Cross-Contamination of a UROtsa Stock with T24 Cells – Molecular Comparison of Different Cell Lines and Stocks

BackgroundUROtsa is an authentic, immortalized human urothelial cell line that is used to study the effects of metals and other toxic substances, mostly in the context of bladder cancer carcinogenesis. Unusual properties on the molecular level of a provided UROtsa cell line stock prompted us to verify its identity.MethodsUROtsa cell line stocks from different sources were tested on several molecular levels and compared with other cell lines. MicroRNA and mRNA expression was determined by Real-Time PCR. Chromosome numbers were checked and PCR of different regions of the large T-antigen was performed. DNA methylation of RARB, PGR, RASSF1, CDH1, FHIT, ESR1, C1QTNF6, PTGS2, SOCS3, MGMT, and LINE1 was analyzed by pyrosequencing and compared with results from the cell lines RT4, T24, HeLa, BEAS-2B, and HepG2. Finally, short tandem repeat (STR) profiling was applied.ResultsAll tested UROtsa cell line stocks lacked large T-antigen. STR analysis unequivocally identified our main UROtsa stock as the bladder cancer cell line T24, which was different from two authentic UROtsa stocks that served as controls. Analysis of DNA methylation patterns and RNA expression confirmed their differences. Methylation pattern and mRNA expression of the contaminating T24 cell line showed moderate changes even after long-term culture of up to 56 weeks, whereas miRNAs and chromosome numbers varied markedly.ConclusionsIt is important to check the identity of cell lines, especially those that are not distributed by major cell banks. However, for some cell lines STR profiles are not available. Therefore, new cell lines should either be submitted to cell banks or at least their STR profile determined and published as part of their initial characterization. Our results should help to improve the identification of UROtsa and other cells on different molecular levels and provide information on the use of urothelial cells for long-term experiments.

背景：UROtsa是一种经鉴定的永生化人尿路上皮细胞系，主要用于研究金属及其他有毒物质的生物学效应，其研究场景多聚焦于膀胱癌致癌过程。本研究中所用的某批次UROtsa细胞系在分子层面表现出异常特性，因此我们对其身份进行了验证。
方法：本研究对不同来源的UROtsa细胞系批次开展了多分子层面的检测，并与其他细胞系进行比对。采用实时定量PCR（Real-Time PCR）检测微小RNA（microRNA, miRNA）与信使RNA（mRNA）的表达水平；统计染色体数目，并对大T抗原（large T-antigen）的不同区域开展PCR扩增检测。采用焦磷酸测序法分析RARB、PGR、RASSF1、CDH1、FHIT、ESR1、C1QTNF6、PTGS2、SOCS3、MGMT及LINE1的DNA甲基化水平，并与RT4、T24、HeLa、BEAS-2B、HepG2细胞系的检测结果进行比对。最终采用短串联重复序列（short tandem repeat, STR）分型技术完成身份鉴定。
结果：所有受试的UROtsa细胞系批次均未检出大T抗原。STR分型结果明确显示，本研究的主批次UROtsa细胞系实为膀胱癌细胞系T24，该结果与作为对照的两批经鉴定的正品UROtsa细胞系存在显著差异。DNA甲基化模式与RNA表达分析进一步证实了二者的差异。即便经过长达56周的长期培养，被污染的T24细胞系的甲基化模式与mRNA表达仅出现中度改变，但其miRNA表达水平与染色体数目则发生了显著变化。
结论：对细胞系的身份进行验证至关重要，尤其是那些未通过主流细胞库分发的细胞系。但部分细胞系尚无公开的STR分型数据，因此新建立的细胞系应要么提交至细胞库进行备案，至少应完成其STR分型检测并将结果作为初始鉴定的一部分予以发表。本研究结果有助于完善多分子层面下UROtsa及其他细胞系的鉴定流程，并为尿路上皮细胞系在长期实验中的应用提供参考依据。