title: Antisense Bcl-2 oligonucleotide uptake in human transitional cell carcinoma.
abstract: OBJECTIVE
  Antisense oligonucleotides (AO) downregulate Bcl-2 protein expression in various tumours if good target cell uptake is achieved. In this study, uptake of FITC labelled AO (FITC-AO) directed at Bcl-2 was examined in: (1) the RT4 bladder tumour cell line; (2) normal pig urothelium, and (3) human superficial bladder tumours.
  
  
  METHODS
  In the RT4 cell line, uptake of FITC-AO, FITC-scrambled and FITC-sense oligonucleotides were quantified by flow cytometry at 4-hour intervals over 24 h. Uptake of FITC-AO was assessed in normal pig urothelium by flow cytometry after FITC-AO was infused for 1 h. Uptake of FITC AO was assessed in samples from 14 human superficial bladder tumours which were maintained in an ex vivo model. In samples from 6 tumours, uptake at 4 h was assessed using fluorescence microscopy. In samples from 8 separate tumours uptake every 4 h within the first 24-hour incubation period was assessed by flow cytometry.
  
  
  RESULTS
  In the RT4 cell line the FITC-AO, FITC-scrambled and FITC-sense oligonucleotide uptake was similar. Disaggregated cells from the normal urothelium of the 3 pigs exhibited 33, 46 and 51% of cells staining positively for FITC-AO as determined by flow cytometry. All 6 tumour samples had detectable intracellular FITC-AO by fluorescence microscopy at 4 h. In the 8 tumours examined over the 24-hour incubation period, there was a range of percentages of positively staining cells. However, most tumours had a monotonic increase in intracellular fluorescence intensity that plateaued 16 h post-infusion.
  
  
  CONCLUSIONS
  Antisense Bcl-2 oligonucleotides were readily taken up by superficial bladder cancer cells but the heterogeneous uptake in tumour samples needs to be considered when assessing the bioavailability of these drugs.
authors.affiliation: Uro-Oncology Research Group, Cancer Research Centre, Department of Pathology, The Queen's University Belfast, Belfast, UK. b.duggan@gub.ac.uk
authors.forename: B J
authors.initials: BJ
authors.lastname: Duggan
authors.forename: F E
authors.initials: FE
authors.lastname: Cotter
authors.forename: J D
authors.initials: JD
authors.lastname: Kelly
authors.forename: P W
authors.initials: PW
authors.lastname: Hamilton
authors.forename: K
authors.initials: K
authors.lastname: McCallion
authors.forename: D
authors.initials: D
authors.lastname: Harkin
authors.forename: T
authors.initials: T
authors.lastname: Gardiner
authors.forename: N
authors.initials: N
authors.lastname: Anderson
authors.forename: P F
authors.initials: PF
authors.lastname: Keane
authors.forename: S R
authors.initials: SR
authors.lastname: Johnston
authors.forename: K E
authors.initials: KE
authors.lastname: Williamson
mesh_terms: D000818:Animals; D017209:Apoptosis; D002295:Carcinoma, Transitional Cell / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D008856:Microscopy, Fluorescence; D020319:Oligodeoxyribonucleotides, Antisense / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013552:Swine; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000378:metabolism*
chemical_list: D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016650:Fluorescein-5-isothiocyanate