Soucek T, Pusch O, Wienecke R, DeClue J E, Hengstschläger M
Obstetrics and Gynecology, University of Vienna, Department of Prenatal Diagnosis and Therapy, Währinger Gürtel 18-20, A-1090 Vienna, Austria.
J Biol Chem. 1997 Nov 14;272(46):29301-8. doi: 10.1074/jbc.272.46.29301.
Tuberous sclerosis is an autosomal dominant disorder characterized by the development of benign growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid tumor suppressor protein, tuberin, that functions as a GTPase-activating protein for Rap1, a member of the superfamily of Ras-related proteins. By immunoblot analyses, we found TSC2 expression to be high in G0 as well as in early small G1 cells. Analyses after different cell synchronization procedures revealed that TSC2 mRNA and protein expression do not fluctuate throughout the cell cycle. Using inducible expression systems we further demonstrated that TSC2 expression is not affected by overexpression of the mitogenic transcription factor E2F-1 or c-Myc. Nevertheless, antisense inhibition of tuberin expression in logarithmically growing cells markedly decreased the percentage of cells in G1. Furthermore, we found that cells exposed to TSC2 antisense oligonucleotides did not undergo G0 arrest after serum withdrawal. Antisense inhibition of TSC2 expression also induced quiescent G0-arrested fibroblasts to reenter the cell cycle. Our data show for the first time that the absence of tuberin can both induce cells to pass through the G1/S transition of the eukaryotic cell cycle and prevent them from entering a quiescent state. These results have clear implications for the tumor suppressor function of TSC2. We further found that reentry into the cell cycle upon loss of TSC2 is dependent on the activity of the G1 cyclin-dependent kinases (CDKs), Cdk2 or Cdk4. Taken together with our finding that antisense inhibition of TSC2 causes up-regulation of cyclin D1 expression, these results provide the first evidence for a connection between tuberin/Rap1 and the G1 CDK-dependent regulation of the transition from G0/G1 to S phase.
结节性硬化症是一种常染色体显性疾病，其特征是在许多组织和器官中出现良性肿瘤。连锁分析揭示了位于9号染色体和16号染色体上的两个致病基因。16号染色体上的TSC2基因编码一种含1784个氨基酸的肿瘤抑制蛋白——结节蛋白，它作为Rap1的GTP酶激活蛋白发挥作用，Rap1是Ras相关蛋白超家族的成员。通过免疫印迹分析，我们发现TSC2在G0期以及早期小G1期细胞中的表达很高。不同细胞同步化程序后的分析表明，TSC2 mRNA和蛋白表达在整个细胞周期中不发生波动。使用诱导表达系统，我们进一步证明TSC2的表达不受促有丝分裂转录因子E2F-1或c-Myc过表达的影响。然而，在对数生长期细胞中对结节蛋白表达进行反义抑制，显著降低了G1期细胞的百分比。此外，我们发现暴露于TSC2反义寡核苷酸的细胞在血清撤除后未进入G0期停滞。对TSC2表达的反义抑制还诱导静止的G0期停滞成纤维细胞重新进入细胞周期。我们的数据首次表明，结节蛋白的缺失既能诱导细胞通过真核细胞周期的G1/S期转换，又能阻止它们进入静止状态。这些结果对TSC2的肿瘤抑制功能具有明确的意义。我们进一步发现，TSC2缺失后重新进入细胞周期依赖于G1期细胞周期蛋白依赖性激酶（CDK）Cdk2或Cdk4的活性。连同我们发现对TSC2的反义抑制导致细胞周期蛋白D1表达上调这一结果，这些结果首次为结节蛋白/Rap1与从G0/G1期到S期转换的G1期CDK依赖性调节之间的联系提供了证据。
