Leung Thomas Ho-Yin, Ching Yick-Pang, Yam Judy Wai Ping, Wong Chun-Ming, Yau Tai-On, Jin Dong-Yan, Ng Irene Oi-Lin
Department of Pathology, SH Ho Foundation Research Laboratories and Hong Kong Jockey Club Clinical Research Centre, Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong.
Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15207-12. doi: 10.1073/pnas.0504501102. Epub 2005 Oct 10.
The deleted in liver cancer 2 (DLC2) gene, located at chromosome 13q12.3, is a recently identified tumor suppressor gene. The gene is frequently underexpressed in human hepatocellular carcinoma, and its chromosomal region shows frequent deletion. DLC2 encodes a unique RhoGTPase-activating protein (RhoGAP) specific for small RhoGTPases, RhoA, and Cdc42. With bioinformatic analysis, we have identified four different isoforms of DLC2, which we named DLC2alpha, DLC2beta, DLC2gamma, and DLC2delta. Three of the isoforms contain the RhoGAP domain, namely, DLC2alpha, DLC2beta, and DLC2gamma. Ectopic expression of these three isoforms in mouse fibroblasts showed cytoplasmic localization. Of interest, overexpression of these isoforms suppressed the lysophosphatidic acid-induced stress fiber formation in mouse fibroblasts and changed the morphology of the transfected cells from angular and spindle to round. Furthermore, the RhoA pull-down assay demonstrated a remarkable reduction in RhoA activity in the DLC2 transiently transfected cells. In contrast, cells transfected with inactive DLC2 GAP-mutant remained unchanged in cell morphology, actin stress fiber formation, and RhoA activity. HepG2 hepatoma cells stably transfected with the DLC2gamma isoform also changed to a round morphology, as in mouse fibroblasts. Of significance, these DLC2gamma stable transfectants showed marked suppression in cell proliferation, motility, and transformation, and there was a remarkable reduction in in vivo RhoA activity in these cells. These results suggest that DLC2 exhibits its tumor suppressor functions in vivo as a GAP specific for RhoA, exerting its effects in suppression of cytoskeleton reorganization, cell growth, cell migration, and transformation.
肝癌缺失基因2（DLC2）位于13号染色体q12.3区域，是最近发现的一种肿瘤抑制基因。该基因在人类肝细胞癌中常表达不足，其染色体区域常出现缺失。DLC2编码一种独特的RhoGTP酶激活蛋白（RhoGAP），对小RhoGTP酶RhoA和Cdc42具有特异性。通过生物信息学分析，我们鉴定出DLC2的四种不同异构体，分别命名为DLC2α、DLC2β、DLC2γ和DLC2δ。其中三种异构体含有RhoGAP结构域，即DLC2α、DLC2β和DLC2γ。这三种异构体在小鼠成纤维细胞中的异位表达显示出胞质定位。有趣的是，这些异构体的过表达抑制了溶血磷脂酸诱导的小鼠成纤维细胞应激纤维形成，并使转染细胞的形态从角形和纺锤形变为圆形。此外，RhoA下拉试验表明，在DLC2瞬时转染的细胞中，RhoA活性显著降低。相比之下，用无活性的DLC2 GAP突变体转染的细胞在细胞形态、肌动蛋白应激纤维形成和RhoA活性方面保持不变。与小鼠成纤维细胞一样，稳定转染DLC2γ异构体的HepG2肝癌细胞也变为圆形形态。重要的是，这些DLC2γ稳定转染体在细胞增殖、运动和转化方面表现出明显的抑制作用，并且这些细胞中的体内RhoA活性显著降低。这些结果表明，DLC2作为一种对RhoA具有特异性的GAP在体内发挥其肿瘤抑制功能，在抑制细胞骨架重组、细胞生长、细胞迁移和转化方面发挥作用。
