scholarly journals Imetelstat Sensitizes Hematopoietic Malignancy Cells to Genotoxic Agent Via Suppression of Telomerase Mediated DNA Repair Process

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3369-3369
Author(s):  
Daisuke Hidaka ◽  
Masahiro Onozawa ◽  
Naohiro Miyashita ◽  
Masao Nakagawa ◽  
Daigo Hashimoto ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Growth ◽  
Telomere Length ◽  
Research Funding ◽  
Hematological Malignancies ◽  
Repair Process ◽  
Telomerase Activity ◽  
Htert Mrna ◽  
Telomerase Inhibition ◽  
Genotoxic Agent

Telomerase is the ribonucleoprotein enzyme that has the main function of extension of telomeric repeat. Telomerase consists of two constructions: telomerase reverse transcriptase protein (hTERT) and RNA template. Inhibition of telomerase activity is considered to be a therapeutic approach for malignant tumors since telomerase is dominantly expressed in germ cells, stem cells, and cancer cells but not in normal cells. We found that (TTAGGG)n repeats could be integrated and repair artificially induced DNA double strand break (DSB) sites (Onozawa M et al. PNAS, 2014). Therefore, we hypothesized that inhibition of telomerase activity may interfere with the DNA repair process and enhance the effects of DNA damaging agents. Imetelstat competitively suppresses telomerase activity. It has a binding site complementary to the RNA template of telomerase and induces specific inhibition. Imetelstat showed clinical benefits in patients with essential thrombocytosis or myelofibrosis used as monotherapy. However, the effectiveness of imetelstat combined with other cytotoxic agent for hematological malignancies is not clear. We therefore evaluated the synergistic anti-tumor effects of imetelstat and cytotoxic agents on hematological malignancies. First, the expression of hTERT mRNA, protein, and activity was confirmed in 8 human cell lines of hematological malignancies (U937, HL60, PALL2, CEM, IM9, RPMI8226, ST1, and SU9T01) but not in peripheral blood mononuclear cells (PBMCs) from a healthy individual. There was a significant correlation between mRNA expression and activity measured by TRAP assay (r=0.839, 95% confidence interval: 0.396-0.965, p=0.00467). The expression of hTERT mRNA in clinical samples was analyzed using primary acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) cells. The expression level of hTERT in ALL cells was significantly higher than that in AML cells (p=0.0093). The telomerase inhibitor, imetelstat had a concentration-dependent effect on telomerase inhibition, while a control oligo did not affect telomerase activity. The expression of hTERT protein was not suppressed by imetelstat treatment. Imetelstat also had a concentration-dependent suppressive effect on cell growth without affecting cell viability. Next, we evaluated the synergistic anti-tumor effects of etoposide and imetelstat at the concentration at which it did not affect cell growth by monotherapy. Cell proliferation at day 4 was inhibited by coadministration of etoposide (1 µM) and imetelstat (1 µM) compared to that with administration of etoposide alone (Figure A). Western blot analysis showed that gamma-H2AX expression level after radiation and imetelstat administration was significantly higher than that of after radiation alone (Figure B). Therefore, imetelstat enhanced DNA-DSB induced by a genotoxic agent. We determined whether the synergistic effect was mediated by shortening of telomere length. Telomere length was analyzed in long-term culture using the flow-FISH method. Imetelstat significantly shortened telomere length compared to a control oligo after day 14, while there was almost no change in telomere length at day 4. We also analyzed the changes of hTERT activity and gamma-H2AX expression after radiation in PBMCs from a healthy individual. Although hTERT activity was not detected in stable state in PBMC, hTERT activity appeared and gradually increased over time and gamma-H2AX expression peaked from 2 to 4 hours after radiation. Images of immunofluorescence staining revealed co-localization of hTERT and gamma-H2AX in the nucleus of PBMCs after radiation (Figure C). Therefore, hTERT induced by DNA damage might be directly involved in the DNA-DSB repair process. We clearly showed synergistic effects of imetelstat and a genotoxic agent in short-term treatment, in which shortening of telomeres was not observed. Potentially, telomerase inhibition could stand as a beneficial addition to other treatment methods when the inhibitor is administered at low non-toxic doses. Our hypothesis needs to be clarified in a clinical trial. Further studies are required to optimize these therapies in a variety of hematopoietic malignancies or other cancers. Figure Disclosures Nakagawa: akeda Pharmaceutical Company Limited: Research Funding. Teshima:Novartis: Honoraria, Research Funding.

