Location of Repository

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity.

By Nikolaos T. Georgopoulos, Lisa A. Kirkwood, Claire L. Varley, Nicola J. Maclaine, Naveed Aziz and Jennifer Southgate

Abstract

BACKGROUND: The development of urothelial malignancy is not solely a consequence of loss of proliferation constraints but also involves loss of cellular differentiation, defined histopathologically as grade. Although tumour grade is an independent prognostic marker for urothelial carcinoma (UC), the molecular events underpinning the loss of urothelial differentiation are poorly understood. \ud \ud \ud OBJECTIVE: To examine the effect of gene alterations implicated in UC development on the ability of human urothelial cells to undergo molecular differentiation and form a functional urothelial barrier. \ud \ud \ud DESIGN, SETTING, AND PARTICIPANTS: Laboratory study. \ud \ud \ud INTERVENTION: Normal human urothelial (NHU) cell cultures were transduced with recombinant retroviruses to produce stable sublines overexpressing wild-type or oncogenic mutated fibroblast growth factor receptor 3 or human telomerase reverse transcriptase (hTERT). Previously generated NHU sublines carrying dominant-negative CDK4 and p53 mutant genes or immortalised with the human papillomavirus 16 E6 oncoprotein were included. \ud \ud \ud MEASUREMENTS: The activity of introduced transgenes was demonstrated by comparing phenotypes of transgene-expressing and isogenic control NHU cells. Modified and control sublines were compared for changes in generational potential (life span) and capacity to respond to differentiation-inducing signals by transcript expression of uroplakins 2 and 3. The ability to form a barrier epithelium was assessed by measuring the transepithelial electrical resistance. \ud \ud \ud RESULTS AND LIMITATIONS: By contrast to tumour suppressor loss of function or oncogene overactivation, hTERT overexpression alone led to life span extension and immortalisation. The hTERT immortalised cells carried no gross genomic alterations but became progressively insensitive to differentiation signals and lost the ability to form an epithelial barrier. Further characterisation of hTERT cells revealed a downregulation of p16 cyclin-dependent kinase inhibitor expression and loss of responsiveness to peroxisome proliferator-activated receptor γ, providing mechanistic explanations for the subjugation of senescence constraints and the abrogation of differentiation capability, respectively. Although immortalised urothelial cell lines without karyotypic aberrations may be generated, such cell lines are compromised in terms of differentiation and functional capacity. CONCLUSIONS: Overexpression of hTERT promotes development of an immortalised differentiation-insensitive urothelial cell phenotype. Although such cells offer a useful insight into the grade/stage paradigm of UC, they have limited value for investigating normal urothelial cell/tissue biology and physiology

Topics: RC0254
Publisher: Elsevier
Year: 2011
OAI identifier: oai:eprints.hud.ac.uk:9907

Suggested articles

Preview

Citations

  1. (2005). A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function. doi
  2. (1997). A survey of telomerase activity in human cancer. doi
  3. (2002). A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status. Mol Cell Biol. doi
  4. (2008). Actions of human telomerase beyond telomeres. Cell Res. doi
  5. (2005). Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro. Exp Cell Res. doi
  6. (2005). CGHExplorer: a program for analysis of array-CGH data. Bioinformatics. doi
  7. (2007). Differentiation potential of urothelium from patients with benign bladder dysfunction. BJU Int. doi
  8. (2005). Effects of loss of p53 and p16 function on life span and survival of human urothelial cells. doi
  9. (2006). Experimental models of human bladder carcinogenesis. Carcinogenesis. doi
  10. (2006). Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway. Oncogene. doi
  11. (2009). FOXA1 and IRF-1 intermediary transcriptional regulators of PPARgamma-induced urothelial cytodifferentiation. Cell Death Differ. doi
  12. (2008). Genes involved in differentiation, stem cell renewal, and tumorigenesis are modulated in telomerase-immortalized human urothelial cells. Mol Cancer Res. doi
  13. (2006). hTERTimmortalized prostate epithelial and stromal-derived cells: an authentic in vitro model for differentiation and carcinogenesis. Cancer Res. doi
  14. (2000). Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol. doi
  15. (2003). Identification of fibroblast growth factor receptor 3 mutations in urine sediment DNA samples complements cytology in bladder tumor detection. doi
  16. (2001). Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation. Mol Biol Cell. doi
  17. (2005). Immortalization of human small airway epithelial cells by ectopic expression of telomerase. Carcinogenesis. doi
  18. (2009). Integrated genomic and transcriptional analysis of the in vitro evolution of telomeraseGeorgopoulos et al Page 21 of 27 immortalized urothelial cells (TERT-NHUC). Genes Chromosomes Cancer. doi
  19. (2007). Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells. Carcinogenesis. doi
  20. (2002). Masters JR and Trejdosiewicz LK: Culture of Human Urothelium, doi
  21. (2010). Molecular pathways of urothelial development and bladder tumorigenesis. Urol Oncol. doi
  22. (1994). Normal human urothelial cells in vitro: proliferation and induction of stratification. Lab Invest.
  23. (2006). PPARgamma-regulated tight junction development during human urothelial cytodifferentiation. J Cell Physiol. doi
  24. (2001). Role of p53 in the responses of human urothelial cells to genotoxic damage. doi
  25. (2004). Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme. J Cell Sci.
  26. (2008). Sensitivity of normal, paramalignant, and malignant human urothelial cells to inhibitors of the epidermal growth factor receptor signaling pathway. Mol Cancer Res. doi
  27. (1997). Telomerase activity: a biomarker of cell proliferation, not malignant transformation. doi
  28. (2001). Telomerase in urological malignancy. doi
  29. (2003). Telomerase therapeutics: telomeres recognized as a DNA damage signal: commentary re: K. Kraemer et al., antisense-mediated hTERT inhibition specifically reduces the growth of human bladder cancer cells.
  30. (1996). Transitional cell carcinoma of the bladder: diagnosis and prognosis. Current Diagnostic Pathology. doi
  31. (2003). Uroplakin gene expression in normal human tissues and locally advanced bladder doi
  32. (2005). Urothelial tumorigenesis: a tale of divergent pathways. Nat Rev Cancer. doi

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.