 domains for IFN- beta-therapy in vivo - Assess IP6K2 mutants grownas xenografts.
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