title: Deoxycytidine kinase expression underpins response to gemcitabine in bladder cancer.
abstract: OBJECTIVE
  In a recent phase II clinical trial, low-dose (100 mg/m(2)) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines.
  
  
  METHODS
  Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay.
  
  
  RESULTS
  Gemcitabine was cytotoxic in the low nanomolar range (10-40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma.
  
  
  CONCLUSIONS
  Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors.
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and.
authors.forename: Martin
authors.initials: M
authors.lastname: Kerr
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.
authors.forename: Helen E
authors.initials: HE
authors.lastname: Scott
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and.
authors.forename: Blaz
authors.initials: B
authors.lastname: Groselj
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and.
authors.forename: Michael R L
authors.initials: MR
authors.lastname: Stratford
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.
authors.forename: Katalin
authors.initials: K
authors.lastname: Karaszi
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom.
authors.forename: Naomi L
authors.initials: NL
authors.lastname: Sharma
authors.affiliation: Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology and anne.kiltie@oncology.ox.ac.uk.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Kiltie
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000235:genetics*; D005260:Female; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; D000093542:Gemcitabine