Hoffmann Michèle J, Koutsogiannouli Evangelia, Skowron Margaretha A, Pinkerneil Maria, Niegisch Günter, Brandt Artur, Stepanow Stefanie, Rieder Harald, Schulz Wolfgang A
Department of Urology, Medical Faculty, Heinrich-Heine-University Düsseldorf , Düsseldorf, Germany.
Institute for Human Genetics, Medical Faculty, Heinrich-Heine-University Düsseldorf , Düsseldorf, Germany.
Bladder Cancer. 2016 Oct 27;2(4):449-463. doi: 10.3233/BLC-160065.
Cell culture models of normal urothelial cells are important for studying differentiation, disease mechanisms and anticancer drug development. Beyond primary cultures with their limitations in lifespan, interindividual heterogeneity and supply, few conditionally immortalized cell lines with limited applicability due to partial transformation or impaired differentiation capacity are available. We describe characteristics of the new spontaneously immortalized cell line HBLAK derived from a primary culture of uroepithelial cells. To characterize utility and limitations of HBLAK cells as an urothelial cell culture model. Differentiation markers were investigated by immunofluorescence and RT-PCR, genetic changes by standard karyotyping, array-CGH, PCR, RT-PCR and exome sequencing; expression of p53 and p21 by Western blotting. HBLAK cells proliferated for >50 passages without senescing. They expressed cytokeratins of basal urothelial cells. Terminal differentiation markers appeared only after induction of differentiation by specific protocols. The karyotype was stable, with few chromosomal changes, especially gains of chromosomes 5 and 20 and a chromosome 9p21 deletion resulting in loss. A C228T promoter mutation was present, but no other mutation typical of urothelial carcinoma. was wild-type and the cell cycle was arrested in response to genomic stress. HBLAK cells retain some differentiation potential and respond to cytotoxic agents similar to normal urothelial cells, but contain genetic changes contributing to immortalization in urothelial tumors. HBLAK may be valuable for evaluating the tumor specificity of novel cancer drugs, but may also be applied as an urothelial carcinogenesis model.
正常尿路上皮细胞的细胞培养模型对于研究细胞分化、疾病机制和抗癌药物开发至关重要。除了原代培养存在寿命有限、个体间异质性和供应问题外，由于部分转化或分化能力受损，适用性有限的条件性永生化细胞系很少。我们描述了源自尿路上皮细胞原代培养的新型自发永生化细胞系HBLAK的特征。以表征HBLAK细胞作为尿路上皮细胞培养模型的效用和局限性。通过免疫荧光和RT-PCR研究分化标志物，通过标准核型分析、阵列比较基因组杂交（array-CGH）、PCR、RT-PCR和外显子测序研究基因变化；通过蛋白质印迹法检测p53和p21的表达。HBLAK细胞增殖超过50代而未衰老。它们表达基底尿路上皮细胞的细胞角蛋白。仅在通过特定方案诱导分化后才出现终末分化标志物。核型稳定，染色体变化很少，尤其是5号和20号染色体增加以及9号染色体p21缺失导致基因丢失。存在C228T启动子突变，但没有尿路上皮癌典型的其他突变。p53是野生型，细胞周期在基因组应激时停滞。HBLAK细胞保留了一些分化潜能，对细胞毒性剂的反应与正常尿路上皮细胞相似，但含有导致尿路上皮肿瘤永生化的基因变化。HBLAK可能对评估新型抗癌药物的肿瘤特异性有价值，但也可作为尿路上皮癌发生模型应用。
