Orit Uziel

Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomerase-expressing cell lines

Imatinib mesylate (IM) is a tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. It has proved beneficial in treating patients with chronic myeloid leukaemia (CML). In addition, IM demonstrates activity against malignant cells expressing c-kit and platelet-derived growth factor receptor (PDGF-R). The activity of IM in the blastic crisis of CML and against various myeloma cell lines suggests that this drug may also target other cellular components. In the light of the important role of telomerase in malignant transformation, we evaluated the effect of IM on telomerase activity (TA) and regulation in various malignant cell lines. Imatinib mesylate caused a dose-dependent inhibition of TA (up to 90% at a concentration of 15 μM IM) in c-kit-expressing SK-N-MC (Ewing sarcoma), SK-MEL-28 (melanoma), RPMI 8226 (myeloma), MCF-7 (breast cancer) and HSC 536/N (Fanconi anaemia) cells as well as in ba/F3 (murine pro-B cells), which do not express c-kit, BCR-ABL or PDGF-R. Imatinib mesylate did not affect the activity of other DNA polymerases. Inhibition of TA was associated with 50% inhibition of proliferation. The inhibition of proliferation was associated with a decrease in the S-phase of the cell cycle and an accumulation of cells in the G2/M phase. No apoptosis was observed. Inhibition of TA was caused mainly by post-translational modifications: dephosphorylation of AKT and, to a smaller extent, by early downregulation of hTERT (the catalytic subunit of the enzyme) transcription. Other steps of telomerase regulation were not affected by IM. This study demonstrates an additional cellular target of IM, not necessarily mediated via known tyrosine kinases, that causes inhibition of TA and cell proliferation.

Lymphoma and Leukemia Cells Possess Fractal Dimensions That Correlate with Their Biological Features

Background: Living cells can be viewed as complex adaptive systems that exhibit non-linear dynamics and fractal features. We investigated the fractal qualities of normal and malignant hematological cells and their potential as a tool for characterizing cell phenotype and clinical behavior. Methods: A mathematical algorithm and an optic tool for fractal analysis of nuclei were developed. A total of 4,713 lymphoid cells derived from 66 patients of five distinct diagnostic groups (normal and reactive lymphocytes, low-grade lymphomas and an aggressive lymphoma) were assessed for their fractal dimension. In addition, in 19 patients fractal analysis of leukemia cells was compared to clinical endpoints. Results: After validating our method, hematological cells possessed fractal dimensions corresponding to their clinical entity. There was a highly significant overall difference in fractal dimensions between various types of hematological malignancies. A preliminary correlation was found between the fractal dimension and the clinical outcome of leukemia patients. Conclusions: Hematological cells possess fractal dimensions that correlate with their biological properties. Measurement of fractal dimension seems to be a sensitive method to assess the hematological cell phenotype and to define a clinical group. This tool may be potentially useful for the evaluation of clinical behavior of hematological diseases.

Ionizing Radiation Up-regulates Telomerase Activity in Cancer Cell Lines by Post-translational Mechanism via Ras/Phosphatidylinositol 3-Kinase/Akt Pathway

Purpose: Telomerase is considered currently as a hallmark of cancer, and its inhibition is expected to become an important anticancer modality. In contrast to abundant data concerning the effect of cytotoxic drugs on telomerase activity (TA), there is scant information on the effect of radiation on telomerase. The mechanism of telomerase regulation by irradiation has never been evaluated in detail. In the present study, we investigated the effect of radiation on TA and its regulation in cancer cells. Experimental Design: The effect of various radiation doses on TA in several malignant and nonmalignant cell lines was evaluated. All malignant cells exhibited similar telomerase response to radiation and its regulation was assessed at transcriptional and post-translational levels in K562 cells. Next step was the evaluation of the upstream signaling pathways leading to changes in TA using kinetics and specific inhibitors. Results: Radiation up-regulated TA in dose-dependent manner only in cancer cells. Telomerase was activated by phosphorylation by Akt and by cytoplasmic-nuclear shift. Transcriptional processes were not involved in TA. This telomerase regulation is mediated by Ras/phosphatidylinositol 3-kinase/Akt pathway. The canonical membrane effectors of irradiation (epidermal growth factor receptor, insulin-like growth factor-I receptor, and Ca2+ influx) were not involved in this process. Conclusions: Radiation up-regulates telomerase activity specifically in cancer cells. This study adds to accumulating evidence pointing to post-translational level as important mode of telomerase regulation. Telomerase activation due to radiation may be detrimental in treatment of cancer. Data described in this study may add to future interventions aiming at inhibition of telomerase activation during irradiation.

