scholarly journals Comparable Effects of the Androgen Derivatives Danazol, Oxymetholone and Nandrolone on Telomerase Activity in Human Primary Hematopoietic Cells from Patients with Dyskeratosis Congenita

2020 ◽  
Vol 21 (19) ◽  
pp. 7196
Author(s):  
Margherita Vieri ◽  
Martin Kirschner ◽  
Mareike Tometten ◽  
Anne Abels ◽  
Benjamin Rolles ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Therapeutic Option ◽  
Telomerase Activity ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Inherited Disease ◽  
Beneficial Effects ◽  
Tert Expression

Dyskeratosis congenita (DKC) is a rare inherited disease of impaired telomere maintenance that progressively leads to multi-organ failure, including the bone marrow. By enhancing telomerase activity, androgen derivatives (ADs) are a potential therapeutic option able to re-elongate previously shortened telomeres. Danazol, oxymetholone, and nandrolone are ADs most frequently used to treat DKC. However, no direct in vitro analyses comparing the efficacy of these ADs have been conducted so far. We therefore treated mononuclear cells derived from peripheral blood and bone marrow of four patients with mutations in telomerase reverse transcriptase (TERT, n = 1),in the telomerase RNA component (TERC, n = 2) and in dyskerin pseudouridine synthase 1 (DKC1, n = 1) and found no substantial differences in the activity of these three agents in patients with TERC/TERT mutations. All AD studied produced comparable improvements of proliferation rates as well as degrees of telomere elongation. Increased TERT expression levels were shown with danazol and oxymetholone. The beneficial effects of all ADs on proliferation of bone marrow progenitors could be reversed by tamoxifen, an estrogen antagonist abolishing estrogen receptor-mediated TERT expression, thereby underscoring the involvement of TERT in AD mechanism of action. In conclusion, no significant differences in the ability to functionally enhance telomerase activity could be observed for the three AD studied in vitro. Physicians therefore might choose treatment based on patients’ individual co-morbidities, e.g., pre-existing liver disease and expected side-effects.

scholarly journals Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3000-3008
Author(s):  
BP Alter ◽  
ME Knobloch ◽  
L He ◽  
AP Gillio ◽  
RJ O'Reilly ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Factor ◽  
Mononuclear Cells ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Normal Numbers ◽  
Erythroid Progenitors ◽  
Bone Marrow Failure Syndromes

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

scholarly journals Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3000-3008 ◽  
Author(s):  
BP Alter ◽  
ME Knobloch ◽  
L He ◽  
AP Gillio ◽  
RJ O'Reilly ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Factor ◽  
Mononuclear Cells ◽  
Bone Marrow Failure ◽  
Dyskeratosis Congenita ◽  
Normal Numbers ◽  
Erythroid Progenitors ◽  
Bone Marrow Failure Syndromes

Abstract Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

scholarly journals In vitro expansion impaired the stemness of early passage mesenchymal stem cells for treatment of cartilage defects

2017 ◽  
Vol 8 (6) ◽  
pp. e2851-e2851 ◽  
Author(s):  
Tongmeng Jiang ◽  
Guojie Xu ◽  
Qiuyan Wang ◽  
Lihui Yang ◽  
Li Zheng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Mononuclear Cells ◽  
Telomerase Activity ◽  
Cartilage Defects ◽  
Cell Based Therapy ◽  
Chondrogenic Potential ◽  
First Choice

Abstract In vitro cultured autologous mesenchymal stem cells (MSCs) within passage 5 have been approved for clinical application in stem cell-based treatment of cartilage defects. However, their chondrogenic potential has not yet been questioned or verified. In this study, the chondrogenic potential of bone marrow MSCs at passage 3 (P3 BMSCs) was investigated both in cartilage repair and in vitro, with freshly isolated bone marrow mononuclear cells (BMMNCs) as controls. The results showed that P3 BMSCs were inferior to BMMNCs not only in their chondrogenic differentiation ability but also as candidates for long-term repair of cartilage defects. Compared with BMMNCs, P3 BMSCs presented a decay in telomerase activity and a change in chromosomal morphology with potential anomalous karyotypes, indicating senescence. In addition, interindividual variability in P3 BMSCs is much higher than in BMMNCs, demonstrating genomic instability. Interestingly, remarkable downregulation in cell cycle, DNA replication and mismatch repair (MMR) pathways as well as in multiple genes associated with telomerase activity and chromosomal stability were found in P3 BMSCs. This result indicates that telomerase and chromosome anomalies might originate from expansion, leading to impaired stemness and pluripotency of stem cells. In vitro culture and expansion are not recommended for cell-based therapy, and fresh BMMNCs are the first choice.