Functional p53 Is Required for Effective Telomerase Inhibition in BCRABL- Positive CML Cells in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 572-572
Author(s):  
Ute Brassat ◽  
Stefan Balabanov ◽  
Melanie Braig ◽  
Daniel Bali ◽  
Kerstin Borgmann ◽  
...  
Keyword(s):  
Telomere Length ◽  
Growth Kinetics ◽  
Blast Crisis ◽  
Telomerase Activity ◽  
Gfp Expression ◽  
Telomere Shortening ◽  
Telomerase Inhibition ◽  
Group A ◽  
Group B

Abstract Telomeres consist of repeat structures such as (TTAGGG)n in vertebrates and are localized at the end of chromosomes. Replication-dependent telomere shortening due to the end-replication problem can be counteracted by upregulation of an endogenous reverse transcriptase called telomerase. Increasing evidence suggests that critical telomere shortening results in genetic instability which may promote tumour evolution and telomerase activation during which critically short telomeres are stabilised and ongoing tumour growth is facilitated. In Chronic myeloid leukemia (CML) the high turnover of the malignant clone is driven by the oncogene BCR-ABL and leads to accelerated telomere shortening in chronic phase (CP) compared to telomere length in healthy individuals. Telomere shortening has been demonstrated to be correlated with disease stage, duration, prognosis and response to molecular targeted treatment. Despite of the accelerated telomere shortening observed, telomerase activity is increased in CP CML and further upregulated with progression of the disease to accelerated phase or blast crisis (AP/BC). To investigate the effect of telomerase inhibition on BCR-ABL-positive cells, we expressed a dominant-negative mutant of hTERT (vector pOS DNhTERT-IRES-GFP) in K562 cells. The cells were single sorted and clones in addition to bulk cultures were long term expanded in vitro. The expression of the transgene DNhTERT was monitored by the expression of GFP and function of DNhTERT was analyzed by measurement of telomere length (by flow-FISH) and telomerase activity (TRAP assay). Evaluation of these parameters showed the following patterns of growth kinetics and telomere biology in individual clones: Two clones lost telomere repeats and were transiently delayed in growth kinetics but eventually escaped from crisis without loss of GFP expression (indicated by a re-increase in telomere length and growth rate, group A) Three other clones lost GFP expression after initial and significant telomere reduction indicating loss of the transgene (group B). Finally, telomere length and growth kinetics of two remaining clones and of the bulk culture cells remained unaffected by expression of DN-hTERT (group C). Of note, none of the clones analyzed either died or entered cell cycle arrest. Further analyses of one clone of group A revealed impaired DNA damage response indicated by two fold increase in number of γH2AX foci in comparison to control cells. Moreover, the expression pattern of genes involved in DNA repair was significantly altered (Dual chip®). Network analysis of the altered genes using MetaCore® software confirmed p53 as a key regulator in signaling of DNA damage in these cells. CML blast crisis cell lines such as K562 are typically negative for functional p53 and p16INK4. Therefore, we went on and investigate if the presence of functional p53 is required for the induction of telomere-mediated apoptosis or senescence in BCR-ABL-positive cells. For this purpose, we restored p53 in telomerase-negative clones by using an inducible system (vector pBABE p53ERtam) in two clones from group A and group B. Induction of p53 in cells with critically short telomeres (telomere length 4–5 kb) lead to immediate induction of apoptosis while vector control cells continued to escape from crisis. These results suggest that the success of strategies aimed at telomerase inhibition in CML is dependent on the presence of functional p53 in BCR-ABL-positive cells which argues in favour of applying these strategies preferentially in CP as opposed to BC.

