Murai Y, Dobashi Y, Okada E, Ishizawa S, Shiota M, Mori S, Takano Y
First Department of Pathology, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan.
Int J Cancer. 2001 Apr 15;92(2):232-9. doi: 10.1002/1097-0215(200102)9999:9999<::aid-ijc1171>3.0.co;2-r.
Five Epstein-Barr virus (EBV)-positive human lymphoma cell lines maintained in severe combined immune deficiency (SCID) mice were used to investigate the role of G1 cyclins in EBV-induced lymphomagenesis. All the primary tumors had been negative for EBV but became positive after establishment in SCID mice, with monoclonal immunoglobulin gene rearrangement and EBV monoclonality. To compare the expression status of G1 cyclins, these EBV-associated lymphoma lines (6 EBV[-] human SCID mouse lymphoma lines, 13 human B cell lymphomas and 8 samples of human tonsil tissue) were examined by reverse transcription-polymerase chain reaction-Southern blotting, Western blotting and immunohistochemistry. mRNA expression of cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin E (CCNE), cyclin-dependent kinase 2 (CDK2) and 4 (CDK4) was found in all 3 types of lymphomas. Western blotting demonstrated identical results. Immunohistochemistry revealed CCND1 to be negative in all lymphomas. CCND2 was positive and restricted to the nuclei in all EBV(+) SCID mouse lymphoma lines, whereas it was limited to the cytoplasm in half of the EBV(-) counterparts. CCNE was positive in the nuclei in all EBV(+) but negative in all EBV(-) SCID mouse lymphoma lines. Immunoprecipitation of EBV(+) and (-) SCID mouse lymphomas for CCND1, CCND2 and CCNE vs. p21, PCNA and CDK2 or CDK4 demonstrated that, in EBV(+) SCID lines, CCND2/CDK4 complexes were present without binding to p21, suggesting independence from p21 regulation. In EBV(-) SCID mouse lymphomas, half of the cases showed complex formation of CCND2/CDK4 without binding of p21. In contrast, CCND1/CDK4 and CCNE/CDK2 were under regulation of p21 in both EBV(+) and (-) lymphomas. These results suggest that differential expression of CCNDs, CCNE and CDKs, as well as variation in their subcellular localization and association with CDK-inhibitor protein, could explain differences in cell proliferation between EBV(+) and EBV(-) lymphomas.
利用5种在严重联合免疫缺陷（SCID）小鼠体内维持生长的爱泼斯坦-巴尔病毒（EBV）阳性人类淋巴瘤细胞系，研究G1细胞周期蛋白在EBV诱导的淋巴瘤发生中的作用。所有原发性肿瘤的EBV检测均为阴性，但在植入SCID小鼠后呈阳性，伴有单克隆免疫球蛋白基因重排和EBV单克隆性。为比较G1细胞周期蛋白的表达状态，通过逆转录-聚合酶链反应- Southern印迹法、蛋白质免疫印迹法和免疫组织化学法对这些EBV相关淋巴瘤细胞系（6个EBV阴性的人类SCID小鼠淋巴瘤细胞系、13个人类B细胞淋巴瘤以及8份人类扁桃体组织样本）进行检测。在所有3种类型的淋巴瘤中均发现细胞周期蛋白D1（CCND1）、细胞周期蛋白D2（CCND2）、细胞周期蛋白E（CCNE）、细胞周期蛋白依赖性激酶2（CDK2）和4（CDK4）的mRNA表达。蛋白质免疫印迹法得出了相同结果。免疫组织化学显示，所有淋巴瘤中的CCND1均为阴性。CCND2呈阳性，且在所有EBV阳性的SCID小鼠淋巴瘤细胞系中局限于细胞核，而在一半的EBV阴性对应细胞系中则局限于细胞质。CCNE在所有EBV阳性细胞系的细胞核中呈阳性，但在所有EBV阴性的SCID小鼠淋巴瘤细胞系中呈阴性。对EBV阳性和阴性SCID小鼠淋巴瘤进行CCND1、CCND2和CCNE与p21、增殖细胞核抗原（PCNA）以及CDK2或CDK4的免疫沉淀实验表明，在EBV阳性的SCID细胞系中，存在CCND2/CDK4复合物，且不与p21结合，提示其不受p21调控。在EBV阴性的SCID小鼠淋巴瘤中，半数病例显示CCND2/CDK4形成复合物，但不与p21结合。相反，在EBV阳性和阴性淋巴瘤中，CCND1/CDK4和CCNE/CDK2均受p21调控。这些结果表明，CCNDs、CCNE和CDKs的差异表达，以及它们在亚细胞定位上的变化和与CDK抑制蛋白的结合情况，可能解释了EBV阳性和EBV阴性淋巴瘤在细胞增殖方面的差异。
