Essner R, Huynh Y, Nguyen T, Rose M, Kojima M, Hoon D S
Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404, USA.
J Gastrointest Surg. 2001 Jan-Feb;5(1):81-90. doi: 10.1016/s1091-255x(01)80017-2.
Interleukin (IL)-2 and IL-4 play a critical role in the regulation of the immune response. Yet both of the receptors for these cytokines have been found on nonhematopoietic cells, including human gastric carcinoma cell lines and tissue specimens. IL-4 causes G1 phase cell cycle arrest of gastric carcinoma; the effect directly correlates with the expression of IL-4 receptor (IL-4R) and is seen within 48 hours after treatment. Cells lacking IL-4R are unaffected by IL-4. We examined signal transduction pathways employed by IL-4 that may account for cell cycle arrest of an established human gastric carcinoma cell line, CRL 1739. Western blot analysis was performed on CRL 1739 cultured in the presence of IL-4 (500 U/ml). Cells were lysed, protein extracted, and electroblotted; blots were then probed with murine mono-clonal antibodies to specific intracellular proteins. Western blotting of CRL 1739 with antiphosphotyrosine antibody (4G10) demonstrated multiple (140 kDa and 65 kDa) phosphoproteins seen only in IL-4-treated CRL 1739. Immunoprecipitation and blotting of CRL 1739 with specific secondary antibodies demonstrated that the 140 kDa phosphoprotein was IL-4R", the 65kDa phosphoprotein was IL-2Rgc, the 130 kDa phosphoprotein was Janus kinase (JAK1), and the 116 kDa phosphoprotein was JAK3. Reverse transcription-polymerase chain reaction with specific primers demonstrated that multiple human gastric tumor specimens expressed IL-4R" and IL-2Rgc but did not express the leukocyte marker CD45. These results suggest that human gastric carcinomas may express functional cytokine receptors, including the IL-2Rgc commonly found in association with the lymphocyte IL-2R. These receptors may represent novel targets for directing cytokine-based therapy.
白细胞介素(IL)-2和IL-4在免疫反应调节中起关键作用。然而,这些细胞因子的两种受体已在非造血细胞上被发现,包括人胃癌细胞系和组织标本。IL-4导致胃癌细胞的G1期细胞周期停滞;这种效应与IL-4受体(IL-4R)的表达直接相关,并且在处理后48小时内可见。缺乏IL-4R的细胞不受IL-4影响。我们研究了IL-4所采用的可能导致已建立的人胃癌细胞系CRL 1739细胞周期停滞的信号转导途径。对在IL-4(500 U/ml)存在下培养的CRL 1739进行蛋白质印迹分析。细胞被裂解、提取蛋白质并进行电印迹;然后用针对特定细胞内蛋白质的鼠单克隆抗体探测印迹。用抗磷酸酪氨酸抗体(4G10)对CRL 1739进行蛋白质印迹显示,仅在经IL-4处理的CRL 1739中可见多种(140 kDa和65 kDa)磷酸化蛋白。用特异性二抗对CRL 1739进行免疫沉淀和印迹表明,140 kDa的磷酸化蛋白是IL-4Rα,65 kDa的磷酸化蛋白是IL-2Rγc,130 kDa的磷酸化蛋白是Janus激酶(JAK1),116 kDa的磷酸化蛋白是JAK3。用特异性引物进行逆转录-聚合酶链反应表明,多个人胃肿瘤标本表达IL-4Rα和IL-2Rγc,但不表达白细胞标志物CD45。这些结果表明,人胃癌可能表达功能性细胞因子受体,包括通常与淋巴细胞IL-2R相关的IL-2Rγc。这些受体可能代表基于细胞因子治疗的新靶点。