Abstract
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.
Highlights
UROtsa cells are a valuable tool to study toxic effects and the development of urothelial cancers
The UROtsa cell line was generated by immortalization of urothelial cells with a construct containing the SV40 large T-antigen [3]
In contrast to cells immortalized with live SV40 virus (SV-HUC-1, SV-HUC-2)
Summary
UROtsa cells are a valuable tool to study toxic effects and the development of urothelial cancers. The UROtsa cell line was generated by immortalization of urothelial cells with a construct containing the SV40 large T-antigen [3]. It is an authentic and well-characterized cell line [3,4,5]. Despite of being derived from the urothelial lining of the ureter UROtsa is considered to be a useful model for normal human bladder urothelium [1,3,4]. UROtsa 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
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