Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, UK.
Eur Urol. 2011 Jul;60(1):141-9. doi: 10.1016/j.eururo.2011.02.022. Epub 2011 Feb 21.
The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood.
To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier.
DESIGN, SETTING, AND PARTICIPANTS: Laboratory study.
Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included.
The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance.
By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity.
Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology.
尿路上皮恶性肿瘤的发生不仅是增殖抑制丧失的结果，还涉及细胞分化的丧失，这在组织病理学上定义为分级。尽管肿瘤分级是尿路上皮癌（UC）的独立预后标志物，但支持尿路上皮分化丧失的分子事件仍知之甚少。
研究参与 UC 发生的基因改变对人尿路上皮细胞进行分子分化并形成功能性尿路上皮屏障的能力的影响。
设计、设置和参与者：实验室研究。
用重组逆转录病毒转导正常的人尿路上皮（NHU）细胞培养物，以产生过表达野生型或致癌突变成纤维细胞生长因子受体 3 或人端粒酶逆转录酶（hTERT）的稳定亚系。包括先前生成的携带显性负 CDKN2A 或 p53 突变基因的 NHU 亚系或通过人乳头瘤病毒 16 E6 癌蛋白永生化的亚系。
通过比较转染基因表达和同基因对照 NHU 细胞的表型来证明引入的转基因的活性。比较修饰和对照亚系在世代潜力（寿命）方面的变化以及通过尿路上皮蛋白 2 和 3 的转录表达对分化诱导信号的反应能力。通过测量跨上皮电阻来评估形成屏障上皮的能力。
与肿瘤抑制基因失活或癌基因过度激活相反，hTERT 过表达仅导致寿命延长和永生化。hTERT 永生化细胞没有明显的基因组改变，但对分化信号逐渐不敏感，并丧失形成上皮屏障的能力。对 hTERT 细胞的进一步表征揭示了 p16 细胞周期蛋白依赖性激酶抑制剂表达的下调和对过氧化物酶体增殖物激活受体 γ 的反应性丧失，分别为衰老限制的征服和分化能力的丧失提供了机制解释。尽管可以生成没有染色体异常的永生化尿路上皮细胞系，但这些细胞系在分化和功能能力方面存在缺陷。
hTERT 的过表达促进了具有永生化、分化不敏感表型的尿路上皮细胞的发展。尽管这些细胞为 UC 的分级/分期范例提供了有用的见解，但它们对研究正常尿路上皮细胞/组织生物学和生理学的价值有限。
