Regulation of CD20 expression by radiation-induced changes in intracellular redox status

Abstract

Increasing the levels of CD20 expression in cells that harbor low CD20 levels may enhance their responsiveness to CD20-specific antibody therapies. Here, we examined the regulation of CD20 expression after treatment with 0.5-2.0 Gy X-irradiation and hydrogen peroxide (H 2 O 2 ), in the presence or absence of known antioxidants, in the Burkitt lymphoma cell lines Daudi and Raji. Irradiation of cells enhanced cell-surface CD20 expression; the kinetics and extent of this change were cell-type specific and time-dependent. The kinetics of reactive oxygen species generation and changes in mitochondrial membrane potential after irradiation were also correlated with changes in CD20 expression. Raji and Daudi cells treated with H 2 O 2 showed a 2-to 2.5-fold increase in CD20 expression at 12 and 20 h, respectively. Buthionine sulfoximine, which depletes glutathione, also increased surface CD20, whereas antioxidants, such as PEG-catalase, PEG-SOD, vitamin C, and amifostine, decreased CD20 expression induced by radiation or H 2 O 2 . The antioxidant-mediated decrease in CD20 expression induced by radiation or H 2 O 2 suggests a mechanism involving redox regulation. These results demonstrate the critical role of radiationinduced oxidative stress in CD20 expression and may have implications for defining and improving the efficacy of CD20-targeted antibody therapy and radioimmunotherapy. Keywords CD20; Ionizing radiation; Reactive oxygen species; Antioxidants; Mitochondria; Mitochondrial membrane potential; Free radicals The CD20 transmembrane protein is exclusively expressed on B cells. It appears during the pre-B-cell stage, but is absent during the earlier or later stages of B cell differentiation, such as in antibody-secreting plasma cells [1,2]. CD20 has received extensive evaluation as an ideal target for immunotherapy and radioimmunotherapy, in part because of its ubiquitous expression on target cells, stable localization within the cell membrane, and absence from normal stem cells [3]. Rituximab, a chimeric monoclonal anti-CD20 antibody, has been used extensively in the treatment of low-grade B cell lymphomas, such as non-Hodgkin lymphoma (NHL) and other related pathologies. In clinical applications, the efficacy of Rituximab seems to fade after a period of months, leading to Rituximab resistance. The Recent studies have reported that bryostatin-1, interleukin-4, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, and interferon-α are all able to induce CD20 surface expression [8][9][10][11][12][13]. However, the mechanisms regulating the expression of CD20 remain poorly understood, even though these cytokines are well known to cause robust intracellular oxidative bursts [14,15]. Recently, it was shown that the bryostatin-1-induced increase of CD20 was regulated at the transcriptional level. The effect of bryostatin-1 on CD20 expression in NHL-derived cells was apparently mediated through the MAPK/ERK signal transduction pathway and involved protein kinase C [13]. Our group has previous...

Bortezomib modulates surface CD20 in B-cell malignancies and affects rituximab-mediated complement-dependent cytotoxicity

“…These include cytokines such as granulocyte-macrophage colonystimulating factor and interleukin-4 as well as several approved or experimental therapeutics. For example, prednisolone, 14 bryostatin-1, 20 radiotherapy, [24][25] and reactive oxygen species 25 were demonstrated to up-regulate CD20 levels in B cells or B-cell tumors. On the other hand, lenalidomide 26 or CD40 ligation in normal B cells 27 down-regulates CD20 levels.…”

Chapter 6 DNA Damage Response and Apoptosis

“…2 Clonogenic assays provide a more general readout for the cumulative effect of various forms of cell death, as well as the ability of cells to proliferate (Gupta et al, 2008;Morrison et al, 2002;Ray et al, 2007). This assay has been extremely useful for epithelial cells and fibroblasts for which it could well predict the clinical tumor response (Brown and Attardi, 2005).…”

Priming Radioimmunotherapy With External Beam Radiation in Patients With Relapsed Low-Grade Non-Hodgkin Lymphoma

“…More recently, radiobiology research has demonstrated that ionizing radiation appears to impact the cellular machinery that regulates CD20 protein expression and trafficking, even at low doses [Gupta et al 2008;Singh et al 2014]. Gupta and colleagues showed that there is an increase in CD20 surface levels through redox signaling after exposure of Burkitt lymphoma cell lines to 0.5-2 Gy of radiation [Gupta et al 2008].…”

“…Gupta and colleagues showed that there is an increase in CD20 surface levels through redox signaling after exposure of Burkitt lymphoma cell lines to 0.5-2 Gy of radiation [Gupta et al 2008]. At least a two-fold enhancement in CD20 surface levels was observed within 12-20 h after radiation.…”

“…Emerging in vitro data have shown that low-dose radiation (0.5-2 Gy) enhances the level of CD20 expression on the cell surface [Gupta et al 2008;Singh et al 2014]. Because the effect of IT-Y90 depends on the surface level of CD20 [Singh et al 2014], we hypothesized there may be a potential synergy between low-dose radiation therapy and RIT.…”