Abstract
BackgroundIncreased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene.Methodology/Principal FindingsWe optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP) assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT.Conclusions/SignificanceThese data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases.
Highlights
The interferon (IFN) family of cytokines exhibits multiple biological activities both in vitro and in vivo [1,2,3,4]
Using our optimized conditions to detect low levels of human telomerase reverse transcriptase (hTERT) mRNA and protein, we found that decreases in the steady-state levels of IFI16 mRNA and protein were inversely correlated with the hTERT mRNA (Fig. 1B) and protein (Fig. 1C) levels
Using a mouse monoclonal antibody 2C4, which has been shown to be specific to the human hTERT protein [28,53] or a specific rabbit polyclonal antibody [54], we could detect the basal low levels of hTERT protein in WI-38 (Fig. 1C) and other young HDFs, such as BJ and IMR-90
Summary
The interferon (IFN) family of cytokines exhibits multiple biological activities both in vitro and in vivo [1,2,3,4]. Studies have suggested that expression of a set of IFN-inducible genes, which encode proteins that mediate the biological activities of IFNs [1,6], is up-regulated during the onset of cellular senescence in a variety of human cells [7,8,9,10,11]. The loss of expression of IFN-inducible genes is correlated with immortalization of cells and the development of certain human cancers [7,11] These observations have suggested a role for IFN-inducible proteins in the regulation of cellular senescence. Increased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. We report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene
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