Abstract C19: DNA demethylation induces upregulation of telomere repeat-containing RNA (TERRA) and downregulation of telomerase activity in human leukemia cells

Abstract

Abstract Background and aim: Telomere elongation mechanisms have been proposed to be regulated by the epigenetic status of the telomeric chromatin. Telomere repeat-containing RNAs (TERRAs), which are RNAs that originate from telomeric DNA transcription, can associate with the telomeric chromatin, where they are proposed to function as negative regulators of telomere length based on their ability to act as potent inhibitors of telomerase in vitro. To elucidate the regulatory mechanism of telomere length in a chromatin-dependent manner, we analyzed factors that influence telomere/telomerase regulation in human leukemia cells treated with 5-azacytidine, a currently available demethylating agent. Methods: Reagents and cells: Human leukemia cell lines (U937, K562, and HL-60) were treated with 0.001 to 10 μM 5-azacytidine (Sigma) for 24, 48, and 72 h; 5-azacytidine was added every 24 h. The antiproliferative effect of 5-azacytidine was measured using the cell counting kit-8 (Wako Chemicals). Telomere, telomerase and TERRA: Telomere length and telomerase activity were measured as reported previously (Ohyashiki JH, Br J Cancer 2005; 92: 1942–1947). The telomeric localization of TERRA was determined by RNA-FISH with strand-specific telomeric DNA probes. Experiments were performed in denaturing conditions with or without RNase, and the number of TERRA signal-positive cells was measured. Methylation: Bisulfite genomic DNA sequencing was performed using primers designed to recognize both methylated and unmethylated forms of the p16/INK4a and p15/INK4b promoter regions. For the genome-wide screening of DNA methylation patterns, the Human Methylation27 BeadChip (Illumina) was used. Results: Demethylating effect of 5-azacytidine: The half maximal inhibitory concentration (IC50) of 5-azacytidine was 3.4 μM in U937, 3.6 μM in K562, and 1 μM in HL-60. Bisulfite genomic DNA sequencing revealed that the p15/INK4b promoter region was markedly demethylated in U937 cells after 72 h treatment with the IC50 dose of 5-azacytidine. Similarly, we noted genome-wide hypomethylation in 5-azacytidine-treated U937 cells using the Human Methylation27 BeadChip, whereas we noted only modest hypomethylation in 5-azacytidine-treated K562 and HL-60 cells. Upregulation of TERRA and telomerase inhibition: Based on the results obtained from the methylation analysis, we identified the localization of TERRA in 5-azacytidine-treated U937 and K562 cells. We observed 0∼2 dots corresponding to TERRA per cell in untreated U937 or K562 cells, and the signals were not detected in RNaseA-treated samples. In U937 cells, TERRA signals significantly increased in accordance with the decrease in telomerase activity. In contrast, neither an increase in TERRA signals nor a decrease in telomerase activity was observed in K562 cells. This indicates that the upregulation of TERRA and telomerase inhibition were closely related to genome-wide hypomethylation. Conclusion: There is emerging evidence for the clinical utility of demethylating agents, such as 5-azacytidine in myelodysplastic syndrome; however, the molecular mechanism of 5-azacytidine has not been fully elucidated. Our results indicate that 5-azacytidine induces the upregulation of TERRA, thereby inhibiting telomerase in U937 cells. Although there is still a long way to go before realizing the diagnostic and therapeutic uses of TERRA in leukemia patients, our study provided novel information on TERRA in the telomere and the telomerase biology of human leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C19.