Telomerase Enzyme Activity In Egyptian Children With Bone Marrow Failure

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4662-4662
Author(s):  
Amal M. El-Beshlawy ◽  
Mona Mohamed Hamdy ◽  
Amina Abd El Salam ◽  
Mervet El Ansary ◽  
Nelly Abulata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Immunosuppressive Therapy ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Control Group ◽  
Egyptian Children

Background Telomeres are structural elements that seal the ends of chromosomes protecting them from recombination and end to end fusion. Maintenance of the integrity of telomeres requires the telomerase ribonucleoprotein complex. Abnormal telomere maintenance is a feature of a variety of human diseases including constitutional aplastic anemia. Predisposition to the development of marrow failure has been conferred by genetic alternations results in low telomerase activity. Short telomeres in leukocytes and reduced hematopoietic function. Aim To evaluate the telomerase functional activity in Egyptian children with inherited and acquired bone marrow failure and its relation to the phenotypic features of the acquired disease and its response to immunosuppressive therapy. Patients and Methods This was a case-control study conducted on unrelated children (n=40) with bone marrow failure syndromes and forty healthy subjects age and sex-matched as controls. The diagnosis of bone marrow failure was based on the bone marrow biopsy and blood-count results. Patients with acquired aplastic anemia (AAA) were considered severe if at least 2 of the following were noted: neutrophil count < 0.5×109/L; platelet count < 20×109/L with hypocellular marrow. Response to immunosuppressive therapy (IST) was evaluated after 6 months of initiation of therapy. Assessment of Telomerase Activity Telomerase activity was measured in mononuclear cells utilizing the Telomeric Repeat Amplification Protocol (TRAP )using the TeloTAGGG Telomerase PCR ELISA. Results Forty patients were included in the study (30 AAA , 6 Fanconi Anemia (FA), 2 Pure Red Cell Aplasia (PRCA), one case with Dyskeratosis Congenita (DCK) and one case with constitutional aplastic anemia. The Mean age was 11.1±4.9 years (range 3.5 to 18 years, median 11 years ) and the duration of follow-up mean; 5.14 (±3.84) years (range 1-13 years). Patients with AAA (n=30) received treatment with cyclosporine A( n =27) and ATG( n=3). Telomerase level The median telomerase level was significantly lower in inherited BMF syndromes when compared to controls [5.05 (4.60 – 8.70 IQR) Vs 11.15 (5.90-16.60 IQR), p=0.04]. In AAA the median telomerase level was insignificantly lower than controls [5.4 (2.3 – 21.0 IQR) Vs 11.15 (5.90-16.60 IQR), p=0.228]. A good inverse correlation was detected between the telomerase level and age of the patients (r=-0.39, p=0.026). No correlation was found between the telomerase level and disease duration in hereditary or AAA (r= -0.303, p=0.111 and r=0.305, p=0.156 respectively). Telomerase activity and clinical variables We classified our cases into two groups according to the median value of the control group (11.5): Low telomerase level group: (n=27): included 18 AAA (66.7%) and 9 hereditary aplastic Anemia (33.3%). Cases with AAA 14/18 (77.8%) with low telomerase activity were severe cases with bone marrow cellularity less than 10 % Vs 6/12 (50%) with normal telomerase. Fifty per cent of cases (9/18) with low telomerase responded partially or completely to IST Vs. 83.4% of the normal telomerase (10/12) group. Patients of AAA who received cyclosporine therapy (n=27), 19 cases (70.4%) were responders versus zero % of patients who received ATG (n=3). The median telomerase of responders was 16.5 + 4.7 Vs 11.6 + 3.8 of none responders. Conclusion • Telomerase activity was affected in hereditary and acquired bone marrow failure. • Evaluation of telomerase activity is essential for therapeutic and prognostic aspects of these diseases. Cyclosporin A can be used as a monotherapy in the treatment of acquired aplastic anemia even in the presence of decreased telomerase activity Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dyskerin Is a Component of the Arabidopsis Telomerase RNP Required for Telomere Maintenance

2008 ◽  
Vol 28 (7) ◽  
pp. 2332-2341 ◽  
Author(s):  
Kalpana Kannan ◽  
Andrew D. L. Nelson ◽  
Dorothy E. Shippen
Keyword(s):  
Telomere Length ◽  
Bone Marrow Failure ◽  
Telomerase Activity ◽  
Dyskeratosis Congenita ◽  
Dominant Negative ◽  
Telomere Maintenance ◽  
Dependent Manner ◽  
Missense Mutations

ABSTRACT Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

scholarly journals Telomerase activity in normal leukocytes and in hematologic malignancies

Blood ◽  
1995 ◽  
Vol 85 (9) ◽  
pp. 2315-2320 ◽  
Author(s):  
CM Counter ◽  
J Gupta ◽  
CB Harley ◽  
B Leber ◽  
S Bacchetti
Keyword(s):  
Bone Marrow ◽  
Hematologic Malignancies ◽  
Telomerase Activity ◽  
Carcinoma Cells ◽  
Lymphocytic Leukemia ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Somatic Tissues ◽  
Low Levels

