Telomerase reverse transcriptase mediated immortalization of human bone marrow stromal cells

Directory of Open Access Journals (Sweden)

Yong Teng

2014-02-01

Full Text Available Primary human bone marrow stromal cells (hMSCs were transfected with human telomerase reverse transcriptase (hTERT gene with lipofection method. The hTERT transfected hMSCs of passage 100 underwent chondrogenesis induction with dexamethasone, transforming the growth factor β and vitamin C, osteogenesis induction with dexamethasone, β glycerophosphoric acid and vitamin C, and cardiomyocyte induction with 5-azacytidine. After 7, 14, 21 and 28 days of induction, immunocytochemistry was performed to detect the expressions of type I and II collagen and osteocalcin, and alizarin red staining was performed to detect the bone nodule formation in osteogenesis induction. Immunocytochemistry was carried out to detect the striated muscle actin expression in cardiomyocytes. The hMSCs undergoing successful transfection were positive for the hTERT. The hTERT transfected cells were grown in vitro successfully and passaged for 136 generations. Results showed that these cells could be induced to differentiate into chondrocytes, bone and myocardial cells. Introduction of exogenous hTERT into hMSCs could achieve immortalized hMSCs with the potential of multi-directional differentiation. Thus, these cells could be applied as seed cells in tissue engineering.

A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

Directory of Open Access Journals (Sweden)

Brittney-Shea Herbert

Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.

Effects of high glucose on mesenchymal stem cell proliferation and differentiation

DEFF Research Database (Denmark)

Li, Yu-Ming; Schilling, Tatjana; Benisch, Peggy

2007-01-01

High glucose (HG) concentrations impair cellular functions and induce apoptosis. Exposition of mesenchymal stem cells (MSC) to HG was reported to reduce colony forming activity and induce premature senescence. We characterized the effects of HG on human MSC in vitro using telomerase-immortalized...

Tumorigenic Heterogeneity in Cancer Stem Cells Evolved from Long-term Cultures of Telomerase-Immortalized

DEFF Research Database (Denmark)

Burns, Jorge S; Abdallah, Basem M; Guldberg, Per

2005-01-01

Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation or if...

Evidence for a relief of repression mechanism for activation of the human telomerase reverse transcriptase promoter.

Science.gov (United States)

Wang, Shuwen; Zhu, Jiyue

2003-05-23

The transcriptional activation of human telomerase reverse transcriptase (hTERT) is an important step during cellular immortalization and tumorigenesis. To study how this activation occurs during immortalization, we have established a set of genetically related pre-crisis cells and their immortal progeny. As expected, hTERT mRNA was detected in our telomerase-positive immortal cells but not in pre-crisis cells or telomerase-negative immortal cells. However, transiently transfected luciferase reporters controlled by hTERT promoter sequences exhibited similar levels of luciferase activity in both telomerase-positive and -negative cells, suggesting that the endogenous chromatin context is likely required for hTERT regulation. Analysis of chromatin susceptibility to DNase I digestion consistently identified a DNase I hypersensitivity site (DHS) near the hTERT transcription initiation site in telomerase-positive cells. In addition, the histone deacetylase inhibitor trichostatin A (TSA) induced hTERT transcription and also a general increase in chromatin sensitivity to DNase treatment in telomerase-negative cells. The TSA-induced hTERT transcription in pre-crisis cells was accompanied by the formation of a DHS at the hTERT promoter. Furthermore, the TSA-induced hTERT transcription and chromatin alterations were not blocked by cycloheximide, suggesting that this induction does not require de novo protein synthesis and that TSA induces hTERT expression through the inhibition of histone deacetylation at the hTERT promoter. Taken together, our results suggest that the endogenous chromatin environment plays a critical role in the regulation of hTERT expression during cellular immortalization.

Isolation of a candidate human telomerase catalytic subunit gene, which reveals complex splicing patterns in different cell types.

Science.gov (United States)

Kilian, A; Bowtell, D D; Abud, H E; Hime, G R; Venter, D J; Keese, P K; Duncan, E L; Reddel, R R; Jefferson, R A

1997-11-01

Telomerase is a multicomponent reverse transcriptase enzyme that adds DNA repeats to the ends of chromosomes using its RNA component as a template for synthesis. Telomerase activity is detected in the germline as well as the majority of tumors and immortal cell lines, and at low levels in several types of normal cells. We have cloned a human gene homologous to a protein from Saccharomyces cerevisiae and Euplotes aediculatus that has reverse transcriptase motifs and is thought to be the catalytic subunit of telomerase in those species. This gene is present in the human genome as a single copy sequence with a dominant transcript of approximately 4 kb in a human colon cancer cell line, LIM1215. The cDNA sequence was determined using clones from a LIM1215 cDNA library and by RT-PCR, cRACE and 3'RACE on mRNA from the same source. We show that the gene is expressed in several normal tissues, telomerase-positive post-crisis (immortal) cell lines and various tumors but is not expressed in the majority of normal tissues analyzed, pre-crisis (non-immortal) cells and telomerase-negative immortal (ALT) cell lines. Multiple products were identified by RT-PCR using primers within the reverse transcriptase domain. Sequencing of these products suggests that they arise by alternative splicing. Strikingly, various tumors, cell lines and even normal tissues (colonic crypt and testis) showed considerable differences in the splicing patterns. Alternative splicing of the telomerase catalytic subunit transcript may be important for the regulation of telomerase activity and may give rise to proteins with different biochemical functions.

Telomere stability and telomerase in mesenchymal stem cells

DEFF Research Database (Denmark)

Serakinci, Nedime; Graakjaer, Jesper; Kølvrå, Steen

2008-01-01

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing...... embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important....... In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall...

Prevalence of Telomerase Activity in Human Cancer

Directory of Open Access Journals (Sweden)

Chi-Hau Chen

2011-05-01

Full Text Available Telomerase activity has been measured in a wide variety of cancerous and non-cancerous tissue types, and the vast majority of clinical studies have shown a direct correlation between it and the presence of cancerous cells. Telomerase plays a key role in cellular immortality and tumorigenesis. Telomerase is activated in 80–90% of human carcinomas, but not in normal somatic cells, therefore, its detection holds promise as a diagnostic marker for cancer. Measurable levels of telomerase have been detected in malignant cells from various samples: tissue from gestational trophoblastic neoplasms; squamous carcinoma cells from oral rinses; lung carcinoma cells from bronchial washings; colorectal carcinoma cells from colonic luminal washings; bladder carcinoma cells from urine or bladder washings; and breast carcinoma or thyroid cancer cells from fine needle aspirations. Such clinical tests for telomerase can be useful as non-invasive and cost-effective methods for early detection and monitoring of cancer. In addition, telomerase activity has been shown to correlate with poor clinical outcome in late-stage diseases such as non-small cell lung cancer, colorectal cancer, and soft tissue sarcomas. In such cases, testing for telomerase activity can be used to identify patients with a poor prognosis and to select those who might benefit from adjuvant treatment. Our review of the latest medical advances in this field reveals that telomerase holds great promise as a biomarker for early cancer detection and monitoring, and has considerable potential as the basis for developing new anticancer therapies.

Inefficiency in macromolecular transport of SCS-based microcapsules affects viability of primary human mesenchymal stem cells but not of immortalized cells

DEFF Research Database (Denmark)

Sanz-Nogués, Clara; Horan, Jason; Thompson, Kerry

2015-01-01

mesenchymal stem cells (hMSCs). Human MSCs are of interest in regenerative medicine applications due to pro-angiogenic, anti-inflammatory and immunomodulatory properties, which result from paracrine effects of this cell type. In the present work we have encapsulated primary hMSCs and hMSC-TERT immortalized...... nutrients and had a more detrimental effect on the viability of primary cell cultures compared to cell lines and immortalized cells. This article is protected by copyright. All rights reserved....

Telomerase activity in gastric cancer.

Science.gov (United States)

Hiyama, E; Yokoyama, T; Tatsumoto, N; Hiyama, K; Imamura, Y; Murakami, Y; Kodama, T; Piatyszek, M A; Shay, J W; Matsuura, Y

1995-08-01

Although many genetic alterations have been reported in gastric cancer, it is not known whether all gastric tumors are capable of indefinite proliferative potential, e.g., immortality. The expression of telomerase and stabilization of telomeres are concomitant with the attainment of immortality in tumor cells; thus, the measurement of telomerase activity in clinically obtained tumor samples may provide important information useful both as a diagnostic marker to detect immortal cancer cells in clinical materials and as a prognostic indicator of patient outcome. Telomerase activity was analyzed in 66 primary gastric cancers with the use of a PCR-based assay. The majority of tumors (85%) displayed telomerase activity, but telomerase was undetectable in 10 tumors (15%), 8 of which were early stage tumors. Most of the tumors with telomerase activity were large and of advanced stages, including metastases. Survival rate of patients of tumors with detectable telomerase activity was significantly shorter than that of those without telomerase activity. Alterations of telomere length (reduced/elongated terminal restriction fragments) were detected in 14 of 66 (21%) gastric cancers, and all 14 had telomerase activity. Cellular DNA contents revealed that all 22 aneuploid tumors had detectable telomerase activity. The present results indicate that telomerase activation may be required as a critical step in the multigenetic process of tumorigenesis, and that telomerase is frequently but not always activated as a late event in gastric cancer progression.

Possible involvement of loss of imprinting in immortalization of human fibroblasts.

Science.gov (United States)

Okamura, Kotaro; Ohno, Maki; Tsutsui, Takeki

2011-04-01

Disruption of the normal pattern of parental origin-specific gene expression is referred to as loss of imprinting (LOI), which is common in various cancers. To investigate a possible role of LOI in the early stage of human cell transformation, we studied LOI in 18 human fibroblast cell lines immortalized spontaneously, by viral oncogenes, by chemical or physical carcinogens, or by infection with a retrovirus vector encoding the human telomerase catalytic subunit, hTERT cDNA. LOI was observed in all the 18 immortal cell lines. The gene most commonly exhibiting LOI was NDN which displayed LOI in 15 of the 18 cell lines (83%). The other genes exhibiting LOI at high frequencies were PEG3 (50%), MAGE-L2 (61%) and ZNF 127 (50%). Expression of NDN that was lost in the immortal cell lines was restored by treatment with 5-aza-2'-deoxycytidine. The ratio of histone H3 lysine 9 methylation to histone H3 lysine 4 methylation of the chromatin containing the NDN promoter in the immortal WI-38VA13 cells was greater than that in the parental cells, suggesting chromatin structure-mediated regulation of NDN expression. We previously demonstrated that inactivation of the p16INK4a/pRb pathway is necessary for immortalization of human cells. Human fibroblasts in the pre-crisis phase and cells with an extended lifespan that eventually senesce, both of which have the normal p16INK4a/pRb pathway, did not show LOI at any imprinted gene examined. Although it is not clear if LOI plays a causal role in immortalization of human cells or is merely coincidental, these findings indicate a possible involvement of LOI in immortalization of human cells or a common mechanism involved in both processes.

Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b

Directory of Open Access Journals (Sweden)

Brosens Jan J

2009-07-01

Full Text Available Abstract Background Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. Methods Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. Results St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82. Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. Conclusion St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural

Long-term culture and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized mesenchymal cells.

Science.gov (United States)

Garba, Abubakar; Acar, Delphine D; Roukaerts, Inge D M; Desmarets, Lowiese M B; Devriendt, Bert; Nauwynck, Hans J

2017-09-01

Mesenchymal cells are multipotent stromal cells with self-renewal, differentiation and immunomodulatory capabilities. We aimed to develop a co-culture model for differentiating hematopoietic cells on top of immortalized mesenchymal cells for studying interactions between hematopoietic and mesenchymal cells, useful for adequately exploring the therapeutic potential of mesenchymal cells. In this study, we investigated the survival, proliferation and differentiation of porcine red bone marrow hematopoietic cells co-cultured with immortalized porcine bone marrow mesenchymal cells for a period of five weeks. Directly after collection, primary porcine bone marrow mesenchymal cells adhered firmly to the bottom of the culture plates and showed a fibroblast-like appearance, one week after isolation. Upon immortalization, porcine bone marrow mesenchymal cells were continuously proliferating. They were positive for simian virus 40 (SV40) large T antigen and the mesenchymal cell markers CD44 and CD55. Isolated red bone marrow cells were added to these immortalized mesenchymal cells. Five weeks post-seeding, 92±6% of the red bone marrow hematopoietic cells were still alive and their number increased 3-fold during five weekly subpassages on top of the immortalized mesenchymal cells. The red bone marrow hematopoietic cells were originally small and round; later, the cells increased in size. Some of them became elongated, while others remained round. Tiny dendrites appeared attaching hematopoietic cells to the underlying immortalized mesenchymal cells. Furthermore, weekly differential-quick staining of the cells indicated the presence of monoblasts, monocytes, macrophages and lymphocytes in the co-cultures. At three weeks of co-culture, flow cytometry analysis showed an increased surface expression of CD172a, CD14, CD163, CD169, CD4 and CD8 up to 37±0.8%, 40±8%, 41±4%, 23±3% and 19±5% of the hematopoietic cells, respectively. In conclusion, continuous mesenchymal cell

Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells

Directory of Open Access Journals (Sweden)

Larivee Siobhan

2006-05-01

Full Text Available Abstract Introduction Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras. Methods HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT. The immortalized cells were passaged in vitro and studied by a combination of G- banding and Spectral Karyotyping (SKY. H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting. Results Immortal HMEC alone were not tumorigenic in γ-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor. Conclusion Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.

The use of hTERT-immortalized cells in tissue engineering

DEFF Research Database (Denmark)

Kassem, Moustapha; Abdallah, Basem; Yu, Zentao

2004-01-01

The use of human telomerase reverse transcriptase (hTERT)-immortalized cells in tissue engineering protocols is a potentially important application of telomere biology. Several human cell types have been created that overexpress the hTERT gene with enhanced telomerase activity, extended life span...... and maintained or even improved functional activities. Furthermore, some studies have employed the telomerized cells in tissue engineering protocols with very good results. However, high telomerase activity allows extensive cell proliferation that may be associated with genomic instability and risk for cell...... transformation. Thus, safety issues should be studied carefully before using the telomerized tissues in the clinic. Alternatively, the development of conditional or intermittent telomerase activation protocols is needed....

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT.

Science.gov (United States)

Haga, Kei; Ohno, Shin-ichi; Yugawa, Takashi; Narisawa-Saito, Mako; Fujita, Masatoshi; Sakamoto, Michiie; Galloway, Denise A; Kiyono, Tohru

2007-02-01

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

International Nuclear Information System (INIS)

Li Hongzhen; Zhou Jianjun; Miki, Jun; Furusato, Bungo; Gu Yongpeng; Srivastava, Shiv; McLeod, David G.; Vogel, Jonathan C.; Rhim, Johng S.

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin α2β1 hi and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 μg/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation

Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation

DEFF Research Database (Denmark)

Abdallah, Basem M; Haack-Sørensen, Mandana; Burns, Jorge S

2005-01-01

Human bone marrow mesenchymal stem cells (hMSC) represent a population of stem cells that are capable of differentiation into multiple lineages. However, these cells exhibit senescence-associated growth arrest and phenotypic changes during long-term in vitro culture. We have recently demonstrated...

Genes involved in immortalization of human mammary cells

Energy Technology Data Exchange (ETDEWEB)

Stampfer, Martha R.; Yaswen, Paul

2001-09-27

Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions--a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). Inappropriate expression of specific oncogenes in a key cellular signaling pathway (Ras, Raf) can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence-like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to overexpressed oncogenic Raf changes from being tumor-suppressive to tumor enhancing, and what are the molecular underpinnings of this response. Our data indicate: (1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of immortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited. (2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor-suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response. Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor-suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means of enforcing a Raf

Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines.

Directory of Open Access Journals (Sweden)

Bao G Vu

Full Text Available BACKGROUND: Human adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin. METHODOLOGY/PRINCIPAL FINDINGS: Primary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8. CONCLUSIONS/SIGNIFICANCE: Endotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to

Reconstitution of active telomerase in primary human foreskin fibroblasts : effects on proliferative characteristics and response to ionizing radiation

NARCIS (Netherlands)

Kampinga, H.H.; Waarde-Verhagen, M.A.W.H. van; Assen-Bolt, A.J. van; Rodemann, H.P.; Prowse, K.R.; Linskens, M.H.K.

2004-01-01

Purpose: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear

Immortalization of canine adipose-derived mesenchymal stem cells and their seminiferous tubule transplantation.

Science.gov (United States)

Fang, Jia; Wei, Yudong; Teng, Xin; Zhao, Shanting; Hua, Jinlian

2018-04-01

Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs. © 2017 Wiley Periodicals, Inc.

Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells.

Science.gov (United States)

Lai, Ruenn Chai; Yeo, Ronne Wee Yeh; Padmanabhan, Jayanthi; Choo, Andre; de Kleijn, Dominique P V; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.

Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.

Science.gov (United States)

Armstrong, Christine A; Tomita, Kazunori

2017-03-01

Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments. © 2017 The Authors.

In Situ Synthesized Silver Nanoclusters for Tracking the Role of Telomerase Activity in the Differentiation of Mesenchymal Stem Cells to Neural Stem Cells.

Science.gov (United States)

Dong, Fangyuan; Feng, Enduo; Zheng, Tingting; Tian, Yang

2018-01-17

Human mesenchymal stem cells (hMSCs) have potential use in cell replacement therapy for central nervous system disorders. However, the factors that impacted the differentiation process are unclear at the present stage because the powerful analytical method is the bottleneck. Herein, a novel strategy was developed for self-imaging and biosensing of telomerase activity in stem cells, using in situ biosynthesized silver nanoclusters (AgNCs) full of C bases. The present AgNCs possess synthetic convenience, long-time stability, and cytocompatibility. The weak fluorescence of these AgNCs is quickly turned on when approaching telomerase because of the strong interaction between C bases on AgNCs and G bases in telomerase, resulting in telomerase-dependent fluorescent signals. The developed method demonstrated high sensitivity and selectivity and broad dynamic linear range with a low detection limit. Using this powerful tool, it was first discovered that telomerase activity plays important roles in the proliferation of hMSCs and neural stem cells (NSCs) as well as during the differentiation processes from hMSCs to NSCs.

Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells.

Directory of Open Access Journals (Sweden)

Raheleh Farahzadi

Full Text Available The use of mesenchymal stem cells (MSCs for cell therapy and regenerative medicine has received widespread attention over the past few years, but their application can be complicated by factors such as reduction in proliferation potential, the senescent tendency of the MSCs upon expansion and their age-dependent decline in number and function. It was shown that all the mentioned features were accompanied by a reduction in telomerase activity and telomere shortening. Furthermore, the role of epigenetic changes in aging, especially changes in promoter methylation, was reported. In this study, MSCs were isolated from the adipose tissue with enzymatic digestion. In addition, immunocytochemistry staining and flow cytometric analysis were performed to investigate the cell-surface markers. In addition, alizarin red-S, sudan III, toluidine blue, and cresyl violet staining were performed to evaluate the multi-lineage differentiation of hADSCs. In order to improve the effective application of MSCs, these cells were treated with 1.5 × 10-8 and 2.99 × 10-10 M of ZnSO4 for 48 hours. The length of the absolute telomere, human telomerase reverse transcriptase (hTERT gene expression, telomerase activity, the investigation of methylation status of the hTERT gene promoter and the percentage of senescent cells were analyzed with quantitative real-time PCR, PCR-ELISA TRAP assay, methylation specific PCR (MSP, and beta-galactosidase (SA-β-gal staining, respectively. The results showed that the telomere length, the hTERT gene expression, and the telomerase activity had significantly increased. In addition, the percentage of senescent cells had significantly decreased and changes in the methylation status of the CpG islands in the hTERT promoter region under treatment with ZnSO4 were seen. In conclusion, it seems that ZnSO4 as a proper antioxidant could improve the aging-related features due to lengthening of the telomeres, increasing the telomerase gene expression

Global gene expression response to telomerase in bovine adrenocortical cells

International Nuclear Information System (INIS)

Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H.

2005-01-01

The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

Directory of Open Access Journals (Sweden)

Gina D Kusuma

Full Text Available Mesenchymal stem/stromal cells (MSCs exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25 human telomerase reverse transcriptase (hTERT transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs and decidua basalis (DMSCs, respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

Science.gov (United States)

Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

2003-09-01

The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for

Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

Directory of Open Access Journals (Sweden)

El-Ayachi Ikbale

2016-03-01

Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown. Keywords: Stem cells, Osteogenic, Adipogenic, Immortalized, hTERT, DPSC

Aneuploidy in immortalized human mesenchymal stem cells with non-random loss of chromosome 13 in culture.

Science.gov (United States)

Takeuchi, Masao; Takeuchi, Kikuko; Ozawa, Yutaka; Kohara, Akihiro; Mizusawa, Hiroshi

2009-01-01

Aneuploidy (an abnormal number of chromosomes) is commonly observed in most human cancer cells, highlighting the need to examine chromosomal instability in tumorigenesis. Previously, the immortalized human mesenchymal stem cell line UE6E7T-3 was shown to undergo a preferential loss of one copy of chromosome 13 after prolonged culture. Here, the loss of chromosome 13 was found to be caused by chromosome missegregation during mitosis, which involved unequal segregation, exclusion of the misaligned chromosome 13 on the metaphase plate, and trapping of chromosome 13 in the midbody region, as observed by fluorescence in situ hybridization. Near-diploid aneuploidy, not tetraploidy, was the direct result. The loss of chromosome 13 was non-random, and was detected by analysis of microsatellites and single nucleotide polymorphism-based loss of heterozygosity (LOH). Of the five microsatellite loci on chromosome 13, four loci showed microsatellite instability at an early stage in culture, and LOH was apparent at a late stage in culture. These results suggest that the microsatellite mutations cause changes in centromere integrity provoking loss of this chromosome in the UE6E7T-3 cell line. Thus, these results support the use of this cell line as a useful model for understanding the mechanism of aneuploid formation in cell cultures.

