Mild hyperoxia limits hTR levels, telomerase activity, and telomere length maintenance in hTERT-transduced bone marrow endothelial cells.
Reactivation of telomerase in endothelial cells (ECs) may be an effective approach to the treatment of vascular disorders associated with telomere attrition and EC senescence. However, overexpression of human telomerase reverse transcriptase (hTERT) does not prevent net telomere loss in ECs grown in standard culture medium with exposure to atmospheric oxygen (21% O(2)). Since these culture conditions are hyperoxic relative to normal tissue in vivo, where oxygen tension is estimated to be 1%-6%, we examined the effects of reduced exposure to oxidative stress (OS) on telomere length maintenance in hTERT-transduced bone marrow endothelial (BMhTERT) cells. Propagation of BMhTERT cells in the free radical scavenger, tert-butylhydroxylamine (tBN), and/or in 5% O(2) increased telomerase enzyme activity and facilitated telomere length maintenance. The enhancement of telomerase activity correlated with higher levels of the telomerase RNA component (hTR). We also investigated the role of the telomere binding protein, TRF1, in telomere length regulation under alternate OS conditions. Inhibition of TRF1 function had no effect on telomere length in BMhTERT cells grown under standard culture conditions. However, alleviation of OS by growth in tBN plus 5% O(2), elevated hTR levels, enhanced telomerase enzyme activity, and enabled progressive telomere lengthening. The direct impact of hTR levels on telomerase-mediated telomere lengthening was demonstrated by overexpression of hTR. BMhTERT cells transduced with hTR exhibited very high telomerase enzyme activity and underwent dramatic telomere lengthening under standard culture conditions. Overall, these results demonstrate that hTR levels are reduced by mild hyperoxia and limit telomerase-mediated telomere lengthening in hTERT-transduced ECs.
Napier
Christine E
CE
Lowy Cancer Research Centre, University of New South Wales, Randwick, NSW, Australia. cnapier@cmri.org.au
Veas
Laura A
LA
Kan
Chin-Yi
CY
Taylor
Lisa M
LM
Yuan
Jun
J
Wen
Victoria W
VW
James
Alexander
A
O'Brien
Tracey A
TA
Lock
Richard B
RB
MacKenzie
Karen L
KL
D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D015687:Cell Hypoxia; D049109:Cell Proliferation; D002478:Cells, Cultured; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D018384:Oxidative Stress; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism*; D035321:Telomeric Repeat Binding Protein 1 / Q000378:metabolism; D014161:Transduction, Genetic; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D006898:Hydroxylamines; D035321:Telomeric Repeat Binding Protein 1; D016159:Tumor Suppressor Protein p53; C422952:telomerase RNA; C072771:N-tert-butylhydroxylamine; D012313:RNA; C509186:TERT protein, human; D019098:Telomerase