Effect of c-MYC and E2F1 gene silencing and of 5-azacytidine treatment on telomerase activity in pancreatic cancer-derived cell lines.
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.


CONCLUSIONS
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.
Kumari
Alpana
A
Department of General Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Srinivasan
Radhika
R
Wig
Jai Dev
JD
D001374:Azacitidine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects; D050687:E2F1 Transcription Factor / Q000235:genetics*; D020868:Gene Silencing; D016259:Genes, myc / Q000187:drug effects*; D006801:Humans; D010190:Pancreatic Neoplasms / Q000188:drug therapy; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034622:RNA Interference; D019098:Telomerase / Q000378:metabolism*
D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C509186:TERT protein, human; D019098:Telomerase; D001374:Azacitidine