Current Perspectives of Telomerase Structure and Function in Eukaryotes with Emerging Views on Telomerase in Human Parasites.

Science.gov (United States)

Dey, Abhishek; Chakrabarti, Kausik

2018-01-24

Replicative capacity of a cell is strongly correlated with telomere length regulation. Aberrant lengthening or reduction in the length of telomeres can lead to health anomalies, such as cancer or premature aging. Telomerase is a master regulator for maintaining replicative potential in most eukaryotic cells. It does so by controlling telomere length at chromosome ends. Akin to cancer cells, most single-cell eukaryotic pathogens are highly proliferative and require persistent telomerase activity to maintain constant length of telomere and propagation within their host. Although telomerase is key to unlimited cellular proliferation in both cases, not much was known about the role of telomerase in human parasites (malaria, Trypanosoma , etc.) until recently. Since telomerase regulation is mediated via its own structural components, interactions with catalytic reverse transcriptase and several factors that can recruit and assemble telomerase to telomeres in a cell cycle-dependent manner, we compare and discuss here recent findings in telomerase biology in cancer, aging and parasitic diseases to give a broader perspective of telomerase function in human diseases.

Re: Role of Telomeres and Telomerase in Cancer

Directory of Open Access Journals (Sweden)

Shay JW

2016-03-01

Full Text Available The most important difference between cancer and normall cells is the ability to continuous proliferation. This activation works due to telomeres and telomerase enzyme. Fifty years ago, Leonard Hayflick discovered that cultured normal humans cells have a limited capacity to divide. Today, this withdrawal from the cell cycle after a certain number of cellular divisions (replicative senescence is known to be triggered as a result of shortened telomeres. Studies on telomeres and telomerase have begun to provide additional information about aging and cancer development and have created new opportunities in the field of regenerative medicine for telomeropathies. Progressive telomere shortening from cell division (replicative aging provides a barrier for tumor progression. Continuous cell growth in malignancy correlates with the reactivation of telomerase. Telomerase is a cellular reverse transcriptase that adds new deoxyribonucleic acid (DNA onto the telomeres that are located at the ends of chromosomes. Telomeres consist of many kilobases of TTAGGG nucleotide repeats. The telomeric nucleotide repeats shorten with each cell division due to replication problems (DNA repair and oxidative damage. Quiescent/senescent state of the cell bypass can be accomplished by abrogating cell cycle checkpoint genes (such as TP53, p16INK4a, pRb. Telomerase is detected in approximately 90% of all malignant tumors. This telomerase activation has emerged as a target for cancer treatment. Telomerase therapeutics are classified as gene therapy (hTERT-telomerase catalytic protein component, hTR-telomerase functional, immunotherapy (Imetalstat-telomerase template antagonist, and small molecule inhibitors. In the near future, more specific researches on telomers and telomerase will contribute to aging/immortality studies (as stem cells and to discover new biomarkers for malignant tissue or anticancer therapeutics.

hTERT gene immortalized human adipose-derived stem cells and its multiple differentiations: a preliminary investigation.

Science.gov (United States)

Wang, L; Song, K; Qu, X; Wang, H; Zhu, H; Xu, X; Zhang, M; Tang, Y; Yang, X

2013-03-01

Human adipose-derived adult stem cells (hADSCs) can express human telomerase reverse transcriptase phenotypes under an appropriate culture condition. Because adipose tissue is abundant and easily accessible, hADSCs offer a promising source of stem cells for tissue engineering application and other cell-based therapies. However, the shortage of cells number and the difficulty to proliferate, known as the "Hayflick limit" in vitro, limit their further clinical application. Here, hADSCs were transfected with human telomerase reverse transcriptase (hTERT) gene by the lentiviral vector to prolong the lifespan of stem cells and even immortalize them. Following to this, the cellular properties and functionalities of the transfected cell lines were assayed. The results demonstrated that hADSCs had been successfully transfected with hTERT gene (hTERT-ADSCs). Then, hTERT-ADSCs were initially selected by G418 and subsequently expanded over 20 passages in vitro. Moreover, the qualitative and quantitative differentiation criteria for 20 passages of hTERT-ADSCs also demonstrated that hTERT-ADSCs could differentiate into osteogenesis, chondrogenesis, and adipogenesis phenotypes in lineage-specific differentiation media. These findings confirmed that this transfection could prolong the lifespan of hADSCs.

Telomerase as a potential anticancer target: growth inhibition and genomic instability.

Science.gov (United States)

Faraoni, Isabella; Graziani, Grazia

2000-02-01

Stabilization of telomere length in chromosomes by an RNA-dependent DNA polymerase (telomerase) appears to be responsible for the replicative immortality of cancer cells. These findings provide the rational basis for generating experimental models to develop anti-telomerase drugs. However, there is conflicting evidence in the literature about the outcome of telomerase inhibition. While tumor cytostatic and cytotoxic effects associated with telomerase inhibition have been described, absence of telomerase has been associated with genetic instability and tumor development. Therefore, a therapeutic strategy based on telomerase inhibition will likely have to cope with problems related to innate or acquired mechanisms of drug resistance and possibly to therapy-related tumors. Copyright 2000 Harcourt Publishers Ltd.

Demonstration of constant upregulation of the telomerase RNA component in human gastric carcinomas using in situ hybridization.

Science.gov (United States)

Heine, B; Hummel, M; Demel, G; Stein, H

1998-06-01

Upregulation of the ribonucleoprotein telomerase seems to be a prerequisite for immortality, a feature of malignant cells. Using a polymerase chain reaction (PCR)-based assay, it is possible to demonstrate telomerase activity (TA) in specimens of most human malignancies, whereas it is absent from most normal tissues. It remains unclear, however, why between 5 and 50 per cent of various malignant tumour samples give negative results when TA is measured by the telomeric repeat amplification protocol (TRAP). The expectation that reverse transcription (RT)-PCR for detection of the telomerase RNA component (hTR) would be able to complement or to replace the TRAP assay failed, since malignant as well as non-malignant tissue samples gave positive results in most instances. In the present study, in situ hybridization (ISH) was developed to demonstrate the RNA component of human telomerase at the single cell level. With this method, 13 specimens of fresh frozen gastric carcinoma and four of normal, dysplastic, or inflamed gastric mucosa were investigated and the results were compared with those obtained by RT-PCR and the TRAP assay. In addition, ISH was performed on formalin-fixed sections of the same cases. The TRAP assay revealed positive results in 8 out of 13 gastric carcinomas and was negative in all non-malignant tissues. RT-PCR led to amplification of the telomerase RNA component in all specimens tested, irrespective of the presence or absence of malignant cells. By ISH, all gastric carcinomas showed strong telomerase RNA component-specific signals over malignant cells, whereas only a few grains were detectable over some types of normal somatic cells, including activated lymphocytes. In conclusion, high expression of the telomerase RNA component was restricted to the malignant cells of all the gastric carcinomas investigated, as shown by ISH. This indicates that the absence of TA in a proportion of carcinomas is due to methodological problems of the TRAP assay and is

Direct induction of hepatocyte-like cells from immortalized human bone marrow mesenchymal stem cells by overexpression of HNF4α

International Nuclear Information System (INIS)

Hu, Xiaojun; Xie, Peiyi; Li, Weiqiang; Li, Zhengran; Shan, Hong

2016-01-01

Hepatocytes from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are expected to be a useful source for cell transplantation. However, relatively low efficiency and repeatability of hepatic differentiation of human BM-MSCs remains an obstacle for clinical translation. Hepatocyte nuclear factor 4 alpha (HNF4α), a critical transcription factor, plays an essential role in the entire process of liver development. In this study, immortalized hBM-MSCs, UE7T-13 cells were transduced with a lentiviral vector containing HNF4α. The typical fibroblast-like morphology of the MSCs changed, and polygonal, epithelioid cells grew out after HNF4α transduction. In hepatocyte culture medium, HNF4α-transduced MSCs (E7-hHNF4α cells) strongly expressed the albumin (ALB), CYP2B6, alpha-1 antitrypsin (AAT), and FOXA2 mRNA and exhibited morphology markedly similar to that of mature hepatocytes. The E7-hHNF4α cells showed hepatic functions such as Indocyanine green (ICG) uptake and release, glycogen storage, urea production and ALB secretion. Approximately 28% of E7-hHNF4α cells expressed both ALB and AAT. Furthermore, these E7-hHNF4α cells via superior mesenteric vein (SMV) injection expressed human ALB in mouse chronic injured liver. In conclusion, this study represents a novel strategy by directly inducing hepatocyte-like cells from MSCs. - Highlights: • We overexpressed HNF4α in immortalized BM-MSCs by lentiviral transduction. • HNF4α-transduced MSCs transdifferentiated into hepatocytes with mature hepatic metabolic functions. • Our study represents a novel strategy by direct induction of hepatocyte-like cells from MSCs.

Detection of telomerase activity using microchip electrophoresis.

Science.gov (United States)

Karasawa, Koji; Arakawa, Hidetoshi

2015-07-01

Telomerase participates in malignant transformation or immortalization of cells and thus has attracted attention as an anticancer drug target and diagnostic tumor marker. The telomeric repeat amplification protocol (TRAP) and improved TRAP methods (TRAP-fluorescence, TRAP-hybridization, etc.) are widely used forms of this telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplification of telomeric repeats by polymerase chain reaction (PCR), making these TRAP methods time consuming and technically demanding. In this study we developed a novel telomerase assay using microchip electrophoresis for rapid and highly sensitive detection of telomerase activity in cancer cells. The mixed gel of 0.8% hydroxypropyl methylcellulose (HPMC) and 0.3% polyethylene oxide (PEO) with SYBR Gold (fluorescent reagent) was used for microchip electrophoresis. As a result, the product amplified by a telomerase-positive cell could be measured in one cell per assay and detected with high reproducibility (CV=0.67%) in the short time of 100s. Copyright © 2015 Elsevier B.V. All rights reserved.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

International Nuclear Information System (INIS)

Tátrai, Péter; Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Buchan, Gyöngyi; Mádi, András; Uher, Ferenc

2012-01-01

Highlights: ► We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. ► hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. ► SV40T introduced along with hTERT abrogates proliferation control and multipotency. ► hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC hTERT , ASC Bmi-1 , ASC Bmi-1+hTERT and ASC SV40T+hTERT were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC Bmi-1 had limited replicative potential, while the rapidly proliferating ASC SV40T+hTERT acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC hTERT and ASC hTERT+Bmi-1 , on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC hTERT also acquired aberrant karyotype and showed signs of transformation after long-term culture. In conclusion, hTERT alone was sufficient to extend the life span of human ASC, but ASC hTERT are prone to transformation during extensive

Telomeres, telomerase and oral cancer (Review).

Science.gov (United States)

Sebastian, Sinto; Grammatica, Luciano; Paradiso, Angelo

2005-12-01

Oral squamous cell carcinoma (oral cancer) and many squamous cell carcinomas of the head and neck arise as a consequence of multiple molecular events induced by the effects of various carcinogens related to tobacco use, environmental factors, and viruses in some instances (e.g., mucosal oncogenic human papillomaviruses), against a background of inheritable resistance or susceptibility. Consequent genetic damage affects many chromosomes and genes, and it is the accumulation of these changes that appears to lead to carcinoma. Telomere maintenance by telomerase or, in its absence, alternative lengthening of telomeres protect this acquired altered genetic information ensuring immortality without losing eukaryotic linear DNA; when this does not occur DNA is lost and end-replication problems arise. Telomerase is reactivated in 80-90% of cancers thus attracting the attention of pathologists and clinicians who have explored its use as a target for anticancer therapy and to develop better diagnostic and prognostic markers. In the last few years, valuable research from various laboratories has provided major insights into telomerase and telomeres leading to their use as diagnostic and prognostic markers in several types of cancer. Moreover, many strategies have emerged which inhibit this complex enzyme for anticancer therapy and are one step ahead of clinical trials. This review explains the basic biology and the clinical implications of telomerase-based diagnosis and prognosis, the prospects for its use in anticancer therapy, and the limitations it presents in the context of oral cancer.

Telomerase and mammalian ageing: a critical appraisal.

Science.gov (United States)

Goyns, M H; Lavery, W L

2000-03-13

The telomeres that occur at the end of chromosomes are maintained by the activity of telomerase and are thought to be important protective factors in maintaining the integrity of chromosomes. It now appears that in vitro replicative senescence, which has been observed in cultured somatic cells, is due to a loss of telomere length in those cells, caused by inactivity of telomerase. This has led to the proposition that telomerase activity is an important determinant in organismal ageing. However, many cells in the body do not proliferate regularly and therefore will not lose telomere length. Cells that do proliferate frequently have now been shown to have active telomerase. Other cells, such as fibroblasts, that do not have telomerase activity but proliferate only occasionally may not reach the Hayflick limit during the lifetime of an animal. There is also no correlation between telomere length and the maximal lifespan exhibited by different species. Studies of telomerase knock-out mice have reported some aspects of accelerated ageing after three generations, but the relevance of these observations to normal ageing remains unconvincing. The role of telomerase in producing immortal tumour cells and the possibility that activation of telomerase is an important event in malignant transformation is similarly controversial and open to alternative interpretations. The significance of these and other observations, and how they define the role of telomerase in ageing, is discussed.

TRAPping telomerase within the intestinal stem cell niche

OpenAIRE

Pech, Matthew F; Artandi, Steven E

2011-01-01

Recent work from Hans Clevers' lab reveals high telomerase activity and telomere length in dividing LGR5-positive intestinal stem cells. They further report random chromosome segregation and thus challenge the ‘immortal strand' hypothesis at least for this stem cell population.

Formation of radiation induced chromosome aberrations: involvement of telomeric sequences and telomerase

International Nuclear Information System (INIS)

Pirzio, L.

2004-07-01

As telomeres are crucial for chromosome integrity; we investigated the role played by telomeric sequences in the formation and in the transmission of radio-induced chromosome rearrangements in human cells. Starting from interstitial telomeric sequences (ITS) as putative region of breakage, we showed that the radiation sensitivity is not equally distributed along chromosomes and. is not affected by ITS. On the contrary, plasmid integration sites are prone to radio-induced breaks, suggesting a possible integration at sites already characterized by fragility. However plasmids do not preferentially insert at radio-induced breaks in human cells immortalized by telomerase. These cells showed remarkable karyotype stability even after irradiation, suggesting a role of telomerase in the genome maintenance despite functional telomeres. Finally, we showed that the presence of more breaks in a cell favors the repair, leading to an increase of transmissible rearrangements. (author)

Generation of human cortical neurons from a new immortal fetal neural stem cell line

International Nuclear Information System (INIS)

Cacci, E.; Villa, A.; Parmar, M.; Cavallaro, M.; Mandahl, N.; Lindvall, O.; Martinez-Serrano, A.; Kokaia, Z.

2007-01-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology

Detection of telomerase activity by the TRAP assay and its variants and alternatives.

Science.gov (United States)

Fajkus, Jirí

2006-09-01

Telomerase activity is closely connected to problems of cellular immortality, proliferative capacity, differentiation, cancer and aging. Correspondingly, techniques for its detection have been essential for progress in telomere biology and are of still increasing importance in molecular diagnostics and therapy of cancer. This article reviews the development of the telomere repeat amplification protocol (TRAP) and its various modifications as the most widespread assay to detect and measure telomerase activity. Alternative possibilities of telomerase activity detection are also discussed which make it possible to omit the PCR-mediated amplification of telomerase products. These approaches are based on recent advances in highly sensitive detection systems.

Elevation of telomerase activity in chronic radiation ulcer of human skin

International Nuclear Information System (INIS)

Li Xiaoying; Zhao Po; Wang Dewen; Yang Zhixiang

1997-01-01

Objective: To investigate the levels of telomerase activity in chronic radiation ulcers of human skin and the possible relationship between the enzyme and cancer transformation. Method: Using nonisotopic telomere repeat amplification protocol (TRAP), detections were performed in 20 cases of chronic radiation ulcers of human skin, 5 cases of normal skin tissues and 5 cases of carcinoma. Results: The positive rates for telomerase activity were 30.0%(6/20), 0(0/5) and 100%(5/5) in chronic radiation ulcers of human skin, normal skin and carcinoma, respectively. The telomerase activity in radiation ulcer was weaker than in carcinoma. Conclusion: The telomerase activity assay might be used as a marker for predicting the prognosis and the effect of treatment in chronic radiation ulcer of human skin

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

Science.gov (United States)

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

Energy Technology Data Exchange (ETDEWEB)

Tatrai, Peter, E-mail: peter.tatrai@biomembrane.hu [Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest (Hungary); Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Szepesi, Aron, E-mail: aron.szepesi@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Matula, Zsolt, E-mail: matula.zsolt@gmail.com [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Szigeti, Anna, E-mail: anna.szigeti@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Buchan, Gyoengyi, E-mail: buchan@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Madi, Andras, E-mail: madi@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Stem Cell, Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Uher, Ferenc, E-mail: uher@biomembrane.hu [Stem Cell Laboratory, Hungarian National Blood Transfusion Service, Dioszegi ut 64, H-1113 Budapest (Hungary); and others

2012-05-25

Highlights: Black-Right-Pointing-Pointer We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. Black-Right-Pointing-Pointer hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. Black-Right-Pointing-Pointer SV40T introduced along with hTERT abrogates proliferation control and multipotency. Black-Right-Pointing-Pointer hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC{sup hTERT}, ASC{sup Bmi-1}, ASC{sup Bmi-1+hTERT} and ASC{sup SV40T+hTERT} were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC{sup Bmi-1} had limited replicative potential, while the rapidly proliferating ASC{sup SV40T+hTERT} acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC{sup hTERT} and ASC{sup hTERT+Bmi-1}, on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC{sup hTERT} also acquired aberrant karyotype and showed signs of transformation after long-term culture

Molecular mechanisms of anti-aging hormetic effects of mild heat stress on human cells

DEFF Research Database (Denmark)

Rattan, Suresh I S; Eskildsen-Helmond, Yvonne E G; Beedholm, Rasmus

2004-01-01

of cellular responsiveness to mild and severe heat stress. Furthermore, we are also undertaking comparative studies using non-aging immortal cell lines, such as SV40-transformed human fibroblasts, spontaneous osteosarcoma cells, and telomerase-immortalized human bone marrow cells for establishing differences...

Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol

International Nuclear Information System (INIS)

Eitsuka, Takahiro; Nakagawa, Kiyotaka; Miyazawa, Teruo

2006-01-01

As high telomerase activity is detected in most cancer cells, inhibition of telomerase by drug or dietary food components is a new strategy for cancer prevention. Here, we investigated the inhibitory effect of vitamin E, with particular emphasis on tocotrienol (unsaturated vitamin E), on human telomerase in cell-culture study. As results, tocotrienol inhibited telomerase activity of DLD-1 human colorectal adenocarcinoma cells in time- and dose-dependent manner, interestingly, with δ-tocotrienol exhibiting the highest inhibitory activity. Tocotrienol inhibited protein kinase C activity, resulting in down-regulation of c-myc and human telomerase reverse transcriptase (hTERT) expression, thereby reducing telomerase activity. In contrast to tocotrienol, tocopherol showed very weak telomerase inhibition. These results provide novel evidence for First time indicating that tocotrienol acts as a potent candidate regulator of telomerase and supporting the anti-proliferative function of tocotrienol

Immortalized porcine mesenchymal cells derived from nasal mucosa, lungs, lymph nodes, spleen and bone marrow retain their stemness properties and trigger the expression of siglec-1 in co-cultured blood monocytic cells.

Science.gov (United States)

Garba, Abubakar; Desmarets, Lowiese M B; Acar, Delphine D; Devriendt, Bert; Nauwynck, Hans J

2017-01-01

Mesenchymal stromal cells have been isolated from different sources. They are multipotent cells capable of differentiating into many different cell types, including osteocytes, chondrocytes and adipocytes. They possess a therapeutic potential in the management of immune disorders and the repair of damaged tissues. Previous work in our laboratory showed an increase of the percentages of CD172a+, CD14+, CD163+, Siglec-1+, CD4+ and CD8+ hematopoietic cells, when co-cultured with immortalized mesenchymal cells derived from bone marrow. The present work aimed to demonstrate the stemness properties of SV40-immortalized mesenchymal cells derived from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow and their immunomodulatory effect on blood monocytes. Mesenchymal cells from nasal mucosa, lungs, spleen, lymph nodes and red bone marrow were isolated and successfully immortalized using simian virus 40 large T antigen (SV40LT) and later, co-cultured with blood monocytes, in order to examine their differentiation stage (expression of Siglec-1). Flow cytometric analysis revealed that the five mesenchymal cell lines were positive for mesenchymal cell markers CD105, CD44, CD90 and CD29, but lacked the expression of myeloid cell markers CD16 and CD11b. Growth analysis of the cells demonstrated that bone marrow derived-mesenchymal cells proliferated faster compared with those derived from the other tissues. All five mesenchymal cell lines co-cultured with blood monocytes for 1, 2 and 7 days triggered the expression of siglec-1 in the monocytes. In contrast, no siglec-1+ cells were observed in monocyte cultures without mesenchymal cell lines. Mesenchymal cells isolated from nasal mucosa, lungs, spleen, lymph nodes and bone marrow were successfully immortalized and these cell lines retained their stemness properties and displayed immunomodulatory effects on blood monocytes.

Correlation between telomerase and mTOR pathway in cancer stem cells.

