Patients
The IC patient was a 28 year old white male who had previously undergone cystoscopy and fulfilled the National Institute of Diabetes, Digestive, and Kidney Diseases (NIDDK) diagnostic criteria for IC which included glomerulations upon cystoscopic examination as well as urgency and dysuria. The control was a 41 year old white female with no history of urinary tract pathology undergoing pelvic surgery. Informed consent was obtained from both subjects in accordance with guidelines of the Institutional Review Board of the University of Maryland School of Medicine.
Explant specimen procurement
Cystoscopy was performed with the patient under general anesthesia using non bacteriostatic normal saline as a bladder irrigant. Rigid cold cup biopsy forceps were used to acquire 4 mm2 pieces of transitional epithelium with submucosa for growth of primary bladder epithelial cells. Tissue specimens were transported from the operating room in sealed sterile containers containing sterile phosphate buffered saline (PBS, Biosource International. Camarillo, CA) at room temperature, removed and placed in DMEM-F12 (Cellgro, Herndon, VA) containing 10% fetal bovine serum, 1% antibiotic/antimycotic solution, 1% glutamine, 5 μg/ml EGF and 1 U/ml insulin (all from Sigma, St. Louis, MO), for growth of bladder epithelial cell explants, characterized by binding of AE-1/AE-3 pancytokeratin antibodies as previously described [13].
Antiproliferative Factor
APF was prepared from the supernatant of bladder epithelial cells explanted from one IC patient and purified using molecular weight fractionation, ion exchange chromatography, hydrophobic interaction chromatography, and reversed phase high performance liquid chromatography (HPLC), as previously described [10]. Mock preparations from culture media of cells from the non-IC control patient were processed using the same purification procedure. Mock preparations do not have any HPLC detectable peptide.
APF treatment
Normal bladder cell explant cultures were plated at 1 × 104 cells per well of a 6 well tissue culture plate in Eagle's minimal essential medium (MEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, 1% antibiotic/antimycotic solution (Sigma, St. Louis, MO) 1% glutamine and incubated overnight at 37°C in a 5% CO2 atmosphere. The following day, cells were serum starved (MEM containing 1% glutamine and 1% antibiotic/antimycotic solution) and incubated overnight at 37°C. On the third day, HPLC-purified APF or mock preparation (10, 20, and 50 μl) was added to the medium and cells were further incubated at 37°C for 48 hours. On day 5, cells were harvested (trypsin/EDTA) and pelleted at 1000 × g centrifugation, resuspended in PBS and fixed using 95% ethanol while vortexing at low speed. Samples were then stored at 4°C until used.
DNA cytometry
All specimens were cytocentrifuged onto slides using a CytoTek Cytocentrifuge at 1,000 RPM for 5 minutes, fixed in 10% Böhm-Springer fixative, hydrolyzed for 45 minutes in 5 N hydrochloric acid and the DNA stained by Feulgen reaction using thionin according to Perceptronix Medical Incorporated (PMI) standard protocol. The Fuelgen stained nuclei were analyzed on a PMI Cytosavant image analysis instrument, which was programmed to scan each slide and acquire 2,000 single nuclei. Debris and cell clumps were rejected using optical density and morphometric features. DNA content was calculated from sum optical density. Diploid control cells were used for validation and identification of G1 cell populations for normalization. Morphometric features for selecting single, whole nuclei included area, sphericity, and eccentricity. After acquisition, cell image galleries were reviewed to ensure that only data from whole, single nuclei were retained in the frequency distribution file. The frequency and cumulative frequency of nuclei were plotted for the calculated sum optical density (DNA content) (Figures 1 and 2). The boundaries of the cell cycle phases were identified by the inflection points in the cumulative frequency distribution and cell subpopulation fractions were calculated.
Statistical analysis
ANOVA analysis was performed using Statview (SAS Institute, Cary, North Caroline) with cell cycle fractions dependence on the independent variables treatment (APF or Mock) and dose (volume of treatment).