Short Telomeres Combined with Low Telomerase Activity Are Associated with a Longer Survival in Patients with Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4262-4262
Author(s):  
Veronique Saada ◽  
Pierre Tran Ba Loc ◽  
Camille Legeai ◽  
Marine Castaing ◽  
Jean-Henri Bourhis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Survival Analysis ◽  
Telomere Length ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Telomerase Activity ◽  
Htert Mrna ◽  
The Impact ◽  
Acute Myeloid

Abstract Telomeres - the terminal regions of human chromosomes, and enzyme telomerase - a ribonucloprotein that synthesizes telomeric DNA onto chromosomal ends, have been thoroughly investigated as potential markers for the prognosis of various cancers including leukemia. However, it is important to consider both parameters and only few studies have investigated the prognostic value of these two combined biomarkers in patients with acute myeloid leukemia (AML). Our work was designed to determine the impact of telomere length together with telomerase activity (TA) on survival in patients with AML. In this current retrospective study, TA (reflected by the quantitative expression of the catalytic subunit of telomerase i.e the hTERT mRNA/18s RNA ratio measured by Q-RT-PCR) and telomere length (determined by southern blot analysis of terminal restriction fragments) were assayed in the bone marrow of 40 patients diagnosed with AML between 1999 and 2003 at Institute Gustave Roussy’s division of Hematology. The patients’ characteristics are shown on table 1. All patients were treated according to standard AML-type chemotherapy protocols. The median of TA (hTERT mRNA/18s RNA ratio) was 0.0458. TA was not detectable in 4 patients. The median of telomere length was 7.6 Kb (range: 3.5–11.2 Kb). No correlation was found between TA and telomere length. A negative correlation existed between telomere length and age (r= −0.42; p=0.0097). The Kaplan-Meier statistical method and logrank test were used for univariate survival analysis and the Cox proportional hazard regression models for multivariate survival analysis. In multivariate analysis, when adjusted for age (>= 50 years versus younger), cytogenetics findings (poor prognosis versus others) and the nature of leukemia (secondary versus de novo), improved survival was found in patients with a combination of short telomere length (<7.6 Kb) and weak TA (<0.09, cut off point separating the upper tertile) and the worse survival was found in patients with long telomere length (>=7.6 Kb) and high TA (>=0.09) (hazard ratio=9.91; 95% CI: 1.75–56.03; p=0.01). Our results suggest that the combination of telomere length and telomerase activity can be considered as an independent prognostic factor for survival in patients with AML. Table 1: Patients characteristics Sex (no. of patients) * AML post solid tumor (n=7), post myelodysplastic syndrome (n=1), post chronic myeloid leukemia (n=1) M 18 F 22 Age (years) Median 50 Range 22–74 Leukocyte count (Giga/L) Median 24.2 Range 1.3–360 Bone marrow blast percentage Median 76.5 Range 20–99 FAB Classification (no. of patients) M0 6 M1 9 M2 5 M4–M5 12 M4Eo 3 M6 1 Biphenotypic AL 3 NK AML 1 Type of leukemia (no. of patients) De novo 31 Secondary 9* Prognosis (based on karyotype) Good 5 Intermediate 21 Poor 10 Missing 4

Telomerase activity level, but not hTERT mRNA and hTR level, regulates telomere length in telomerase-reconstituted primary fibroblasts

2004 ◽  
Vol 297 (2) ◽  
pp. 434-443 ◽  
Author(s):  
Susan J.J Swiggers ◽  
H.Arno J Nibbeling ◽  
Annelieke Zeilemaker ◽  
Marianne A Kuijpers ◽  
Karin A Mattern ◽  
...  
Keyword(s):  
Telomere Length ◽  
Telomerase Activity ◽  
Activity Level ◽  
Htert Mrna ◽  
Primary Fibroblasts

Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event

2012 ◽  
Vol 112 (5) ◽  
pp. 773-781 ◽  
Author(s):  
Matthew J. Laye ◽  
Thomas P. J. Solomon ◽  
Kristian Karstoft ◽  
Karin K. Pedersen ◽  
Susanne D. Nielsen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Telomere Length ◽  
Mononuclear Cells ◽  
Physiological Stress ◽  
Telomerase Activity ◽  
Mrna Levels ◽  
Htert Mrna ◽  
Peripheral Blood Mononuclear ◽  
Shelterin Complex ◽  
Blood Mononuclear Cells

Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects ( n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 ( P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4+ or CD8+ cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.

