Koyama Maho, Oka Takashi, Ouchida Mamoru, Nakatani Yoko, Nishiuchi Ritsuo, Yoshino Tadashi, Hayashi Kazuhiko, Akagi Tadaatsu, Seino Yoshiki
Department of Pediatrics, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan.
Lab Invest. 2003 Dec;83(12):1849-58. doi: 10.1097/01.lab.0000106503.65258.2b.
Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.
此前我们通过Northern印迹、逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹及免疫组化分析，发现淋巴瘤/白血病细胞系及患者标本中SHP1基因的蛋白质和信使核糖核酸（mRNA）表达降低。在本研究中，通过甲基化特异性PCR、亚硫酸氢盐测序及限制性内切酶介导的PCR分析，在许多B细胞白血病/淋巴瘤细胞系以及患者标本中检测到SHP1基因启动子的异常甲基化，包括弥漫性大B细胞淋巴瘤（甲基化频率93%）、黏膜相关淋巴组织（MALT）淋巴瘤（82%）、套细胞淋巴瘤（75%）、浆细胞瘤（100%）及滤泡性淋巴瘤（96%）。高级别MALT淋巴瘤病例（100%）的甲基化频率显著高于低级别MALT淋巴瘤病例（70%），这与高级别（80%）和低级别MALT淋巴瘤（54%）中SHP1蛋白无表达的频率密切相关。这表明异常的CpG甲基化导致的SHP1基因沉默与淋巴瘤进展相关。在用去甲基化试剂5-氮杂-2'-脱氧胞苷处理后，B细胞系中SHP1蛋白表达得以恢复。将完整的SHP1基因转染至不表达SHP1基因的造血培养细胞中，可诱导生长抑制，这表明异常甲基化导致的SHP1基因沉默在恶性淋巴瘤/白血病细胞获得生长优势中起重要作用。B细胞淋巴瘤/白血病患者标本中SHP1基因异常高频率（75%至100%）的CpG甲基化表明，SHP1基因沉默是恶性淋巴瘤/白血病发病的关键事件之一，对诊断或预后标志物以及基因治疗靶点也具有重要意义。这些数据支持SHP1基因是肿瘤抑制基因之一的可能性。
