Antisense Bcl–2 Oligonucleotide Uptake in Human Transitional Cell Carcinoma

Abstract

Objectives: 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 superificial 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. Conclusion: 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.