Science.gov (United States)

Dogan, Fatma; Biray Avci, Cigir

2018-01-30

Cancer stem cells (CSCs), which are defined as a subset of tumor cells, are able to self-renew, proliferate, differentiate similar to normal stem cells. Therefore, targeting CSCs has been considered as a new approach in cancer therapy. The mammalian target of rapamycin (mTOR) is a receptor tyrosine kinase which plays an important role in regulating cell proliferation, differentiation, cell growth, self-renewal in CSCs. On the other hand, hTERT overactivation provides replicative feature and immortality to CSCs, so the stemness and replicative properties of CSCs depend on telomerase activity. Therefore hTERT/telomerase activity may become a universal biomarker for anticancer therapy and it is an attractive therapeutic target for CSCs. It is known that mTOR regulates telomerase activity at the translational and post-translational level. Researchers show that mTOR inhibitor rapamycin reduces telomerase activity without changing hTERT mRNA activity. Correlation between mTOR and hTERT is important for survival and immortality of cancer cells. In addition, the PI3K/AKT/mTOR signaling pathway and hTERT up-regulation are related with cancer stemness features and drug resistance. mTOR inhibitor and TERT inhibitor combination may construct a novel strategy in cancer stem cells and it can make a double effect on telomerase enzyme. Consequently, inhibition of PI3K/AKT/mTOR signaling pathway components and hTERT activation may prohibit CSC self-renewal and surpass CSC-mediated resistance in order to develop new cancer therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

Immortalization of chicken preadipocytes by retroviral transduction of chicken TERT and TR.

Directory of Open Access Journals (Sweden)

Wei Wang

Full Text Available The chicken is an important agricultural animal and model for developmental biology, immunology and virology. Excess fat accumulation continues to be a serious problem for the chicken industry. However, chicken adipogenesis and obesity have not been well investigated, because no chicken preadipocyte cell lines have been generated thus far. Here, we successfully generated two immortalized chicken preadipocyte cell lines through transduction of either chicken telomerase reverse transcriptase (chTERT alone or in combination with chicken telomerase RNA (chTR. Both of these cell lines have survived >100 population doublings in vitro, display high telomerase activity and have no sign of replicative senescence. Similar to primary chicken preadipocytes, these two cell lines display a fibroblast-like morphology, retain the capacity to differentiate into adipocytes, and do not display any signs of malignant transformation. Isoenzyme analysis and PCR-based analysis confirmed that these two cell lines are of chicken origin and are free from inter-species contamination. To our knowledge, this is the first report demonstrating the generation of immortal chicken cells by introduction of chTERT and chTR. Our established chicken preadipocyte cell lines show great promise as an in vitro model for the investigation of chicken adipogenesis, lipid metabolism, and obesity and its related diseases, and our results also provide clues for immortalizing other avian cell types.

Immortalization of chicken preadipocytes by retroviral transduction of chicken TERT and TR

Science.gov (United States)

Wang, Wei; Zhang, Tianmu; Wu, Chunyan; Wang, Shanshan; Wang, Yuxiang; Wang, Ning

2017-01-01

The chicken is an important agricultural animal and model for developmental biology, immunology and virology. Excess fat accumulation continues to be a serious problem for the chicken industry. However, chicken adipogenesis and obesity have not been well investigated, because no chicken preadipocyte cell lines have been generated thus far. Here, we successfully generated two immortalized chicken preadipocyte cell lines through transduction of either chicken telomerase reverse transcriptase (chTERT) alone or in combination with chicken telomerase RNA (chTR). Both of these cell lines have survived >100 population doublings in vitro, display high telomerase activity and have no sign of replicative senescence. Similar to primary chicken preadipocytes, these two cell lines display a fibroblast-like morphology, retain the capacity to differentiate into adipocytes, and do not display any signs of malignant transformation. Isoenzyme analysis and PCR-based analysis confirmed that these two cell lines are of chicken origin and are free from inter-species contamination. To our knowledge, this is the first report demonstrating the generation of immortal chicken cells by introduction of chTERT and chTR. Our established chicken preadipocyte cell lines show great promise as an in vitro model for the investigation of chicken adipogenesis, lipid metabolism, and obesity and its related diseases, and our results also provide clues for immortalizing other avian cell types. PMID:28486516

Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin

Science.gov (United States)

Gardano, Laura; Holland, Linda; Oulton, Rena; Le Bihan, Thierry; Harrington, Lea

2012-01-01

Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3′ terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions. PMID:22187156

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

International Nuclear Information System (INIS)

Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

2011-01-01

Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

In vitro reconstitution of the active T. castaneum telomerase.

Science.gov (United States)

Schuller, Anthony P; Harkisheimer, Michael J; Skordalakes, Emmanuel

2011-07-14

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro. Telomerase is a specialized reverse transcriptase that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit. Additionally, telomerase is dormant in most somatic cells in adults, but is active in cancer cells where it promotes cell immortality. The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres. Prior to 2008, only structures for individual telomerase domains had been solved. A major breakthrough in this field came from the determination of the crystal structure of the active, catalytic subunit of T. castaneum telomerase, TcTERT. Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

Introduction of a normal human chromosome 8 corrects abnormal phenotypes of Werner syndrome cells immortalized by expressing an hTERT gene

International Nuclear Information System (INIS)

Ariyoshi, Kentaro; Kodama, Seiji; Suzuki, Keiji; Goto, Makoto; Oshimura, Mitsuo; Ishizaki, Kanji; Watanabe, Masami

2009-01-01

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging and caused by mutations of the WRN gene mapped at 8p12. To examine functional complementation of WS phenotypes, we introduced a normal human chromosome 8 into a strain of WS fibroblasts (WS3RGB) immortalized by expressing a human telomerase reverse transcriptase subunit (hTERT) gene. Here, we demonstrate that the abnormal WS phenotypes including cellular sensitivities to 4-nitroquinoline-1-oxide (4NQO) and hydroxy urea (HU), and chromosomal radiosensitivity at G 2 phase are corrected by expression of the WRN gene mediated by introducing a chromosome 8. This indicates that those multiple abnormal WS phenotypes are derived from a primary, but not secondary, defect in the WRN gene. (author)

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Ren, He, E-mail: herenrh@yahoo.com.cn [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Hao, Jihui, E-mail: jihuihao@yahoo.com [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China)

2010-03-26

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

Directory of Open Access Journals (Sweden)

Gokhan Yildiz

Full Text Available Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal" by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15

Decellularized matrix from tumorigenic human mesenchymal stem cells promotes neovascularization with galectin-1 dependent endothelial interaction.

Directory of Open Access Journals (Sweden)

Jorge S Burns

Full Text Available BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC strain (hMSC-TERT20 immortalized by retroviral vector mediated human telomerase (hTERT gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+ and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1

Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction

Science.gov (United States)

Burns, Jorge S.; Kristiansen, Malthe; Kristensen, Lars P.; Larsen, Kenneth H.; Nielsen, Maria O.; Christiansen, Helle; Nehlin, Jan; Andersen, Jens S.; Kassem, Moustapha

2011-01-01

Background Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. Methodology/Principal Findings Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. Conclusions Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was

Immortalization of normal human fibroblasts by treatment with 4-nitroquinoline 1-oxide.

Science.gov (United States)

Bai, L; Mihara, K; Kondo, Y; Honma, M; Namba, M

1993-02-01

Normal human fibroblasts (the OUMS-24 strain), derived from a 6-week-old human embryo, were transformed (into the OUMS-24F line) and immortalized by repeated treatments (59 times) with 4-nitroquinoline 1-oxide (4NQO). Treatment began during primary culture and ended at the 51st population doubling level (PDL). At the 57th PDL (146 days after the last treatment), morphologically altered, epithelial-type cells appeared, began to grow and became immortal (now past the 100th PDL). However, the control fibroblasts, which were not treated with 4NQO, senesced at the 62nd PDL. The finding that extensive, repeated treatments with 4NQO are required for the immortalization of normal human cells, indicates that multiple mutational events are involved in the immortalization of human cells in general. In other words, immortalization itself seems to be a multi-step process. Karyotypic analysis showed that many cells were hypodiploid before immortalization, but that afterwards chromosomes were distributed broadly in the diploid to tetraploid regions. The immortalized cells showed amplification and enhanced expression of c-myc. Two-dimensional electrophoretic analysis showed that the number of disappearing cellular proteins was greater than the number of the newly appearing ones after the cells became immortalized. Since the immortalized cells showed neither anchorage-independent growth nor tumorigenicity, they are useful for studying factors that can contribute to multi-step carcinogenesis in human cells. In addition, genetically matched normal (OUMS-24) and immortalized (OUMS-24F) cells will be useful for analyzing the genes related to cellular mortality and immortalization.

Generation and Characterization of an Immortalized Human Esophageal Myofibroblast Line.

Directory of Open Access Journals (Sweden)

Chao Niu

Full Text Available Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD. We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.

Regulation of Telomere Homeostasis during Epstein-Barr virus Infection and Immortalization.

Science.gov (United States)

Kamranvar, Siamak A; Masucci, Maria G

2017-08-09

The acquisition of unlimited proliferative potential is dependent on the activation of mechanisms for telomere maintenance, which counteracts telomere shortening and the consequent triggering of the DNA damage response, cell cycle arrest, and apoptosis. The capacity of Epstein Barr virus (EBV) to infect B-lymphocytes in vitro and transform the infected cells into autonomously proliferating immortal cell lines underlies the association of this human gamma-herpesvirus with a broad variety of lymphoid and epithelial cell malignancies. Current evidence suggests that both telomerase-dependent and -independent pathways of telomere elongation are activated in the infected cells during the early and late phases of virus-induced immortalization. Here we review the interaction of EBV with different components of the telomere maintenance machinery and the mechanisms by which the virus regulates telomere homeostasis in proliferating cells. We also discuss how these viral strategies may contribute to malignant transformation.

GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)

Science.gov (United States)

Cao, Hui; Chu, Yuankui; Lv, Xiao; Qiu, Pubin; Liu, Chao; Zhang, Huiru; Li, Dan; Peng, Sha; Dou, Zhongying; Hua, Jinlian

2012-01-01

Background The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present. Results To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis. Conclusions These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs. PMID:22384031

[Methods of measuring telomere length and telomerase activity--practice and problems].

Science.gov (United States)

Saito, Y; Suda, T; Hatakeyama, K

1998-05-01

The development of a highly sensitive method for detection of telomerase activity, telomeric repeat amplification protocol (TRAP), has provided knowledge on telomerase activity in normal and cancer tissues. Subsequent several modifications have been achieved, including an introduction of the internal standard and hybridization protection technique that leads to simplicity and improvement of reproducibility and linearity of this method, and application of TRAP to in situ analysis to identify the cells responsible for telomerase activity. As for measurement of telomere length, fluorescence in situ hybridization technique appeared to give an information of telomere length on an individual chromosome in contrast to analysis of terminal restriction fragment, a conventional method which can express mean telomere length of all chromosomes. Further methodological improvement in this field is ongoing and showing a new sight on cell mortality and immortality.

Human telomerase activity regulation

OpenAIRE

Wojtyla, Aneta; Gladych, Marta; Rubis, Blazej

2010-01-01

Telomerase has been recognized as a relevant factor distinguishing cancer cells from normal cells. Thus, it has become a very promising target for anticancer therapy. The cell proliferative potential can be limited by replication end problem, due to telomeres shortening, which is overcome in cancer cells by telomerase activity or by alternative telomeres lengthening (ALT) mechanism. However, this multisubunit enzymatic complex can be regulated at various levels, including expression control b...

Telomerase Protects Werner Syndrome Lineage-Specific Stem Cells from Premature Aging

Directory of Open Access Journals (Sweden)

Hoi-Hung Cheung

2014-04-01

Full Text Available Werner syndrome (WS patients exhibit premature aging predominantly in mesenchyme-derived tissues, but not in neural lineages, a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here, we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging, we differentiated iPSCs to mesenchymal stem cells (MSCs and neural stem/progenitor cells (NPCs. We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs, but not in NPCs. We postulate this “aging” discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC, whereas inhibition of telomerase sensitized NPCs to DNA damage. Our findings unveil a role for telomerase in the protection of accelerated aging in a specific lineage of stem cells.

Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I

Energy Technology Data Exchange (ETDEWEB)

Rosendahl, Ann H., E-mail: ann.rosendahl@med.lu.se [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden); Lund University and Skåne University Hospital, Department of Clinical Sciences Lund, Division of Oncology and Pathology, Lund (Sweden); Gundewar, Chinmay; Said Hilmersson, Katarzyna [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden); Ni, Lan; Saleem, Moin A. [University of Bristol, School of Clinical Sciences, Children' s Renal Unit and Academic Renal Unit, Bristol (United Kingdom); Andersson, Roland [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden)

2015-01-15

Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer. - Highlights: • Generation of human conditionally immortalized human pancreatic stellate cell lines. • Temperature-sensitive SV40LT allows switch to primary PSC phenotype characteristics. • Proteome profiling revealed distinct expression patterns between TPSC and NPSC. • Enhanced IGF-I-stimulated proliferation and motility by TPSC compared to NPSC.

Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

Science.gov (United States)

Taylor, Lisa M; James, Alexander; Schuller, Christine E; Brce, Jesena; Lock, Richard B; Mackenzie, Karen L

2004-10-15

Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

Zoning of mucosal phenotype, dysplasia, and telomerase activity measured by telomerase repeat assay protocol in Barrett's esophagus

NARCIS (Netherlands)

Going, JJ; Fletcher-Monaghan, AJ; Neilson, L; Wisman, BA; van der Zee, A; Stuart, RC; Keith, WN

2004-01-01

Glandular dysplasia in Barrett's esophagus may regress spontaneously but can also progress to cancer. The human telomerase RNA template and the human telomerase reverse transcriptase enzyme which do not, of themselves, correlate strongly with telomerase activity, are too often overexpressed in

Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

Directory of Open Access Journals (Sweden)

Kee Hang Lee

Full Text Available Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs immortalized by the human telomerase reverse transcriptase (hTERT gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM cells were injected into adult (4-6-week-old Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL, they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases.

Dioxin exerts anti-estrogenic actions in a novel dioxin-responsive telomerase-immortalized epithelial cell line of the porcine oviduct (TERT-OPEC).

Science.gov (United States)

Hombach-Klonisch, Sabine; Pocar, Paola; Kauffold, Johannes; Klonisch, Thomas

2006-04-01

Oviduct epithelial cells are important for the nourishment and survival of ovulated oocytes and early embryos, and they respond to the steroid hormones estrogen and progesterone. Endocrine-disrupting polyhalogenated aromatic hydrocarbons (PHAH) are environmental toxins that act in part through the ligand-activated transcription factor arylhydrocarbon receptor (AhR; dioxin receptor), and exposure to PHAH has been shown to decrease fertility. To investigate effects of PHAHs on the oviduct epithelium as a potential target tissue of dioxin-type endocrine disruptors, we have established a novel telomerase-immortalized oviduct porcine epithelial cell line (TERT-OPEC). TERT-OPEC exhibited active telomerase and the immunoreactive epithelial marker cytokeratin but lacked the stromal marker vimentin. TERT-OPEC contained functional estrogen receptor (ER)-alpha and AhR, as determined by the detection of ER-alpha- and AhR-specific target molecules. Treatment of TERT-OPEC with the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant increase in the production of the cytochrome P-450 microsomal enzyme CYP1A1. Activated AhR caused a downregulation of ER nuclear protein fraction and significantly decreased ER-signaling in TERT-OPEC as determined by ERE-luciferase transient transfection assays. In summary, the TCDD-induced and AhR-mediated anti-estrogenic responses by TERT-OPEC suggest that PHAH affect the predominantly estrogen-dependent differentiation of the oviduct epithelium within the fallopian tube. This action then alters the local endocrine milieu, potentially resulting in a largely unexplored cause of impaired embryonic development and female infertility.

Human cardiac stem cells exhibit mesenchymal features and are maintained through Akt/GSK-3β signaling

International Nuclear Information System (INIS)

Tateishi, Kento; Ashihara, Eishi; Honsho, Shoken; Takehara, Naofumi; Nomura, Tetsuya; Takahashi, Tomosaburo; Ueyama, Tomomi; Yamagishi, Masaaki; Yaku, Hitoshi; Matsubara, Hiroaki; Oh, Hidemasa

2007-01-01

Recent evidence suggested that human cardiac stem cells (hCSCs) may have the clinical application for cardiac repair; however, their characteristics and the regulatory mechanisms of their growth have not been fully investigated. Here, we show the novel property of hCSCs with respect to their origin and tissue distribution in human heart, and demonstrate the signaling pathway that regulates their growth and survival. Telomerase-active hCSCs were predominantly present in the right atrium and outflow tract of the heart (infant > adult) and had a mesenchymal cell-like phenotype. These hCSCs expressed the embryonic stem cell markers and differentiated into cardiomyocytes to support cardiac function when transplanted them into ischemic myocardium. Inhibition of Akt pathway impaired the hCSC proliferation and induced apoptosis, whereas inhibition of glycogen synthase kinase-3 (GSK-3) enhanced their growth and survival. We conclude that hCSCs exhibit mesenchymal features and that Akt/GSK-3β may be crucial modulators for hCSC maintenance in human heart

Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

International Nuclear Information System (INIS)

Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko; Hiratsuka, Masaharu; Sano, Akiko; Osawa, Kanako; Okazaki, Akiyo; Katoh, Motonobu; Kazuki, Yasuhiro; Oshimura, Mitsuo; Tomizuka, Kazuma

2008-01-01

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-β-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells

Highly sensitive electrochemical detection of human telomerase activity based on bio-barcode method.

Science.gov (United States)

Li, Ying; Liu, Bangwei; Li, Xia; Wei, Qingli

2010-07-15

In the present study, an electrochemical method for highly sensitive detection of human telomerase activity was developed based on bio-barcode amplification assay. Telomerase was extracted from HeLa cells, then the extract was mixed with telomerase substrate (TS) primer to perform extension reaction. The extension product was hybridized with the capture DNA immobilized on the Au electrode and then reacted with the signal DNA on Au nanoparticles to form a sandwich hybridization mode. Electrochemical signals were generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to the DNA on Au nanoparticles via electrostatic interaction. This method can detect the telomerase activity from as little as 10 cultured cancer cells without the polymerase chain reaction (PCR) amplification of telomerase extension product. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Targeting telomerase and DNA repair in human cancers

International Nuclear Information System (INIS)

Prakash Hande, M.

2014-01-01

Telomerase reactivation is essential for telomere maintenance in human cancer cells ensuring indefinite proliferation. Targeting telomere homeostasis has become one of the promising strategies in the therapeutic management of tumours. One major potential drawback, however, is the time lag between telomerase inhibition and critically shortened telomeres triggering cell death, allowing cancer cells to acquire drug resistance. Numerous studies over the last decade have highlighted the role of DNA repair proteins such as Poly (ADP-Ribose) Polymerase-1 (PARP-1), and DNA-dependent protein kinase (DNA-PKcs) in the maintenance of telomere homoeostasis. Dysfunctional telomeres, resulting from the loss of telomeric DNA repeats or the loss of function of telomere-associated proteins trigger DNA damage responses similar to that observed for double strand breaks. We have been working on unravelling such synthetic lethality in cancer cells and this talk would be on one such recently concluded study that demonstrates that inhibition of DNA repair pathways, i.e., NHEJ pathway and that of telomerase could be an alternative strategy to enhance anti-tumour effects and circumvent the possibility of drug resistance. (author)

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs

Directory of Open Access Journals (Sweden)

Choo Andre

2011-04-01

Full Text Available Abstract Background Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. Methods The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. Results MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. Conclusion Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time

Premature aging in telomerase-deficient zebrafish

Directory of Open Access Journals (Sweden)

Monique Anchelin

2013-09-01

The study of telomere biology is crucial to the understanding of aging and cancer. In the pursuit of greater knowledge in the field of human telomere biology, the mouse has been used extensively as a model. However, there are fundamental differences between mouse and human cells. Therefore, additional models are required. In light of this, we have characterized telomerase-deficient zebrafish (Danio rerio as the second vertebrate model for human telomerase-driven diseases. We found that telomerase-deficient zebrafish show p53-dependent premature aging and reduced lifespan in the first generation, as occurs in humans but not in mice, probably reflecting the similar telomere length in fish and humans. Among these aging symptoms, spinal curvature, liver and retina degeneration, and infertility were the most remarkable. Although the second-generation embryos died in early developmental stages, restoration of telomerase activity rescued telomere length and survival, indicating that telomerase dosage is crucial. Importantly, this model also reproduces the disease anticipation observed in humans with dyskeratosis congenita (DC. Thus, telomerase haploinsufficiency leads to anticipation phenomenon in longevity, which is related to telomere shortening and, specifically, with the proportion of short telomeres. Furthermore, p53 was induced by telomere attrition, leading to growth arrest and apoptosis. Importantly, genetic inhibition of p53 rescued the adverse effects of telomere loss, indicating that the molecular mechanisms induced by telomere shortening are conserved from fish to mammals. The partial rescue of telomere length and longevity by restoration of telomerase activity, together with the feasibility of the zebrafish for high-throughput chemical screening, both point to the usefulness of this model for the discovery of new drugs able to reactivate telomerase in individuals with DC.

Malignant transformation potentials of human umbilical cord mesenchymal stem cells both spontaneously and via 3-methycholanthrene induction.

Directory of Open Access Journals (Sweden)

Qiuling Tang

Full Text Available Human umbilical cord mesenchymal stem cells (HUMSCs are highly proliferative and can be induced to differentiate into advanced derivatives of all three germ layers. Thus, HUMSCs are considered to be a promising source for cell-targeted therapies and tissue engineering. However there are reports on spontaneous transformation of mesenchymal stem cells (MSCs derived from human bone marrows. The capacity for HUMSCs to undergo malignant transform spontaneously or via induction by chemical carcinogens is presently unknown. Therefore, we isolated HUMSCs from 10 donors and assessed their transformation potential either spontaneously or by treating them with 3-methycholanthrene (3-MCA, a DNA-damaging carcinogen. The malignant transformation of HUMSCs in vitro was evaluated by morphological changes, proliferation rates, ability to enter cell senescence, the telomerase activity, chromosomal abnormality, and the ability to form tumors in vivo. Our studies showed that HUMSCs from all 10 donors ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUMSCs from two of the 10 donors treated with 3-MCA displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. When these cells (tHUMSCs were injected into immunodeficient mice, they gave rise to sarcoma-like or poorly differentiated tumors. Moreover, in contrast to HUMSCs, tHUMSCs showed a positive expression of human telomerase reverse transcriptase (hTERT and did not exhibit a shortening of the relative telomere length during the long-term culture in vitro. Our studies demonstrate that HUMSCs are not susceptible to spontaneous malignant transformation. However, the malignant transformation could be induced by chemical carcinogen 3-MCA.

