scholarly journals Interleukin-6, -8, and TGF-β Secreted from Mesenchymal Stem Cells Show Functional Role in Reduction of Telomerase Activity of Leukemia Cell Via Wnt5a/β-Catenin and P53 Pathways

2020 ◽  
Vol 10 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Ezzatollah Fathi ◽  
Behnaz Valipour ◽  
Zohreh Sanaat ◽  
Hojjatollah Nozad Charoudeh ◽  
Raheleh Farahzadi
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Real Time Pcr ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Telomerase Activity ◽  
Htert Gene ◽  
Gene And Protein Expression

Purpose: The effect of mesenchymal stem cells (MSCs) on the immortality features of malignant cells, such as hematologic cancerous cells, are controversial, and the associated mechanisms are yet to be well understood. The aim of the present study was to investigate the in vitro effect of bone marrow-derived MSCs (BMSCs) on the chronic myeloid leukemia cell line K562 through telomere length measurements, telomerase activity assessments, and hTERT gene expression. The possible signaling pathways involved in this process, including Wnt-5a/β-catenin and P53, were also evaluated. Methods: Two cell populations (BMSCs and K562 cell line) were co-cultured on transwell plates for 7 days. Next, K562 cells were collected and subjected to quantitative real-time PCR, PCR-ELISA TRAP assay, and the ELISA sandwich technique for telomere length, hTERT gene expression, telomerase activity assay, and cytokine measurement, respectively. Also, the involvement of the mentioned signaling pathways in this process was reported by real-time PCR and Western blotting through gene and protein expression, respectively. Results: The results showed that BMSCs caused significant decreases in telomere length, telomerase activity, and the mRNA level of hTERT as a regulator of telomerase activity. The significant presence of interleukin (IL)-6, IL-8, and transforming growth factor beta (TGF-β) was obvious in the co-cultured media. Also, BMSCs significantly decreased and increased the gene and protein expression of β-catenin and P53, respectively. Conclusion: It was concluded that the mentioned effects of IL-6, IL-8, and TGF-β cytokines secreted from MSCs on K562 cells as therapeutic agents were applied by Wnt-5a/β-catenin and P53 pathways

Telomerase activity, mTert gene expression and the telomere length in mouse mesenchymal stem cells in the late period after γ- and γ,n-irradiation and in the tumors developed from these cells

2020 ◽  
Vol 66 (3) ◽  
pp. 265-273
Author(s):  
O.V. Vysotskaya ◽  
A.I. Glukhov ◽  
Yu.P. Semochkina ◽  
S.A. Gordeev ◽  
E.Yu. Moskaleva
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cell Lines ◽  
Telomerase Activity ◽  
Mouse Bone Marrow ◽  
Fibrosarcoma Cell

In proliferating normal and tumor cells, the telomere length (TL) is maintained by high telomerase activity (TA). In the absence of TA the TL maintenance involves a mechanism of alternative lengthening of telomeres (ALT). The aim of this study was to investigate the level of TA, the mTert expression and TL in cultured normal and transformed by γ- and γ,n-irradiation mesenchymal stem cells (MSCs) from mouse bone marrow, in sarcomas that developed after the transplantation of these cells into syngeneic mice, and in fibrosarcoma cell lines obtained from these tumors to find out the role of AT or ALT in maintaining TL in these cells. During prolonged cultivation of normal and transformed under the influence of γ- (1 Gy and 6 Gy) and γ,n-irradiation (0.05 Gy, 0.5 Gy, and 2 Gy) MSCs from mouse bone marrow, a decrease in TA was detected in irradiated cells. Even deeper decrease in TA was found in sarcomas developed after administration of transformed MSCs to syngeneic mice and in fibrosarcoma cell lines isolated from these tumors in which TA was either absent or was found to be at a very low level. TL in three of the four lines obtained was halved compared to the initial MSCs. With absent or low TA and reduced TL, the cells of all the obtained fibrosarcoma lines successfully proliferated without signs of a change in survival. The mechanism of telomere maintainance in fibrosarcoma cell lines in the absence of TA needs further investigation and it can be assumed that it is associated with the use of the ALT. The detected decrease or absence of TA in transformed under the action of irradiation MSCs with the preservation or even an increase in the telomerase gene expression may be associated with the formation of inactive splicing variants, and requires further study. The obtained lines of transformed MSCs and fibrosarcomas with TA and without the activity of this enzyme can be a useful model for studying the efficacy of TA and ALT inhibitors in vitro and in vivo.