scholarly journals GDF-15 as a biomarker of aging

2020 ◽  
Author(s):  
Huan Liu ◽  
Yun Huang ◽  
Yongnan Lyu ◽  
Wen Dai ◽  
Yongqing Tong ◽  
...  
Keyword(s):  
Telomere Length ◽  
Hematological Parameters ◽  
Negative Relationship ◽  
Multivariate Regression Analysis ◽  
Telomerase Activity ◽  
Cross Sectional Study ◽  
Htert Mrna ◽  
Human Telomerase Reverse Transcriptase ◽  
Cross Sectional ◽  
Biomarkers Of Aging

Abstract Background: The aging process is accompanied by the gradual development of chronic systemic inflammation (inflamm-aging). Growth differentiation factor-15 (GDF-15) is associated with inflammation and known to be a stress-induced factor. The present study aimed to explore the association of GDF-15 with aging.Methods: In this cross-sectional study, serum GDF-15, hematological parameters, and biomedical parameters were determined in 120 healthy individuals (23-83 years old, males). Three telomere related parameters, including telomere length, telomerase activity, and the expression of human telomerase reverse transcriptase (hTERT) mRNA were also quantified.Results: The older group has a higher levels of GDF-15 and lower expression of hTERT mRNA, and PBMC telomerase activity (p<0.001). In individuals with high GDF-15 levels, they were older, and presented with the lower level of hTERT mRNA and T/S ratio (p<0.01). Spearman correlation analysis shows that GDF-15 positively correlated with age (r=0.664, p<0.001), and negatively correlated with telomere length (r=-0.434, p<0.001), telomerase activity (r=-0.231, p=0.012), and hTERT mRNA (r=-0.206, p=0.024). Furthermore, in multivariate regression analysis, GDF-15 levels showed a statistically significant linear and negative relationship with PBMC telomerase activity (β-coefficient=-0.583, 95% CI -1.044 to -0.122, p=0.014), telomere length (β-coefficient=-0.200, 95% CI -0.305 to -0.094, p<0.001), and hTERT mRNA (β-coefficient=-0.207, 95% CI -0.312 to -0.102, p<0.001) after adjusting for confounders.Conclusions: In conclusion, our results show that circulating GDF-15 is the potential biomarkers of aging that may influence the risk and progression of multiple aging conditions.

scholarly journals Comparison of senescence-related changes between three- and two-dimensional cultured adipose-derived mesenchymal stem cells

2020 ◽  
Author(s):  
Qiliang Yin ◽  
Na Xu ◽  
Dong sheng Xu ◽  
Ming xin Dong ◽  
Xiu min Shi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Energy Metabolism ◽  
Cell Growth ◽  
Telomere Length ◽  
Cell Morphology ◽  
Telomerase Activity ◽  
Two Dimensional ◽  
3D Cultures ◽  
2D And 3D

Abstract Background: Adipose-derived mesenchymal stem cells ( ADMSCs ) have attracted widespread interest as cell-based tissue repair systems. To obtain adequate quantities of ADMSCs for therapeutic applications, extensive in vitro expansion is required. However, under current two-dimensional (2D) approaches , ADMSCs rapidly undergo replicative senescence , and cell growth is impeded and stem cell properties are eliminated by mechanisms that are poorly understood . These issues limit the extensive applications of ADMSCs . In this study, we investigated senescence-related changes in mesenchymal stem cells (MSCs) isolated from human adipose tissue in 2D and three-dimensional (3D) cultures. Methods: We studied cell growth over a given period (21 days) to determine if modes of culture were associated with ADMSCs senescence . ADMSCs were isolated from healthy females by liposuction surgery and then were grew in 2D and 3D cultures. The cell morphology was observed during cell culture. Every other time of culture, senescence-associated β-galactosidase (SA-β-gal) expression, cell viability, proliferation, and differentiation potential of ADMSCs from 2D and 3D cultures were detected. Also, senescence and stemness related genes expression, telomere length, telomerase activity, and energy metabolism of ADMSCs for different culture time were evaluated. Results: With long-term propagation, we observed significant changes in cell morphology, proliferation, differentiation abilities and energy metabolism, which were associated with increases in SA-β-gal activity, and decreases in telomere length and telomerase activity . Notably, when cultured in 3D, these changes were improved. Conclusions: Our results indicate that 3D culture is able to ameliorate senescence-related changes in ADMSCs.

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