Loss of Expression of the Pro-Apoptotic Bcl-2 Family Proteins Bak and Bax in Rituximab- and Chemotherapy-Resistant Non-Hodgkin’s Lymphoma Cells.

Abstract

Abstract Sustained remissions following standard chemotherapy are achieved in less than half of patients diagnosed with non-Hodgkin’s lymphoma (NHL). A major case of treatment failure is the development of resistance to current therapies. Early studies demonstrated that rituximab was a safe and effective monotherapy for patients with indolent B-cell lymphoma refractory or relapsed following prior chemotherapy. In one study, Davis et al. demonstrated a loss of responsiveness upon rituximab re-treatment in 60% of patients suggesting the development of antibody resistance. To study mechanisms by which cells become resistant to rituximab we induced rituximab resistance in several well characterized B-cell lymphoma cell lines (Raji, RL, SU-DHL-4) by exposing them to increasing concentrations of rituximab or rituximab plus human serum as a source of complement. Individual clones were than generated from rituximab-resistant cell lines (RRCL) by limiting dilution. Characterization of the cell lines and clones generated by repeated exposure to rituximab revealed that in addition to gaining a rituximab-resistant phenotype they also developed concurrent chemotherapy resistance. In rituximab-sensitive parental cell lines, chemotherapy induced apoptosis via the intrinsic pathway. However, apoptotic cell death was completely blocked in resistant cell lines and clones. Upon investigation of potential mediators of both rituximab and chemotherapy resistance, we observed a significant down-regulation of the pro-apoptotic Bcl-2 family proteins Bax and Bak. We therefore hypothesize that resistance to chemotherapy- and rituximab-induced apoptosis is due to the down-regulation of Bax and Bak observed in all rituximab-resistant cell lines and clones characterized to date. We are currently investigating the mechanism(s) underlying the down-regulation of Bax and Bak protein in RRCL. Preliminary data suggests that wild-type Bax and Bak mRNA is expressed at a comparable level in RRCL and parental cells. This strongly suggests that Bax and Bak protein expression may be differentially controlled via post-transcriptional mechanisms in resistant cells. Currently, we are evaluating expression of Bax and Bak in archived lymphoma biopsy specimens and their correlation to treatment response or resistance. If a clinical correlation is identified, therapies aimed at restoring Bak and/or Bax expression may someday prove useful in circumventing clinical resistance to currently used immuno +/− chemotherapy-based regimens.