title: Cross-contamination of a UROtsa stock with T24 cells--molecular comparison of different cell lines and stocks.
abstract: BACKGROUND
  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.
  
  
  METHODS
  UROtsa 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.
  
  
  RESULTS
  All 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.
  
  
  CONCLUSIONS
  It 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.
authors.affiliation: Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr University Bochum, IPA, Bochum, Germany. johnen@ipa-dguv.de
authors.forename: Georg
authors.initials: G
authors.lastname: Johnen
authors.forename: Peter
authors.initials: P
authors.lastname: Rozynek
authors.forename: Yvonne
authors.initials: Y
authors.lastname: von der Gathen
authors.forename: Oleksandr
authors.initials: O
authors.lastname: Bryk
authors.forename: Ricarda
authors.initials: R
authors.lastname: Zdrenka
authors.forename: Christian
authors.initials: C
authors.lastname: Johannes
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Weber
authors.forename: O Brien
authors.initials: OB
authors.lastname: Igwilo-Okuefuna
authors.forename: Irina
authors.initials: I
authors.lastname: Raiko
authors.forename: Jörg
authors.initials: J
authors.lastname: Hippler
authors.forename: Thomas
authors.initials: T
authors.lastname: Brüning
authors.forename: Elke
authors.initials: E
authors.lastname: Dopp
mesh_terms: D000957:Antigens, Viral, Tumor / Q000235:genetics; D002460:Cell Line / Q000166:cytology; D045744:Cell Line, Tumor / Q000166:cytology; D059018:DNA Contamination; D019175:DNA Methylation; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D018895:Microsatellite Repeats / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D019459:Urothelium / Q000166:cytology*
chemical_list: D000957:Antigens, Viral, Tumor; D035683:MicroRNAs; D012333:RNA, Messenger