BACKGROUND: The gene promoter region of human telomerase reverse transcriptase (hTERT) contains binding sites for c-myc and E2F1 as well as CpG islands, suggesting regulation by genetic factors and epigenetically by methylation. Hence, the effect of the demethylating agent 5-azacytidine and silencing of c-MYC and E2F1 genes on its expression and consequently on telomerase activity were studied in pancreatic cancer-derived cell lines. METHODS: MIaPaCa-2 and PANC-1 cell lines were transfected with SiRNA against E2F1 and c-MYC genes separately as well as along with 5-azacytidine treatment. The hTERT gene methylation status was determined by methylation-specific PCR and telomerase activity quantitated by TRAP-PCR-ELISA. RESULTS: Demethylation by 5-azacytidine resulted in hTERT inhibition with a reduction in telomerase activity to 37-49% of controls. Silencing of E2F-1 or c-MYC also decreased the hTERT transcript and telomerase activity with a more pronounced effect with respect to c-MYC silencing. There was a synergistic effect of demethylation and gene silencing on the inhibition of hTERT mRNA expression which resulted in undetectable levels of telomerase activity. CONCLUSION: Telomerase activity, which is necessary for cellular immortalization, can be shut down by a combined approach using SiRNA-mediated gene silencing and demethylating agents, which has therapeutic implications. Copyright 2009 S. Karger AG, Basel.
BACKGROUND: The gene promoter region of human telomerase reverse transcriptase (hTERT) contains binding sites for c-myc and E2F1 as well as CpG islands, suggesting regulation by genetic factors and epigenetically by methylation. Hence, the effect of the demethylating agent 5-azacytidine and silencing of c-MYC and E2F1 genes on its expression and consequently on telomerase activity were studied in pancreatic cancer-derived cell lines. METHODS: MIaPaCa-2 and PANC-1 cell lines were transfected with SiRNA against E2F1 and c-MYC genes separately as well as along with 5-azacytidine treatment. The hTERT gene methylation status was determined by methylation-specific PCR and telomerase activity quantitated by TRAP-PCR-ELISA. RESULTS: Demethylation by 5-azacytidine resulted in hTERT inhibition with a reduction in telomerase activity to 37-49% of controls. Silencing of E2F-1 or c-MYC also decreased the hTERT transcript and telomerase activity with a more pronounced effect with respect to c-MYC silencing. There was a synergistic effect of demethylation and gene silencing on the inhibition of hTERT mRNA expression which resulted in undetectable levels of telomerase activity. CONCLUSION: Telomerase activity, which is necessary for cellular immortalization, can be shut down by a combined approach using SiRNA-mediated gene silencing and demethylating agents, which has therapeutic implications. Copyright 2009 S. Karger AG, Basel.
Authors: Jean-Pierre Issa; Ignacio I Wistuba; Razelle Kurzrock; David J Stewart; Maria I Nunez; Jaroslav Jelinek; David Hong; Sanjay Gupta Journal: Clin Epigenetics Date: 2014-01-09 Impact factor: 6.551
Authors: Nina Milutin Gašperov; Ivan Sabol; Pavao Planinić; Goran Grubišić; Ivan Fistonić; Ante Ćorušić; Magdalena Grce Journal: PLoS One Date: 2015-06-09 Impact factor: 3.240