C80
Hypoxia induces a switch from hTR to hTERT dependent regulation of telomerase activity in the Ewing’s sarcoma family of tumour (ESFT) cell lines
Andrew Proctor1, Samantha Brownhill1, Ian Lewis2, Susan Burchill1
1Children's Cancer Group, Leeds Institute of Molecular Medicine, Leeds, UK, 2Department of Paediatric Oncology and Haematology, St James Hospital, Leeds, UK
Telomerase is the primary mechanism of telomere maintenance in ESFT. Since both the hTR and hTERT promoters contain hypoxia-response elements and telomerase activity (TA) reportedly increases when cells are placed in hypoxia, we have investigated the relationship between TA, hTR and hTERT in normoxia and hypoxia. TA was measured using an optimised TRAP assay and hTR, wild type (WT) and alpha deletion (α-) splice variants of hTERT were quantified using QRT-PCR. ESFT cell lines were maintained either in normoxia or hypoxia up to 48h. Basal TA was heterogeneous across the ESFT cell lines studied (n=9), the range of activity was similar to that in other malignant cells (n=6). A673 cells had high levels of TA (2.4TPGunits/ng protein), whereas TTC-466 and STA-ET-I cells had relatively low activity (0.11 and 0.12TPGunits/ng protein respectively). There was no statistical correlation between TA and expression of hTERT WT or -α mRNA. However, there was a correlation between TA and expression of hTR RNA (p=0.03), suggesting that hTR may modulate TA in normoxia. TA was increased by 65% and 66% respectively in SK-N-MC and TTC-466 cells after 48h in hypoxia. This increase in TA was not associated with changes in hTR RNA. Preceding the hypoxia-induced increase in TA, expression of both WT and α- variants of hTERT mRNA was increased. These observations suggest that expression of hTERT WT and –α but not hTR are regulated by hypoxia, and that increased expression of hTERT mRNAs may increase TA in ESFT cells maintained in hypoxia.