Malignant Transformation Potentials of Human Umbilical Cord Mesenchymal Stem Cells Both Spontaneously and via 3-Methycholanthrene Induction

Science.gov (United States)

Lai, Xiulan; Liu, Sizheng; Chen, Yezeng; Zheng, Zexin; Xie, Qingdong; Maldonado, Martin; Cai, Zhiwei; Qin, Shan; Ho, Guyu; Ma, Lian

2013-01-01

Human umbilical cord mesenchymal stem cells (HUMSCs) are highly proliferative and can be induced to differentiate into advanced derivatives of all three germ layers. Thus, HUMSCs are considered to be a promising source for cell-targeted therapies and tissue engineering. However there are reports on spontaneous transformation of mesenchymal stem cells (MSCs) derived from human bone marrows. The capacity for HUMSCs to undergo malignant transform spontaneously or via induction by chemical carcinogens is presently unknown. Therefore, we isolated HUMSCs from 10 donors and assessed their transformation potential either spontaneously or by treating them with 3-methycholanthrene (3-MCA), a DNA-damaging carcinogen. The malignant transformation of HUMSCs in vitro was evaluated by morphological changes, proliferation rates, ability to enter cell senescence, the telomerase activity, chromosomal abnormality, and the ability to form tumors in vivo. Our studies showed that HUMSCs from all 10 donors ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUMSCs from two of the 10 donors treated with 3-MCA displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. When these cells (tHUMSCs) were injected into immunodeficient mice, they gave rise to sarcoma-like or poorly differentiated tumors. Moreover, in contrast to HUMSCs, tHUMSCs showed a positive expression of human telomerase reverse transcriptase (hTERT) and did not exhibit a shortening of the relative telomere length during the long-term culture in vitro. Our studies demonstrate that HUMSCs are not susceptible to spontaneous malignant transformation. However, the malignant transformation could be induced by chemical carcinogen 3-MCA. PMID:24339974

Antiproliferative Effect of the Isoquinoline Alkaloid Papaverine in Hepatocarcinoma HepG-2 Cells — Inhibition of Telomerase and Induction of Senescence

Directory of Open Access Journals (Sweden)

Sakineh Kazemi Noureini

2014-08-01

Full Text Available Cancer cells are often immortal through up-regulation of the hTERT gene, which encodes the catalytic subunit of a special reverse transcriptase to overcome end-replication problem of chromosomes. This study demonstrates that papaverine, an isoquinoline alkaloid from the Papaveraceae, can overcome telomerase dependent immortality of HepG-2 cells that was used as a model of hepatocarcinoma. Although this alkaloid does not directly interact with telomeric sequences, papaverine inhibits telomerase through down-regulation of hTERT, which was analysed using thermal FRET and qRT-PCR, respectively. The IC50 values for the reduction of both telomerase activity and hTERT expression was 60 µM, while IC50 for cytotoxicity was 120 µM. Repeated treatments of the cells with very low non-toxic concentrations of papaverine resulted in growth arrest and strong reduction of population doublings after 40 days. This treatment induced senescent morphology in HepG-2 cells, which was evaluated by beta-galactosidase staining. Altogether, papaverine can be regarded as a promising model compound for drug design targeting cancer development.

Antimetastatic Effects of a Novel Telomerase Inhibitor, GRN163L, on Human Prostate Cancer

Science.gov (United States)

2010-05-01

Human Papilloma Virus Type 18 (HPV-18) DNA. PZ-HPV-7 cells are generally considered as non-tumorigenic in subcutaneous xenograft animal models...6481. [39] H.J. Sommerfeld, A.K. Meeker, M.A. Piatyszek, G.S. Bova, J.W. Shay, D.S. Coffey, Telomerase activity: a prevalent marker of malignant human ...6:192–8. 31. Sommerfeld HJ, Meeker AK, Piatyszek MA, Bova GS, Shay JW, Coffey DS. Telomerase activity: a prevalent marker of malignant human prostate

Rearrangement of Upstream Sequences of the hTERT Gene During Cellular Immortalization

Science.gov (United States)

Zhao, Yuanjun; Wang, Shuwen; Popova, Evgenya Y.; Grigoryev, Sergei A.; Zhu, Jiyue

2010-01-01

Telomerase expression, resulting from transcriptional activation of the hTERT gene, allows cells to acquire indefinite proliferative potential during cellular immortalization and tumorigenesis. However, mechanisms of hTERT gene activation in many immortal cell lines and cancer cells are poorly understood. Here, we report our studies on hTERT activation using genetically related pairs of telomerase-negative (Tel−) and -positive (Tel+) fibroblast lines. First, whereas transiently transfected plasmid reporters did not recapitulate the endogenous hTERT promoter, the promoter in chromosomally integrated bacterial artificial chromosome (BAC) reporters was activated in a subset of Tel+ cells, indicating that activation of the hTERT promoter required native chromatin context and/or distal regulatory elements. Second, the hTERT gene, located near the telomere of chromosome 5p, was translocated in all three Tel+ cell lines but not in their parental pre-crisis cells and Tel− immortal siblings. The breakage points were mapped to regions upstream of the hTERT promoter, indicating that the hTERT gene was the target of these chromosomal rearrangements. In two Tel+ cell lines, translocation of the endogenous hTERT gene appeared to be the major mechanism of its activation as the activity of hTERT promoter in many chromosomally integrated BAC reporters, with intact upstream and downstream neighboring loci, remained relatively low. Therefore, our results suggest that rearrangement of upstream sequences is an important new mechanism of hTERT promoter activation during cellular immortalization. The chromosomal rearrangements likely occurred during cellular crisis and facilitated by telomere dysfunction. Such translocations allowed the hTERT promoter to escape from the native condensed chromatin environment. PMID:19672873

Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders

Directory of Open Access Journals (Sweden)

Nora Urraca

2015-11-01

Full Text Available A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease.

Culture models of human mammary epithelial cell transformation

Energy Technology Data Exchange (ETDEWEB)

Stampfer, Martha R.; Yaswen, Paul

2000-11-10

Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

The Telomerase Inhibitor MST-312 Interferes with Multiple Steps in the Herpes Simplex Virus Life Cycle.

Science.gov (United States)

Haberichter, Jarod; Roberts, Scott; Abbasi, Imran; Dedthanou, Phonphanh; Pradhan, Prajakta; Nguyen, Marie L

2015-10-01

The life cycle of herpes simplex virus (HSV) has the potential to be further manipulated to yield novel, more effective therapeutic treatments. Recent research has demonstrated that HSV-1 can increase telomerase activity and that expression of the catalytic component of telomerase, telomerase reverse transcriptase (TERT), alters sensitivity to HSV-dependent apoptosis. Telomerase is a cellular enzyme that synthesizes nucleotide repeats at the ends of chromosomes (telomeres), which prevents shortening of the 3' ends of DNA with each cell division. Once telomeres reach a critical length, cells undergo senescence and apoptosis. Here, we used a cell-permeable, reversible inhibitor of the telomerase enzyme, MST-312, to investigate telomerase activity during HSV infection. Human mammary epithelial cells immortalized through TERT expression and human carcinoma HEp-2 cells were infected with the KOS1.1 strain of HSV-1 in the presence of MST-312. MST-312 treatment reduced the number of cells displaying a cytopathic effect and the accumulation of immediate early and late viral proteins. Moreover, the presence of 20 μM to 100 μM MST-312 during infection led to a 2.5- to 5.5-log10 decrease in viral titers. MST-312 also inhibited the replication of HSV-2 and a recent clinical isolate of HSV-1. Additionally, we determined that MST-312 has the largest impact on viral events that take place prior to 5 h postinfection (hpi). Furthermore, MST-312 treatment inhibited virus replication, as measured by adsorption assays and quantification of genome replication. Together, these findings demonstrate that MST-312 interferes with the HSV life cycle. Further investigation into the mechanism for MST-312 is warranted and may provide novel targets for HSV therapies. Herpes simplex virus (HSV) infections can lead to cold sores, blindness, and brain damage. Identification of host factors that are important for the virus life cycle may provide novel targets for HSV antivirals. One such factor

Parameters in three-dimensional osteospheroids of telomerized human mesenchymal (stromal) stem cells grown on osteoconductive scaffolds that predict in vivo bone-forming potential

DEFF Research Database (Denmark)

Burns, Jorge S; Hansen, Pernille Lund; Larsen, Kenneth H

2010-01-01

Osteoblastic differentiation of human mesenchymal stem cells (hMSC) in monolayer culture is artefactual, lacking an organized bone-like matrix. We present a highly reproducible microwell protocol generating three-dimensional ex vivo multicellular aggregates of telomerized hMSC (hMSC-telomerase re......Osteoblastic differentiation of human mesenchymal stem cells (hMSC) in monolayer culture is artefactual, lacking an organized bone-like matrix. We present a highly reproducible microwell protocol generating three-dimensional ex vivo multicellular aggregates of telomerized hMSC (h......, was deposited in the scaffold concavities. Here, mature osteoblasts stained positively for differentiated osteoblast markers TAZ, biglycan, osteocalcin, and phospho-AKT. Quantification of collagen birefringence and relatively high expression of genes for matrix proteins, including type I collagen, biglycan...

Human RTEL1 stabilizes long G-overhangs allowing telomerase-dependent over-extension.

Science.gov (United States)

Porreca, Rosa M; Glousker, Galina; Awad, Aya; Matilla Fernandez, Maria I; Gibaud, Anne; Naucke, Christian; Cohen, Scott B; Bryan, Tracy M; Tzfati, Yehuda; Draskovic, Irena; Londoño-Vallejo, Arturo

2018-05-18

Telomere maintenance protects the cell against genome instability and senescence. Accelerated telomere attrition is a characteristic of premature aging syndromes including Dyskeratosis congenita (DC). Mutations in hRTEL1 are associated with a severe form of DC called Hoyeraal-Hreidarsson syndrome (HHS). HHS patients carry short telomeres and HHS cells display telomere damage. Here we investigated how hRTEL1 contributes to telomere maintenance in human primary as well as tumor cells. Transient depletion of hRTEL1 resulted in rapid telomere shortening only in the context of telomerase-positive cells with very long telomeres and high levels of telomerase. The effect of hRTEL1 on telomere length is telomerase dependent without impacting telomerase biogenesis or targeting of the enzyme to telomeres. Instead, RTEL1 depletion led to a decrease in both G-overhang content and POT1 association with telomeres with limited telomere uncapping. Strikingly, overexpression of POT1 restored telomere length but not the overhang, demonstrating that G-overhang loss is the primary defect caused by RTEL1 depletion. We propose that hRTEL1 contributes to the maintenance of long telomeres by preserving long G-overhangs, thereby facilitating POT1 binding and elongation by telomerase.

Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy

DEFF Research Database (Denmark)

Bruedigam, Claudia; Bagger, Frederik Otzen; Heidel, Florian H.

2014-01-01

(-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia...... progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs....

Telomerase Activity Impacts on Epstein-Barr Virus Infection of AGS Cells

Science.gov (United States)

Rac, Jürgen; Haas, Florian; Schumacher, Andrina; Middeldorp, Jaap M.; Delecluse, Henri-Jacques; Speck, Roberto F.

2015-01-01

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva. PMID:25856387

Telomerase – future drug target enzyme?

Directory of Open Access Journals (Sweden)

Tomaž Langerholc

2012-06-01

Full Text Available Eucaryotic chromosome endings (telomeres replication problem was solved in the 1980’s by discovery of the telomerase enzyme. The Nobel Prize in Physiology or Medicine was awarded in 2009 for the discovery of telomerase. Altered telomerase expression in cancer, and human dream of eternal youth have accelerated the development of pharmacological telomerase inhibitors and activators. However, after 15 years of development they are still not available on the market. In the present article we reviewed pharmacological agents that target telomerase activity, which have entered clinical trials. Current drugs in development are mostly not intended to be used alone, as telomerase inhibitors under clinical trials are used in combination with the existing chemotherapeutics and anti-telomerase vaccines in combination with immuno-stimulants. Apart from cancer and aging, there are other diseases linked to deregulated activity of telomerase/telomeres and we also discuss technical and legal problems that researchers encounter in developing anti-telomerase therapy. Given the pace of development, first anti-telomerase drugs might appear on the market in the next 5 years.

Telomerase Inhibition by a New Synthetic Derivative of the Aporphine Alkaloid Boldine

Directory of Open Access Journals (Sweden)

Sakineh Kazemi Noureini

2018-04-01

Full Text Available Telomerase, the enzyme responsible for cell immortality, is an important target in anti-cancer drug discovery. Boldine, an abundant aporphine alkaloid of Peumus boldus, is known to inhibit telomerase at non-toxic concentrations. Cytotoxicity of N-benzylsecoboldine hydrochloride (BSB, a synthetic derivative of boldine, was determined using the MTT method in MCF7 and MDA-MB231 cells. Aliquots of cell lysates were incubated with various concentrations of BSB in qTRAP (quantitative telomere repeat amplification protocol-ligand experiments before substrate elongation by telomerase or amplification by hot-start Taq polymerase. The crystal structure of TERT, the catalytic subunit of telomerase from Tribolium castaneum, was used for docking and molecular dynamics analysis. The qTRAP-ligand data gave an IC50 value of about 0.17 ± 0.1 µM for BSB, roughly 400 times stronger than boldine, while the LD50 in the cytotoxicity assays were 12.5 and 21.88 µM, respectively, in cells treated for 48 h. Although both compounds interacted well with the active site, MD analysis suggests a second binding site with which BSB interacts via two hydrogen bonds, much more strongly than boldine. Theoretical analyses also evaluated the IC50 for BSB as submicromolar. BSB, with greater hydrophobicity and flexibility than boldine, represents a promising structure to inhibit telomerase at non-toxic concentrations.

Telomerase as an emerging target to fight cancer--opportunities and challenges for nanomedicine.

Science.gov (United States)

Philippi, C; Loretz, B; Schaefer, U F; Lehr, C M

2010-09-01

Telomerase as an enzyme is responsible for the renewal of the chromosomal ends, the so-called telomeres. By preventing them from shortening with each cell cycle, telomerase is able to inhibit cellular senescence and apoptosis. Telomerase activity, which is detectable in the majority of cancer cells, allows them to maintain their proliferative capacity. The thus obtained immortality of those cells again is a key to their malignancy. Based on these discoveries, it is obvious that telomerase inhibitors would represent an innovative approach to fight cancer, and a variety of such candidate molecules are currently in the pipeline. Telomerase inhibitors largely fall in two classes of compounds: small synthetic molecules and nucleotide-based biologicals. For several candidates, some proof of concept studies have been demonstrated, either on cell cultures or in animal models. But the same studies also revealed that inefficient delivery is largely limiting the translational step into the clinic. The most appealing feature of telomerase inhibitors, which distinguishes them from conventional anticancer drugs, is probably seen in their intrinsic non-toxicity to normal cells. Nevertheless, efficient delivery to the target cells, i.e. to the tumor, is still required. Here, some well-known biopharmaceutical problems such as insufficient solubility, permeability or even metabolic stability are frequently encountered. To address these challenges, there is a clear need for adequate delivery technologies, for example by using nanomedicines, that would allow to overcome their biopharmaceutical shortcomings and to warrant a sufficient bioavailability at the target side. This review first briefly explains the concept of telomerase and telomerase inhibition in cancer therapy. It secondly aims to provide an overview of the different currently known telomerase inhibitors. Finally, the biopharmaceutical limitations of these molecules are discussed as well as the possibilities to overcome

Dynamic telomerase gene suppression via network effects of GSK3 inhibition.

Directory of Open Access Journals (Sweden)

Alan E Bilsland

2009-07-01

Full Text Available Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression.In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3'-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFkappaB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc.Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.

Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders.

Science.gov (United States)

Urraca, Nora; Memon, Rawaha; El-Iyachi, Ikbale; Goorha, Sarita; Valdez, Colleen; Tran, Quynh T; Scroggs, Reese; Miranda-Carboni, Gustavo A; Donaldson, Martin; Bridges, Dave; Reiter, Lawrence T

2015-11-01

A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs) are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT) and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

Energy Technology Data Exchange (ETDEWEB)

Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Jhaveri, Hiral M. [Department of Periodontics and Oral Implantology, Dr. D.Y. Patil Dental College and Hospital, Pune (India); Mishra, Gyan C. [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India); Wani, Mohan R., E-mail: mohanwani@nccs.res.in [National Center for Cell Science, University of Pune Campus, Pune 411 007 (India)

2010-03-12

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine

International Nuclear Information System (INIS)

Tomar, Geetanjali B.; Srivastava, Rupesh K.; Gupta, Navita; Barhanpurkar, Amruta P.; Pote, Satish T.; Jhaveri, Hiral M.; Mishra, Gyan C.; Wani, Mohan R.

2010-01-01

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into multiple cell lineages. Presently, bone marrow is considered as a prime source of MSCs; however, there are some drawbacks and limitations in use of these MSCs for cell therapy. In this study, we demonstrate that human gingival tissue-derived MSCs have several advantages over bone marrow-derived MSCs. Gingival MSCs are easy to isolate, homogenous and proliferate faster than bone marrow MSCs without any growth factor. Importantly, gingival MSCs display stable morphology and do not loose MSC characteristic at higher passages. In addition, gingival MSCs maintain normal karyotype and telomerase activity in long-term cultures, and are not tumorigenic. Thus, we reveal that human gingiva is a better source of MSCs than bone marrow, and large number of functionally competent clinical grade MSCs can be generated in short duration for cell therapy in regenerative medicine and tissue engineering.

Comparison of telomerase activity in prostate cancer, prostatic intraepithelial neoplasia and benign prostatic hyperplasia

Directory of Open Access Journals (Sweden)

Soleiman Mahjoub

2006-11-01

Full Text Available BACKGROUND: Telomerase is a reverse transcriptase enzyme that synthesizes telomeric DNA on chromosome ends. The enzyme is important for the immortalization of cancer cells because it maintains the telomeres. METHODS: Telomerase activity (TA was measured by fluorescence-based telomeric repeat amplification protocol (FTRAP assay in prostate carcinoma and benign prostatic hyperplasia (BPH. RESULTS: TA was present in 91.4% of 70 prostate cancers, 68.8% of 16 prostatic intraepithelial neoplasia (PIN, 43.3% of 30 BPH*, 21.4% of 14 atrophy and 20% of 15 normal samples adjacent to tumor. There was not any significant correlation between TA, histopathological tumor stage or gleason score. In contrast to high TA in the BPH* tissue from the cancer-bearing gland, only 6.3% of 32 BPH specimens from patients only diagnosed with BPH were telomerase activity-positive. CONCLUSIONS: These results indicate that TA is present in most prostate cancers. The high rate of TA in tissue adjacent to tumor may be attributed either to early molecular alteration of cancer that was histologically unapparent, or to the presence of occult cancer cells. Our findings suggest that the re-expression of telomerase activity could be one step in the transformation of BPH to PIN. KEY WORDS: Telomerase activity, prostate cancer, prostatic intraepithelial neoplasia, benign prostatic hyperplasia.

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

Science.gov (United States)

Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

2017-11-15

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells

DEFF Research Database (Denmark)

Stiehler, Maik; Bünger, Cody; Baatrup, Anette

2009-01-01

Ex vivo engineering of autologous bone tissue as an alternative to bone grafting is a major clinical need. In the present study, we evaluated the effect of 3-D dynamic spinner flask culture on the proliferation, distribution, and differentiation of human mesenchymal stem cells (MSCs). Immortalized...... human MSCs were cultured on porous 75:25 PLGA scaffolds for up to 3 weeks. Dynamically cultured cell/scaffold constructs demonstrated a 20% increase in DNA content (21 days), enhanced ALP specific activity (7 days and 21 days), a more than tenfold higher Ca2+ content (21 days), and significantly...

Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

International Nuclear Information System (INIS)

Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R.

1990-01-01

Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV

A human beta cell line with drug inducible excision of immortalizing transgenes

Science.gov (United States)

Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

2015-01-01

Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

AZT as a telomerase inhibitor

International Nuclear Information System (INIS)

Gomez, Daniel E.; Armando, Romina G.; Alonso, Daniel F.

2012-01-01

Telomerase is a highly specialized reverse transcriptase (RT) and the maintenance of telomeric length is determined by this specific enzyme. The human holoenzyme telomerase is a ribonucleoprotein composed by a catalytic subunit, hTERT, an RNA component, hTR, and a group of associated proteins. Telomerase is normally expressed in embryonic cells and is repressed during adulthood. The enzyme is reexpressed in around 85% of solid tumors. This observation makes it a potential target for developing drugs that could be developed for therapeutic purposes. The identification of the hTERT as a functional catalytic RT prompted studies of inhibiting telomerase with the HIV RT inhibitor azidothymidine (AZT). Previously, we have demonstrated that AZT binds preferentially to telomeres, inhibits telomerase and enhances tumor cell senescence, and apoptosis after AZT treatment in breast mammary adenocarcinoma cells. Since then, several studies have considered AZT for telomerase inhibition and have led to potential clinical strategies for anticancer therapy. This review covers present thinking of the inhibition of telomerase by AZT and future treatment protocols using the drug.