Granulocyte colony-stimulating factor administration upregulates telomerase activity in CD34+ haematopoietic cells and may prevent telomere attrition after chemotherapy

Summary. Hematopoietic reconstitution could be associated with premature ageing of the transplanted cells and a high frequency of myelodysplastic syndrome and secondary leukaemia. Telomere length decreases with cell divisions and age, and at a crucial length it is associated with chromosomal instability and cell senescence. Telomerase is a reverse transcriptase enzyme that adds nucleotides to chromosomal ends. Most somatic cells lack telomerase activity yet haematopoietic stem cells retain low levels of telomerase. Some studies have found that chemotherapy and stem cell transplantation lead to the accelerated shortening of telomere length. As granulocyte colony‐stimulating factor (G‐CSF) is routinely used in the mobilization of stem cells for transplantation, we evaluated its effects on telomerase activity and regulation, and on telomere dynamics, in normal donors and selected lymphoma patients. Administration of G‐CSF increased telomerase activity in CD34+ haematopoietic cells compared with controls. In marrow‐derived CD34+ cells, telomerase activity increased sevenfold, compared with a 14‐fold increase in peripheral‐blood‐mobilized CD34+ cells. A parallel increase in the expression of human telomerase enzyme reverse transcriptase RNA and protein kinase C α occurred. In addition, G‐CSF administration to five lymphoma patients after consecutive courses of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy, resulted in telomere length preservation or elongation, as opposed to marked attrition in patients who did not receive growth factors. We conclude that the in vivo administration of G‐CSF prevents or attenuates telomere attrition associated with chemotherapy administration. This attenuation may contribute to the preservation of telomere integrity inG‐CSF‐primed transplanted stem cells.

Oxidative stress causes telomere damage in Fanconi anaemia cells - a possible predisposition for malignant transformation.

Fanconi anaemia (FA) is an autosomal recessive and X‐linked disease characterized by severe genetic instability and increased incidence of cancer. One explanation for this instability may be the cellular hypersensitivity to oxidative stress leading to chromosomal breaks. This study explored the possible oxidative damage to telomeres of FA lymphocyte cell line, HSC536/N, and its possible effect on telomere function. We postulated that combination of oxidative damage with overexpression of telomerase may provide a possible model for malignant transformation in FA. The cells were grown in the presence of telomerase inhibitor and exposed for 1 month to H2O2 combined with various antioxidants. This exposure caused shortening of telomere length and damage to the telomere single stranded overhang, which was prevented by several oxidants. This shortening was associated with development of severe telomere dysfunction. Control cells did not exhibit this sensitivity to H2O2. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal P53 upregulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxidative damage. These results suggest a scenario in which oxidative stress causes telomere shortening and ensuing telomere dysfunction may form the basis for malignant transformation in FA cells. Upregulation of telomerase activity in sporadic FA cells may perpetuate that process, thus explaining the malignant character of FA cells in vivo.

Nonmyeloablative conditioning does not prevent telomere shortening after allogeneic stem cell transplantation

Background. Stem cell transplantation (SCT) may be associated with premature aging of the hematopoietic stem cells. Telomere length reflects the proliferative history of a cell. In most studies published so far on telomere dynamics after myeloablative allogeneic SCT, recipients had shorter telomeres than their respective donors, thus reflecting “accelerated aging” of hematopoietic cells. We evaluated telomere dynamics in patients who underwent transplantation with nonmyeloablative protocols, assuming that the decreased intensity of chemotherapy might prevent telomere attrition. Methods. Telomere length was measured using FISH-FACS method. Telomeres of recipients were compared to their respective donors. Twenty-three consecutive patients after nonmyeloablative SCT were evaluated. A control group consisted of 10 donor-recipient pairs after conventional myeloablative transplantation. Results. There was significant telomere shortening in both recipients of nonmyeloablative and myeloablative conditioning (0.487±0.65 kb, P=0.003; 0.361±0.50 kb, P=0.047 respectively). The extent of telomere shortening in the two groups was not different (P=0.64). There was no correlation between the degree of shortening and parameters such as time interval from transplant, age of donor or recipient, and the number of infused cells. Conclusions. This is the first study on telomere dynamics after nonmyeloablative conditioning SCT. The study demonstrates significant shortening of telomeres in recipients in spite of decreased intensity conditioning. Results of this study suggest that the main mechanism following transplantation is the proliferative stress imposed upon the stem cells and not direct damage by cytotoxic drugs. The different kinetics of restoration of hematopoiesis and the probable ongoing process of graft-versus-leukemia in the bone marrow do not prevent the attrition of telomeric ends of chromosomes.

