Objective To develop a method of determining the characteristics of epirubicin resistance and to study the reversal of such resistance in the intravesical treatment of superficial bladder cancer, using sensitive and resistant derivatives of a bladder cancer cell line in vitro. Materials and methods Epirubicin fluorescence and flow cytometry were used to measure the intracellular levels of epirubicin in both sensitive and resistant live cultured bladder tumour cells, with and without different doses of the resistance‐reversing agent verapamil. Results There was a reliable, highly significant and consistent difference in intracellular epirubicin concentration between the resistant and sensitive bladder tumour cells. In addition, it was possible to substantially reverse the features of resistant cell subline with additional verapamil. Conclusion Application of this assay to clinical specimens should allow better targeting of epirubicin intravesical chemotherapy and avoid the premature termination of such treatment in patients whose tumours remain sensitive to this agent. Furthermore, the addition of verapamil to intravesical epirubicin may permit effective treatment of those patients whose tumours have inherent or acquired resistance to epirubicin.

Objective To evaluate the use of confocal microscopy in cytoplasmic and granular in appearance. When incubated at 0°C, both cell lines showed no nuclear uptake the study of resistance to epirubicin and to determine the effect of temperature, viability and a resistance-and thus resembled resistant cells at 37°C. However, dead cells rapidly acquired brightly fluorescent nuclei. reversing agent on the intracellular distribution of this drug in sensitive and resistant derivatives of aThe resistance-reversing agent verapamil appeared to cause reversion of the resistant to the sensitive superficial bladder cancer cell line. Materials and methods Viable and non-viable adherent phenotype. Conclusion Confocal microscopy allows epirubicin-cells were incubated in epirubicin solutions under various conditions. After incubation, the distribution sensitive and resistant cultured tumour cells to be differentiated reliably and provides information about of intracellular epirubicin fluorescence was visualized using confocal microscopy and a ×50 water-the mechanisms of action of, and resistance to, epirubicin. Applying this technique to clinical specimens immersion lens. Results There was a striking and consistent difference should enable patients who have the resistant phenotype to be detected and the efficacy of intravesical between resistant and sensitive cells in the intracellular distribution of the drug. In addition to having resistance-reversing agents to be evaluated in such cases. greater overall levels of epirubicin fluorescence, sensitive cells accumulated epirubicin predominantly in the Keywords Bladder cancer, confocal microscopy, epirubicin, verapamil, multidrug resistance nucleus. Epirubicin fluorescence in resistant cells was used to construct a high-definition image of the intracel-

Objective To assess, using epirubicin-sensitive and multidrug resistant (MDR) derivatives of human bladder cancer cell lines in vitro , the probable effect of intravesical pH changes, with and without the MDR antagonist verapamil, on the uptake, intracellular distribution and cytotoxicity of epirubicin during intravesical chemotherapy. Materials and methods Incubations for cytotoxicity testing were carried out in buffered medium containing epirubicin, at pH values of 6.0-8.5, with verapamil where appropriate. The cytotoxicity of epirubicin, with and without verapamil, was determined using the tetrazolium cytotoxicity assay. Intracellular epirubicin fluorescence was assessed using flow cytometry and confocal microscopy. Flow cytometric total intracellular epirubicin fluorescence was measured at pH 6.0, 6.4, 6.8, 7.2, and 7.6, and confocal microscopy was carried out at pH 6.0 and 8.0. The MDR-reversing agent verapamil was added at 100 m g/mL to some incubations. Results Epirubicin cytotoxicity in resistant cell lines appears considerably enhanced by adding verapamil and further improved, especially in MDR cells, by alkalinization of the drug solution to pH 8.0. Flow cytometry results showed striking and consistent differences in epirubicin handling with pH. Sensitive cells can be induced to absorb considerably more drug at alkaline pH, whilst resistant cells show no such behaviour. Nuclear drug fluorescence was greater in sensitive cells at alkaline pH, but cytoplasmic drug fluorescence in the resistant cells was little changed by pH. Adding verapamil to resistant cells restored the sensitive phenotype of drug handling. Conclusion Buffering epirubicin to an alkaline pH before intravesical application should increase its intrinsic cytotoxicity. The potential for synergy at certain drug combinations will be enhanced by applying these findings. MDR reversal and fatty acid augmentation of drug uptake are discussed as examples.

Summary Idarubicin is a highly lipophilic anthracycline and appears effective against tumours resistant to conventional anthracyclines. Confocal microscopy demonstrates predominantly cytoplasmic idarubicin accumulation. This distribution is unaltered by resistance status or the resistance reversing agent verapamil. Our results contrast with studies on conventional anthracycines and suggest that nuclear accumulation may not be a prerequisite for anthracycline cytotoxicity.

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live and in situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.