The effect of β-ionone on telomerase activity in the human leukemia cell line K562

Directory of Open Access Journals (Sweden)

Zohreh Faezizadeh

2015-06-01

Full Text Available Background: Telomerase is highly activated in most human cancer cells, therefore, its inhibition has been proposed as a novel and promising strategy for cancer therapy. Many plant-derived anticancer agents act through inhibition of telomerase activity and induction of apoptosis. β-ionone, a carotenoid compound isolated from Roseaceae, has been reported to possess anticancer properties. The present study was undertaken to examine the mechanism of β-ionone-induced apoptosis in human leukemia cell line K562 with special emphasis on its role in telomerase inhibition. Method: In this study the anti-proliferation effect of β-ionone on K562 cells was evaluated by MTT assay. Apoptosis rate was detected by Hoechst staining and flow cytometry analysis. Telomerase activity was measured by (TRAP ELISA assay. Results: Exposure of K562 cells to β-ionone caused a dose-dependent decrease in proliferation. Flow cytometry analysis and Hoechst staining showed that percentage of apoptotic cells markedly increased with an increase in β-ionone concentration. Compared to control cells, treatment of K562 cells with β-ionone resulted in a significant decrease of telomerase activity. Moreover, a positive correlation was detected between telomerase inhibition and apoptosis induction in the treated K562 cells. Conclusion: Based on these results, β-ionone is an appropriate candidate for inhibiting telomerase activity in K562 cells. Therefore, it may be utilized as a novel drug against some leukemia cell lines.

Telomerase-Deficient Mice Exhibit Bone Loss Owing to Defects in Osteoblasts and Increased Osteoclastogenesis by Inflammatory Microenvironment

DEFF Research Database (Denmark)

Saeed, H.; Abdallah, B. M.; Ditzel, N.

2011-01-01

Telomere shortening owing to telomerase deficiency leads to accelerated senescence of human skeletal (mesenchymal) stem cells (MSCs) in vitro, whereas overexpression leads to telomere elongation, extended life span, and enhanced bone formation. To study the role of telomere shortening in vivo, we...... studied the phenotype of telomerase-deficient mice (Terc(-/-)).Terc(-/-) mice exhibited accelerated age-related bone loss starting at 3 months of age and during 12 months of follow-up revealed by dual-energy X-ray absorptiometric (DXA) scanning and by micro-computed tomography (mu CT). Bone...... histomorphometry revealed decreased mineralized surface and bone-formation rate as well as increased osteoclast number and size in Terc(-/-) mice. Also, serum total deoxypyridinoline (tDPD) was increased in Terc(-/-) mice. MSCs and osteoprogenitors isolated from Terc(-l-) mice exhibited intrinsic defects...

A Smart DNA Tweezer for Detection of Human Telomerase Activity.

Science.gov (United States)

Xu, Xiaowen; Wang, Lei; Li, Kan; Huang, Qihong; Jiang, Wei

2018-03-06

Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) n . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.

Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

Energy Technology Data Exchange (ETDEWEB)

Woodworth, C.D.; Doniger, J.; DiPaolo, J.A.

1989-01-01

Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review

Directory of Open Access Journals (Sweden)

Takahiro Eitsuka

2018-02-01

Full Text Available Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol, and the other downregulates the expression of human telomerase reverse transcriptase (hTERT, the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol. In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart

DEFF Research Database (Denmark)

Serakinci, Nedime; Christensen, Rikke; Graakjaer, Jesper

2007-01-01

-detected alkaline DNA unwinding assay together with Western blot analyses for phosphorylated H2AX and Q-FISH was applied for investigation of telomeric damage. Our results indicate that hMSC and TERT-immortalized hMSCs can cope with relatively high doses of gamma-rays and that overall DNA repair is similar...... in the two cell lines. The telomeres were extensively destroyed after irradiation in both cell types suggesting that telomere caps are especially sensitive to radiation. The TERT-immortalized hMSCs showed higher stability at telomeric regions than primary hMSCs indicating that cells with long telomeres...

Whole-Genome Expression Analysis of Human Mesenchymal Stromal Cells Exposed to Ultrasmooth Tantalum vs. Titanium Oxide Surfaces

DEFF Research Database (Denmark)

Stiehler, C.; Bunger, C.; Overall, R. W.

2013-01-01

to titanium (Ti) surface. The aim of this study was to extend the previous investigation of biocompatibility by monitoring temporal gene expression of MSCs on topographically comparable smooth Ta and Ti surfaces using whole-genome gene expression analysis. Total RNA samples from telomerase-immortalized human...... MSCs cultivated on plain sputter-coated surfaces of Ta or Ti for 1, 2, 4, and 8 days were hybridized to n = 16 U133 Plus 2.0 arrays (Affymetrix(A (R))). Functional annotation, cluster and pathway analyses were performed. The vast majority of genes were differentially regulated after 4 days...... of cultivation and genes upregulated by MSCs exposed to Ta and Ti were predominantly related to the processes of differentiation and transcription, respectively. Functional annotation analysis of the 1,000 temporally most significantly regulated genes suggests earlier cellular differentiation on Ta compared...

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

NARCIS (Netherlands)

Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

Universe, human immortality and future human evaluation

CERN Document Server

Bolonkin, Alexander

2011-01-01

This book debates the universe, the development of new technologies in the 21st century and the future of the human race. Dr Bolonkin shows that a human soul is only the information in a person's head. He offers a new unique method for re-writing the main brain information in chips without any damage to the human brain. This is the scientific prediction of the non-biological (electronic) civilization and immortality of the human being. Such a prognosis is predicated upon a new law, discovered by the author, for the development of complex systems. According to this law, every self-copying system tends to be more complex than the previous system, provided that all external conditions remain the same. The consequences are disastrous: humanity will be replaced by a new civilization created by intellectual robots (which Dr Bolonkin refers to as "E-humans" and "E-beings"). These creatures, whose intellectual and mechanical abilities will far exceed those of man, will require neither food nor oxygen to sustain their...

Telomerase Activation in Atherosclerosis and Induction of Telomerase Reverse Transcriptase Expression by Inflammatory Stimuli in Macrophages

Science.gov (United States)

Gizard, Florence; Heywood, Elizabeth B.; Findeisen, Hannes M.; Zhao, Yue; Jones, Karrie L.; Cudejko, Cèline; Post, Ginell R.; Staels, Bart; Bruemmer, Dennis

2010-01-01

Objective Telomerase serves as a critical regulator of tissue renewal. Although telomerase activity is inducible in response to various environmental cues, it remains unknown whether telomerase is activated during the inflammatory remodeling underlying atherosclerosis formation. To address this question, we investigated in the present study the regulation of telomerase in macrophages and during atherosclerosis development in LDL-receptor-deficient mice. Methods and Results We demonstrate that inflammatory stimuli activate telomerase in macrophages by inducing the expression of the catalytic subunit telomerase reverse transcriptase (TERT). Reporter and chromatin immunoprecipitation assays identified a previously unrecognized NF-κB response element in the TERT promoter, to which NF-κB is recruited during inflammation. Inhibition of NF-κB signaling completely abolished the induction of TERT expression, characterizing TERT as a bona fide NF-κB target gene. Furthermore, functional experiments revealed that TERT-deficiency results in a senescent cell phenotype. Finally, we demonstrate high levels of TERT expression in macrophages of human atherosclerotic lesions and establish that telomerase is activated during atherosclerosis development in LDL-receptor-deficient mice. Conclusion These results characterize TERT as a previously unrecognized NF-κB target gene in macrophages and demonstrate that telomerase is activated during atherosclerosis. This induction of TERT expression prevents macrophage senescence and may have important implications for the development of atherosclerosis. PMID:21106948

Telomere 1 (POT1) gene expression and its association with telomerase activity in colorectal tumor samples with different pathological features.

Science.gov (United States)

Izgi, Ahu; Gunal, Armagan; Yalcin, Serap; Gunduz, Ufuk

2014-09-01

The ends of chromosoms, telomeres are bound with a number of proteins which protect and stabilize telomeres against degredation, end to end fusion and aberrant recombinations. Telomeric DNA is bound of two groups of proteins, which are double-stranded telomeric DNA bindings proteins, and single stranded telomeric binding proteins. Among telomere binding proteins, protections of telomere 1 protein is a single stranded telomere binding proteins and suggested to be a significant player for telomere elongation and has an association with an enzyme called as telomerase which is an intrinsic reverse transcriptase. Telomerase synthesizes hexameric telomeric repeats onto the chromosomes thereby compansating telomere loss in immortal cells, such as tumor cells, whereas telomeres are shorthened with each division in normal cells. PCR-based TRAP (telomeric repeat amplification protocol) assay is a very sensitive assay for the detection of enzymatic activity of telomerase even if a few numbers of cancerous cells are available. The association between telomerase activity and hPOT1 expression in colorectal cancer is still unclear. Protein extraction was performed from specimens of matched normal and colorectal cancer specimens. Protein concentrations were determined by Bradford assay. Optimized protein concentrations were used for TRAP Assay. TRAP products were seperated by vertical gel electrophoresis on 12.5% polyacrylamide gels and visualized by silver staining. Gene expression of hPOT1 was determined by qPCR analysis. The results demonstrated that all tumor tissues were telomerase positive whereas all corresponding normal tissue was telomerase negative. Among clinicopathological findings, telomerase activity was found to be associated with stage, histology, localization, distant metastasis and lymph node metastasis of tumor in the current study. Although all of the clinicopathological findings differed in the expression of hPOT1 compared to normal tissues, they did not

Dose-Dependent Cytotoxic Effects of Boldine in HepG-2 Cells—Telomerase Inhibition and Apoptosis Induction

Directory of Open Access Journals (Sweden)

Sakineh Kazemi Noureini

2015-02-01

Full Text Available Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT gene (p < 0.01 and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002. However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02 and p21 (p < 0.01 genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations.

Proteome alteration induced by hTERT transfection of human fibroblast cells.

Science.gov (United States)

Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

Proteome alteration induced by hTERT transfection of human fibroblast cells

Directory of Open Access Journals (Sweden)

Riou Jean-François

2008-04-01

Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

Identification of senescence-associated genes in human bone marrow mesenchymal stem cells

International Nuclear Information System (INIS)

Ryu, Eunsook; Hong, Su; Kang, Jaeku; Woo, Junghoon; Park, Jungjun; Lee, Jongho; Seo, Jeong-Sun

2008-01-01

Human bone marrow mesenchymal stem cells (hBMMSCs) are multipotent stem cells that can differentiate into several specialized cell types, including bone, cartilage, and fat cells. The proliferative capacity of hBMMSCs paves the way for the development of regenerative medicine and tissue engineering. However, long-term in vitro culture of hBMMSCs leads to a reduced life span of the cells due to senescence, which leads eventually to growth arrest. To investigate the molecular mechanism behind the cellular senescence of hBMMSCs, microarray analysis was used to compare the expression profiles of early passage hBMMSCs, late passage hBMMSCs and hBMMSCs ectopically expressing human telomerase reverse transcriptase (hTERT). Using an intersection analysis of 3892 differentially expressed genes (DEGs) out of 27,171 total genes analyzed, we identified 338 senescence-related DEGs. GO term categorization and pathway network analysis revealed that the identified genes are strongly related to known senescence pathways and mechanisms. The genes identified using this approach will facilitate future studies of the mechanisms underlying the cellular senescence of hBMMSCs

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

Science.gov (United States)

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F.

2017-01-01

ABSTRACT Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. PMID:28188264

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

Directory of Open Access Journals (Sweden)

Ludovic Arandel

2017-04-01

Full Text Available Myotonic dystrophy type 1 (DM1 and type 2 (DM2 are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

Science.gov (United States)

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F; Furling, Denis

2017-04-01

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. © 2017. Published by The Company of Biologists Ltd.

[Telomerase in lung cancer. Testing the activity of the "immortaligy enzyme" bronchial biopsies increases the diagnostic yield in cases of suspected peripheral bronchogenic carcinomas].

Science.gov (United States)

Freitag, L; Litterst, P; Obertrifter, B; Velehorschi, V; Kemmer, H P; Linder, A; Brightman, I

2000-11-01

The proliferative capability is time-limited in normal somatic cells by the shortening of their chromosomal ends, the telomeres (Hayflick limit). An important feature of malignant cells is their immortality. The probably most common mechanism of tumour cells to achieve unlimited replicability is the activation of the enzyme telomerase. The reverse transcriptase can compensate the loss of telomeres. Using a PCR-based TRAP assay we found telomerase activity in tumour biopsies, exsudates and bronchial washings in various thoracic malignancies. In 38 of 47 patients with suspected peripheral lung cancer eventually surgery or invasive procedures proved a malignancy. In fluoroscopically guided bronchial brushings from 25 of these 38 patients (66%) the TRAP assay revealed telomerase activity. There was a single false positive case (tuberculosis) and with a single exception, the simultaneously taken brushes of the contralateral lobes were all telomerase negative. In 23 patients (61%) tumour cells were found in the cytological examination. In 33 patients at least one marker was positive. Thus the combination of cytology and telomerase test in bronchial brush biopsies attained a diagnostic yield of 87%.

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

Science.gov (United States)

Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

1990-01-01

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

Mixed Integer Linear Programming based machine learning approach identifies regulators of telomerase in yeast.

Science.gov (United States)

Poos, Alexandra M; Maicher, André; Dieckmann, Anna K; Oswald, Marcus; Eils, Roland; Kupiec, Martin; Luke, Brian; König, Rainer

2016-06-02

Understanding telomere length maintenance mechanisms is central in cancer biology as their dysregulation is one of the hallmarks for immortalization of cancer cells. Important for this well-balanced control is the transcriptional regulation of the telomerase genes. We integrated Mixed Integer Linear Programming models into a comparative machine learning based approach to identify regulatory interactions that best explain the discrepancy of telomerase transcript levels in yeast mutants with deleted regulators showing aberrant telomere length, when compared to mutants with normal telomere length. We uncover novel regulators of telomerase expression, several of which affect histone levels or modifications. In particular, our results point to the transcription factors Sum1, Hst1 and Srb2 as being important for the regulation of EST1 transcription, and we validated the effect of Sum1 experimentally. We compiled our machine learning method leading to a user friendly package for R which can straightforwardly be applied to similar problems integrating gene regulator binding information and expression profiles of samples of e.g. different phenotypes, diseases or treatments. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

Energy Technology Data Exchange (ETDEWEB)

Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

2009-04-20

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells.

Science.gov (United States)

Chung, C M; Man, C; Jin, Y; Jin, C; Guan, X Y; Wang, Q; Wan, T S K; Cheung, A L M; Tsao, S W

2005-07-01

Immortalization is an early and essential step of human carcinogenesis. Amplification of chromosome 20q has been shown to be a common event in immortalized cells and cancers. We have previously reported that gain and amplification of chromosome 20q is a non-random and common event in immortalized human ovarian surface epithelial (HOSE) cells. The chromosome 20q harbors genes including TGIF2 (20q11.2-q12), AIB1 (20q12), PTPN1 (20q13.1), ZNF217 (20q13.2), and AURKA (20q13.2-q13.3), which were previously reported to be amplified and overexpressed in ovarian cancers. Some of these genes may be involved in immortalization of HOSE cells and represent crucial premalignant changes in ovarian surface epithelium. Investigation of the involvement of these genes was examined in four pairs of pre-crisis (preimmortalized) and post-crisis (immortalized) HOSE cells. Overexpression of AURKA (Aurora kinase A), also known as BTAK and STK15, by both real time-quantitative polymerase chain reaction (RT-QPCR) and Western blotting was detected in all the four immortalized HOSE cells examined while overexpression of AIB1 and ZNF217 was observed in two of four immortalized HOSE cells examined. Overexpression of TGIF2 and PTPN1 was not significant in our immortalized HOSE cell systems. The degree of overexpression of AURKA was shown to be closely associated with the amplification of chromosome 20q in immortalized HOSE cells. Fluorescence in situ hybridization (FISH) with labeled P1 artificial clone (PAC) confirmed the amplification of the chromosomal region (20q13.2-13.3) where AURKA resides. DNA amplification of AURKA was also confirmed using semi-quantitative PCR. Our study showed that amplification and overexpression of AURKA is a common and significant event during immortalization of HOSE cells and may represent an important premalignant change in ovarian carcinogenesis. Copyright (c) 2005 Wiley-Liss, Inc.

Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells

DEFF Research Database (Denmark)

Simonsen, Janne Lytoft; Rosada, Cecilia; Serakinci, Nedime

2002-01-01

Human bone marrow stromal cells (hMSCs) were stably transduced by a retroviral vector containing the gene for the catalytic subunit of human telomerase (hTERT). Transduced cells (hMSC-TERTs) had telomerase activity, and the mean telomere length was increased as compared with that of control cells....... The transduced cells have now undergone more than 260 population doublings (PD) and continue to proliferate, whereas control cells underwent senescence-associated proliferation arrest after 26 PD. The cells maintained production of osteoblastic markers and differentiation potential during continuous subculturing......, did not form tumors, and had a normal karyotype. When implanted subcutaneously in immunodeficient mice, the transduced cells formed more bone than did normal cells. These results suggest that ectopic expression of telomerase in hMSCs prevents senescence-associated impairment of osteoblast functions....

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

Directory of Open Access Journals (Sweden)

Ryo Kurita

Full Text Available Transfusion of red blood cells (RBCs is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

Expression of human telomerase reverse transcriptase protein in oral epithelial dysplasia and oral squamous cell carcinoma: An immunohistochemical study

Science.gov (United States)

Raghunandan, Bangalore Nagarajachar; Sanjai, Karpagaselvi; Kumaraswamy, Jayalakshmi; Papaiah, Lokesh; Pandey, Bhavna; Jyothi, Bellur MadhavaRao

2016-01-01

Background: Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA sequences and almost universally provides the molecular basis for unlimited proliferative potential. The telomeres become shorter with each cycle of replication and reach a critical limit; most cells die or enter stage of replicative senescence. Telomere length maintenance by telomerase is required for all the cells that exhibit limitless replicative potential. It has been postulated that reactivation of telomerase expression is necessary for the continuous proliferation of neoplastic cells to attain immortality. Use of immunohistochemistry (IHC) is a useful, reliable method of localizing the human telomerase reverse transcriptase (hTERT) protein in tissue sections which permits cellular localization. Although there exists a lot of information on telomerase in oral cancer, little is known about their expression in oral epithelial dysplasia and their progression to oral squamous cell carcinoma (OSCC) compared to normal oral mucosa. This study addresses this lacuna. Aims: To compare the expression of hTERT protein in oral epithelial dysplasia and OSCC with normal oral mucosa by Immunohistochemical method. Subjects and Methods: In this preliminary study, IHC was used to detect the expression of hTERT protein in OSCC (n = 20), oral epithelial dysplasia (n = 21) and normal oral mucosa (n = 10). The tissue localization of immunostain, cellular localization of immunostain, nature of stain, intensity of stain, percentage of cells stained with hTERT protein were studied. A total number of 100 cells were counted in each slide. Statistical Analysis: All the data were analyzed using SPSS software version 16.0. The tissue localization, cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity was compared across the groups using Pearson's Chi-square test. The mean percentage of cells stained for oral epithelial dysplasia, OSCC and normal oral mucosa were

Multistep carcinogenesis of normal human fibroblasts. Human fibroblasts immortalized by repeated treatment with Co-60 gamma rays were transformed into tumorigenic cells with Ha-ras oncogenes.

Science.gov (United States)

Namba, M; Nishitani, K; Fukushima, F; Kimoto, T

1988-01-01

Two normal mortal human fibroblast cell strains were transformed into immortal cell lines, SUSM-1 and KMST-6, by treatment with 4-nitroquinoline 1-oxide (4NQO) and Co-60 gamma rays, respectively. These immortalized cell lines showed morphological changes of cells and remarkable chromosome aberrations, but neither of them grew in soft agar or formed tumors in nude mice. The immortal cell line, KMST-6, was then converted into neoplastic cells by treatment with Harvey murine sarcoma virus (Ha-MSV) or the c-Ha-ras oncogene derived from a human lung carcinoma. These neoplastically transformed cells acquired anchorage-independent growth potential and developed tumors when transplanted into nude mice. All the tumors grew progressively without regression until the animals died of tumors. In addition, the tumors were transplantable into other nude mice. Normal human fibroblasts, on the other hand, were not transformed into either immortal or tumorigenic cells by treatment with Ha-MSV or c-Ha-ras alone. Our present data indicate that (1) the chemical carcinogen, 4NQO, or gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to immortality, and (2) the ras gene played a role in the progression of the immortally transformed cells to more malignant cells showing anchorage-independent growth and tumorigenicity. In other words, the immortalization process of human cells seems to be a pivotal or rate-limiting step in the carcinogenesis of human cells.

Reptin is required for the transcription of telomerase reverse transcriptase and over-expressed in gastric cancer

Directory of Open Access Journals (Sweden)

Liu Tiantian

2010-05-01

Full Text Available Abstract Background Telomerase is activated in oncogenesis, which confers an immortal phenotype to cancer cells. The AAA + ATPase Reptin is required for telomerase biogenesis by maintaining telomerase RNA (hTER stability and is aberrantly expressed in certain cancers. Given its role in chromatin remodeling and transcription regulation, we determined the effect of Reptin on the transcription of the telomerase reverse transcriptase (hTERT gene, a key component of the telomerase complex and its expression in gastric cancer. Results Knocking down Reptin or its partner Pontin using small interfering RNA in gastric and cervical cancer cells led to significant decreases in hTERT mRNA, but hTERT promoter activity was inhibited in only Reptin-depleted cells. Reptin interacted with the c-MYC oncoprotein and its stimulatory effect on the hTERTpromoter was significantly dependent on functional E-boxes in the promoter. Moreover, Reptin bound to the hTERT proximal promoter and the loss of the Reptin occupancy led to dissociation of c-MYC from the hTERT promoter in Reptin-depleted cells. Reptin inhibition dramatically impaired clonogenic potential of gastric cancer cells by inducing cell growtharrest and over-expression of Reptin was observed in primary gastric cancer specimens. Conclusions The hTERT gene is a direct target of Reptin, and hTERT transcription requires constitutive expression of Reptin and its cooperation with c-MYC. Thus, Reptin regulates telomerase at two different levels. This finding, together with the requirementof Reptin for the clonogenic potential of cancer cells and its over-expression in gastriccancer and other solid tumors, suggests that Reptin may be a putative therapeutic target.

