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Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells
T Kiyono1, S A Foster, J I Koop
1Cancer Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
Abstract:
Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases and telomere shortening. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.
Insights
Telomerase alone does not immortalize epithelial cells. Combining telomerase activity with Rb/p16 pathway inactivation efficiently immortalizes human keratinocyte and mammary epithelial cells, revealing key mechanisms for cellular longevity.
Area of Science:
- Cell Biology
- Molecular Oncology
- Gerontology
Background:
- Normal human cells enter replicative senescence after limited divisions.
- Senescence involves cyclin-dependent kinase inhibitors and telomere shortening.
- Viral oncogenes like HPV E6/E7 target p53 and Rb pathways to reverse senescence.
Purpose of the Study:
- To investigate the sufficiency of telomerase activity for immortalizing human epithelial cells.
- To identify critical pathways, beyond telomere maintenance, required for cellular immortalization.
Main Methods:
- Expression of human telomerase reverse transcriptase (hTERT) in human fibroblasts, keratinocytes, and mammary epithelial cells.
- Introduction of human papillomavirus (HPV) E6 and E7 oncoproteins.
- Analysis of Rb/p16 pathway inactivation and its effect on cell proliferation and senescence.
Main Results:
- hTERT expression alone extended fibroblast lifespan but did not immortalize keratinocytes or mammary epithelial cells.
- Neither hTERT addition nor E6-induced telomerase activity led to immortalization.
- Inactivation of the Rb/p16 pathway by E7, combined with telomerase activity, efficiently immortalized epithelial cells.
- Elimination of p53 or the G1 checkpoint was not required for immortalization.
Conclusions:
- Telomerase activity is necessary but not sufficient for epithelial cell immortalization.
- Inactivation of the Rb/p16 pathway is a critical cooperating event with telomerase for achieving cellular immortality in epithelial cells.
- This study elucidates key molecular requirements for overcoming replicative senescence and achieving sustained cell proliferation.