Reptin is required for the transcription of telomerase reverse transcriptase and over-expressed in gastric cancer

Abstract

Background: Telomerase is activated in oncogenesis, which confers an immortal phenotype to cancer cells. The AAA + ATPase Reptin is required for telomerase biogenesis by maintaining telomerase RNA (hTER) stability and is aberrantly expressed in certain cancers. Given its role in chromatin remodeling and transcription regulation, we determined the effect of Reptin on the transcription of the telomerase reverse transcriptase (hTERT) gene, a key component of the telomerase complex and its expression in gastric cancer.

Results: Knocking down Reptin or its partner Pontin using small interfering RNA in gastric and cervical cancer cells led to significant decreases in hTERT mRNA, but hTERT promoter activity was inhibited in only Reptin-depleted cells. Reptin interacted with the c-MYC oncoprotein and its stimulatory effect on the hTERTpromoter was significantly dependent on functional E-boxes in the promoter. Moreover, Reptin bound to the hTERT proximal promoter and the loss of the Reptin occupancy led to dissociation of c-MYC from the hTERT promoter in Reptin-depleted cells. Reptin inhibition dramatically impaired clonogenic potential of gastric cancer cells by inducing cell growtharrest and over-expression of Reptin was observed in primary gastric cancer specimens.

Conclusions: The hTERT gene is a direct target of Reptin, and hTERT transcription requires constitutive expression of Reptin and its cooperation with c-MYC. Thus, Reptin regulates telomerase at two different levels. This finding, together with the requirementof Reptin for the clonogenic potential of cancer cells and its over-expression in gastriccancer and other solid tumors, suggests that Reptin may be a putative therapeutic target.

Figure 1

Reptin depletion leads to reduced hTERT mRNA expression and telomerase activity in gastric and cervical cancer cells. C, P and R: Control, Pontin and Reptin siRNA, respectively. Bars: Standard deviations. The results were obtained from three to four independent experiments. (A) Reptin protein expression in gastric and cervical cancer cells and efficiency of Reptin knocking down by siRNA. Cells were treated with control, Reptin or Pontin siRNA for 72 hours and then analyzed for Reptin expression by immunoblotting assay. (B) Reduced hTERT mRNA expression in Reptin or Pontin siRNA-treated cells. The same sets of cells from as above were used to determine hTERT mRNA using rqPCR. Relative levels of hTERT mRNA are shown. (C) Reduced telomerase activity in Reptin or Pontin siRNA-treated cells. Cellular proteins were extracted from the cells above and telomerase activity was assessed using a Telomerase ELISA kit. The enzymatic activity was expressed as absorbance [optimal density (OD) in arbitrary units]. (D) Additive inhibitory effect on hTERT mRNA expression in cells treated with combination of Reptin and Pontin siRNAs. HGC-27 and HeLa cells were treated with Reptin or Pontin siRNA alone or both of them at the half concentration used in (B) and hTERT mRNA abundance was analyzed with rqPCR. Relative levels of hTERT mRNA are shown.

Figure 2

Reptin cooperates with c-MYC to regulate the hTERT promoter activity. (A and B) c-MYC-dependent inhibition of the hTERT promoter activity by Reptin depletion. AGS and HGC-27 cells were first treated with control (C), Reptin (R) or Pontin (P) siRNA and then transfected with either p181^wt or its c-MYC mutant variant p181^MYC-. Luciferase activity was assessed using a dual luciferase detection kit 48 hours after transfection and relative luciferase activity was obtained from its normalization by co-transfected thymidine kinase renilla activity and expressed as percentage of that in control cells. Bars: Standard deviation. * and **: P < 0.05 and 0.01, respectively. Bars: Standard deviations. NS: Not significant. Three independent experiments were performed. (C) Concomitant presence or absence of Reptin and c-MYC on the hTERT promoter. AGS and HGC-27 cells were treated with control or Reptin siRNA and chromatin immunoprecipitation was then performed to examine the association between the hTERT promoter sequence and Reptin, c-MYC, or acetylated histone H3. (D) c-MYC expression in Reptin-depleted gastric cancer cells as determined by using rqPCR (Left panel) and western blot (Right panel). NS: Not significant and **: P < 0.01. (E) Physical interaction between Reptin and c-MYC as demonstrated using immunoprecipitation. Cellular proteins derived from AGS and BGC-823 cells were precipitated by a c-MYC antibody followed by detection using the antibody against Reptin. Rabbit IgG was used as a negative control. Two independent experiments were performed with similar results.

Figure 3

Reptin depletion severely impairs the clonogenic potential and growth of gastric cancer cells. (A) Cells were transfected with Reptin siRNA for 72 hours and then seeded into six-well plates (300 cells/well). After ten days, Giemsa staining was performed and the foci number was documented. Six independent experiments were performed. (B) AGS and HGC-27 cells treated with Control and Reptin siRNA were analyzed for cell cycle distribution using flow cytometry with ModFit. Two independent experiments were performed with similar results.

Figure 4

Reptin is over-expressed in primary gastric cancer. Immunohistochemistry was performed to determine Reptin expression. (A) Overall Reptin-positive cells in gastric cancer specimens and their matched normal tissues from 20 patients. (B) and (C) Representative of Reptin staining on normal and cancerous gastric tissues, respectively (Magnification × 400). Left panels: The enlarged areas of right panels in the rectangles. (D) Positive correlation between Reptin and hTERT mRNA levels in primary gastric tissues. The abundance of Reptin and hTERT mRNA in 20 normal and cancerous gastric tissues was assessed using rqPCR as described in Materials and methods.