Specific Inhibition of hTERT by shRNA Sensitizes for Imatinib Mesylate in BCR-ABL-Positive Cells.

In the normal somatic cells, loss of telomere length leads to cell senescence appearing chromosome end-to-end fusion, and rearrangements. Most cancer cells exhibit constitutive telomerase activation and maintain telomere stability. Therefore, telomerase produces telomere repeats and represents the anti-cancer therapeutic target. Imatinib mesylate is a tyrosine kinase inhibitor, which inhibits BCR/ABL signal transductions. Despite of high remission rate of imatinib, the resistance to imatinib has been recognized as the major problem in treatment of the advanced or refractory CML. We have demonstrated that a dominant negative form of hTERT presents telomere shortening followed by proliferation arrest and apoptosis in leukemia cell lines. In the present study, we investigated the biological activity of hTERT specific short hairpin RNA (hTERT-shRNA) (kindly gifted from Dr. William C. Hahn, Dana-Farber Cancer Institute) in CML blastic-crisis cell line. We first disrupted the function of a catalytic subunit of telomerase complex, hTERT by expressing the hTERT-shRNA in the telomerase expressing K562 cell line, and found the inhibition of telomerase activity after single cell selections. We also found cumulative telomere shortenings in the hTERT-shRNA-expressing K562 cells. The hTERT-shRNA-expressing K562 cells showed increasing rate of apoptosis, and morphologically hyper-diploid cells followed by proliferation arrest. The hTERT-shRNA-expressing K562 cells reduced number of colony formation. The hTERT-shRNA-expressing K562 cells (population doubling 30) cytogenetically demonstrated end-to-end fusions and multi-centric chromosomes, which might be equivalent to disruptions of telomeric structure composed by repetitive DNA and telomere binding proteins. These results demonstrated that hTERT-shRNA effectively reduced the telomerase activity and induced telomere dysfunction in leukemia cells. Further we evaluated the combination efficacy of hTERT-shRNA and imatinib. The hTERT-shRNA-expressing K562 cells demonstrated significantly higher rate of apoptotic cells, and reduced proliferation activity compared with the parental K562 cells in the dose dependent manner of imatinib. These results suggest that the telomerase inhibition by hTERT-shRNA combined with imatinib is effective in BCR-ABL-positive leukemia.