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

Science.gov (United States)

Behjati, Mohaddeseh; Hashemi, Mohammad; Kazemi, Mohammad; Salehi, Mansoor; Javanmard, Shaghayegh Haghjooy

2017-01-01

Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis. Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured. ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels ( P = 0.09) and vWF ( P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue. The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Trend of telomerase activity change during human iPSC self-renewal and differentiation revealed by a quartz crystal microbalance based assay

Science.gov (United States)

Zhou, Yitian; Zhou, Ping; Xin, Yinqiang; Wang, Jie; Zhu, Zhiqiang; Hu, Ji; Wei, Shicheng; Ma, Hongwei

2014-11-01

Telomerase plays an important role in governing the life span of cells for its capacity to extend telomeres. As high activity of telomerase has been found in stem cells and cancer cells specifically, various methods have been developed for the evaluation of telomerase activity. To overcome the time-consuming procedures and complicated manipulations of existing methods, we developed a novel method named Telomeric Repeat Elongation Assay based on Quartz crystal microbalance (TREAQ) to monitor telomerase activity during the self-renewal and differentiation of human induced pluripotent stem cells (hiPSCs). TREAQ results indicated hiPSCs possess invariable telomerase activity for 11 passages on Matrigel and a steady decline of telomerase activity when differentiated for different periods, which is confirmed with existing golden standard method. The pluripotency of hiPSCs during differentiation could be estimated through monitoring telomerase activity and compared with the expression levels of markers of pluripotency gene via quantitative real time PCR. Regular assessment for factors associated with pluripotency or stemness was expensive and requires excessive sample consuming, thus TREAQ could be a promising alternative technology for routine monitoring of telomerase activity and estimate the pluripotency of stem cells.

A Cajal body-independent pathway for telomerase trafficking in mice

International Nuclear Information System (INIS)

Tomlinson, Rebecca L.; Li, Jian; Culp, Bradley R.; Terns, Rebecca M.; Terns, Michael P.

2010-01-01

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.

A Cajal body-independent pathway for telomerase trafficking in mice

Energy Technology Data Exchange (ETDEWEB)

Tomlinson, Rebecca L.; Li, Jian; Culp, Bradley R.; Terns, Rebecca M., E-mail: rterns@bmb.uga.edu; Terns, Michael P., E-mail: mterns@bmb.uga.edu

2010-10-15

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.

Immortalization of human neural stem cells with the c-myc mutant T58A.

Directory of Open Access Journals (Sweden)

Lidia De Filippis

Full Text Available Human neural stem cells (hNSC represent an essential source of renewable brain cells for both experimental studies and cell replacement therapies. Their relatively slow rate of proliferation and physiological senescence in culture make their use cumbersome under some experimental and pre-clinical settings. The immortalization of hNSC with the v-myc gene (v-IhNSC has been shown to generate stem cells endowed with enhanced proliferative capacity, which greatly facilitates the study of hNSCs, both in vitro and in vivo. Despite the excellent safety properties displayed by v-IhNSCs--which do not transform in vitro and are not tumorigenic in vivo--the v-myc gene contains several mutations and recombination elements, whose role(s and effects remains to be elucidated, yielding unresolved safety concerns. To address this issue, we used a c-myc T58A retroviral vector to establish an immortal cell line (T-IhNSC from the same hNSCs used to generate the original v-IhNSCs and compared their characteristics with the latter, with hNSC and with hNSC immortalized using c-myc wt (c-IhNSC. T-IhNSCs displayed an enhanced self-renewal ability, with their proliferative capacity and clonogenic potential being remarkably comparable to those of v-IhNSC and higher than wild type hNSCs and c-IhNSCs. Upon growth factors removal, T-IhNSC promptly gave rise to well-differentiated neurons, astrocytes and most importantly, to a heretofore undocumented high percentage of human oligodendrocytes (up to 23%. Persistent growth-factor dependence, steady functional properties, lack of ability to generate colonies in soft-agar colony-forming assay and to establish tumors upon orthotopic transplantation, point to the fact that immortalization by c-myc T58A does not bring about tumorigenicity in hNSCs. Hence, this work describes a novel and continuous cell line of immortalized human multipotent neural stem cells, in which the immortalizing agent is represented by a single gene which, in

Nutrition and lifestyle in healthy aging: the telomerase challenge.

Science.gov (United States)

Boccardi, Virginia; Paolisso, Giuseppe; Mecocci, Patrizia

2016-01-01

Nutrition and lifestyle, known to modulate aging process and age-related diseases, might also affect telomerase activity. Short and dysfunctional telomeres rather than average telomere length are associated with longevity in animal models, and their rescue by telomerase maybe sufficient to restore cell and organismal viability. Improving telomerase activation in stem cells and potentially in other cells by diet and lifestyle interventions may represent an intriguing way to promote health-span in humans.

The People Paradox: Self-Esteem Striving, Immortality Ideologies, and Human Response to Climate Change

Directory of Open Access Journals (Sweden)

Janis L. Dickinson

2009-06-01

Full Text Available In 1973, Ernest Becker, a cultural anthropologist cross-trained in philosophy, sociology, and psychiatry, invoked consciousness of self and the inevitability of death as the primary sources of human anxiety and repression. He proposed that the psychological basis of cooperation, competition, and emotional and mental health is a tendency to hold tightly to anxiety-buffering cultural world views or "immortality projects" that serve as the basis for self-esteem and meaning. Although he focused mainly on social and political outcomes like war, torture, and genocide, he was increasingly aware that materialism, denial of nature, and immortality-striving efforts to control, rather than sanctify, the natural world were problems whose severity was increasing. In this paper I review Becker's ideas and suggest ways in which they illuminate human response to global climate change. Because immortality projects range from belief in technology and materialism to reverence for nature or belief in a celestial god, they act both as barriers to and facilitators of sustainable practices. I propose that Becker's cross-disciplinary "science of man," and the predictions it generates for proximate-level determinants of social behavior, add significantly to our understanding of and potential for managing the people paradox, i.e., that the very things that bring us symbolic immortality often conflict with our prospects for survival. Analysis of immortality projects as one of the proximate barriers to addressing climate change is both cautionary and hopeful, providing insights that should be included in the cross-disciplinary quest to uncover new pathways toward rational, social change.

Telomerase Repeated Amplification Protocol (TRAP).

Science.gov (United States)

Mender, Ilgen; Shay, Jerry W

2015-11-20

Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al. , 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase-consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC - counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al. , 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

International Nuclear Information System (INIS)

Schrader, Mark; Burger, Angelika M; Müller, Markus; Krause, Hans; Straub, Bernd; Schostak, Martin; Schulze, Wolfgang; Lauke, Heidrun; Miller, Kurt

2002-01-01

The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT). Telomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression. These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

Directory of Open Access Journals (Sweden)

Schulze Wolfgang

2002-11-01

Full Text Available Abstract Background The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT, which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT. Methods Telomerase activity (TA was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. Results High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome showed no telomerase activity and only minimal hTERT expression. Conclusions These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status.

Network signatures of cellular immortalization in human lymphoblastoid cell lines

Energy Technology Data Exchange (ETDEWEB)

Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: jaepiljeon@hanmail.net [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)

2013-11-15

Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

Tiptoeing to chromosome tips: facts, promises and perils of today's human telomere biology.

Science.gov (United States)

Fajkus, J; Simícková, M; Maláska, J

2002-04-29

The past decade has witnessed an explosion of knowledge concerning the structure and function of chromosome terminal structures-telomeres. Today's telomere research has advanced from a pure descriptive approach of DNA and protein components to an elementary understanding of telomere metabolism, and now to promising applications in medicine. These applications include 'passive' ones, among which the use of analysis of telomeres and telomerase (a cellular reverse transcriptase that synthesizes telomeres) for cancer diagnostics is the best known. The 'active' applications involve targeted downregulation or upregulation of telomere synthesis, either to mortalize immortal cancer cells, or to rejuvenate mortal somatic cells and tissues for cellular transplantations, respectively. This article reviews the basic data on structure and function of human telomeres and telomerase, as well as both passive and active applications of human telomere biology.

The Emerging Roles for Telomerase in the Central Nervous System

Directory of Open Access Journals (Sweden)

Meng-Ying Liu

2018-05-01

Full Text Available Telomerase, a specialized ribonucleoprotein enzyme complex, maintains telomere length at the 3′ end of chromosomes, and functions importantly in stem cells, cancer and aging. Telomerase exists in neural stem cells (NSCs and neural progenitor cells (NPCs, at a high level in the developing and adult brains of humans and rodents. Increasing studies have demonstrated that telomerase in NSCs/NPCs plays important roles in cell proliferation, neuronal differentiation, neuronal survival and neuritogenesis. In addition, recent works have shown that telomerase reverse transcriptase (TERT can protect newborn neurons from apoptosis and excitotoxicity. However, to date, the link between telomerase and diseases in the central nervous system (CNS is not well reviewed. Here, we analyze the evidence and summarize the important roles of telomerase in the CNS. Understanding the roles of telomerase in the nervous system is not only important to gain further insight into the process of the neural cell life cycle but would also provide novel therapeutic applications in CNS diseases such as neurodegenerative condition, mood disorders, aging and other ailments.

Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

Science.gov (United States)

2014-01-01

Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts.

Science.gov (United States)

Hara, H; Kaji, H

1987-02-01

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.

Expression of telomerase reverse transcriptase in radiation-induced chronic human skin ulcer

International Nuclear Information System (INIS)

Zhao Po; Li Zhijun; Lu Yali; Zhong Mei; Gu Qingyang; Wang Dewen

2001-01-01

Objective: To investigate the expression of the catalytic subunit of telomerase, telomerase reverse transcriptase (TRT) and the possible relationship between the TRT and cancer transformation or poor healing in radiation-induced chronic ulcer of human skin. Methods: Rabbit antibody against human TRT and SP immunohistochemical method were used to detect TRT expression in 24 cases of formalin-fixed, paraffin-embed human skin chronic ulcer tissues induced by radiation, 5 cases of normal skin, 2 of burned skin, and 8 of carcinoma. Results: The positive rate for TRT was 58.3%(14/24) in chronic radiation ulcers, of which the strongly positive rate was 41.7%(10/24) and the weakly positive 16.7%(4/24), 0% in normal (0/5) and burned skin (0/2), and 100% in carcinoma (8/8). The strongly positive expression of TRT was observed almost always in the cytoplasm and nucleus of squamous epithelial cells of proliferative epidermis but the negative and partly weakly positive expression in the smooth muscles, endothelia of small blood vessels and capillaries, and fibroblasts. Chronic inflammtory cells, plasmacytes and lymphocytes also showed weakly positive for TRT. Conclusion: TRT expression could be involved in the malignant transformation of chronic radiation ulcer into squamous carcinoma, and in the poor healing caused by sclerosis of small blood vessels and lack of granulation tissue consisting of capillaries and fibroblasts

High levels of telomere dysfunction bestow a selective disadvantage during the progression of human oral squamous cell carcinoma.

Science.gov (United States)

Gordon, Katrina E; Ireland, Hazel; Roberts, Meryl; Steeghs, Karen; McCaul, James A; MacDonald, D Gordon; Parkinson, E Kenneth

2003-01-15

Human epithelial cells experience multiple barriers to cellular immortality in culture (mortality mechanisms 0, 1, and 2). Mortality mechanism 2 (M2) is termed crisis and involves telomere dysfunction due to lack of telomerase. However, proliferating normal keratinocytes in vivo can express telomerase, so it is unclear whether human squamous cell carcinomas (SCCs), which usually have high telomerase levels, develop from preexisting telomerase-positive precursors or by the activation of telomerase in telomerase-deficient somatic cells. We show that 6 of 29 oral SCCs show characteristics of M2 crisis in vivo, as indicated by a high anaphase bridge index (ABI), which is a good correlate of telomere dysfunction, and that 25 of 29 tumors possess some anaphase bridges. ABIs in excess of 0.2 in the primary tumor showed a decrease in the corresponding lymph node metastases. This suggests that high levels of telomere dysfunction (>0.2) and, by inference, M2 crisis bestow a selective disadvantage on SCCs during progression stages of the disease. Supporting this, SCCs with high levels of telomere dysfunction grow poorly in culture, and the ectopic expression of telomerase corrects this, together with other features of M2 crisis. Our data suggest that a substantial proportion of oral SCCs in vivo ultimately arise from telomerase-deficient keratinocytes rather than putative telomerase-proficient cells in the undifferentiated parts of the epithelium. Furthermore, the presence of significant levels of telomere dysfunction in a high proportion of SCCs at diagnosis but not in the normal epithelium implies that the therapeutic inhibition of telomerase should selectively compromise the growth of such tumors.

Shwachman-Diamond Syndrome Protein SBDS Maintains Human Telomeres by Regulating Telomerase Recruitment

Directory of Open Access Journals (Sweden)

Yi Liu

2018-02-01

Full Text Available Shwachman-Diamond syndrome (SDS is a rare pediatric disease characterized by various systemic disorders, including hematopoietic dysfunction. The mutation of Shwachman-Bodian-Diamond syndrome (SBDS gene has been proposed to be a major causative reason for SDS. Although SBDS patients were reported to have shorter telomere length in granulocytes, the underlying mechanism is still unclear. Here we provide data to elucidate the role of SBDS in telomere protection. We demonstrate that SBDS deficiency leads to telomere shortening. We found that overexpression of disease-associated SBDS mutants or knockdown of SBDS hampered the recruitment of telomerase onto telomeres, while the overall reverse transcriptase activity of telomerase remained unaffected. Moreover, we show that SBDS could specifically bind to TPP1 during the S phase of cell cycle, likely functioning as a stabilizer for TPP1-telomerase interaction. Our findings suggest that SBDS is a telomere-protecting protein that participates in regulating telomerase recruitment.

ORIGINAL ARTICLE Detection of human telomerase reverse ...

African Journals Online (AJOL)

salah

currently remains the gold standard procedure for diagnosis, yet, it is invasive and costly. Urinary cytopathology remains to be the only non-invasive alter- native method for diagnosis. Although it is tumour specific, yet it has a poor sensitivity, especially for low grade tumours. Detection of Telomerase enzyme in exfoliated ...

Human mesenchymal stem cells

DEFF Research Database (Denmark)

Abdallah, Basem; Kassem, Moustapha

2008-01-01

Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being...... introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided....

Estrogen induction of telomerase activity through regulation of the mitogen-activated protein kinase (MAPK dependent pathway in human endometrial cancer cells.

Directory of Open Access Journals (Sweden)

Chunxiao Zhou

Full Text Available Given that prolonged exposure to estrogen and increased telomerase activity are associated with endometrial carcinogenesis, our objective was to evaluate the interaction between the MAPK pathway and estrogen induction of telomerase activity in endometrial cancer cells. Estradiol (E2 induced telomerase activity and hTERT mRNA expression in the estrogen receptor (ER-α positive, Ishikawa endometrial cancer cell line. UO126, a highly selective inhibitor of MEK1/MEK2, inhibited telomerase activity and hTERT mRNA expression induced by E2. Similar results were also found after transfection with ERK 1/2-specific siRNA. Treatment with E2 resulted in rapid phosphorylation of p44/42 MAPK and increased MAPK activity which was abolished by UO126. The hTERT promoter contains two estrogen response elements (EREs, and luciferase assays demonstrate that these EREs are activated by E2. Exposure to UO126 or ERK 1/2-specific siRNA in combination with E2 counteracted the stimulatory effect of E2 on luciferase activity from these EREs. These findings suggest that E2-induction of telomerase activity is mediated via the MAPK pathway in human endometrial cancer cells.

[Telomerase activity in uveal melanomas].

Science.gov (United States)

Rohrbach, J M; Riedinger, C; Wild, M; Partsch, M

2000-05-01

The maximum number of cell divisions of a certain cell population is genetically fixed so that aging cells become non-dividing (senescent) at least. This replicative life span, also known as "Hayflick limit", is probably defined by a "critical" length of the telomeres. Telomeres are special DNA-sequences located at the four ends of the chromosomes which are shortened with each cell cycle. Cells of most, but not all malignant tumours have been shown to reactivate the enzyme telomerase so that telomeres can be reconstructed, "Hayflick limit" can be overcome, and unlimited cell division can be established. This study was undertaken to elucidate whether telomerase reactivation is used by uveal melanoma cells. Fresh tumour tissue was removed from 10 untreated uveal melanomas after enucleation. Telomerase activity was determined using a PCR ELISA according to the Telomeric Repeat Amplification Protocol (TRAP). Normal tissue of the skin and the conjunctiva served as control. Telomerase activity was detectable in 90% of the investigated uveal melanomas. All control specimens were telomerase negative. Uveal melanoma growth seems to depend on telomerase reactivation. Thus, telomerase inhibition could offer a new principle for uveal melanoma therapy in the future.

Immortality in the Christian Physicalistic Theology: A Critical Survey

Directory of Open Access Journals (Sweden)

Hasan Ahmadizade

2017-07-01

Full Text Available Physicalistic Theology is a term that has no exact definition in theologian views. In the 20th century some of Christian thinkers on theology, like Nancy Murphy and Peter van Inwagen, by accepting a Physicalistic approach on human being, tried to analyze the Christian beliefs about human identity and his immortality. This approach today is called Physicalistic Theology. According to this approach, human is not but this physical body itself and so we can simply analyze the immortality problem. In this article we try to by an analytic and descriptive method, analyze the immortality of human according to the view of Physicalistic Theology. We will analyze the most important reasoning of Physicalistic Theology that is: no-interaction between the material and the immaterial, interaction between the person and the body, and the physicalism in Christian beliefs. One of the conclusions of this article is that according to Physicalistic view, the person that at some time has not been in the world, must exists any time to destroyed forever because the Christians believe to things that cannot justify rationally. The problem of immortality is one of these matters. Physicalistic Theology try to prove the immortality based on the miracles and the absolute power of God.

Curcumin Regulates Low-Linear Energy Transfer γ-Radiation-Induced NFκB-Dependent Telomerase Activity in Human Neuroblastoma Cells

International Nuclear Information System (INIS)

Aravindan, Natarajan; Veeraraghavan, Jamunarani; Madhusoodhanan, Rakhesh; Herman, Terence S.; Natarajan, Mohan

2011-01-01

Purpose: We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NFκB regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NFκB-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells. Methods and Materials: SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NFκB-dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, or pUSE-IκBα-Luc, p50/p65, or RelA siRNA-transfected cells. NFκB activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay. Results: Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NFκB becomes functionally activated after IR and mediates TA upregulation by binding to the κB-binding region in the promoter region of the TERT gene. Consistently, elimination of the NFκB-recognition site on the telomerase promoter or inhibition of NFκB by the IκBα mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NFκB overexpressed cells. Furthermore, curcumin enhanced the IR-induced inhibition of cell survival. Conclusions: These results

Telomerases: chemistry, biology, and clinical applications

National Research Council Canada - National Science Library

Lue, Neal F; Autexier, Chantal

2012-01-01

.... Other topics include telomerase biogenesis, transcriptional and post-translational regulation, off-telomere functions of telomerase and the role of telomerase in cellular senescence, aging and cancer...

Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice

Science.gov (United States)

Li, Runqin; Zhang, Yinglin

2016-01-01

Background and Objective: Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer. Materials and Methods: The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin. Results: The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53. Conclusion: The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells. PMID:27402632

Progressive Increase in Telomerase Activity From Benign Melanocytic Conditions to Malignant Melanoma

Directory of Open Access Journals (Sweden)

Ruben D. Ramirez

1999-04-01

Full Text Available The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction—based assay (TRAP. High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastasess = 54.5, lymph node metastasess = 56.5. Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9. Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Telomerase activity as a marker for malignancy in feline tissues.

Science.gov (United States)

Cadile, C D; Kitchell, B E; Biller, B J; Hetler, E R; Balkin, R G

2001-10-01

To establish the diagnostic significance of the telomeric repeat amplification protocol (TRAP) assay in detecting feline malignancies. Solid tissue specimens collected from 33 client-owned cats undergoing diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital between July 1997 and September 1999 and an additional 20 tissue samples were collected from 3 clinically normal control cats euthanatized at the conclusion of an unrelated study. The TRAP assay was used for detection of telomerase activity. Each result was compared to its respective histopathologic diagnosis. Twenty-nine of 31 malignant and 1 of 22 benign or normal tissue samples had telomerase activity, indicating 94% sensitivity and 95% specificity of the TRAP assay in our laboratory. The diagnostic significance of telomerase activity has been demonstrated in humans and recently in dogs by our laboratory. We tested feline samples to determine whether similar patterns of telomerase activity exist. On the basis of our results, the TRAP assay may be clinically useful in providing a rapid diagnosis of malignancy in cats. The telomerase enzyme may also serve as a therapeutic target in feline tumors.

Immortal ethics.

Science.gov (United States)

Harris, John

2004-06-01

This article draws on ideas published in my "Intimations of Immortality" essay in Science (Vol. 288, No. 5463, p. 59, April 7, 2000) and my "Intimations of Immortality-The Ethics and Justice of Life Extending Therapies" in editor Michael Freeman's Current Legal Problems (Oxford University Press 2002: 65-97). This article outlines the ethical issues involved in life-extending therapies. The arguments against life extension are examined and found wanting. The consequences of life extension are explored and found challenging but not sufficiently daunting to warrant regulation or control. In short, there is no doubt that immortality would be a mixed blessing, but we should be slow to reject cures for terrible diseases that may be an inextricable part of life-extending procedures even if the price we have to pay for those cures is increasing life expectancy and even creating immortals. Better surely to accompany the scientific race to achieve immortality with commensurate work in ethics and social policy to ensure that we know how to cope with the transition to parallel populations of mortals and immortals as envisaged in mythology.

Telomerase activity and its association with psychological stress, mental disorders, lifestyle factors and interventions: A systematic review.

