0022-534 7/86/1355-1091$02.00/0 THE JOURNAL OF UROLOGY Copyright © 1986 by The Williams & Wilkins Co. Vol.135, May Printed in U.S.A. PROLIFERATION, ESTERASE ACTIVITY, AND PROPIDIUM IODIDE EXCLUSION IN UROLOGIC TUMOR CELLS AFTER IN VITRO EXPOSURE TO CHEMOTHERAPEUTIC AGENTS ROBERT C. FLANIGAN,* EDWARD J. PAVLIK, JOHN R. VAN NAGELL, KATHRYN KEATON AND DANIEL E. KENADY From the Lexington Veterans Administration Hospital and the Departments of Urology, Obstetrics and Gynecology, Biochemistry and Surgery, University of Kentucky College of Medicine, Lexington, Kentucky ABSTRACT After exposing urological tumor cells to anticancer agents in vitro, cellular esterase activity and the ability to exclude propidium iodide (PI) were examined as dual indicators of functionality or "viability." High esterase activity/PI exclusion was observed in assays in which anticancer agents failed to inhibit cellular proliferation, while low esterase activity /PI exclusion was often observed when proliferation had been significantly inhibited. In a number of instances, exposure to anticancer agents did produce significant inhibition of proliferation without lowering viability. In this setting, the recovery of proliferative capacity could be demonstrated with several transitional cell carcinoma cell lines, and this recovery was always associated with high esterase activity /PI exclusion. When the proliferation of primary urological tumor preparations was inhibited by drug exposure, estimates of elevated viability were obtained in 27 per cent of the determinations. Thus, viability estimates may be an indicator of the potential for tumor-cell recovery from exposure to anticancer agents. Moreover, the potential for recovery may explain differences between the results of chemosensitivity testing and actual clinical events by reconciling clinical failures with elevated viabilities indicative of this potential. The development of accurate chemosensitivity testing meth- odologies for selecting effective chemotherapies for patients with various malignancies has been a long-sought goal by cancer researchers. 1 -B An agar clonogenic assay which measures the ability of anticancer agents to inhibit the in vitro proliferation of tumor stem cells has been developed by Salmon and collaborators 9 and enjoys widespread use with various modifi- cations. Several types of chemosensitivity assays have been applied to urological malignancies. 1 0- 20 These assays have ap- peared to successfully identify drug resistance, but have been able to identify clinical sensitivity only 40 to 75 per cent of the time. 21 - 29 Aside from an extensive number of technical ques- tions that might contribute explanations for this performance is the general failure of the clonogenic assay to provide an estimate of the potential recovery from proliferative arrest. Cells which experience reproductive arrest and remain viable after drug exposure are not distinguished from tumor cells that have experienced drug-related death. As long as cells remain viable the potential for recovery exists. In the present report, we have monitored cellular function- ality and integrity after drug exposure as a measure of "viabil- ity." With this approach, cellular esterases present in viable cells convert colorless fluorescein diacetate to green fluorescent fluorescein. 31 - 34 In addition, propidium iodide is normally ex- cluded from viable cells but is incorporated into non-viable cells producing red fluorescence and thus permitting their identification. 34 Flow cytometry has been used for the quanti- fication of these fluorescence signals so that simple, repro- ducible determinations are used here in conjunction with stand- ard determination of cellular proliferation after drug exposure. Herein we show that after in vitro exposure of urologic tumor cells to anticancer agents, considerable cellular esterase activity Accepted for publication December 9, 1985. * Requests for reprints: Division of Urology, Dept. of Surgery, Uni- versity of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536-0084. Supported by the Veterans Administration. and ability to exclude propidium iodide may be maintained despite significant inhibitory effects on proliferation. These observations are related to demonstrable proliferative recovery which is a parameter relevant to drug ineffectiveness in vivo. MATERIALS AND METHODS Cell lines. Dr. Jorgen Fogh (Memorial-Sloan Kettering) pro- vided cell lines originating from transitional cell carcinomas (TCC) of the urinary bladder (253J, T24, SW-1710, TCCSUP, 639V, 647V, J82, SW780, and SWl 738). Dr. George Moore (Denver General Hospital) provided a TCC cell line (COL232) and another was obtained from the American Type Culture Collection (CRL1472: "HT-1376"). Cell culture. Cells were grown as specified by the originating source in the required nutrient media (Dulbecco's MEM, McCoy's 5A, L-15, or RPMI (1640)) containing 10 per cent fetal bovine serum, 100 U/ml. penicillin, 100 mg./ml. strepto- mycin, with bicarbonate and 10 mM Hepes buffering. Where appropriate, additional supplementation consisted of insulin (0.01 U/ml.), cortisone (10 μg./ml.), and nonessential amino acids (obtained from Grand Island Biologicals Co. (1 X)). Cells were subcultured by disrupting attachment through the use of Hank's balanced salt solution (HBSS) containing EDTA (2 mM, pH 7.4). Cells were grown in plastic tissue culture vessels after plating at 3 to 4 X 10 3 cell/cm. 2 and were renourished with fresh liquid media every three days (0.4 to 0.6 ml. media/ cm. 2 ). Tumor cell counting was performed on Coulter counters (Models ZBi and ZF, Coulter Electronics). Primary tumor cell preparations were obtained from sterile minces of recently resected tumor. Tumor fragments were suspended in HBSS containing collagenase (6 μg./ml.), dis- pensed to "trypsinization" flasks, and horizontally agitated in a shaking water bath at 37C for 100 to 180 min. The superna- tant, which contained dispersed tumor cells, was poured through a "Cellector" stainless-steel sieve (Bellco Glass, Inc.), pelleted by centrifugation at 100 g (10 min.), resuspended, and plated in Dulbecco's MEM containing 10 per cent fetal bovine 1091