Puthenveettil J A, Burger M S, Reznikoff C A
University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Adv Exp Med Biol. 1999;462:83-91. doi: 10.1007/978-1-4615-4737-2_7.
Normal human uroepithelial cells (HUCs) proliferate rapidly in culture during early passage and then spontaneously undergo replicative senescence. We previously reported that the cyclin D1-CDK4/6 inhibitor, p16INK4a, is elevated at senescence in HUCs. Hence, we proposed that p16INK4a may play a critical role in mediating senescence in this cell type. In the current study, we further characterized the senescent state in HUCs. We also tested the possible roles of changes in other cell cycle proteins, including p53, p21WAF1, pRb, and cyclin D1 in HUC senescence.
Normal HUCs cultured from explants of ureteral mucosa were used for these studies. Senescence associated-beta-galactosidase activity (SA-beta-gal) was used to identify cells in senescence. Flow cytometric analysis was used to determine changes in cell cycle distribution at senescence. Response of cells to serum stimulation was determined by Northern analysis of c-fos. Western analysis was used to assess changes in p53, p21WAF, p16INK4a, cyclin D1 and plasminogen activator inhibitor-1 (PAI-1) levels at senescence.
beta-gal-positive HUCs were blocked at G1/S in senescence and failed to show c-fos induction in response to serum stimulation. As previously reported, senescent HUCs also showed elevated p16INK4a. However, unlike human fibroblasts, neither p53 nor p21WAF1 elevation accompanied HUCs senescence. PAI-1 levels were also not elevated in HUC senescence.
These findings support a model in which elevation of p16INK4a, but not p53 or p21WAF1 plays a critical role in HUC replicative senescence. These findings elucidate the tumor suppressor mechanism of p16INK4a and the frequent loss of either p16INK4a or pRb in invasive human bladder tumors.
正常人类尿道上皮细胞（HUCs）在早期传代培养时增殖迅速，随后自发进入复制性衰老。我们之前报道过，细胞周期蛋白D1 - CDK4/6抑制剂p16INK4a在HUCs衰老时水平升高。因此，我们提出p16INK4a可能在介导这种细胞类型的衰老中起关键作用。在当前研究中，我们进一步对HUCs的衰老状态进行了特征描述。我们还测试了其他细胞周期蛋白变化，包括p53、p21WAF1、pRb和细胞周期蛋白D1在HUCs衰老中的可能作用。
从输尿管黏膜外植体培养的正常HUCs用于这些研究。衰老相关β - 半乳糖苷酶活性（SA - β - gal）用于鉴定衰老细胞。流式细胞术分析用于确定衰老时细胞周期分布的变化。通过对c - fos的Northern分析确定细胞对血清刺激的反应。Western分析用于评估衰老时p53、p21WAF、p16INK4a、细胞周期蛋白D1和纤溶酶原激活物抑制剂 - 1（PAI - 1）水平的变化。
β - gal阳性的HUCs在衰老时被阻滞在G1/S期，对血清刺激无c - fos诱导反应。如先前报道，衰老的HUCs也显示p16INK4a水平升高。然而，与人类成纤维细胞不同，HUCs衰老时p53和p21WAF1水平均未升高。HUCs衰老时PAI - 1水平也未升高。
这些发现支持一种模型，即p16INK4a水平升高而非p53或p21WAF1在HUCs复制性衰老中起关键作用。这些发现阐明了p16INK4a的肿瘤抑制机制以及侵袭性人类膀胱肿瘤中p16INK4a或pRb的频繁缺失。