Science.gov (United States)

Deng, W; Cheung, S T; Tsao, S W; Wang, X M; Tiwari, A F Y

2016-02-01

To summarise and discuss the association between telomerase activity and psychological stress, mental disorders and lifestyle factors. A systematic review was carried out to identify prospective or retrospective studies and interventions published up to June 2015 that reported associations between telomerase activity and psychological stress, mental disorders and lifestyle factors. Electronic data bases of PubMed, ProQuest, CINAHL and Google Scholar were searched. Twenty six studies on humans measured telomerase activity in peripheral blood mononuclear cells (PBMCs) or leukocytes and examined its association with psychological stress, mental disorders and lifestyle factors. Of those studies, three reported significantly decreased telomerase activity in individuals under chronic psychological stress. Interestingly, one of the three studies found that acute laboratory psychological stress significantly increased telomerase activity. Nine studies reported mixed results on association between mental disorders and telomerase activity. Of the nine studies, five reported that major depressive disorder (MDD) was associated with significantly increased telomerase activity. In thirteen out of fourteen studies on lifestyle factors, it was reported that physical exercise, diet micronutrient supplementation, mindfulness meditation, Qigong practice or yoga mediation resulted in increase in telomerase activity. In addition, two studies on animal models showed that depression-like behaviour was associated with decreased hippocampus telomerase activity. Five animal studies showed that physical exercise increased telomerase activity by cell-type-specific and genotype-specific manners. Although multi-facet results were reported on the association between telomerase activity and psychological stress, mental disorders and lifestyle factors, there were some consistent findings in humans such as (1) decreased telomerase activity in individuals under chronic stress, (2) increased

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines.

Science.gov (United States)

Touat-Hamici, Zahia; Bulteau, Anne-Laure; Bianga, Juliusz; Jean-Jacques, Hélène; Szpunar, Joanna; Lobinski, Ryszard; Chavatte, Laurent

2018-04-13

Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76 Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture. Copyright © 2018 Elsevier B.V. All rights reserved.

Telomerase activity-independent function of telomerase reverse transcriptase is involved in acrylamide-induced neuron damage.

Science.gov (United States)

Zhang, P; Pan, H; Wang, J; Liu, X; Hu, X

2014-07-01

Polyacrylamide is used widely in industry, and its decomposition product, acrylamide (ACR), readily finds its way into commonly consumed cosmetics and baked and fried foods. ACR exerts potent neurotoxic effects in human and animal models. Telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase, traditionally has been considered to play an important role in maintaining telomere length. Emerging evidence has shown, however, that TERT plays an important role in neuroprotection by inhibiting apoptosis and excitotoxicity, and by promoting angiogenesis, neuronal survival and neurogenesis, which are closely related to the telomere-independent functions of TERT. We investigated whether and how the TERT pathway is involved in ACR induced neurotoxicity in rat cortical neurons. We found that ACR 1) significantly reduced the viability of cortical neurons as measured by MTT assay, 2) induced neuron apoptosis as revealed by FITC-conjugated Annexin V/PI double staining and flow cytometry (FACS) analysis, 3) elevated expression of cleaved caspase-3, and 4) decreased bcl-2 expression of cortical neurons. ACR also increased intracellular ROS levels in cortical neurons, increased MDA levels and reduced GSH, SOD and GSH-Px levels in mitochondria in a dose-dependent manner. We found that TERT expression in mitochondria was increased by ACR at concentrations of 2.5 and 5.0 mM, but TERT expression was decreased by 10 mM ACR. Telomerase activity, however, was undetectable in rat cortical neurons. Our results suggest that the TERT pathway is involved in ACR induced apoptosis of cortical neurons. TERT also may exert its neuroprotective role in a telomerase activity-independent way, especially in mitochondria.

The Roles of Telomerase in the Generation of Polyploidy during Neoplastic Cell Growth

Directory of Open Access Journals (Sweden)

Agni Christodoulidou

2013-02-01

Full Text Available Polyploidy contributes to extensive intratumor genomic heterogeneity that characterizes advanced malignancies and is thought to limit the efficiency of current cancer therapies. It has been shown that telomere deprotection in p53-deficient mouse embryonic fibroblasts leads to high rates of polyploidization. We now show that tumor genome evolution through whole-genome duplication occurs in ∼15% of the karyotyped human neoplasms and correlates with disease progression. In a panel of human cancer and transformed cell lines representing the two known types of genomic instability (chromosomal and microsatellite, as well as the two known pathways of telomere maintenance in cancer (telomerase activity and alternative lengthening of telomeres, telomere dysfunction-driven polyploidization occurred independently of the mutational status of p53. Depending on the preexisting context of telomere maintenance, telomerase activity and its major components, human telomerase reverse transcriptase (hTERT and human telomerase RNA component (hTERC, exert both reverse transcriptase-related (canonical and noncanonical functions to affect tumor genome evolution through suppression or induction of polyploidization. These new findings provide a more complete mechanistic understanding of cancer progression that may, in the future, lead to novel therapeutic interventions.

Immortalization of Werner syndrome and progeria fibroblasts

International Nuclear Information System (INIS)

Saito, H.; Moses, R.E.

1991-01-01

Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents

When Telomerase Causes Telomere Loss.

Science.gov (United States)

Glousker, Galina; Lingner, Joachim

2018-02-05

Telomerase counteracts telomere shortening, preventing cellular senescence. Telomerase deficiency causes telomere syndromes because of premature telomere exhaustion in highly proliferative cells. Paradoxically, in a recent issue of Cell, Margalef et al. (2018) demonstrate that telomerase causes telomere loss in cells lacking the RTEL1 helicase, which is defective in Hoyeraal-Hreidarsson syndrome (HHS). Copyright © 2018 Elsevier Inc. All rights reserved.

Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562, and HL-60.

Science.gov (United States)

Deezagi, Abdolkhaleg; Manteghi, Sanaz; Khosravani, Pardis; Vaseli-Hagh, Neda; Soheili, Zahra-Soheila

2009-09-01

The purpose of this research was to understand the effect of hyperthermia on the telomerase activity in human leukemic cell lines (HL-60, K562, and TF-1). The cells were treated by hyperthermia at the range of 41-44 degrees C for 120 min and incubated for 96 h. Then telomerase activity, cell proliferation, and apoptosis were assessed. The results indicated that hyperthermia significantly induced apoptosis on the cells. The cells exhibited pre-apoptotic pattern at 41 and 42 degrees C at 60-120 min and apoptotic pattern at 43 and 44 degrees C over 30 min after hyperthermia. Telomerase activity (that was assayed immediately after hyperthermia) was stable at 41-42 degrees C for 60 min but decreased to 35-40% at 120 min. However, at severe hyperthermia (43-44 degrees C) telomerase activity was decreased in a time- and dose-dependent manner. Following hyperthermia (41-44 degrees C up to 120 min), the cells were incubated for 96 h. In these conditions, the telomerase activity was decreased by about 60-80% in comparison with that untreated control cells.

Mesenchymal stem cells: cell biology and potential use in therapy

DEFF Research Database (Denmark)

Kassem, Moustapha; Kristiansen, Malthe; Abdallah, Basem M

2004-01-01

Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods...... are currently available for isolation of the mesenchymal stem cells based on their physical and immunological characteristics. Because of the ease of their isolation and their extensive differentiation potential, mesenchymal stem cells are among the first stem cell types to be introduced in the clinic. Recent...... studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells...

Telomerase in lung cancer diagnostics

International Nuclear Information System (INIS)

Kovkarova, E.; Stefanovski, T.; Dimov, A.; Naumovski, J.

2003-01-01

Background. Telomerase is a ribonucleoprotein that looks after the telomeric cap of the linear chromosomes maintaining its length. It is over expressed in tumour tissues, but not in normal somatic cells. Therefore the aim of this study was to determine the telomerase activity in lung cancer patients as novel marker for lung cancer detection evaluating the influence of tissue/cell obtaining technique. Material and methods. Using the TRAP (telomeric repeat amplification protocol), telomerase activity was determined in material obtained from bronchobiopsy (60 lung cancer patients compared with 20 controls) and washings from transthoracic fine needle aspiration biopsy performed in 10 patients with peripheral lung tumours. Results. Telomerase activity was detected in 75% of the lung cancer bronchobyopsies, and in 100% in transthoracic needle washings. Conclusions. Measurement of telomerase activity can contribute in fulfilling the diagnosis of lung masses and nodules suspected for lung cancer. (author)

Behaviour of telomere and telomerase during aging and regeneration in zebrafish.

Science.gov (United States)

Anchelin, Monique; Murcia, Laura; Alcaraz-Pérez, Francisca; García-Navarro, Esther M; Cayuela, María L

2011-02-09

Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.

In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

International Nuclear Information System (INIS)

Liu Hua; Luan Fang; Ju Ying; Shen Hongyu; Gao Lifen; Wang Xiaoyan; Liu Suxia; Zhang Lining; Sun Wensheng; Ma Chunhong

2007-01-01

The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation

In vitro transfection of the hepatitis B virus PreS2 gene into the human hepatocarcinoma cell line HepG2 induces upregulation of human telomerase reverse transcriptase

Energy Technology Data Exchange (ETDEWEB)

Hua, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Fang, Luan [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Ying, Ju [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Hongyu, Shen [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lifen, Gao [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Xiaoyan, Wang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Suxia, Liu [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Lining, Zhang [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Wensheng, Sun [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Chunhong, Ma [Institute of Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan 250012 (China); Key Laboratory for Experimental Teratology, Ministry of Education (China)]. E-mail: machunhong@sdu.edu.cn

2007-04-06

The preS2 domain is the minimal functional unit of transcription activators that is encoded by the Hepatitis B virus (HBV) surface (S) gene. It is present in more than one-third of the HBV-integrates in HBV induced hepatocarcinoma (HCC). To further understand the functional role of PreS2 in hepatocytes, a PreS2 expression plasmid, pcS2, was constructed and stably transfected into HepG2 cells. We conducted growth curve and colony-forming assays to study the impact of PreS2 expression on cell proliferation. Cells transfected with PreS2 proliferated more rapidly and formed colonies in soft agar. PreS2 expressing cells also induced upregulation of human telomerase reverse transcriptase (hTERT) and telomerase activation by RT-PCR and the modified TRAP assay. Blocking expression of hTERT with antisense oligonuleotide reversed the growth rate in cells stably transfected with PreS2. Our data suggest that PreS2 may increase the malignant transformation of human HCC cell line HepG2 by upregulating hTERT and inducing telomerase activation.

Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.

Science.gov (United States)

Berman, Andrea J; Gooding, Anne R; Cech, Thomas R

2010-10-01

The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

A novel peptide-nucleotide dual vaccine of human telomerase reverse transcriptase induces a potent cytotoxic T-cell response in vivo

International Nuclear Information System (INIS)

Guo, Hong; Hao, Jia; Wu, Chao; Shi, Yun; Zhao, Xiao-yan; Fang, Dian-chun

2007-01-01

Human telomerase reverse transcriptase (hTERT) is highly expressed in over 85% of human cancers, which makes it a broadly applicable molecular target for cancer therapy. Several groups have demonstrated that hTERT can efficiently evoke specific cytotoxic T lymphocytes (CTL) responses for malignant tumors. In the present study, we developed a novel virus-like particulate peptide-nucleotide dual vaccine (PNDV) of hTERT, which was composed of a low-affinity epitope variant with encoding full-length gene in the same virus-size particulate. We verified the formation of PNDV by DNA retarding assay, DNase I protection assay and transmission electron microscopy, and confirmed its immunogenicity and transfection activities in mammalian cells. Furthermore, in vivo immunization of HLA-A2.1 transgenic mice generated efficient IFN-γ secretion and hTERT-specific CTLs which are known to cause selective cell death of telomerase positive gastrointestinal cancer cells. To our knowledge, this represents the first report on collocating a low-affinity epitope variant with a full-length hTERT gene for anti-cancer vaccine design. This novel strategy for vaccine design not only enables enhanced immunity to a universal tumor antigen, but also has the potential to generate CTLs effective in telomerase-positive tumor cells of diverse tissue origins. Therefore, our findings bear significant implications for immunotherapy of human cancers

Establishment, characterization and immortalization of a fibroblast cell line from the Chinese red belly toad Bombina maxima skin.

Science.gov (United States)

Xiang, Yang; Gao, Qian; Su, Weiting; Zeng, Lin; Wang, Jinhuan; Hu, Yi; Nie, Wenhui; Ma, Xutong; Zhang, Yong; Lee, Wenhui; Zhang, Yun

2012-01-01

The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n = 28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.

Inhibition of telomerase by linear-chain fatty acids: a structural analysis.

Science.gov (United States)

Oda, Masako; Ueno, Takamasa; Kasai, Nobuyuki; Takahashi, Hirotada; Yoshida, Hiromi; Sugawara, Fumio; Sakaguchi, Kengo; Hayashi, Hideya; Mizushina, Yoshiyuki

2002-01-01

In the present study, we have found that mono-unsaturated linear-chain fatty acids in the cis configuration with C(18) hydrocarbon chains (i.e. oleic acid) strongly inhibited the activity of human telomerase in a cell-free enzymic assay, with an IC(50) value of 8.6 microM. Interestingly, fatty acids with hydrocarbon chain lengths below 16 or above 20 carbons substantially decreased the potency of inhibition of telomerase. Moreover, the cis-mono-unsaturated C(18) linear-chain fatty acid oleic acid was the strongest inhibitor of all the fatty acids tested. A kinetic study revealed that oleic acid competitively inhibited the activity of telomerase ( K (i)=3.06 microM) with respect to the telomerase substrate primer. The energy-minimized three-dimensional structure of the linear-chain fatty acid was calculated and modelled. A molecule width of 11.53-14.26 A (where 1 A=0.1 nm) in the C(16) to C(20) fatty acid structure was suggested to be important for telomerase inhibition. The three-dimensional structure of the telomerase active site (i.e. the substrate primer-binding site) appears to have a pocket that could bind oleic acid, with the pocket being 8.50 A long and 12.80 A wide. PMID:12121150

Telomere lengthening and other functions of telomerase.

Science.gov (United States)

Rubtsova, M P; Vasilkova, D P; Malyavko, A N; Naraikina, Yu V; Zvereva, M I; Dontsova, O A

2012-04-01

Telomerase is an enzyme that maintains the length of the telomere. The telomere length specifies the number of divisions a cell can undergo before it finally dies (i.e. the proliferative potential of cells). For example, telomerase is activated in embryonic cell lines and the telomere length is maintained at a constant level; therefore, these cells have an unlimited fission potential. Stem cells are characterized by a lower telomerase activity, which enables only partial compensation for the shortening of telomeres. Somatic cells are usually characterized by the absence of telomerase activity. Telomere shortening leads to the attainment of the Hayflick limit, the transition of cells to a state of senescence. The cells subsequently enter a state of crisis, accompanied by massive cell death. The surviving cells become cancer cells, which are capable both of dividing indefinitely and maintaining telomere length (usually with the aid of telomerase). Telomerase is a reverse transcriptase. It consists of two major components: telomerase RNA (TER) and reverse transcriptase (TERT). TER is a non-coding RNA, and it contains the region which serves as a template for telomere synthesis. An increasing number of articles focussing on the alternative functions of telomerase components have recently started appearing. The present review summarizes data on the structure, biogenesis, and functions of telomerase.

DETECTION OF TELOMERASE ACTIVITY IN BREAST CARCINOMA

Institute of Scientific and Technical Information of China (English)

Yang Wentao; Xu Liangzhong; Zhang Taiming; Zhu weiping; Li Xiaomei; Jin Aiping

1998-01-01

Objective:To investigate the significance of telomerase activity in breast carcinoma with its respect to axillary lymph node status. Methods: Telomerase activity was analyzed in 88 breast carcinomas and 16benign breast lesions, using polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay. Results: Telomerase activity was detected in 75 (85%) of 88 breast carcinomas (including three breast carcinomas in situ which were all positive for telomerase activity), whereas in benign breast lesions analyzed only 2(12.5%) of 16 cases were positive for telomerase activity. The difference between the two groups was statistically significant (P＜0.001). Besides,telomerase activity was expressed significantly higher in node-positive breast carcinoma (93%) than in nodenegative ones (77%) (P＜0.05). Conclusion: Our results suggest that telomerase activation plays an important role during breast carcinoma development. It is possible that this enzyme may serve as an early indication of breast carcinoma.

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

OpenAIRE

Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Na?ra; Rau, Fr?d?rique; Jollet, Arnaud; Edom-Vovard, Fr?d?rique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois

2017-01-01

International audience; Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded rep...

Improved Inhibition of Telomerase by Short Twisted Intercalating Nucleic Acids under Molecular Crowding Conditions

DEFF Research Database (Denmark)

Agarwal, Tani; Pradhan, Devranjan; Géci, Imrich

2012-01-01

Human telomeric DNA has the ability to fold into a 4-stranded G-quadruplex structure. Several G-quadruplex ligands are known to stabilize the structure and thereby inhibit telomerase activity. Such ligands have demonstrated efficient telomerase inhibition in dilute conditions, but under molecular...

Immortalization protocols used in cell culture models of human breast morphogenesis

DEFF Research Database (Denmark)

Gudjonsson, T; Villadsen, R; Rønnov-Jessen, L

2004-01-01

of the tissue of origin. In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern. This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application...

Leptin as a critical regulator of hepatocellular carcinoma development through modulation of human telomerase reverse transcriptase

Directory of Open Access Journals (Sweden)

Stefanou Nikolaos

2010-08-01

Full Text Available Abstract Background Numerous epidemiological studies have documented that obesity is associated with hepatocellular carcinoma (HCC. The aim of this study was to investigate the biological actions regulated by leptin, the obesity biomarker molecule, and its receptors in HCC and the correlation between leptin and human telomerase reverse transcriptase (hTERT, a known mediator of cellular immortalization. Methods We investigated the relationship between leptin, leptin receptors and hTERT mRNA expression in HCC and healthy liver tissue samples. In HepG2 cells, chromatin immunoprecipitation assay was used to study signal transducer and activator of transcription-3 (STAT3 and myc/mad/max transcription factors downstream of leptin which could be responsible for hTERT regulation. Flow cytometry was used for evaluation of cell cycle modifications and MMP1, 9 and 13 expression after treatment of HepG2 cells with leptin. Blocking of leptin's expression was achieved using siRNA against leptin and transfection with liposomes. Results We showed, for the first time, that leptin's expression is highly correlated with hTERT expression levels in HCC liver tissues. We also demonstrated in HepG2 cells that leptin-induced up-regulation of hTERT and TA was mediated through binding of STAT3 and Myc/Max/Mad network proteins on hTERT promoter. We also found that leptin could affect hepatocellular carcinoma progression and invasion through its interaction with cytokines and matrix mettaloproteinases (MMPs in the tumorigenic microenvironment. Furthermore, we showed that histone modification contributes to leptin's gene regulation in HCC. Conclusions We propose that leptin is a key regulator of the malignant properties of hepatocellular carcinoma cells through modulation of hTERT, a critical player of oncogenesis.

Behaviour of telomere and telomerase during aging and regeneration in zebrafish.

Directory of Open Access Journals (Sweden)

Monique Anchelin

Full Text Available Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.

MNS16A tandem repeats minisatellite of human telomerase gene: a risk factor for colorectal cancer.

Science.gov (United States)

Hofer, Philipp; Baierl, Andreas; Feik, Elisabeth; Führlinger, Gerhard; Leeb, Gernot; Mach, Karl; Holzmann, Klaus; Micksche, Michael; Gsur, Andrea

2011-06-01

Telomerase reactivation and expression of human telomerase gene [human telomerase reverse transcriptase (hTERT)] are hallmarks of unlimited proliferation potential of cancer cells. A polymorphic tandem repeats minisatellite of hTERT gene, termed MNS16A was reported to influence hTERT expression. To assess the role of MNS16A as potential biomarker for colorectal cancer (CRC), we investigated for the first time the association of MNS16A genotypes with risk of colorectal polyps and CRC. In the ongoing colorectal cancer study of Austria (CORSA), 3842 Caucasian participants were recruited within a large screening project in the province Burgenland including 90 CRC cases, 308 high-risk polyps, 1022 low-risk polyps and 1822 polyp free controls verified by colonoscopy. MNS16A genotypes were determined by polymerase chain reaction from genomic DNA. Associations of MNS16A genotypes with CRC risk were estimated by logistic regression analysis computing odds ratios (ORs) and 95% confidence intervals (CIs). We identified five different variable number of tandem repeats (VNTRs) of MNS16A including VNTR-364, a newly discovered rare variant. VNTR-274 allele was associated with a 2.7-fold significantly increased risk of CRC compared with the VNTR-302 wild-type (OR = 2.69; 95% CI = 1.11-6.50; P = 0.028). In our CORSA study, the medium length VNTR-274 was identified as risk factor for CRC. Although, this population-based study herewith reports the largest cohort size concerning MNS16A thus far, further large-scale studies in diverse populations are warranted to confirm hTERT MNS16A genotype as potential biomarker for assessment of CRC risk.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

OpenAIRE

Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

2007-01-01

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipa...

Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

Science.gov (United States)

2007-06-01

human mammary epithelial cell types by human papilloma virus 16 e6 or e7. Proc Nat Acad Sci USA 1995; 92:3687-91. 54. Shay JW, Pereira-Smith OM, Wright...Liu X-L, Chu Q, Gao Q, Band V. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 e6 or e7. Proc Nat Acad

Radiation-induced progressive decreasing in the expression of reverse transcriptase gene of hEST2 and telomerase activity

International Nuclear Information System (INIS)

Zhu Hanneng; Chen Wenying; Xiong Sidong

2000-01-01

Telomerase is a ribonucleoprotein complex that adds heximeric repeats called telomeres to the growing ends of chromosomal DNA. Telomerase activity is present in a vast majority of tumors but is repressed in most normal tissues. Human telomerase catalytic subunit gene (hEST2) reverse transcriptase (RT) segment was cloned by PCR according to the sequence published in GeneBank. PCR was used to investigate the expression of the hEST2 RT segment in diverse tumors as well as in various normal tissues. Results indicated that hEST2 RT segment was detectable in tumor cells lines but not in normal cells and tissues. In order to identify the relationship between telomerase and the biological effect of radiation injury, HeLa cells, KB cells and A431 cells were employed to measure the change in telomerase activity after 60 Co-ray irradiation at RNA level and protein level. Quantitative PCR determined that expression of hEST2 RT segment that encodes seven motifs of the human telomeras decreased with increasing dosage of radiation. In addition, a PCR-based telomeric repeat amplification protocol was used to assay telomerase activity after exposure to radiation. The results strongly support the experiments we had made: Telomerase activity decreases with increasing dosage of radiation. We conclude that detection of the hEST2 RT segment by Northern blotting is a new method for detecting telomerase activity. Furthermore, radiation can cause a dose-dependent decrease in telomerase activity. The effect of radiation on telomerase is one possible reason for the death of cancer cells after irradiation. (author)

Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas.

