Brian J. Duggan

Morphological expression of KIT positive interstitial cells of Cajal in human bladder.

Purpose We investigated the 3-dimensional morphological arrangement of KIT positive interstitial cells of Cajal in the human bladder and explored their structural interactions with neighboring cells. Materials and Methods Human bladder biopsy samples were prepared for immunohistochemistry/confocal or transmission electron microscopy. Results Whole mount, flat sheet preparations labeled with anti-KIT (Merck, Darmstadt, Germany) contained several immunopositive interstitial cell of Cajal populations. A network of stellate interstitial cells of Cajal in the lamina propria made structural connections with a cholinergic nerve plexus. Vimentin positive cells of several morphologies were present in the lamina propria, presumably including fibroblasts, interstitial cells of Cajal and other cells of mesenchymal origin. Microvessels were abundant in this region and branched, elongated KIT positive interstitial cells of Cajal were found discretely along the vessel axis with each perivascular interstitial cell of Cajal associated with at least 6 vascular smooth muscle cells. Detrusor interstitial cells of Cajal were spindle-shaped, branched cells tracking the smooth muscle bundles, closely associated with smooth muscle cells and vesicular acetylcholine transferase nerves. Rounded, nonbranched KIT positive cells were more numerous in the lamina propria than in the detrusor and were immunopositive for anti-mast cell tryptase. Transmission electron microscopy revealed cells with the ultrastructural characteristics of interstitial cells of Cajal throughout the human bladder wall. Conclusions The human bladder contains a network of KIT positive interstitial cells of Cajal in the lamina propria, which make frequent connections with a cholinergic nerve plexus. Novel perivascular interstitial cells of Cajal were discovered close to vascular smooth muscle cells, suggesting interstitial cells of Cajal-vascular coupling in the bladder. KIT positive detrusor interstitial cells of Cajal tracked smooth muscle bundles and were associated with nerves, perhaps showing a functional tri-unit controlling bladder contractility.

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.

Molecular markers for predicting recurrence, progression and outcomes of bladder cancer (do the poster boys need new posters?).

Purpose of review Molecular markers for bladder cancer recurrence and progression continue to drive many research programmes. Translating the laboratory findings into the clinical environment where these markers are used in clinical decision making has proved problematic. In the clinical arena, stage and grade are still the main focus for decisions about patient management. There is however an evolution in bladder cancer research from single-marker/single-pathway research to a more global assessment of the tumour cell with DNA microarrays and proteomics. Recent findings In the last year, DNA microarray assessment has revealed several interesting molecular markers such as p33ING1 and DEK. Parallel ‘conventional’ single-pathway research has focused on new novel markers such as HER2/neu, survivin and matrix metalloproteinase 2 (MMP-2). Molecular markers that have a long-standing association with bladder cancer progression such as p53, E-cadherin and Ki-67 have been reviewed by both single-marker studies and by microarray studies and their status remains important. Summary It is an exciting time in the molecular biology research of bladder cancer as the focus changes to assess the global genetic and protein expression within tumour cells. From such a wealth of information it is likely that molecular markers will make the translation from benchside to bedside.

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.

Spontaneous Rupture of Adrenocortical Carcinoma

A 52-year-old male presented with acute severe abdominal pain and signs of shock. Computerize tomography (CT) of the abdomen showed a large retroperitoneal mass on the left side (fig. 1). Contrast enhanced CT suggested a mixed density lesion originating from the left adrenal gland with hemorrhage within the mass (fig. 2). Preliminary diagnosis was ruptured adrenal carcinoma. At exploration via a thoracoabdominal approach a large tumor mass and associated hematoma were identified in the retroperitoneum extending from the diaphragm to the left renal pedicle. The mass was resected together with the spleen, with preservation of the left kidney and pancreas. Small nodules found within the liver were confirmed on frozen section to represent metastatic adrenal carcinoma. Histological examination of the resected specimen confirmed primary adrenocortical carcinoma. Postoperative recovery was uneventful and the patient subsequently received mitotane chemotherapy. DISCUSSION Primary adrenocortical carcinoma is an extremely rare tumor with an annual incidence of 1 to 2 cases per million population. The tumor displays a bimodal age incidence, peaking in the first and fourth decades of life with an apparent worse prognosis associated with the latter. These tumors may be classified as functional when there are clinical and biochemical manifestations of hormone production present, or nonfunctional when no excess hormone release can be

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.