Jin F, Wienecke R, Xiao G H, Maize J C, DeClue J E, Yeung R S
Division of Medical Science, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9154-9. doi: 10.1073/pnas.93.17.9154.
The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.
在一些结节性硬化症家族的生殖系中发生突变失活的Tsc2基因，编码一种名为结节蛋白的大型膜相关GTP酶激活蛋白（GAP）。对遗传性癌症的艾克大鼠模型的研究有力地支持了Tsc2作为肿瘤抑制基因的作用。在本研究中，通过在缺乏功能性结节蛋白的肿瘤细胞系以及内源性结节蛋白水平正常的大鼠成纤维细胞中表达野生型Tsc2基因，评估了结节蛋白的生物学活性。重新引入野生型Tsc2后，艾克大鼠来源的肾癌细胞的集落形成效率显著降低。表达转染Tsc2基因的肿瘤细胞变得更依赖贴壁生长，并失去了在严重联合免疫缺陷小鼠中形成肿瘤的能力。在细胞水平上，结节蛋白表达的恢复导致形态学变化，其特征为细胞增大和接触抑制增加。与全长Tsc2基因一样，一个仅编码结节蛋白C末端（氨基酸1049 - 1809，包括GAP结构域）的克隆，当转染到艾克大鼠肿瘤细胞中时，能够降低集落形成和体内致瘤性。在正常的大鼠1成纤维细胞中，结节蛋白的条件性过表达也抑制了体外集落形成和细胞生长。这些结果为Tsc2的肿瘤抑制功能提供了直接的实验证据，并表明结节蛋白C末端在该活性中起重要作用。
