The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. Genetic experiments using a dominant-negative form of human telomerase (DN-hTERT) have demonstrated that telomerase inhibition can result in telomere shortening followed by proliferation arrest and cell death by apoptosis in leukemia cells (

Tauchi et al. Clin Cancer Res, 8; 3341, 2002
). Recently, we have demonstrated that treatment with a G-quadruplex-interactive agent, telomestatin reproducibly inhibited telomerase activity in the BCR-ABL positive leukemic cell lines (
Tauchi et al. Oncogene, 22; 5338, 2003
). In the present study, we investigated the mechanisms of apotosis induced by telomerase inhibition in acute leukemia. We first examined the levels of activated p38MAP kinase, MKK3/6, ASK1 and JNK in DN-hTERT-expressing U937 cells (PD, population doubling, 25) and telomestatin-treated U937 cells (PD25). Activation of p38MAP kinase and MKK3/6 was found in DN-hTERT-expressing U937 cells (PD25) and telomestatin-treated U937 cells (PD25), however, activation of JNK and ASK1 was not detected in these cells. We have also found the activation of caspase-3 cascades and Bax translocation in DN-hTERT-expressing U937 cells (PD25) and telomestatin-treated U937 cells (PD25). To examine the effect of p38MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580, which specifically inhibits p38MAP kinase but not the Erk or JNK pathway. DN-hTERT-expressing U937 cells stopped proliferation on PD25, however, in the presence of SB203580, a significant increase in growth rate was observed. Treatment of SB203580 also reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25). These results suggest that p38MAP kinase has a critical role for the induction of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the effect of telomestatin on the growth of U937 cells in xenograft mouse model. Systemic intraperiotoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited marked apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity. Taken together, these results lay the foundations for a program of drug development to achieve the dual aims of efficacy and selectivity in vivo.

Topics:
leukemia, acute, telomerase, phosphotransferases, neoplasms, leukemia, transplantation, heterologous, bcr-abl tyrosine kinase, cancer, caspase-3, leukemic cells

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2005, The American Society of Hematology
2004
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