Telomerase activation by histone deacetylase inhibitor in normal cells

Abstract

Although telomerase activity is known to be regulated mainly at the level of transcription of the human telomerase catalytic subunit (hTERT) gene, the molecular mechanism underlying tumor-specific expression of telomerase remains unclear. Emerging evidence suggests that reversible acetylation of nucleosomal histones and the resultant changes in the chromatin structure are important processes in gene transcription. In particular, histone deacetylase (HDAC) inhibitors activate the transcription of certain genes by altering the acetylation status of nucleosomal histones. The present study examines the effects of HDAC inhibitor on hTERT gene transcription. Treatment with tricostatin A (TSA) induced significant activation of hTERT mRNA expression and telomerase activity in normal cells, but not in cancer cells. Transient expression assays revealed that TSA activates the hTERT promoter. Furthermore, the proximal 181 bp core promoter of hTERT, which contains two c-Myc and five Sp1 sites, was determined to be the responsible element. Overexpression of Sp1 enhanced responsiveness to TSA, and mutation of Sp1 sites, but not c-Myc sites, of the core promoter of hTERT abrogated this activation. Introduction of the dominant-negative form of the Sp family inhibited TSA activation. These results indicate that HDAC inhibitor activates the hTERT promoter in normal cells, in which Sp1 plays a key role. This finding suggests one way whereby histone deacetylation may be involved in silencing the hTERT gene in normal cells.

Figure 1

TRAP assays to detect change in telomerase activity in cells treated with TSA. (A) TSA induced telomerase activity in normal HRCE cells. (B) No change in telomerase activity was observed in C33A cervical cancer cells. N, negative control using lysis buffer only. P, positive control using extracts from cancer cell lines with telomerase activity.

Figure 1

TRAP assays to detect change in telomerase activity in cells treated with TSA. (A) TSA induced telomerase activity in normal HRCE cells. (B) No change in telomerase activity was observed in C33A cervical cancer cells. N, negative control using lysis buffer only. P, positive control using extracts from cancer cell lines with telomerase activity.

Figure 2

RT–PCR assays to detect change in hTERT mRNA expression in cells treated with TSA. TSA significantly induced hTERT mRNA expression in normal HRCE cells. No change in expression was observed in C33A cancer cells.

Figure 3

Transient expression assays to examine the transcriptional regulation of hTERT promoter by TSA. (A) Luciferase reporter plasmids used in the assays. c-Myc and Sp1 sites within a core promoter of hTERT are shown. Mutated sites are described as crossed-out. LUC, luciferase gene. HRCE (B) and C33A (C) cells were transfected with luciferase reporter plasmids and incubated with or without TSA. pGL3-control reporter plasmid was used to confirm the promoter specificity of TSA. At 24 h after treatment, cells were harvested and luciferase assays were performed. Relative luciferase activity of each reporter plasmid is shown, which is normalized to that of the pGL3-control untreated with TSA (given as 1.0). Bars, SD.

Figure 3

Transient expression assays to examine the transcriptional regulation of hTERT promoter by TSA. (A) Luciferase reporter plasmids used in the assays. c-Myc and Sp1 sites within a core promoter of hTERT are shown. Mutated sites are described as crossed-out. LUC, luciferase gene. HRCE (B) and C33A (C) cells were transfected with luciferase reporter plasmids and incubated with or without TSA. pGL3-control reporter plasmid was used to confirm the promoter specificity of TSA. At 24 h after treatment, cells were harvested and luciferase assays were performed. Relative luciferase activity of each reporter plasmid is shown, which is normalized to that of the pGL3-control untreated with TSA (given as 1.0). Bars, SD.

Figure 3

Transient expression assays to examine the transcriptional regulation of hTERT promoter by TSA. (A) Luciferase reporter plasmids used in the assays. c-Myc and Sp1 sites within a core promoter of hTERT are shown. Mutated sites are described as crossed-out. LUC, luciferase gene. HRCE (B) and C33A (C) cells were transfected with luciferase reporter plasmids and incubated with or without TSA. pGL3-control reporter plasmid was used to confirm the promoter specificity of TSA. At 24 h after treatment, cells were harvested and luciferase assays were performed. Relative luciferase activity of each reporter plasmid is shown, which is normalized to that of the pGL3-control untreated with TSA (given as 1.0). Bars, SD.

Figure 4

Transient expression assays to examine the role of Sp1 in TSA regulation of promoter. (A) HRCE cells were transfected with pG5-Luc containing Gal4 sites or pGL3-control together with pM-Sp1 effector plasmids, which expresses Sp1/Gal4 fusion proteins, and were incubated with or without TSA. Relative luciferase activity of each reporter plasmid is shown, which is normalized to that of pGL3-control untreated with TSA (given as 1.0). (B) HRCE cells were transfected with hTERT-promoter reporter plasmids together with pCMV-DNSp3, which expresses the dominant-negative form of Sp3, and were incubated with or without TSA. At 24 h after treatment, cells were harvested and luciferase assays were performed. Relative luciferase activity is shown, which is normalized to that of pGL3-3328 untreated with TSA (given as 1.0). Bars, SD.

Figure 4

Transient expression assays to examine the role of Sp1 in TSA regulation of promoter. (A) HRCE cells were transfected with pG5-Luc containing Gal4 sites or pGL3-control together with pM-Sp1 effector plasmids, which expresses Sp1/Gal4 fusion proteins, and were incubated with or without TSA. Relative luciferase activity of each reporter plasmid is shown, which is normalized to that of pGL3-control untreated with TSA (given as 1.0). (B) HRCE cells were transfected with hTERT-promoter reporter plasmids together with pCMV-DNSp3, which expresses the dominant-negative form of Sp3, and were incubated with or without TSA. At 24 h after treatment, cells were harvested and luciferase assays were performed. Relative luciferase activity is shown, which is normalized to that of pGL3-3328 untreated with TSA (given as 1.0). Bars, SD.