Hoechst 33342 side population identification is a conserved and unified mechanism in urological cancers.

Mutation within the adult human stem cell (SC) compartment has been proposed as a factor in the initiation and promotion of carcinogenesis. Isolation of these cancer stem cells (CSCs) has proven difficult, limiting their subsequent phenotypic, functional, and genetic characterization. We have used the Hoechst 33342 dye efflux technique to isolate an epithelial side population (SP) from genitourinary (GU) cancers, which is enriched for cells with SC traits. With informed consent, samples were taken from patients with primary tumors and undergoing surgery for prostatic (CaP), invasive bladder transitional cell (TCC), and renal cell carcinomas (RCC). Single cell epithelial suspensions were extracted from these and incubated with Hoechst 33342. Hoechst SP/non-SP profiles were then generated by flow cytometry using standardized protocols. SP/non-SP cell cycle status was established by Hoechst 33342 and Pyronin Y staining. Immunocytochemistry staining was performed for markers suggested as stem markers as well as lineage-specific markers. Functionality was determined using colony-forming assays and long-term monolayer culture. A characteristic verapamil-sensitive SP was isolated from all 3 urological malignancies and represented 0.57% +/- 0.11% (CaP), 0.52% +/- 0.49% (TCC), and 5.9% +/- 0.9% (RCC) of the total epithelial population. Cell cycle analysis showed that the SP had enhanced numbers of cells in G(0) as compared to the total cell population (CaP 12.4% +/- 3.2 vs. 3.8% +/- 1.0, RCC 23.2% +/- 3.4 vs. 1.8% +/- 0.9, and TCC 28.5% +/- 4.9 vs. 4% +/- 1.3). Immunocytochemistry demonstrated an increased expression of proliferative and putative stem markers within the SP fraction. Cultures confirmed significant enhancement of colony-forming ability and proliferative capacity of the SP fraction. A characteristic SP enriched for stem-like cells has been isolated from the 3 most common urological malignancies. This provides strong evidence that Hoechst 33342 efflux is a conserved and unified mechanism in GU cancer.

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.

The natural history of postoperative renal function in patients undergoing ileal conduit diversion for cancer measured using serial isotopic glomerular filtration rate and 99m technetium-mercaptoacetyltriglycine renography.

PURPOSE: There is little consensus regarding long-term followup of renal function in patients who undergo urinary diversion. We established the usefulness of combined serial isotopic glomerular filtration rate measurement and diuresis renography in the early identification of patients at risk for deterioration of renal function following ileal conduit diversion. MATERIALS AND METHODS: A total of 340 patients with ileal conduit diversion who were followed between 1990 and 2000 were identified. We analyzed data on 178 patients who had more than 4 years of followup. Renal function was assessed by serial estimation of serum creatinine, isotopic glomerular filtration rate and diuresis renographic measurements. RESULTS: Of the patients 52 (29%) demonstrated a worsening glomerular filtration rate. Mean followup +/- SEM was 8.2 +/- 0.4 years (range 4 to 30) and 67% of patients had more than 6 years of followup. In this group we found that hypertension, recurrent urinary sepsis and an initial post-diversion glomerular filtration rate of less than 50 ml per minute per 1.73 m were prevalent risk factors. Hypertension was an independent predictor of a decreased glomerular filtration rate in this group. Of 52 patients with a deteriorating glomerular filtration rate 19 had type II or IIIb curves on followup renography, of whom 13 underwent revision surgery. Renal function subsequently stabilized or improved in this group. CONCLUSIONS: Of patients with an ileal conduit 29% have renal function deterioration in the long term. No surgical cause for glomerular filtration rate deterioration was found in 18%. Important predisposing factors in nonobstructed cases were hypertension, recurrent urinary sepsis and a glomerular filtration rate of less than 50 ml per minute per 1.73 m. Hypertension was an independent predictor of a decreased glomerular filtration rate in the group with worsening glomerular filtration rates. In 11% of patients deterioration was due to upper tract obstruction. This was identifiable using renography and the glomerular filtration rate. A type IIIb curve was an early indicator of obstruction.