Impaired Ca++ mobilization in rituximab-resistant cells is associated with changes in the structure of CD20 antigen and downregulation of Bax/Bak pro-apoptotic proteins
Authors: Peter S. Kim, Hong Song, Matthew E. Wolpoe, Lanquing Huang, George Sgouros, Leisha A. Emens, Elizabeth M. Jaffee, Richard T. Reilly
Journal: Cancer Research (2006)

647  The use of rituximab as a single agent or in combination with systemic chemotherapy is commonly used in the majority of B-cell lymphomas. Biologically rituximab induces apoptosis via Ca++ dependent mechanisms. Therapeutic strategies to improve rituximab activity are currently been explored in multiple clinical trials. Positive selective pressure by extending treatment with rituximab by using “maintenance” regimens could potentially result in the emergence of resistance to rituximab. Studying and understanding the mechanisms of resistance (MOR) to rituximab could give us valuable information in developing and using rituximab-based therapies that limit its development and/or finding ways to overcome this condition. To this end, we developed several rituximab-resistant cell lines (RRCL) by exposing Raji cells to escalating doses of rituximab with (4RH) or without (2R) concurrent exposure to human complement. follow by limited dilutiion cloning. Functional assays were performed and demonstrated decreased rituximab sensitivity in 2R and 4RH cells. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 [C-terminal] and 1439 [N-terminal]) and external domain (B1) of CD20. Expression of pro- or anti-apoptotic proteins was also determined by Western blotting. Extra- and intracellular Ca++ mobilization was measured by flow cytometric analysis using FLUO-3 AM (acetoxymethyl ester). Pluronic acid F-127 was utilized to load FLUO-3 AM into NHL cells. Optimization of the Ca++ indicator and calibration curves was performed for each cell line. Raji and RRCL were labeled under optimal conditions with FLUO-3 AM/Pluronic Acid F-127. Subsequently, cells were then re-suspended in HANKS media with or without Ca++ and exposed to rituximab or isotype antibody (10μg/ml) +/- human serum (25%). Surface CD20 expression was similar between Raji cells and RRCL. Significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. Also, downregulation of Bax/Bak was demonstrated in RRCL. In vitro exposure of Raji cells to rituximab + HS resulted in Ca++ mobilization even in Ca++-depleted media. Notably, Ca++ mobilization was impaired in RRCL when compared to Raji parental cells. Our data suggest that the acquirement of rituximab resistance in B-cell lymphoma cell lines is associated with changes in the intracellular domain of CD20 and Ca++ regulator pro-apoptotic proteins Bax/Bak resulting in a decrease in the intracellular mobilization of Ca++ in responses to rituximab therapy. Evaluation of lymphoma clinical samples for similar changes is currently underway in an attempt to validate our laboratory model.