Telomeres are essential for function and stability of eukaryotic chromosomes. In the absence of telomerase, the enzyme that synthesizes telomeric DNA, telomeres shorten with cell division, a process thought to contribute to cell senescence and the proliferative crisis of transformed cells. We reported telomere stabilization concomitant with detection of telomerase activity in cells immortalized in vitro and in ovarian carcinoma cells, and suggested that telomerase is essential for unlimited cell proliferation. We have now examined the temporal pattern of telomerase expression in selected hematologic malignancies. We found that, unlike other somatic tissues, peripheral, cord blood, and bone marrow leukocytes from normal donors expressed low levels of telomerase activity. In leukocytes from chronic lymphocytic leukemia (CLL) patients, activity was lower than in controls in early disease, and comparable with controls in late disease. Relative to bone marrow, telomerase activity was enhanced in myelodysplastic syndrome (MDS) and more significantly so in acute myeloid leukemia (AML). Regardless of telomerase levels, telomeres shortened with progression of the diseases. Our results suggest that early CLL and MDS cells lack an efficient mechanism of telomere maintenance and that telomerase is activated late in the progression of these cancers, presumably when critical telomere loss generates selective pressure for cell immortality.

Phenotypic Rescue Of Induced Pluripotent Stem Cells From Dyskeratosis Congenita Patients By Ectopic Expression Of DKC1 But Not TERC

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2468-2468 ◽  
Author(s):  
Bai-wei Gu ◽  
Jason A. Mills ◽  
Marisa Apicella ◽  
Jian-meng Fan ◽  
Deborah L. French ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Pluripotent Stem Cells ◽  
Bone Marrow Failure ◽  
Telomerase Activity ◽  
Ips Cells ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Direct Role ◽  
Induced Pluripotent

Abstract Telomerase is a ribonucleoprotein that adds telomeric repeats onto the chromosome ends, preventing the replication-dependent loss of telomere repeats and cellular senescence in highly proliferative germ-line cells and in stem cells and cancer cells. Dyskeratosis Congenita (DC) is a rare bone marrow failure syndrome, which affects tissues that need constant renewal by stem cell activity. So far 8 genes have been found whose mutation causes DC and they all encode products that play a role in telomere maintenance. About 35% of DC patients show X-linked-recessive inheritance due to mutations in the DKC1gene encoding dyskerin, a protein important in telomere maintenance and ribosomal RNA biogenesis. Mutant dyskerin can destabilize telomerase RNA leading to rapidly shortening telomeres, accelerated stem cell aging and bone marrow failure. However the precise mechanism by which this occurs is not known and some results suggest dyskerin may play a more direct role in telomerase action. So far studies of the cell biology of DC stem cells have been hampered by their scarcity in patients and their short life span and attempts to create mouse models have suffered from differences in both telomere biology and hematopoiesis between mouse and human. In this study, to investigate disease pathogenesis of X-linked DC, we generated induced pluripotent stem cells (iPSC) from patients’ skin fibroblast cell carrying DKC1 mutations Q31E, Δ37 and A353V. The recurrent A353V mutation accounts for about 40% of DKC1 mutations and usually causes a severe clinical phenotype while patients carrying Q31E and Δ37 mutations show a milder phenotype. We found that dyskerin protein expression in all of these dyskerin mutant iPS cells is decreased in agreement with our mouse studies that show mutant proteins are relatively unstable. These iPS cells show dramatically decreased TERC RNA levels and telomerase activity. Telomere length measurement revealed that mutant iPS cells lose the ability to elongate telomeres during the reprogramming processing; telomere erosion was particularly rapid in A353V cells. To further investigate whether dyskerin protein could play direct role in regulating telomerase activity other than stabilization of TERC RNA during the processing of assembly, we tested if the defect in telomerase function in these iPS cells could be rescued by expressing wild type dyskerin or TERC RNA. We expressed the rescuing genes by using the zinc-finger nuclease-mediated method to insert them into the safe harbor AAVs1 site, initially of DKC1Δ37 cells. After testing the telomerase function, we found that expressing WT-dyskerin protein in Δ37 iPS cells fully restores the mature Terc RNA expression level and the telomerase activity to normal levels. However, although Δ37 iPS with over-expressed WT TERC RNA can accumulate normal level of TERC RNA, these cells fail to increase telomerase activity. These results suggest that defective telomerase activity cause by DKC1 mutations can only reversed by expressing WT dyskerin but not by TERC RNA. These data suggest that, as one of three core components of the telomerase complex, dyskerin may play a direct role in telomerase activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cardioprotective Effects of Puerarin-V on Isoproterenol-Induced Myocardial Infarction Mice Is Associated with Regulation of PPAR-Υ/NF-κB Pathway

