Abstract B63: Apaf-1 mislocalization is a strong indicator of drug resistance in B cell lymphomas

Abstract

Abstract Resistance to chemotherapy remains a challenge in the clinical management of diffuse B cell lymphomas despite aggressive chemotherapy such as CHOP and monoclonal CD20. We previously reported that sequestration of Apaf-1 to membrane lipid rafts was responsible for apoptosis resistance in B-cell lymphoma cell lines1. Here, we extended our studies to 60 clinical biopsies from patients with B-cell lymphomas, T-cell lymphomas and reactive lymphadenopathy, to investigate if the resistance to drug-induced apoptosis was, indeed, a function of Apaf-1 mislocalization. Firstly, cells were separated from these biopsies and their sensitivity to a variety of apoptosis inducing agents was assessed. Whereas, most T cell lymphomas as well as reactive lymphadenopathy cells were sensitive to apoptotic stimuli, B cell lymphomas exhibited strong resistance. We then investigated the expression of Apaf-1 and its intracellular localization in these clinical biopsies. To do so, cell fraction was performed to separate cytosol from membrane-enriched fractions. The latter were further subjected to density gradient centrifugation to obtain lipid raft fractions. We show that Apaf-1 was expressed in total cell lysates from B and T cell lymphomas, however upon fractionation the localization was strikingly different. In T cell lymphoma samples as well as in cells derived from reactive lymphadenopathy biopsies, Apaf-1 expression was prominently detected in the cytosol, which correlated with the sensitivity of the cells to apoptotic stimuli. In contrast, whereas cytosolic Apaf-1 expression was significantly lower or absent in almost all B cell lymphomas analyzed, increased localization of the protien was detected in membrane lipid rafts. The latter was confirmed by immunohistochemical analysis of tissues from the same biopsy specimens. Interestingly, the resistance of B cell lymphomas to apoptotic execution (drug-induced or death receptor-mediated) was significantly bypassed upon incubation of cells with pharmacological agents that facilitated the dissociation of Apaf-1 from the lipid rafts to the cytosol. Taken together, our results implicate Apaf-1 mislocalization as a potential diagnostic marker for B-cell lymphomas as well as a predictor of response to therapeutic management. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B63.