Rituximab-Mediated Sensitization of B-NHL Cells Lines to TRAIL-Induced Apoptosis: Involvement of YY1 Suppression and DR5 Upregulation.

Abstract

There has been considerable interest in the treatment of drug-resistant tumor cells with TRAIL or agonist monoclonal antibody directed against the TRAIL receptors DR4 and DR5. TRAIL has been shown to be largely non-toxic to normal tissues and cytotoxic to transformed tumor cells. However, many tumors, including B-NHL, are resistant to TRAIL-induced apoptosis. We have reported that rituximab signals B-NHL cells and inhibits several cell survival signaling pathways leading to chemosensitization (Jazirehi and Bonavida, Oncogene ; 2004: 2121, 2005). In addition, we have recently reported that rituximab sensitizes B-NHL cells to Fas-ligand-induced apoptosis, via inhibition of the transcription repressor Yin Yang 1 (YY1) (Vega et al. The Journal of Immunology;175:2174 2005). We have found that YY1 negatively regulates DR5 transcription and expression and thus, we hypothesized that rituximab-mediated inhibition of YY1 may sensitize TRAIL-resistant B-NHL cells lines to TRAIL-induced apoptosis. The B-NHL cells lines, Ramos and Daudi, were treated with rituximab (20μg/ml for 6h) and were then exposed to various concentrations of recombinant TRAIL (2.5–10 ng/ml for 24h). Following incubation, the cells were examined for apoptosis by assessing activation of caspase-3 and by Annexin V/PI. The findings revealed that the cell lines were relatively resistant to TRAIL but following treatment with rituximab significant potentiation of apoptosis and synergy were achieved. Optimal apoptosis was observed with a concentration of TRAIL of 10ng/ml. The rituximab-treated cells showed a 2 fold upregulation of cell surface DR5 expression as compared to untreated cells. In addition, rituximab treated cells showed significant inhibition of YY1 expression as determined by Western and EMSA. We have also examined the expression of YY1 in tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma, obtained from the Aids and Cancer Specimen Resource of the NCI. These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma and 6 Small Cell Lymphoma and were examined for YY1 by immunhistochemistry. The findings revealed that YY1 was overexpressed as compared to normal tissues. Currently, we are examining the effect of rituximab-mediated sensitization of patients derived B-NHL cells to TRAIL-induced apoptosis. The present findings demonstrate that drug-resistant and TRAIL-resistant B-NHL cells can be sensitized by rituximab to TRAIL-induced apoptosis. Further, the studies revealed a potential novel mechanism of rituximab-mediated effect in vivo by recruiting host cells expressing/secreting TRAIL to exert a cytotoxic activity on the rituximab-treated cells. The findings also suggest the potential therapeutic efficacy, in vivo, of combination of rituximab and either recombinant TRAIL or agonist monoclonal antibodies against DR4 or DR5 in the treatment of resistant cells. We propose that inhibitors of YY1 can serve as a sensitizing agent for TRAIL-induced apoptosis in rituximab-resistant B-NHL cells.