Samuel R. Johnston

The effect of antisense Bcl-2 oligonucleotides on Bcl-2 protein expression and apoptosis in human bladder transitional cell carcinoma

PURPOSE Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder. MATERIALS AND METHODS Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model. RESULTS Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death. CONCLUSIONS Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.

Targeting death receptors in bladder, prostate and renal cancer

PURPOSE We describe key components of normal and aberrant death receptor pathways, the association of these abnormalities with tumorigenesis in bladder, prostate and renal cancer, and their potential application in novel therapeutic strategies targeted toward patients with cancer. MATERIALS AND METHODS A MEDLINE literature search of the key words death receptors, TRAIL (tumor necrosis factor related apoptosis inducing ligand), FAS, bladder, prostate, renal and cancer was done to obtain information for review. A brief overview of the TRAIL and FAS death receptor pathways, and their relationship to apoptosis is described. Mechanisms that lead to nonfunction of these pathways and how they may contribute to tumorigenesis are linked. Current efforts to target death receptor pathways as a therapeutic strategy are highlighted. RESULTS Activation of tumor cell expressing death receptors by cytotoxic immune cells is the main mechanism by which the immune system eliminates malignant cells. Death receptor triggering induces a caspase cascade, leading to tumor cell apoptosis. Receptor gene mutation or hypermethylation, decoy receptor or splice variant over expression, and downstream inhibitor interference are examples of the ways that normal pathway functioning is lost in cancers of the bladder and prostate. Targeting death receptors directly through synthetic ligand administration and blocking downstream inhibitor molecules with siRNA or antisense oligonucleotides represent novel therapeutic strategies under development. CONCLUSIONS Research into the death receptor pathways has demonstrated the key role that pathway aberrations have in the initiation and progression of malignancies of the bladder, prostate and kidney. This new understanding has resulted in exciting approaches to restore the functionality of these pathways as a novel therapeutic strategy.

Lipid Peroxidation Assessment of Free Radical Production Following Release of Obstructive Uropathy

PURPOSE To identify if free radicals were released following relief of ureteric obstruction. MATERIALS AND METHODS Free radical production was assessed by a high performance liquid chromatography assay of malondialdehyde (MDA) in a 24-hour complete unilateral ureteric obstruction model in Wistar rats. RESULTS In the first experiment, a rise greater than 20% above prerelease levels in renal venous malondialdehyde levels was observed at 8, 15, 22, and 30 minutes following release. At 45 and 60 minutes, levels were similar to the prerelease levels. In the second experiment, renal venous samples taken before release and at 10 and 20 minutes after release in control and obstructed groups (n = 7) identified a significant difference (T = 10 minutes p = 0.006 and T = 20 minutes p = 0.045). A significant MDA rise following release occurred only in the obstructed group (obstruction group; MDA level T = 0 min. 1.01 mumol/l. +/- 0.13, T = 10 min. 2.12 mumol/l. +/- 0.3 p = 0.016, T = 20 min. 2.09 mumol/l. +/- 0.28 p = 0.016. CONTROL GROUP T = 0 min. 1.13 mumol/l. +/- 0.23, T = 10 min. 1.05 mumol/l. +/- 0.18 p = 0.95, T = 20 min. 1.23 mumol/l. +/- 0.19 p = 0.67). In the third experiment, an attempt was made to block free radical release with allopurinol. No statistical difference between the control and obstructed groups with allopurinol was noted at 20 minutes after release. A Kruskal Wallace two-way analysis identified a significant difference between experiments two and three at both time periods (p = 0.049). CONCLUSION This study suggests, for the first time, that free radicals are released after relief of ureteric obstruction and that this release is partially attenuated by allopurinol pretreatment.

Molecular targets for the therapeutic manipulation of apoptosis in bladder cancer.

The philosophical assertion that death is an essential part of life is often used to justify the need for the stepwise process of apoptosis or programmed cell death. However, just as apoptosis is a normal part of embryogenesis and normal development, its deregulation is implicated in various clinical disorders, including cancer. The failure of apoptosis allows mutated cells to continue to enter the cell cycle, perpetuating the cycle of mutation and oncogenesis. This cycle of mutational activity results in the accumulation of active oncogenes and defective tumor suppressor genes within cells, increasing proliferative ability. 1, 2 Apoptosis is segregated into petitioner, judgment and execution phases, and the death switch is irreversibly activated in the execution phase (see figure). After the cell death machinery (the caspases) is activated, it cannot be reversed and cell death is unstoppable. 3, 4 Despite an abundance of research into apoptosis, the fine details of the complex web of actions and interactions of each pro-apoptotic and antiapoptotic protein remain to be fully elucidated. The increasing array of molecular biology techniques allows cancer researchers to scan tumor cells for molecular targets that are suitable for therapeutic manipulation. 5 Applying these techniques for investigating the genotype of apoptotic resistant cells would aid diagnosis, and predict treatment response and prognosis. The impetus for such studies into molecular and genetic markers of apoptosis is generated by the hypothesis that the phenotype of bladder cancer cells is largely determined by the cell genotype. In other words, if a tumor cell is highly resistant to apoptosis, the cell genotype has determined this resistance. The increasing array of molecular biology techniques has provided a basis for identifying molecular targets for novel therapy. We outline the main molecular targets that have a role in abnormal bladder tumor cell apoptosis and discuss various options for overcoming it.

Antisense Bcl-2 oligonucleotide uptake in human transitional cell carcinoma.

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.

Validation of a Rapid Method to Quantify Apoptosis in Superficial Bladder Cancer

OBJECTIVES To derive and validate a rapid method for calculating apoptotic indices in superficial transitional cell carcinoma (TCC) as a measure of chemosensitivity to mitomycin. MATERIALS AND METHODS Apoptotic cells, identified by light microscopy in 20 superficial TCC specimens, were expressed as an index of the total tumour cell population within defined fields. For a given field, the total cell population was estimated by: (i) an exhaustive count of the total number of cells in the field and (ii) an abbreviated method in which the number of cells in a subfield was multiplied to provide an estimate of the total field number. Field and specimen estimates were compared using agreement statistics and the intra- and inter-observer reproducibility of apoptotic indices calculated. RESULTS Cellularity and apoptotic indices obtained using method (ii) were correlated significantly with the true cell counts (P < 0.001). Agreement statistics showed that only 9.4% of counts fell outside two standard deviations (SD) from the mean in field analysis, and only 10% of counts fell outside 2 SD from the mean in specimen analysis. There was a fivefold variation in tumour cell counts among individual fields. CONCLUSIONS The reported variation in cellularity among fields shows that the calculation of apoptosis must use the total cell population as the reference. The limits of agreement for the estimated and true cell counts are small enough to be confident that the shorter method to estimate cellularity can be used in place of counting all cells.