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3322 ◽  
Author(s):  
Xuguang Li ◽  
Tianyi Yuan ◽  
Di Chen ◽  
Yucai Chen ◽  
Shuchan Sun ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Inflammatory Responses ◽  
Therapeutic Option ◽  
Crystal Form ◽  
Human Coronary Artery ◽  
Beneficial Effects ◽  
Ppar Γ ◽  
Cardioprotective Effects

Puerarin is a well-known traditional Chinese medicine which has been used for the treatment of cardiovascular diseases. Recently, a new advantageous crystal form of puerarin, puerarin-V, has been developed. However, the cardioprotective effects of puerarin-V on myocardial infarction (MI) heart failure are still unclear. In this research, we aim to evaluate the cardioprotective effects of puerarin-V on the isoproterenol (ISO)-induced MI mice and elucidate the underlying mechanisms. To induce MI in C57BL/6 mice, ISO was administered at 40 mg/kg subcutaneously every 12 h for three times in total. The mice were randomly divided into nine groups: (1) control; (2) ISO; (3) ISO + puerarin injection; (4–9) ISO + puerarin-V at different doses and timings. After treatment, cardiac function was evaluated by electrocardiogram (ECG), biochemical and histochemical analysis. In vitro inflammatory responses and apoptosis were evaluated in human coronary artery endothelial cells (HCAECs) challenged by lipopolysaccharide (LPS). LPS-induced PPAR-Υ/NF-κB and subsequently activation of cytokines were assessed by the western blot and real-time polymerase chain reaction (PCR). Administration of puerarin-V significantly inhibits the typical ST segment depression compared with that in MI mice. Further, puerarin-V treatment significantly improves ventricular wall infarction, decreases the incidence of mortality, and inhibits the levels of myocardial injury markers. Moreover, puerarin-V treatment reduces the inflammatory milieu in the heart of MI mice, thereby blocking the upregulation of proinflammatory cytokines (TNF-α, IL-1β and IL-6). The beneficial effects of puerarin-V might be associated with the normalization in gene expression of PPAR-Υ and PPAR-Υ/NF-κB /ΙκB-α/ΙΚΚα/β phosphorylation. In the in vitro experiment, treatment with puerarin-V (0.3, 1 and 3 μM) significantly reduces cell death and suppresses the inflammation cytokines expression. Likewise, puerarin-V exhibits similar mechanisms. The cardioprotective effects of puerarin-V treatment on MI mice in the pre + post-ISO group seem to be more prominent compared to those in the post-ISO group. Puerarin-V exerts cardioprotective effects against ISO-induced MI in mice, which may be related to the activation of PPAR-γ and the inhibition of NF-κB signaling in vivo and in vitro. Taken together, our research provides a new therapeutic option for the treatment of MI in clinic.

scholarly journals Investigation of Mitochondrial Transfer between Human Erythroblasts

2021 ◽  
Author(s):  
◽  
Brittany Lewer
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Qpcr Assay ◽  
Dna Polymorphisms ◽  
Mitochondrial Transfer ◽  
Hel Cells ◽  
Allele Specific

<p>The increasingly studied phenomenon of mitochondria transferring between cells contrasts the popular belief that mitochondria reside permanently within their cells of origin. Research has identified this process occurring in many tissues such as brain, lung and more recently within the bone marrow. This project aimed to investigate if mitochondria could be transferred between human erythroblasts, a context not previously studied.  Tissue microenvironments can be modelled using co-culture systems. Fluorescence activated cell sorting and a highly sensitive Allele-Specific-Blocker qPCR assay were used to leverage mitochondrial DNA polymorphisms between co-cultured populations. Firstly, HL-60ρ₀ bone marrow cells, without mitochondrial DNA, deprived of essential nutrients pyruvate and uridine were co-cultured in vitro with HEL cells, a human erythroleukemia. Secondly, HEL cells treated with deferoxamine or cisplatin, were cocultured with parental HL-60 cells in vitro. Lastly, ex vivo co-cultures between erythroblasts differentiated from mononuclear cells in peripheral blood were conducted, where one population was treated with deferoxamine.  Co-culture was able to improve recovery when HL-60ρ₀ cells were deprived of pyruvate and uridine. Improved recovery was similarly detected for HEL cells treated with deferoxamine after co-culture with HL-60 cells. Transfer of mitochondrial DNA did not occur at a detectable level in any co-culture condition tested. The high sensitivity of the allele-specific-blocker qPCR assay required completely pure populations to analyse, however this was not achieved using FACS techniques. In conclusion, results have not demonstrated but cannot exclude the possibility that erythroid cells transfer mitochondria to each other.</p>

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