HIF-1-mediated activation of telomerase in cervical cancer cells

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of O 2 homeostasis, which regulates the expression of several genes linked to angiogenesis and energy metabolism. Tumor hypoxia has been shown to be associated with poor prognosis in a variety of tumors, and HIF-1 induced by hypoxia plays pivotal roles in tumor progression. The presence of putative HIF-1-binding sites on the promoter of human telomerase reverse transcriptase gene (hTERT) prompted us to examine the involvement of HIF-1 in the regulation of hTERT and telomerase in tumor hypoxia. The telomeric repeat amplification protocol (TRAP) assay revealed that hypoxia activated telomerase in cervical cancer ME180 cells, with peak induction at 24-48 h of hypoxia. Notably, hTERT mRNA expression was upregulated at 6-12 h of hypoxia, concordant with the elevation of HIF-1 protein levels at 6 h. hTERT protein levels were subsequently upregulated at 24 h and later. Luciferase assays using reporter plasmids containing hTERT core promoter revealed that hTERT transcription was significantly activated in hypoxia and by HIF-1 overexpression, and that the two putative binding sites within the core promoter are responsible for this activation. Chromatin immunoprecipitation assay identified the specific binding of HIF-1 to these sites (competing with c-Myc), which was enhanced in hypoxia. The present findings suggest that hypoxia activates telomerase via transcriptional activation of hTERT, and that HIF-1 plays a critical role as a transcription factor. They also suggest the existence of novel mechanisms of telomerase activation in cancers, and have implications for the molecular basis of hypoxia-induced tumor progression and HIF-1-based cancer gene therapy.

The Opposing Effect of Hypoxia-Inducible Factor-2α on Expression of Telomerase Reverse Transcriptase

“…The hTERT gene has previously been identified as a direct target of HIF-1a (13,16). We therefore sought to further determine the effect of both HIF-1a and HIF-2a on the hTERT promoter activity in RCC cells.…”

“…We therefore sought to further determine the effect of both HIF-1a and HIF-2a on the hTERT promoter activity in RCC cells. It is known that p181, a reporter construct with an insert of the core hTERT promoter sequence, carries the hypoxia-responsible element (HRE), and that HIF-1a activates the hTERT transcription by binding this HRE (13,16). When p181 was cotransfected with either HIF1a or HIF-2a expression plasmids into A498 and Caki-2 cells, we observed substantial increases in luciferase activity driven by the hTERT core promoter in a dose-dependent manner Hypoxia-induced HIF-1a and/or HIF-2a expression and accompanied changes in hTERT mRNA and its variants in RCC (Caki-2, TK-10 , and A498 ) and glioma (U563, U373 , and U251 ) cell lines.…”

“…By promoting angiogenesis, HIF-1 plays important roles in progression, invasiveness, or metastasis of malignant diseases (7,8). Furthermore, recent studies have suggested that hypoxic treatment or overexpression of HIF-1a affects hTERT and telomerase expression (10)(11)(12)(13)(14)(15)(16). Most observations showed that the incubation of human cancer cells at lower levels of oxygen enhanced hTERT mRNA expression and telomerase activity via HIF-1a -mediated trans-activation of the hTERT gene.…”

“…(a) Koshiji et al observed that hypoxia-induced HIF-1a significantly inhibited hTERT expression in human colon cancer cells, in sharp contrast to other reports (11). (b) Different mechanisms, including transcriptional and post-transcriptional approaches, have been implicated in hypoxia-regulated hTERT expression (10,(12)(13)(14)16). It remains to be defined whether these regulatory pathways contribute to the enhanced hTERT expression or telomerase activity in all human cancer cells.…”

Mechanisms of telomere maintenance and attrition: linking cancer and ageing

“…However, environmental factors such as cigarette smoking and further DNA damaging substances can also accelerate telomere attrition and senescence (Acosta and Gil 2009;Finkel et al 2007), thereby linking Toxicology to telomere and ageing research. On the other hand, genetic instability and DNA breakage may lead to ampliWcation and increased expression of telomerase, although the involved mechanisms are not yet fully understood (Yatabe et al 2004;Verdun and Karlseder 2007). Genetic instability (Florl and Schulz 2008;Bolt 2007, 2008;Stoiber et al 2008;Beyersmann and Hartwig 2008) and DNA damage induction (Bolt 2008;Periyakaruppan et al 2007;Borza et al 2008;Bolt and Hengstler 2008;Zhang et al 2008;Wong et al 2008;Glahn et al 2008;Krishnamurthi et al 2008;Ullmann et al 2008;Wozniak et al 2007;Schmid et al 2007;Nishimura et al 2008;Schug et al 2008 (Liew and Norbury 2009, this issue).…”