The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. In order to determine whether G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), might have effects on telomere dynamics and to evaluate the clinical utility, we assessed the effects of telomestatin on BCR-ABL-positive human leukemia cells. We found that treatment with telomestatin reproducibly inhibited telomerase activity in the BCR-ABLpositive leukemic cell lines OM9;22 and K562, resulting in telomere shortening. Inhibition of telomerase activity by telomestatin disrupts telomere maintenance and ultimately results in telomere dysfunction. Telomestatin completely suppressed the plating efficiency of K562 cells at 1 lm; however, telomestatin had less effects on BFU-Es and CFU-GMs colony formation from normal bone marrow CD34-positive cells. Enhanced chemosensitivity toward imatinib and chemotherapeutic agents was also observed in telomestatin-treated K562 cells. Further, the combination of telomestatin plus imatinib more effectively inhibited hematopoietic colony formation by primary human chronic myelogenous leukemia cells. Last, telomestatin induced the activation of ATM and Chk2, and subsequently increased the expression of p21 CIP1 and p27 KIP1 . These results demonstrate that telomere dysfunction induced by telomestatin activates the ATM-dependent DNA damage response. We conclude that telomerase inhibitors combined with the use of imatinib and other chemotherapeutic agents may be very useful for the treatment of human leukemia.
The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. 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. In the present study, we investigated the mechanisms of apoptosis induced by telomerase inhibition in acute leukemia. We have found the activation of caspase-3 and poly-(ADPribose) polymerase in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25). Activation of p38 mitogen-activated protein (MAP) kinase and MKK3/6 was also found in telomestatin-treated U937 cells (PD20) and dominantnegative DN-hTERT-expressing U937 cells (PD25); however, activation of JNK and ASK1 was not detected in these cells. To examine the effect of p38 MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580. Dominant-negative-hTERT-expressing U937 cells stopped proliferation on PD25; however, a significant increase in growth rate was observed in the presence of SB203580. Treatment of SB203580 also reduced the induction of apoptosis in DNhTERT-expressing U937 cells (PD25). These results suggest that p38 MAP 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 intraperitoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatintreated 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.
To evaluate the effect of deferasirox in human myeloid leukemia cells, and to identify the moleclular pathways responsible for antiproliferative effects on leukemia cells during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anticancer effect of deferasirox. The inhibitory concentration (IC 50 ) of deferasirox was 17-50 mM in three human myeloid cell lines (K562, U937, and HL60), while those in fresh leukemia cells obtained from four patients it varied from 88 to 172 mM. Gene expression profiling using Affymerix GeneChips (U133 Plus 2.0) revealed up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) encoding p21 (1) Evidence suggests that iron is required for cell survival and proliferation, and perturbation in cellular iron uptake can arrest cell growth both in vitro and in vivo.(2) As a part of ribonucleotide reductase, the enzyme responsible for deoxyribonucleotides synthesis, iron is an essential growth factor and ratelimiting trace element in DNA synthesis.(3) Dysregulaton of iron metabolism leads to iron overloading associated with deleterious effects on cells and tissues.(3) Numerous iron chelators have been synthesized in order to treat iron overload diseases, especially thalassemia. Evidence suggests the hyperproliferative effect of iron overload in a subset of cancer cells and iron depletion by chelators inhibits the proliferation of cancer cells, including leukemia cells.(4-8) Among the different molecules synthesized, hexadenate deferoxamine (DFO) is the major molecule used for the treatment of iron overload. However, it is highly hydrophilic, and inactive if taken perorally. For this reason, the perorally active iron chelator, deferasirox, is of special interest, since recent reports demonstrated that it acts as a potent nuclear factor kappa-lightchain-enhancer of activated B cell (NF-kappa-B) inhibitor and improves hematological data in a subset of patients with myelodysplastic syndromes (MDS). (9,10) To evaluate the effect of deferasirox (also knows as ICL670, Novartis, Basel, Switzerland), and to identify molecular pathways responsible for the observed reduced transfusion requirement during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anticancer effect of deferasirox. Materials and MethodsReagents and cell cultures. The oral iron chelator, deferasirox was donated by Novartis. We purchased three human myeloid leukemia cell lines, K562, U937, and HL-60 from Health Science Research Resources Bank (Osaka, Japan) for this study. Cells were grown in RPMI1640 with 10% fetal bovine serum. After obtaining written informed consent, peripheral blood mononuclear cells (PBMCs) were isolated from four patients with acute myeloid leukemia (AML) by the Ficoll-Hypaque technique. This study was approved by our institutional medical ethics committee.Cell viability and apoptosis assay. The inhibitory effect of deferasirox on cell growth was assessed by a Cell Counting Kit-8 (Wako Chemicals, Tokyo, Japan)....
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