Regulation of CD20 in rituximab-resistant cell lines and B-cell non-Hodgkin lymphoma.
Regulation of CD20 in rituximab-resistant cell lines and B-cell non-Hodgkin lymphoma.
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DOI：
10.1158/1078-0432.ccr-11-1429
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发表时间：
2012-02-15
期刊：
影响因子：
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通讯作者：
Czuczman MS
中科院分区：
文献类型：
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作者：
Tsai PC;Hernandez-Ilizaliturri FJ;Bangia N;Olejniczak SH;Czuczman MS
本研究的目的是进一步研究CD20抗原表达对利妥昔单抗活性的影响，并确定在利妥昔单抗耐药细胞系(RRCL)中CD20下调的机制。通过Chromium51释放实验、ImageStream图像分析、免疫组织化学染色、流式细胞仪分析、CD20基因敲除、启动子活性、CD20启动子染色质免疫沉淀(ChIP)分析和CD20质粒转染实验，对利妥昔单抗敏感、利妥昔单抗耐药细胞系和原代肿瘤B细胞进行鉴定，以确定与CD20调控相关的机制。随着利妥昔单抗的反复作用，RRCL的CD20表面表达逐渐丧失。我们确定了有效的利妥昔单抗相关补体介导的细胞毒作用(CMC)所需的CD20抗原表面阈值水平。然而，CD20表面表达与利妥昔单抗-CMC之间的直接相关性仅在利妥昔单抗敏感细胞系(RSCL)中观察到。RRCL中CD20启动子活性降低。对各种CD20启动子片段的详细分析表明，RRCL中缺乏积极的调控因子。芯片分析显示RRCL中几个关键的阳性调控蛋白与CD20启动子的结合减少。白介素4(IL-4)诱导RRCL和原代B细胞淋巴瘤细胞CD20启动子活性和CD20表达增加，但美妥昔单抗活性略有提高。强制表达CD20可恢复胞浆内CD20表达，但不能恢复表面CD20表达，提示RRCL存在CD20蛋白转运缺陷。我们确定了几种改变RRCL中CD20表达的机制，并证明，虽然CD20表达对美罗华单抗活性很重要，但其他因素可能会导致B细胞淋巴瘤对美罗华单抗的敏感性。
The aim of this research was to further investigate the contribution of CD20 antigen expression to rituximab activity and define the mechanisms responsible for CD20 downregulation in rituximab-resistant cell lines (RRCL). Rituximab-sensitive, rituximab-resistant cell lines, and primary neoplastic B-cells were evaluated by Chromium51-release assays, ImageStream image analysis, immunohistochemical staining, flow cytometric analysis, CD20 knockdown, promoter activity, chromatin immunoprecipitation (ChIP) analysis of CD20 promoter, and CD20 plasmid transfection experiments in order to identify mechanisms associated with CD20 regulation in rituximab-resistant cells. RRCL exhibited a gradual loss ofCD20 surface expressionwith repeated exposure to rituximab. We identified a CD20 antigen surface threshold level required for effective rituximab-associated complement-mediated cytotoxicity (CMC). However, a direct correlation between CD20 surface expression and rituximab-CMC was observed only in rituximab-sensitive cell lines (RSCL). CD20 promoter activity was decreased in RRCL. Detailed analysis of various CD20 promoter fragments suggested a lack of positive regulatory factors in RRCL. ChIP analysis showed reduced binding of several key positive regulatory proteins on CD20 promoter in RRCL. Interluekin-4(IL-4)induced higher CD20 promoter activityand CD20 expression, but modestly improving rituximab activity in RRCL and in primary B-cell lymphoma cells. Forced CD20 expression restored cytoplasmic but not surface CD20, suggesting the existence of a defect in CD20 protein transport in RRCL. We identified several mechanisms that alter CD20 expression in RRCL and demonstrated that, while CD20 expression is important for rituximab activity, additional factors likely contribute torituximab sensitivityin B-cell lymphoma.