Liu Ching-Ann, Wang Mei-Jung, Chi Chin-Wen, Wu Chew-Wun, Chen Jeou-Yuan
Graduate Institute of Life Sciences, National Defense Medical Center, Veterans General Hospital, Taipei, Taiwan, ROC.
J Biomed Sci. 2004 Sep-Oct;11(5):661-70. doi: 10.1007/BF02256132.
Like many epithelial-derived cancers, gastric cancer (GC) results from a multistep tumorigenic process. However, the detailed mechanisms involved in GC formation are poorly characterized. Using an ordered differential display method, we have identified rhotekin (RTKN), the gene coding for the Rho effector, RTKN, as one of the genes differentially expressed in human GC. Northern analysis using human multiple tissue blots showed that RTKN is predominantly expressed in the kidney and spinal cord, and, to a lesser degree, in the thyroid, tongue, liver, brain, prostate, trachea, and stomach. RT-PCR analysis confirmed that RTKN was overexpressed in most (5/7; 71%) GC examined. By analyzing the Stanford Microarray Database for the expression profiles of gastric tissues, we also found a progressional increase in RTKN expression in nonneoplastic mucosa, GC, and then lymph node metastases (p < 0.005 by Jonckheere-Terpstra test), suggesting that RTKN expression correlates with GC progression. The role of RTKN in the pathogenic development of GC was investigated by transfection and expression of RTKN in AGS gastric cells, which express endogenous RTKN at a low basal level. Flow-cytometric analysis showed that RTKN-transfected AGS cells were significantly more resistant than vector-transfected cells to apoptosis upon treatment with sodium butyrate. To explore the mechanisms underlying RTKN-mediated cell survival, a reporter assay was performed. Since the NF-kappaB activation is known to promote cell survival and Rho GTPase may lead to NF-kappaB activation, we transfected AGS cells with the RTKN expression vector along with a pNF-kappaB-Luc reporter plasmid. Our results showed that overexpression of RTKN induced robust activation of NF-kappaB, and RTKN-mediated NF-kappaB activation was suppressed significantly by C3 transferase, an inhibitor of the small GTPase Rho. We conclude that Rho/RTKN-mediated NF-kappaB activation leading to cell survival may play a key role in gastric tumorigenesis. This study provides original documentation for the overrepresentation of the Rho GTPase effector rhotekin in human cancer and its links to cancer formation.
与许多上皮来源的癌症一样，胃癌（GC）源于一个多步骤的致瘤过程。然而，参与胃癌形成的详细机制仍不清楚。我们使用一种有序差异显示方法，鉴定出编码Rho效应蛋白rhotekin（RTKN）的基因RTKN，它是在人类胃癌中差异表达的基因之一。使用人类多组织印迹进行的Northern分析表明，RTKN主要在肾脏和脊髓中表达，在甲状腺、舌头、肝脏、大脑、前列腺、气管和胃中表达程度较低。RT-PCR分析证实，在大多数（5/7；71%）检测的胃癌中RTKN过表达。通过分析斯坦福微阵列数据库中胃组织的表达谱，我们还发现RTKN在非肿瘤性黏膜、胃癌以及随后的淋巴结转移中的表达呈逐步增加（Jonckheere-Terpstra检验，p < 0.005），这表明RTKN表达与胃癌进展相关。通过在低基础水平表达内源性RTKN的AGS胃癌细胞中转染并表达RTKN，研究了RTKN在胃癌致病发展中的作用。流式细胞术分析表明，用丁酸钠处理后，转染RTKN的AGS细胞比转染载体的细胞对凋亡具有显著更高的抗性。为了探索RTKN介导的细胞存活的潜在机制，进行了一项报告基因检测。由于已知NF-κB激活可促进细胞存活，且Rho GTP酶可能导致NF-κB激活，我们用RTKN表达载体和pNF-κB-Luc报告质粒转染AGS细胞。我们的结果表明，RTKN的过表达诱导了NF-κB的强烈激活，并且小GTP酶Rho的抑制剂C3转移酶显著抑制了RTKN介导的NF-κB激活。我们得出结论，Rho/RTKN介导的NF-κB激活导致细胞存活可能在胃癌发生中起关键作用。本研究为Rho GTP酶效应蛋白rhotekin在人类癌症中的过度表达及其与癌症形成的联系提供了原始证据。