218 DONOR-MATCHED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF CANINE MESENCHYMAL STEM CELLS DERIVED FROM DIFFERENT ORIGINS

2012 ◽  
Vol 24 (1) ◽  
pp. 221
Author(s):  
S. A. Ock ◽  
G. H. Maeng ◽  
Y. M. Lee ◽  
T. H. Kim ◽  
B. M. Kumar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Rt Pcr ◽  
Cytochemical Staining ◽  
Single Donor ◽  
Cell Capacity

Canine mesenchymal stem cells (cMSC) have been successfully isolated from several adult tissue sources. However, differences in the biological properties of MSC have been shown to be associated with donor variability. Further, the stem cell capacity of cMSC of various tissues isolated from a single donor is currently unclear. Therefore, this study investigated the functional and molecular characteristics of cMSC derived from bone marrow (cBM-MSC), adipose tissue (cA-MSC) and dermal skin (cDS-MSC) of a single donor. Three kinds of cMSC were isolated by following previously published protocols. AP activity was assessed with a chromogen kit (Abcam Inc., Cambridge, MA, USA). Expression of CD markers (CD45, 90 and 105) and stem cell transcription factors (Oct3/4, Nanog and Sox2) was analysed by immunocytochemical staining. All cells were induced into osteogenesis and adipogenesis by following protocols described earlier and confirmed by cytochemical staining and the detection of lineage specific genes by RT-PCR. Chromosomal stability was assessed by a method described earlier (Ock and Rho 2008 J. Vet. Med. Sci. 70, 1165–1172) and cell cycle status was determined by a flow cytometry. Telomere length was analysed by Telo TAGGG Telomere Length Assay kit (Roche, Mannheim, Germany) and telomerase activity was evaluated by semiquantitative nested RT-PCR. Statistical analysis was performed by ANOVA using SPSS 12.0 and significance was tested when P < 0.05. Expressions of AP activity and the transcription factors, such as Oct3/4, Nanog and Sox2 were absent in all cMSC. All 3 types of cMSC positively expressed the surface markers CD90 and 105 but not CD45. Exposure of all cell lines to osteogenic and adipogenic induction medium resulted in the calcium deposition evidenced by Alizarin red S staining and the accumulation of fat globules indicated by Oil red O staining, respectively. Differentiation was further confirmed by the detection of marker genes, such as Runx2 and Pparγ. However, the degree of osteogenic or adipogenic differentiation among the 3 kinds of cMSC was different and particularly, cA-MSC had enhanced cytochemical staining associated with expression of specific genes, Runx2 and Pparγ. Ploidy analysis showed that the diploid rate was high with over 90% in all cMSC and indicated no noticeable chromosomal abnormalities. Further, less than 52% of cells were found at G1 phase in all cMSC, with lowest percentage observed in cDS-MSC (33.3%). Regardless of varied tissue sources, cMSC from a single donor showed no differences in telomere lengths (∼18–19 kbp), but the telomerase activity was different with significantly higher levels found in cBM-MSC. In conclusion, the above results suggest that tissue specific cMSC derived from a single donor possess differences in stem cell capacity and support the consideration of tissue source before judging the suitability of cells for therapeutic applications. This work was supported by grant from Basic Science Research Program through NRF funded by the Ministry of Education, Science and Technology (2009-0064229).

scholarly journals Silver nanoparticles induce the cardiomyogenic differentiation of bone marrow derived mesenchymal stem cells via telomere length extension

2021 ◽  
Vol 12 ◽  
pp. 786-797
Author(s):  
Khosro Adibkia ◽  
Ali Ehsani ◽  
Asma Jodaei ◽  
Ezzatollah Fathi ◽  
Raheleh Farahzadi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Silver Nanoparticles ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cardiac Markers ◽  
Ag Nps ◽  
Cardiomyogenic Differentiation

