Schönthal A H, Hwang J J, Stevenson D, Trousdale M D
Department of Molecular Microbiology and Immunology and Kenneth Norris Jr Comprehensive Cancer Center, The Gene Therapy Laboratories, Los Angeles, CA, 90033, USA.
Exp Eye Res. 1999 May;68(5):531-9. doi: 10.1006/exer.1998.0634.
Corneal endothelial cells have a limited capacity for proliferation. Upon transformation with the SV40 large T antigen, however, these cells undergo division and grow rapidly. In order to gain insight into the control mechanisms that determine this proliferative switch, we investigated the expression level and activity of various known cell cycle-regulatory proteins in these cells. Primary human and rabbit corneal endothelial cells were transduced in vitro with a replication-defective adenovirus containing SV40 large T antigen, and subsequently the expression and activity of cell cycle-regulatory proteins was analyzed. Cells transduced with large T antigen exhibited strongly increased activity of cyclin-dependent kinases. This increase correlated with the elevated expression of various cyclin-dependent kinase subunits, such as cyclin A, and to a lesser extent, cyclin D, cdk2, and cdk4. Furthermore, the expression of two cyclin-dependent kinase inhibitors, p21(WAF1) and p27(KIP1), which was high in primary human cells (but not in primary rabbit cells), was strongly reduced in large T-antigen transduced cells. Thus, the remarkably low proliferative activity of normal human corneal endothelial cells appears to be regulated at two levels: the expression of certain cell cycle-regulatory proteins that are essential for cell cycle progression is extremely low (cyclin A) or somewhat low (cdk2 and cdk4); but the amount of p21 and p27, inhibitors of cell cycle progression, is very high. As a consequence, the enzymatic activity of cyclin-dependent kinase is below detectable levels. However, the growth-inhibitory status of these components is clearly reversible: upon transduction with large T antigen, the expression of cyclin A, cyclin D, cdk2, and cdk4 is induced, whereas the expression of p21 and p27 is inhibited, and the cells proliferate. Thus, our study provides insight into the molecular basis of the attenuated proliferation of corneal endothelial cells and suggests potential targets that could be manipulated for the purpose of therapeutic interventions aimed at renewed cell growth.
角膜内皮细胞的增殖能力有限。然而，在用猿猴病毒40大T抗原转化后，这些细胞会进行分裂并快速生长。为了深入了解决定这种增殖转变的控制机制，我们研究了这些细胞中各种已知细胞周期调节蛋白的表达水平和活性。将含有猿猴病毒40大T抗原的复制缺陷型腺病毒在体外转导原代人角膜内皮细胞和兔角膜内皮细胞，随后分析细胞周期调节蛋白的表达和活性。用大T抗原转导的细胞表现出细胞周期蛋白依赖性激酶的活性显著增加。这种增加与各种细胞周期蛋白依赖性激酶亚基的表达升高相关，如细胞周期蛋白A，以及程度较轻的细胞周期蛋白D、细胞周期蛋白依赖性激酶2（cdk2）和细胞周期蛋白依赖性激酶4（cdk4）。此外，两种细胞周期蛋白依赖性激酶抑制剂p21（WAF1）和p27（KIP1）在原代人细胞中表达较高（但在原代兔细胞中不高），在用大T抗原转导的细胞中其表达显著降低。因此，正常人角膜内皮细胞极低的增殖活性似乎在两个水平上受到调节：对于细胞周期进程至关重要的某些细胞周期调节蛋白的表达极低（细胞周期蛋白A）或略低（cdk2和cdk4）；但细胞周期进程抑制剂p21和p27的量非常高。结果，细胞周期蛋白依赖性激酶的酶活性低于可检测水平。然而，这些成分的生长抑制状态显然是可逆的：在用大T抗原转导后，细胞周期蛋白A、细胞周期蛋白D、cdk2和cdk4的表达被诱导，而p21和p27的表达被抑制，细胞开始增殖。因此，我们的研究深入了解了角膜内皮细胞增殖减弱的分子基础，并提出了一些潜在的靶点，为旨在促进细胞重新生长的治疗干预措施提供了可操作的方向。
