Down-Regulation of Bax and Bak in Immuno-Chemotherapy Resistant Non-Hodgkin's Lymphoma Cells.

Abstract

The balance between pro-apoptotic (Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1) Bcl-2 family proteins is essential for the maintenance of B-cell homeostasis. Disruption of this critical balance occurs in the majority of B-cell neoplasms. Clinically, high Bcl-2/Bak and Bcl-2/Bax ratios have been associated with decreased median survival (7.3 years and 3.8 years, respectively) in follicular lymphoma patients1. Recent work in mouse embryonic fibroblasts deficient for the multi-domain pro-apoptotic Bcl-2 family proteins Bax and Bak has shown that they are essential for induction of cell death following apoptotic stimuli that act through the mitochondrial (intrinsic) pathway2. The vast majority of clinically available anti-neoplastic agents, including rituximab, are known to induce cell death via this pathway and therefore likely rely on Bax and/or Bak to exert their anti-tumor effects. Alteration in expression of Bax and/or Bak could therefore underlie acquired resistance to rituximab and chemotherapy in NHL patients. To study the phenomenon of rituximab resistance we developed several rituximab-resistant cell lines (RRCL) that we subsequently showed were also resistant to chemotherapy. RRCL were generated by exposing Raji, SU-DHL-4 and RL cells to escalating doses of rituximab +/− human serum and subsequently cloning by limited dilution. In our present work we studied the efficacy of clinically-applicable chemotherapeutic agents against RRCL. Additionally we studied the intrinsic apoptotic pathway in an attempt to explain shared mechanisms of resistance to chemotherapy and rituximab. We found that RRCL have dramatically reduced levels of both Bax and Bak proteins by Western blot while levels of Bcl-2, Bcl-xL and Mcl-1 protein were comparable to parental cells. Transfection of RRCL with Bax or Bak sensitized them to apoptotic cell death. Currently, we are attempting to validate previous studies that have shown that down-regulation of Bax and/or Bak correlates with a poor prognosis in NHL. Additionally, we are exploring the mechanism(s) by which Bax and Bak are down-regulated in RRCL and primary patient samples.