Integrated genomic and transcriptional analysis of the in vitro evolution of telomerase‐immortalized urothelial cells (TERT‐NHUC)

Abstract

Much progress has been made in identifying the molecular genetic alterations that occur in bladder cancer. However, in many cases the genes targeted by these alterations are not known. Telomerase immortalized human urothelial cells (TERT-NHUC) are a useful resource for in vitro studies of genes involved in urothelial transformation. When cultured under standard conditions they remain genetically stable but when cultured under low-density conditions they exhibit genetic instability and acquire chromosomal alterations. TERT-NHUC from three donors were cultured at low plating density and examined at four time-points during a culture period of 600 days. Analyses included population doubling kinetics, array-based CGH (aCGH), chromosome counts, fluorescence in situ hybridization (FISH), mutation analysis, Affymetrix gene expression analysis, Western blotting for p16, anchorage-independent growth and tumorigenicity assays. Alterations acquired during continued culture of TERT-NHUC at low density (TERT-NHUC-L) included some observed in urothelial carcinoma (UC) cell lines and primary UC. Examination of gene expression in TERT-NHUC with distinct acquired genetic aberrations may pinpoint genes targeted by these alterations. Data from an aCGH study of UC cell lines and primary tumors were examined for changes in chromosomal regions that also showed alterations in TERT-NHUC-L. Loss of a region on 2q including BOK was identified in UC cell lines and primary tumors. DNER and FRAS1 were identified as potential candidate genes, whose expression is altered independently of the acquisition of any genetic event.

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

“…Our findings are in agreement with a previous report demonstrating hTERT-mediated immortalisation of NHU cells [24]. In accordance with our study, Chapman et al reported that immortalisation of NHU cells was not accompanied by any detectable genetic instability when the HU-hTERT derivatives were maintained under conditions optimised for the culture of NHU cells, although they did observe a predisposition to genetic aberration when hTERT over-expressers were stressed under sub-optimal culture conditions [26].…”

Diagnostic and prognostic value of long noncoding RNAs as biomarkers in urothelial carcinoma

“…Cell lines were regularly verified by DNA fingerprint analysis and checked for mycoplasm contamination. The TERT-immortalized normal human urothelial cell line TERT-NHUC was obtained from Dr. M. A. Knowles [21] and cultured in keratinocyte serum-free medium (Gibco, life technologies; Carlsbad, CA, USA) supplemented with 0.25 ng/ml epidermal growth factor, 12.5 μg/ml bovine pituitary extract and 1:100 ITS (Gibco), 0.35 μg/ml (-)N-epinephrine and 0.33 mg/ml hydrocortisone (Sigma Aldrich; St. Louis, MO, USA).…”

Spectrum of Phosphatidylinositol 3-Kinase Pathway Gene Alterations in Bladder Cancer

“…TSC1 was screened in 27, RHEB in 8, and LKB1 in 9 fragments. PIK3CA, FGFR3, HRAS, KRAS, and NRAS were analyzed with the use of high-resolution melting curve analysis as described (25). PIK3CA was analyzed in 20 fragments covering all coding sequences.…”