Tumor cells derived exosomes contain hTERT mRNA and transform nonmalignant fibroblasts into telomerase positive cells

Exosomes are small (30-100nm) vesicles secreted from all cell types serving as inter-cell communicators and affecting biological processes in “recipient” cells upon their uptake. The current study demonstrates for the first time that hTERT mRNA, the transcript of the enzyme telomerase, is shuttled from cancer cells via exosomes into telomerase negative fibroblasts, where it is translated into a fully active enzyme and transforms these cells into telomerase positive, thus creating a novel type of cells; non malignant cells with telomerase activity. All tested telomerase positive cells, including cancer cells and non malignant cells with overexpressed telomerase secreted exosomal hTERT mRNA in accordance with the endogenous levels of their hTERT mRNA and telomerase activity. Similarly exosomes isolated from sera of patients with pancreatic and lung cancer contained hTERT mRNA as well. Telomerase activity induced phenotypic changes in the recipient fibroblasts including increased proliferation, extension of life span and postponement of senescence. In addition, telomerase activity protected the fibroblasts from DNA damage induced by phleomycin and from apoptosis, indicating that also telomerase “extracurricular” activities are manifested in the recipient cells. The shuttle of telomerase from cancer cells into fibroblasts and the induction of these changes may contribute to the alterations of cancer microenvironment and its role in cancer. The described process has an obvious therapeutic potential which will be explored in further studies.

The effect of chemotherapy on telomere dynamics: clinical results and possible mechanisms

Abstract Telomeres are the chromosomal end components, and their length in hematopoietic stem cells correlates with the bone marrow proliferative reserve. There are few data regarding telomere dynamics in hematopoietic stem cells after exposure to chemotherapy. We show that the attrition of telomeres after cytotoxic treatment correlates with the intensity of chemotherapy. Using cytotoxic drugs with differential effects on hematopoietic stem cells, our data imply that chemotherapy-induced telomere shortening results from direct damage to hematopoietic stem cells and/or the induction of proliferative stress on bone marrow while sparing repopulating stem cells. These results gain importance considering the current long survival of patients with cancer.

Effect of imatinib on the signal transduction cascade regulating telomerase activity in K562 (BCR-ABL-positive) cells sensitive and resistant to imatinib.

OBJECTIVE Imatinib mesylate (IM) is a tyrosine kinase inhibitor selective for BCR-ABL and indicated for the treatment of chronic myeloid leukemia. It has recently been demonstrated that IM also targets other cellular components. Considering the significant role of telomerase in malignant transformation, we studied the effect of IM on telomerase activity (TA) and regulation in BCR-ABL-positive and -negative cells, sensitive and resistant to IM. MATERIALS AND METHODS Through combining telomeric repeat amplification protocol for detecting TA, reverse transcription polymerase chain reaction and Western blots for detecting RNA and protein levels of telomerase regulating proteins and fluorescence-activated cell sorting analysis, we showed that IM targets telomerase and the signal transduction cascade upstream of it. RESULTS IM significantly inhibited TA in BCR-ABL-positive and -negative cells and in chronic myeloid leukemia patients. TA inhibition was also observed in BCR-ABL positive cells resistant to IM at drug concentrations that did not lead to a reduction in BCR-ABL expression. In addition, a reduction in phosphorylated AKT and phosphorylated PDK-1 was also detected following IM incubation. CONCLUSIONS We demonstrate an inhibitory effect of IM on TA and on the AKT/PDK pathway. Because this effect was observed in cell expressing the BCR-ABL protein as well as cells not expressing it, and in cells sensitive as well as resistant to IM, it is reasonable to assume that the inhibitory effect of IM on TA is not mediated through known IM targets. The results of this study show that cells resistant to IM with regard to its effect on BCR-ABL could still be sensitive to IM treatment regarding other cellular components.

Differential downregulation of telomerase activity by bortezomib in multiple myeloma cells-multiple regulatory pathways in vitro and ex vivo

Background:The importance of telomerase in multiple myeloma (MM) is well established; however, its response to bortezomib has not been addressed.Methods:The effect of bortezomib on telomerase activity and cell proliferation was evaluated in four MM cell lines and in myeloma cells obtained from eight patients. The mechanism of telomerase regulation on epigenetic, transcriptional, and post-translational levels was further assessed in two selected cell lines: ARP-1 and CAG. Clinical data were correlated with the laboratory findings.Results:Bortezomib downregulated telomerase activity and decreased proliferation in all cell lines and cells obtained from patients, albeit in two different patterns of kinetics. ARP-1 cells demonstrated higher and earlier sensitivity than CAG cells due to differential phosphorylation of hTERT by PKCα. Methylation of hTERT promoter was not affected. Transcription of hTERT was similarly inhibited in both lines by decreased binding of SP-1 and not of C-Myc and NFκB. The ex vivo results confirmed the in vitro findings and suggested existence of clinical relevance.Conclusion:Bortezomib downregulates telomerase activity in MM cells both transcriptionally and post-translationally. MM cells, both in vitro and in patients, exhibit different sensitivity to the drug due to different post-translational response. The effect of bortezomib on telomerase activity may correlate with resistance to bortezomib in patients, suggesting its potential utility as a pre-treatment assessment.