mot-2-mediated cross talk between nuclear factor-κb and p53 is involved in arsenite-induced tumorigenesis of human embryo lung fibroblast cells

Abstract

Background: Inactivation of p53 is involved in arsenite-induced tumorigenesis; however, the molecular mechanisms remain poorly understood. Objective: We investigated the molecular mechanisms underlying the inactivation of p53 and neoplastic transformation induced by arsenite in human embryo lung fibroblast (HELF) cells. Methods: Anchorage-independent growth assays were performed, and tumorigenicity in intact animals was assessed to confirm arsenite-induced neoplastic transformation. We determined the levels and functions of p53, nuclear factor-kappa B (NF-κB; a key transcriptional regulator), and mot-2 (a p53 inhibitor) and their relationships in arsenite-induced transformed HELF cells by two-dimensional electrophoresis, reverse-transcriptase polymerase chain reaction, Western blot, immunofluorescence, and co-immunoprecipitation assays. Results: Exposure of HELF cells to low levels of arsenite increased their proliferation rate and anchorage-independent growth and disrupted normal contact inhibition. When introduced into nude mice, transformed cells were tumorigenic. We used proteomic analysis to identify proteins with altered expression between untreated and arsenite-exposed cells. We found decreased expression of NF-κB repressing factor (NKRF; an inhibitor of NF-κB-mediated gene transcription), increased expression of mot-2, and increased activation of NF-κB. Changes in cells exposed to 1.0 μM arsenite were more marked than changes in cells exposed to 0.5 or 2.0 μM arsenite. Inactivation of NF-κB prevented malignant transformation induced by 1.0 μM arsenite. Moreover, we also identified a mechanism whereby NF-κB regulated p53. Specifically, activation of NF-κB up-regulated mot-2 expression, which prevented nuclear translocation of p53 and switched the binding preference of the p53 and NF-κB coactivator CBP [cyclic AMP-responsive element binding protein (CREB) binding protein] from p53 to NF-κB. Conclusions: mot-2-mediated cross talk between NF-κB and p53 appears to be involved in arsenite-induced tumorigenesis of HELF cells.