title: A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.
abstract: OBJECTIVE
  Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut).
  
  
  METHODS
  Microarray analysis, quantitative reverse transcription polymerase chain reaction, Western blot analysis and immunocytochemistry were used to identify and evaluate candidate genes involved in CDDP resistance of p53(mut_c) cells. RNAi knockdown or treatment with inhibitors together with flow cytometry-based methods was used for functional assessment of the identified candidate genes. Cellular metabolic activity was assessed with the XTT assay, and the redox capacity of cells was evaluated by measuring cellular glutathione (GSH) levels.
  
  
  RESULTS
  Upregulation of ABCC2 and ABCG2 transporters was observed in CDDP-resistant p53(mut_c) HNSCC cells. Furthermore, p53(mut_c) cells exhibited a pronounced side population that could be suppressed by RNAi knockdown of ABCG2 as well as treatment with the ATP-binding-cassette transporter inhibitors imatinib, MK571 and tariquidar. Metabolic activity and cellular GSH levels were higher in CDDP-resistant p53(mut_c) cells, consistent with a higher capacity to fend off cytotoxic oxidative effects such as those caused by CDDP treatment. Finally, ABCC2/G2 inhibition of HNSCC cells with MK571 markedly enhanced CDDP sensitivity of HNSCC cells.
  
  
  CONCLUSIONS
  The observations in this study point to a major role of p53(mut_c) in conferring a stem cell like phenotype to HNSCC cells that is associated with ABCC2/G2 overexpression, high GSH and metabolic activity levels as well as CDDP resistance.
authors.affiliation: Department of Otolaryngology, Head and Neck Surgery, University Hospital Giessen and Marburg, Campus Marburg, Baldingerstrasse, 35033, Marburg, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Tonigold
authors.forename: Annette
authors.initials: A
authors.lastname: Rossmann
authors.forename: Marie
authors.initials: M
authors.lastname: Meinold
authors.forename: Michael
authors.initials: M
authors.lastname: Bette
authors.forename: Melanie
authors.initials: M
authors.lastname: Märken
authors.forename: Katharina
authors.initials: K
authors.lastname: Henkenius
authors.forename: Anne C
authors.initials: AC
authors.lastname: Bretz
authors.forename: Gavin
authors.initials: G
authors.lastname: Giel
authors.forename: Chengzhong
authors.initials: C
authors.lastname: Cai
authors.forename: Fiona R
authors.initials: FR
authors.lastname: Rodepeter
authors.forename: Vladimir
authors.initials: V
authors.lastname: Beneš
authors.forename: Reidar
authors.initials: R
authors.lastname: Grénman
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Carey
authors.forename: Hermann
authors.initials: H
authors.lastname: Lage
authors.forename: Thorsten
authors.initials: T
authors.lastname: Stiewe
authors.forename: Andreas
authors.initials: A
authors.lastname: Neubauer
authors.forename: Jochen A
authors.initials: JA
authors.lastname: Werner
authors.forename: Cornelia
authors.initials: C
authors.lastname: Brendel
authors.forename: Robert
authors.initials: R
authors.lastname: Mandic
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D005978:Glutathione / Q000378:metabolism*; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D015854:Up-Regulation
chemical_list: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D002945:Cisplatin; D005978:Glutathione; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D016159:Tumor Suppressor Protein p53; C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human