Effects of Interleukin 4 (IL-4) on CD20 expression in rituximab-sensitive (RSCL) and rituximab resistant cell lines (RRCL) Free

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While the use of rituximab in combination with chemotherapy has resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. Decrease in CD20 antigen density on the tumor cell surface is a potential mechanism that may explain rituximab resistance. Several investigators are focused on understanding the mechanisms that regulate CD20 expression in order to develop therapeutic strategies to upregulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin) and therefore rituximab activity. In an attempt to characterize the mechanisms responsible for rituximab resistance, we developed several Rituximab Resistant Cell Lines (RRCL) derived from rituximab-sensitive RL and Raji cells. Of interest we demonstrate down-regulation of CD20 mRNA and surface antigen expression in RRCL when compared to RSCL. In our present work we examined transcription factors involved in CD20 expression and tested if IL-4 co-stimulation could enhance CD20 antigen expression and sensitivity to rituximab. To this end, we evaluated differences in the expression of B-cell specific transcription factors (PU.1, Oct-2, Pax5, E2A and EBF) in RSCL RL and RRCL RL-4RH after 24, 48 and 72 hrs exposure to IL-4 (5ng/ml) or control. Changes in surface CD20 were measured by flow cytometry and western blotting while rituximab sensitivity was determined by standardized ⁵¹Chromium-release assays. As previously demonstrated, we found a significant down-regulation of CD20 antigen in the RRCL. In addition, a higher expression of Oct-2, Pax5, PU.1, and EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of Pax5 and EBF in nuclear extracts from RL or RL-4RH when compared with untreated cells. In addition, an up-regulation of cytosolic and surface CD20 was detected in RL-4RH exposed to IL-4. However, rituximab-associated complement mediated cytotoxicity (CMC) was not improved in RRCL pre-exposed to IL-4 when compared to untreated RRCL. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors Oct-2, PU.1, Pax5, EBF and concomitant suppression of CD20 mRNA/antigen. While in vitro exposure to IL-4 results in CD20 re-expression in RRCL, rituximab associated CMC was not improved. These data suggest that either a threshold of CD20 antigen density at the cell surface may be necessary for effective complement-mediated lysis or that there exists alternative mechanisms responsible for rituximab resistance that are not influenced by IL-4 (up-regulation of complement inhibitory proteins).