Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells - PubMed (original) (raw)
Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor beta growth inhibition in p16INK4A(-) human mammary epithelial cells
M R Stampfer et al. Proc Natl Acad Sci U S A. 2001.
Abstract
Failures to arrest growth in response to senescence or transforming growth factor beta (TGF-beta) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16(INK4A) resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-beta. The ability to maintain growth in TGF-beta was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-beta resistance may explain our previously described gain of TGF-beta resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-beta resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.
Figures
Figure 1
Schematic chart of hTERT transduction of (A) postselection p16(−) 184 HMEC at passages 11 and 18; (B) conditional immortal 184A1 (preconversion at passage 12, early conversion at passage 22); (C) preselection p16(+) 184 HMEC at passage 3.
Figure 2
Growth of postselection HMEC ± hTERT and ± TGF-β. 184 HMEC infected at passage 11 with either hTERT-containing [hTERT-184(11p)] or control retroviruses [LXSN-184(11p)] and uninfected 184 HMEC were assayed for growth ± TGF-β at the indicated passages.
Figure 3
hTERT expression induces TGF-β resistance in (A) postselection and (B) conditionally immortal HMEC. Data from Tables 1 and 2 and from ref. and unpublished work for the p53(+) line 184A1 and the p53(−/−) line 184AA3 are plotted to illustrate the rapid gain of TGF-β resistance in postselection HMEC vs. the gradual gain of resistance, with similar kinetics after telomerase activation, in p53(+) and (−/−) immortal HMEC lines and in hTERT-transduced 184A1. Closed arrows indicate passage of infection; open arrows indicate passage when telomerase activity was first detected in the uninfected lines.
Figure 4
Ectopic hTERT causes telomeres to lengthen in normal and conditionally immortal HMEC, whereas telomeres become critically short in immortal HMEC undergoing conversion. (A) Postselection 184 HMEC; hTERT-184(11p) shows rapid telomere lengthening whereas LXSN-184(11p) telomeres continue to shorten to a mean TRF of ≈5 kb at senescence. Both (B) conditional immortal 184A1 transduced at passage 12 and (C) conditional immortal 184A1 transduced at passage 22 with hTERT show rapid telomere lengthening, whereas LSXN-184A1 undergoing conversion show continued telomere erosion to faint critically short mean TRF of ≈2 kb, followed by stabilization.
Figure 5
Rare hTERT immortalization of preselection 184 HMEC is associated with gradual down-regulation of p16 and acquisition of TGF-β resistance. (A) Decreasing p16 expression (brown precipitate) in individual cells with increasing passage in the rare preselection HMEC that gained immortality after hTERT transduction at passage 3 [hTERT-184(3p)]. p16(−) postselection HMEC transduced with hTERT [184-TERT(11p)] are shown for comparison. (Bar = 100 μm.) (B) Western blot analysis showing decreasing p16 expression with increasing passage in hTERT-184(3p). (C) Increasing ability to grow in TGF-β correlates with decreasing p16 expression in hTERT-184(3p). Growth was assayed as described in_Methods_; LI was determined from five separate fields at the indicated days postseeding.
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