Finding new strategies for the treatment of heart failures using stem cells has attracted a lot of attention. Meanwhile, nanotechnology-based approaches to regenerative medicine hypothesize a possible combination of stem cells and nanotechnology in the treatment of diseases. This study aims to investigate the in vitro effect of silver nanoparticles (Ag-NPs) on the cardiomyogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) through detection of cardiac markers. For this purpose, MSCs were isolated from bone marrow resident and differentiated to the cardiac cells using a dedicated medium with Ag-NPs. Also, the cardiomyogenic differentiation of BM-MSCs was confirmed using immunocytochemistry. Then, real-time PCR and western blotting assay were used for measuring absolute telomere length (TL) measurement, and gene and protein assessment of the cells, respectively. It was found that 2.5 µg/mL Ag-NPs caused elongation of the telomeres and altered VEGF, C-TnI, VWF, SMA, GATA-4, TERT, and cyclin D protein and gene expression in the cardiomyogenically differentiated BM-MSCs. Also, there was a significant increase in the protein and gene expression of Wnt3 and β-catenin as main components of pathways. We concluded that Ag-NPs could change the in vitro expression of cardiac markers of BM-MSCs via the Wnt3/β-catenin signaling pathway.

Bromodomain Inhibition By OTX015 Regulates c-MYC and HEXIM1 in a Panel of Human Acute Leukemia Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5957-5957
Author(s):  
Marie-Magdelaine Coudé ◽  
Thorsten Braun ◽  
Jeannig Berrou ◽  
Mélanie Dupont ◽  
Raphael Itzykson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Mrna Expression ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Antileukemic Activity ◽  
Protein Levels ◽  
Gene And Protein Expression

Abstract Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models. Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software. Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells. Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. Figure 1: Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Figure 1:. Heatmap of gene expression after exposure to 500 nM OTX015 for 24 or 48h in AML and ALL cell lines. Repression in blue. Overexpression in red. Disclosures Riveiro: OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes

2004 ◽  
Vol 82 (1) ◽  
pp. 49-55 ◽  
Author(s):  
J�rg Fellenberg ◽  
Tim H. Br�mmendorf ◽  
Anna-Maria Eschlbeck ◽  
Wiltrud Richter ◽  
Dominik Parsch
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Human Mesenchymal Stem Cells ◽  
Telomerase Activity

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.

scholarly journals ID: 1051 Telomerase activity, telomere length and P53 mutation detection on cellular senescence of Human Amnion Mesenchymal Stem Cells (HAMCs)

2017 ◽  
Vol 4 (S) ◽  
pp. 131
Author(s):  
Fiona Macniesia Thomas ◽  
Vijay Kumar ◽  
Siti Fatimah Simat ◽  
Helen Benedict Lasimbang
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Telomere Length ◽  
Cellular Senescence ◽  
Mutation Detection ◽  
Telomerase Activity ◽  
P53 Mutation ◽  
Human Amnion ◽  
Long Term Culture

A fundamental understanding of senescence in human amnion mesenchymal stem cells (HAMCs) is crucial for its application in cellular therapy. Previous findings strongly support that HAMCs undergoes cellular senescence after long term in-vitro culture, with evidence of significant morphological changes and the presence of the senescent associated β-galactosidase (SA-β-Gal) marker. The telomere length and the telomerase activity have been linked with cellular aging and they are important in regulating cell proliferation. In addition, p53 gene has been associated with cell senescence. The aim of this study was to investigate the telomerase activity, telomere length in senescent HAMCs, and to detect p53 mutations in these cells. Samples were obtained from amnion placenta and then cultured for long term. Prolong-cultured HAMCs was isolated at passages 5, 10 and 15 and then analysed via telomeric repeat amplification protocol (TRAP), telomere length assay and p53 mutation detection assay. The results showed that after long term culture of HAMCs, there was a decrease in telomere length and telomerase activity from passages 5, 10 to 15. Telomerase controls the telomere’s length which maintains the cells proliferation. The decrease of telomere length and telomerase activity may suggest that the proliferation of HAMCs has slowed down due to HAMCs entering senescence after long term culture. P53 mutation detection study indicated that HAMCs at all passage did not have altered sequences. Thus, the cells did not undergo uncontrollable replication due to the effect of long-term culture. Further studies on senescence in HAMCs will be assessed by investigating the expression level of p53, p21, p16, pRB and GADD45 genes in long term culture of HAMCs via RT-qPCR. The findings will help us understand the associations between gene expressions and the process of senescence

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