Antisense Bcl-2 oligonucleotide uptake in human transitional cell carcinoma.
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.
Duggan
B J
BJ
Uro-Oncology Research Group, Cancer Research Centre, Department of Pathology, The Queen's University Belfast, Belfast, UK. b.duggan@gub.ac.uk
Cotter
F E
FE
Kelly
J D
JD
Hamilton
P W
PW
McCallion
K
K
Harkin
D
D
Gardiner
T
T
Anderson
N
N
Keane
P F
PF
Johnston
S R
SR
Williamson
K E
KE
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*
D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016650:Fluorescein-5-isothiocyanate