Modulation in CD20 Surface Levels
A number of CD20 mAbs are now used in clinical practice or are in different stages of development (Table 3). Most of them such as rituximab, 90Y-Ibritumomab, tositumomab, ofatumumab and Obinutuzumab (GA 101) have been FDA approved for use in NHLs and RA. All anti-CD20 mAbs are biochemically and functionally divided into two distinct subtypes such as rituximab-like type I and tositumomab-like type II as shown in Table 2 [75,76].
In clinical applications, the efficacy of anti-CD20 mAbs seems to be decline after a period of months of treatments due to therapeutic resistance. Actually the explanation for this therapeutic resistance is not clear. The possible mechanisms of this resistance of B-cell NHLs against anti-CD20 mAbs therapy may be include three patterns: (I) Protection of the tumor cells from mAbs mediated depletion of B-cell lymphoma by ADCC, CDC and apoptotic stimulation (II) Inadequate binding of mAbs to the CD20 molecule and (III) Low levels of CD20 antigens on cells surface or reduce CD20 surface levels on cells.
Although, some investigators provide information that decreased levels of CD20 expression and/ or harbor low levels of CD20 on surface of malignant B-cells may be one of the major contributing factors for antibody response [103,104]. However, there is general agreement that diseases such as chronic lymphocytic leukemia display the CD20 cell surface molecules in fairly low levels and respond proportionally less as compared to others low grade B-cell malignancies [30,104–106]. Some studies are strongly suggested that cytokines, some inhibitors and radiation exposure showed strong ability to significantly induced expression of CD20, HER2 and EFGR at both total protein levels as well as availability on cell surface specifically in malignant cells, not on normal cell lineages. In relation to CD20 expression some reports provide strong evident that bryostatin-1, interleukin-4, granulocyte macrophage colony stimulating factor, tumor necrosis factor-α, interferon-α and γ radiation have strong ability to induce changes in CD20 expression at transcription, translation and epigenetically as well as their associated transcription factors as showing in Table 4 [107–113].
The bryostatin-1 induced increases in CD20 expression were found at the transcriptional level. The effects 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 [111]. An increase in CD20 transcription was also shown to be triggered by CpG independently of PU.1 transcription factor in CLL cells [128]. Recently, it was also showed that L-744,832 induced inhibition of farnesyltransferase activity leads to up-regulation of CD20 levels and to improved human tumor cell killing activity followed by anti-CD20 mAbs. Moreover, the inhibition of farnesyltransferase activity was found to be associated with increased binding affinity of PU.1 and Oct-2 to the CD20 promoter sequences [117]. Bortezomib a proteasome inhibitor have potential to induced expression of COOH-terminal region of the internal domain of CD20 but not the whole CD20 molecule [118]. Recent study addressed the potential activity of bortezomib in more detail that the unexpected negative influence of proteasome inhibitors on the CD20 levels as well as rituximab mediated CDC and ADCC toward CD20 positive B-cell malignancies [119]. The CD20 expression is also regulated by epigenetic mechanisms. For example 5-azacytidine (inhibitor of DNA methyltransferase activity) can significantly increases the CD20 expression in B-cell lymphoma [120] and trichostatin-A (a modulator of histone-acetylation status) also have ability to increases CD20 mRNA and protein levels in RRBL1 cells, a B-cell lymphoma cell line [121,122]. Two other HDAC inhibitors such as valproic acid (VPA) and romidepsin both have ability to increased CD20 expression at protein and mRNA levels in B-cell lymphoma cell lines. The VPA-mediated increase in CD20 expression is clinically achievable and safe, but insufficient for inducing cell death. Moreover, it is also revealed that HDAC inhibitors trans-activated the CD20 gene promoter through hyper-acetylation and Sp1 recruitment [123]. Whereas, other reports are exploited that CD20 antigens is down-regulated by anti-CD20 mAb rituximab treatment. It is a well-recognized phenomenon in patients with non-Hodgkin’s lymphomas particular in chronic lymphocytic leukemia (CLL). In CLL, rituximab mediated down modulation of CD20 is associated with reduced levels of CD20 mRNA at in vitro and in vivo indicating regulation of CD20 expression at the level of transcription [129,130]. Recently it is also reported that initially CD20 antigens disappeared in patients with CLL treated with rituximab containing salvage regimens occurred in 4 out of 8 (50%) tested patients after some time CD20 levels returned at progression or recovered. Half of whom developed Richter’s syndrome [131]. One more report indicated that lenalidomide or CD40 ligation in normal B-cells down regulates CD20 levels [132,133]. Radiation induced changes in CD20 expression on B-cell lymphoma were identified first time in 1997 by Philippe et al. [131]. Later on, Kunala et al. was also suggested that exposure of ionizing radiation (10Gy) can significantly increases CD20 surface expression in a dose and time dependent manner in IM9, IM9/Bcl-2 and Ramos neoplastic B-cell lines. In contrast, he was also investigated that CD20 expression was not induced in CD20-negative Molt-4 cell line whereas it was increases only about 25% in the GM1310B normal B-cell line. Moreover, the overexpression of Bcl-2 protein does not inhibited radiation induced CD20 expression. In addition, the treatment of cells with actinomycin-D is known to inhibit RNA synthesis followed by 10Gy γ-radiation. This suggests a transcriptional regulation of CD20 expression rather than a simple alteration in cell surface morphology or surface level of CD20 on the targets cells [126,127]. Gupta et al. strongly suggested that the significant increases in cell surface expression of CD20 were transient and cell type dependent manner in logarithmically growing Daudi and Raji cells followed by 0.5 and/or 1.5Gy radiation exposure. The enhanced expression of CD20 antigen was associated with transcriptional up-regulation of CD20 mRNA and CD20 regulatory transcription factors. Moreover, the changes in CD20 surface levels were found to be correlated with overall changes in oxidative stress and mitochondrial membrane potential [112]. Recently, Singh et al. demonstrated that sub-lethal dose (0.5Gy) of γ-radiation can induce ~3 fold CD20 levels on Burkitt’s lymphoma cell line ‘Daudi’ and it was also associated with changes in oxidative condition in intracellular milieu [124,125]. Moreover, cytokines which involved in CD20 expression also cause robust intracellular oxidative bursts. Accumulating evidence indicates that CD20 expression in malignant cells can be modulated at transcriptional, transcriptional, posttranscriptional and even posttranslational levels and their occurrence and significance may be vary depending on the type of malignancies. However, the precise mechanisms of changes in CD20 expression still unclear and further need to be investigation.