Directory of Open Access Journals (Sweden)

Yuwei Zhang

Full Text Available Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT and telomerase RNA component (TERC in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.Sequencing analysis revealed one deletion involving TERC (TERC del 341-360, and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous; A1062T (4 heterozygous]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01. Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs following Zeocin�

Telomere dynamics and cytogenetic changes in human hematologic neoplasias: a working hypothesis.

Science.gov (United States)

Ohyashiki, K; Ohyashiki, J H

1997-03-01

Chromosome termini, termed telomeres, provide important protection to avoid loss of master gene(s) that may exist at subtelomeric regions. Moreover, erosion of telomeres by cell division through end-replication problems resulted in telomeric-associated cytogenetic aberrations. To maintain a telomere length related to cell immortality, telomerase activity is upregulated in cancer cells, therefore, telomerase is considered to be a new marker of neoplasias. In this paper, we review and make suggestions regarding key aspects of telomere dynamics in both normal hematopoiesis and in malignant hematologic diseases.

Meningiomas, dicentric chromosomes, gliomas, and telomerase activity.

Science.gov (United States)

Carroll, T; Maltby, E; Brock, I; Royds, J; Timperley, W; Jellinek, D

1999-08-01

Lack of telomere maintenance during cell replication leads to telomere erosion and loss of function. This can result in telomere associations which probably cause the dicentric chromosomes seen in some tumour cells. One mechanism of telomere maintenance in dividing cells is the action of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening during cell division. Over 90 per cent of extracranial malignant neoplasms have been found to have telomerase activity. This study sought to determine if there was a relationship between absence of telomerase activity and presence of dicentric chromosomes in meningiomas and to what extent the other main group of central nervous system tumours, the gliomas, expressed telomerase activity. Telomerase activity was measured on 25 meningiomas and 29 gliomas. Four of the meningiomas were atypical variants and 11 were positive for dicentric chromosomes. Twenty-five of 29 gliomas were glioblastoma multiforme tumours. Measures were taken to ensure absence of false positives due to primer-dimer interaction and false negatives due to protein degradation or the presence of Taq polymerase inhibitors. All 25 meningiomas and the four low-grade gliomas (WHO grade II) were telomerase activity-negative. Seven (28 per cent) of the 25 glioblastoma multiforme tumours showed telomerase activity. The absence of telomerase activity in meningiomas and the high frequency of telomere associations support the hypothesis that these tumours are benign, transformed but pre-crisis. The relatively low frequency of telomerase activity in the malignant glioblastoma multiforme suggests that most of these tumours may have other mechanisms of telomere maintenance and that the potentially therapeutic telomerase inhibitors will not be of great value in the future management of the majority of patients suffering from these tumours. Copyright 1999 John Wiley & Sons, Ltd.

Isolation and characterization of exosome from human embryonic stem cell-derived c-myc-immortalized mesenchymal stem cells

NARCIS (Netherlands)

Lai, Ruenn Chai; Yeo, Ronne Wee Yeh; Padmanabhan, Jayanthi; Choo, Andre; De Kleijn, Dominique P V; Lim, Sai Kiang

2016-01-01

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular

Analysis of proviral integration in human mammary epithelial cell lines immortalized by retroviral infection with a temperature-sensitive SV40 T-antigen construct.

Science.gov (United States)

Stamps, A C; Davies, S C; Burman, J; O'Hare, M J

1994-06-15

A panel of eight conditionally immortal lines derived by infection of human breast epithelial cells with an amphotropic retrovirus transducing a ts mutant of SV40 large T-antigen was analyzed with respect to individual retroviral integration patterns. Each line contained multiple integration sites which were clonal and stable over extended passage. Similar integration patterns were observed between individual lines arising separately from the same stock of pre-immortal cells, suggesting a common progenitor. Retroviral integration analysis of pre-immortal cells at different stages of pre-crisis growth showed changes indicative of a progressive transition from polyclonality to clonality as the cells approached crisis. Each of the immortal lines contained a sub-set of the integration sites of their pre-immortal progenitors, with individual combinations and copy numbers of sites. Since all the cell lines appeared to originate from single foci in separate flasks, it is likely that each set arose from a common clone of pre-immortal cells as the result of separate genetic events. There was no evidence from this analysis to suggest that specific integration sites played any part either in the selection of pre-crisis clones or in the subsequent establishment of immortal lines.

Human MLH1 suppresses the insertion of telomeric sequences at intra-chromosomal sites in telomerase-expressing cells

Science.gov (United States)

Jia, Pingping; Chastain, Megan; Zou, Ying; Her, Chengtao

2017-01-01

Abstract Aberrant formation of interstitial telomeric sequences (ITSs) promotes genome instabilities. However, it is unclear how aberrant ITS formation is suppressed in human cells. Here, we report that MLH1, a key protein involved in mismatch repair (MMR), suppresses telomeric sequence insertion (TSI) at intra-chromosomal regions. The frequency of TSI can be elevated by double-strand break (DSB) inducer and abolished by ATM/ATR inhibition. Suppression of TSI requires MLH1 recruitment to DSBs, indicating that MLH1's role in DSB response/repair is important for suppressing TSI. Moreover, TSI requires telomerase activity but is independent of the functional status of p53 and Rb. Lastly, we show that TSI is associated with chromosome instabilities including chromosome loss, micronuclei formation and chromosome breakage that are further elevated by replication stress. Our studies uncover a novel link between MLH1, telomerase, telomere and genome stability. PMID:28180301

Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts

Directory of Open Access Journals (Sweden)

Easton Marilyn J

2010-02-01

Full Text Available Abstract Background Simian Virus 40 (SV40 immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC or SV40-immortalized and then 3-MC-transformed (HUC-TC. Results To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-γ and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-γ metabolic responses were functional in both cell lines, but IFN-γ anti-proliferative responses functioned only in tumor cells. Conclusions Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.

Telomerase Inhibitors from Natural Products and Their Anticancer Potential

Directory of Open Access Journals (Sweden)

Kumar Ganesan

2017-12-01

Full Text Available Telomeres and telomerase are nowadays exploring traits on targets for anticancer therapy. Telomerase is a unique reverse transcriptase enzyme, considered as a primary factor in almost all cancer cells, which is mainly responsible to regulate the telomere length. Hence, telomerase ensures the indefinite cell proliferation during malignancy—a hallmark of cancer—and this distinctive feature has provided telomerase as the preferred target for drug development in cancer therapy. Deactivation of telomerase and telomere destabilization by natural products provides an opening to succeed new targets for cancer therapy. This review aims to provide a fundamental knowledge for research on telomere, working regulation of telomerase and its various binding proteins to inhibit the telomere/telomerase complex. In addition, the review summarizes the inhibitors of the enzyme catalytic subunit and RNA component, natural products that target telomeres, and suppression of transcriptional and post-transcriptional levels. This extensive understanding of telomerase biology will provide indispensable information for enhancing the efficiency of rational anti-cancer drug design.

Telomerase activity and apoptosis genes as parameters of ...

African Journals Online (AJOL)

Ekram Abdel-Salam

2013-01-23

Jan 23, 2013 ... ORIGINAL ARTICLE. Telomerase ... The Egyptian Journal of Medical Human Genetics www.ejmhg.eg.net ... membrane protein that belongs to the tumor necrosis factor superfamily and ... revision of the 1975 Helsinki Declaration. Methods ... Determination of Soluble Fas was in duplicate plasma sam- ples.

Inhibition of UBE2D3 expression attenuates radiosensitivity of MCF-7 human breast cancer cells by increasing hTERT expression and activity.

Directory of Open Access Journals (Sweden)

Wenbo Wang

Full Text Available The known functions of telomerase in tumor cells include replenishing telomeric DNA and maintaining cell immortality. We have previously shown the existence of a negative correlation between human telomerase reverse transcriptase (hTERT and radiosensitivity in tumor cells. Here we set out to elucidate the molecular mechanisms underlying regulation by telomerase of radiosensitivity in MCF-7 cells. Toward this aim, yeast two-hybrid (Y2H screening of a human laryngeal squamous cell carcinoma radioresistant (Hep2R cDNA library was first performed to search for potential hTERT interacting proteins. We identified ubiquitin-conjugating enzyme E2D3 (UBE2D3 as a principle hTERT-interacting protein and validated this association biochemically. ShRNA-mediated inhibition of UBE2D3 expression attenuated MCF-7 radiosensitivity, and induced the accumulation of hTERT and cyclin D1 in these cells. Moreover, down-regulation of UBE2D3 increased hTERT activity and cell proliferation, accelerating G1 to S phase transition in MCF-7 cells. Collectively these findings suggest that UBE2D3 participates in the process of hTERT-mediated radiosensitivity in human breast cancer MCF-7 cells by regulating hTERT and cyclin D1.

Telomerer og telomerase

DEFF Research Database (Denmark)

Bendix, Laila; Kølvraa, Steen

2010-01-01

In 2009 the Nobel Prize in Medicine was awarded to EH Blackburn, CW Greider and JW Szostak for their work on "How chromosomes are protected by telomeres and the enzyme telomerase". Telomeres are specialized DNA structures localized at the end of linear chromosomes. Telomeres are known as the biol......In 2009 the Nobel Prize in Medicine was awarded to EH Blackburn, CW Greider and JW Szostak for their work on "How chromosomes are protected by telomeres and the enzyme telomerase". Telomeres are specialized DNA structures localized at the end of linear chromosomes. Telomeres are known...

Change alone is eternal, perpetual, immortal : pharmacological immortality in science fiction

OpenAIRE

Grech, Victor E.; Vassallo, Clare; Callus, Ivan

2012-01-01

Immortality is a common feature in science-fiction (SF). This paper lists the ways in which the pharmacological induction of immortality has been depicted in SF, and the resultant outcomes. Immortality or extreme longevity are often melded with infertility in order to eliminate the overpopulation issues that would inevitably arise. This is only one way in which theoretical utopias which afford life extension become dystopias, cautionary tales that admonish against hubris. In this fashion, SF ...

Apoptosis and reduced cell proliferation of HL-60 cell line caused by human telomerase reverse transcriptase inhibition by siRNA.

Science.gov (United States)

Miri-Moghaddam, Ebrahim; Deezagi, Abdolkhaleg; Soheili, Zahra Sohaila; Shariati, Parvin

2010-01-01

The close correlation between telomerase activity and human telomerase reverse transcriptase (hTERT) expression has made hTERT to be considered as a selective molecular target for human cancer therapy. In this study, the ability of short-interfering RNA (siRNA) to downregulate hTERT expression and its correlation with cell growth and apoptosis in the promyelocytic cell line HL-60 was evaluated. hTERT siRNA was designed and transfected to HL-60. hTERT mRNA expression, cell proliferation and apoptotic cells were measured. The results indicated that hTERT siRNA resulted in 97.2 ± 0.6% downregulation of the hTERT mRNA content; inhibition of the cell proliferation rate was about 52.8 ± 2.3% and the apoptotic index of cells was 30.5 ± 1.5%. hTERT plays an essential role in cell proliferation and control of the viability of leukemic cells, thus promising the development of drugs for leukemia. Copyright © 2010 S. Karger AG, Basel.

Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types

DEFF Research Database (Denmark)

Dabelsteen, Sally; Hercule, Paula; Barron, Patricia

2009-01-01

hESderK cells and keratinocytes a substantially extended lifespan. When exposed to transforming growth factor beta or to an incompletely processed form of Laminin-332, three lifespan-extended or immortalized hESderK lines that we studied became directionally hypermotile, a wound healing and invasion......(+)/K14(+) urothelial and tracheobronchial epithelial cells. Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase...

Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

Energy Technology Data Exchange (ETDEWEB)

Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James; Takenaka, Yasuhiro; Stampfer, Martha R.; Gilley, David; Yaswen, Paul

2004-12-23

We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.

Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

Energy Technology Data Exchange (ETDEWEB)

Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

2010-12-10

Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

International Nuclear Information System (INIS)

Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

2010-01-01

Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

Telomerase and the search for the end of cancer.

Science.gov (United States)

Mocellin, Simone; Pooley, Karen A; Nitti, Donato

2013-02-01

Many of the fundamental molecular mechanisms underlying tumor biology remain elusive and, thus, developing specific anticancer therapies remains a challenge. The recently discovered relationships identified among telomeres, telomerase, aging, and cancer have opened a new avenue in tumor biology research that may revolutionize anticancer therapy. This review summarizes the critical aspects of telomerase biology that underpin the development of novel telomerase-targeting therapies for malignant diseases, and special regard is given to the aspects of telomerase that make it such an appealing target, such as the widespread expression of telomerase in cancers. Despite significant progress, issues remain to be addressed before telomerase-based therapies are truly effective and we include critical discussion of the results obtained thus far. Copyright © 2012 Elsevier Ltd. All rights reserved.

Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

DEFF Research Database (Denmark)

Harbo, M.; Koelvraa, S.; Serakinci, N.

2012-01-01

mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells......A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just...... estimated by senescence-associated beta-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage...

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

International Nuclear Information System (INIS)

Kadivar, Mehdi; Khatami, Shohreh; Mortazavi, Yousef; Shokrgozar, Mohammad Ali; Taghikhani, Mohammad; Soleimani, Masoud

2006-01-01

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric α-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty

Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

Directory of Open Access Journals (Sweden)

Arnouk Hilal

2009-08-01

Full Text Available Abstract Background Infection with high-risk type human papilloma viruses (HPVs is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Results Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE. The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23% of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2% were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1; and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27. Conclusion This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

Differences in telomerase activity between colon and rectal cancer.

Science.gov (United States)

Ayiomamitis, Georgios D; Notas, George; Zaravinos, Apostolos; Zizi-Sermpetzoglou, Adamantia; Georgiadou, Maria; Sfakianaki, Ourania; Kouroumallis, Elias

2014-06-01

Colorectal cancer is one of the most common cancers and the third leading cause of cancer death in both sexes. The disease progresses as a multistep process and is associated with genetic alterations. One of the characteristic features of cancer is telomerase activation. We sought to evaluate the differences in telomerase activity between colon cancer and adjacent normal tissue and to correlate the differences in telomerase activity between different locations with clinicopathological factors and survival. Matched colon tumour samples and adjacent normal mucosa samples 10 cm away from the tumour were collected during colectomy. We assessed telomerase activity using real time polymerase chain reaction. Several pathological characteristics of tumours, including p53, Ki-67, p21, bcl2 and MLH1 expression were also studied. We collected samples from 49 patients. There was a significantly higher telomerase activity in colon cancer tissue than normal tissue. Adenocarcinomas of the right colon express significantly higher telomerase than left-side cancers. Colon cancers and their adjacent normal tissue had significantly more telomerase and were more positive to MLH1 than rectal cancers. The expression of p53 negatively correlated to telomerase activity and was linked to better patient survival. Colon and rectal cancers seem to have different telomerase and MLH1 profiles, and this could be another factor for their different biologic and clinical behaviour and progression. These results support the idea that the large bowel cannot be considered a uniform organ, at least in the biology of cancer.

Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

Czech Academy of Sciences Publication Activity Database

Garbe, J.C.; Vrba, Lukáš; Sputova, K.; Fuchs, L.; Novák, Petr; Brothman, A.R.; Jackson, M.; Chin, K.; LaBarge, M.A.; Watts, G.; Futscher, B. W.; Stampfer, M.R.

2014-01-01

Roč. 13, č. 21 (2014), s. 3423-3435 ISSN 1538-4101 Institutional support: RVO:60077344 Keywords : genomic instability * human mammary epithelial cells * telomerase Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 4.565, year: 2014

The People Paradox: Self-Esteem Striving, Immortality Ideologies, and Human Response to Climate Change

OpenAIRE

Janis L. Dickinson

2009-01-01

In 1973, Ernest Becker, a cultural anthropologist cross-trained in philosophy, sociology, and psychiatry, invoked consciousness of self and the inevitability of death as the primary sources of human anxiety and repression. He proposed that the psychological basis of cooperation, competition, and emotional and mental health is a tendency to hold tightly to anxiety-buffering cultural world views or "immortality projects" that serve as the basis for self-esteem and meaning. Although he focused m...

SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium.

Directory of Open Access Journals (Sweden)

Ya'an Kang

Full Text Available Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT. The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs. Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGFβ-SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at -3790 bp to -3795 bp within the promoter region of CDH2 was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.

Telomere Elongation and Naive Pluripotent Stem Cells Achieved from Telomerase Haplo-Insufficient Cells by Somatic Cell Nuclear Transfer

Directory of Open Access Journals (Sweden)

Li-Ying Sung

2014-12-01

Full Text Available Summary: Haplo-insufficiency of telomerase genes in humans leads to telomere syndromes such as dyskeratosis congenital and idiopathic pulmonary fibrosis. Generation of pluripotent stem cells from telomerase haplo-insufficient donor cells would provide unique opportunities toward the realization of patient-specific stem cell therapies. Recently, pluripotent human embryonic stem cells (ntESCs have been efficiently achieved by somatic cell nuclear transfer (SCNT. We tested the hypothesis that SCNT could effectively elongate shortening telomeres of telomerase haplo-insufficient cells in the ntESCs with relevant mouse models. Indeed, telomeres of telomerase haplo-insufficient (Terc+/− mouse cells are elongated in ntESCs. Moreover, ntESCs derived from Terc+/− cells exhibit naive pluripotency as evidenced by generation of Terc+/− ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency. These data suggest that SCNT could offer a powerful tool to reprogram telomeres and to discover the factors for robust restoration of telomeres and pluripotency of telomerase haplo-insufficient somatic cells. : Sung et al. demonstrate in a mouse model that telomeres of telomerase haplo-insufficient cells can be elongated by somatic cell nuclear transfer. Moreover, ntESCs derived from Terc+/− cells exhibit pluripotency evidenced by generation of Terc+/−ntESC clone pups by tetraploid embryo complementation, the most stringent test of naive pluripotency.

Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma.

Directory of Open Access Journals (Sweden)

Flávia S Donaires

Full Text Available Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3% in patients as compared to controls (p = 0.02. Three of the four variants (T726M, A1062T, and V1090M were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis. A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

A telomerase em células-tronco hematopoéticas Telomerase in hematopoietic stem cells

Directory of Open Access Journals (Sweden)

Silvana Perini

2008-02-01

Full Text Available A proliferação das células-tronco hematopoéticas sofre a perda dos telômeros a cada divisão celular. Alguns autores discordam quanto à perda ou não do potencial proliferativo e capacidade de auto-renovação das células mais diferenciadas. Revisaremos aqui o papel da telomerase na biologia do sistema hematopoético, na diferenciação normal ou maligna, assim como no envelhecimento das células-tronco hematopoéticas. A constante renovação celular requerida pela hematopoese confere às células-tronco embrionárias, assim como à maioria das células tumorais, um aumento da capacidade proliferativa marcada pela detecção da enzima telomerase e possível manutenção dos telômeros. Estudos clínicos se farão necessários para esclarecer melhor a atividade da telomerase em células-tronco hematopoéticas, seu possível uso como marcador de diagnóstico e seu uso a fim de propósitos prognósticos.Hematopoietic stem cell proliferation leads to telomere length decreases at each cellular division. Some authors disagree about the telomere influence on the reduction of the proliferative potential and capacity of self renewal. Here we review telomerase function in the biology of the hematopoietic system, in normal or differentiation and its influence on the ageing of hematopoietic stem cells. The constant cellular renewal required to maintain the hematopoietic system, provides embryonic stem cells, as well as malignant cells, an increased proliferative capacity. This is marked by the detection of telomerase enzyme activity and possible telomere maintenance. Clinical trials will be required to clarify telomerase activity in hematopoietic stem cells, its possible use as a diagnostic marker and its use for prognostic purposes.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

OpenAIRE

Romain Barbet; Isabelle Peiffer; Antoinette Hatzfeld; Pierre Charbord; Jacques A. Hatzfeld

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs ...

Telomeres and Telomerase in the Radiation Response: implications for instability, reprogramming, and carcinogenesis

Directory of Open Access Journals (Sweden)

Brock James Sishc

2015-11-01

Full Text Available Telomeres are nucleoprotein complexes comprised of tandem arrays of repetitive DNA sequence that serve to protect chromosomal termini from inappropriate degradation, as well as to prevent these natural DNA ends from being recognized as broken DNA (double-strand breaks; DSBs and triggering of inappropriate DNA damage responses. Preservation of telomere length requires telomerase, the specialized reverse transcriptase capable of maintaining telomere length via template-mediated addition of telomeric repeats onto the ends of newly synthesized chromosomes. Loss of either end-capping function or telomere length maintenance has been associated with genomic instability or senescence in a variety of settings; therefore telomeres and telomerase have well-established connections to cancer and aging. It has long been recognized that oxidative stress promotes shortening of telomeres, and that telomerase activity is a radiation-inducible function. However, the effects of ionizing radiation (IR exposure on telomeres per se are much less well understood and appreciated. To gain a deeper understanding of the roles telomeres and telomerase play in the response of human cells to ionizing radiations of different qualities, we tracked changes in telomeric end-capping function, telomere length, and telomerase activity in panels of mammary epithelial and hematopoietic cell lines exposed to low linear energy transfer (LET gamma(γ-rays or high LET high charge, high energy (HZE particles, delivered either acutely or at low dose rates (LDR. In addition to demonstrating that dysfunctional telomeres contribute to IR-induced mutation frequencies and genome instability, we reveal non-canonical roles for telomerase, in that telomerase activity was required for IR-induced enrichment of mammary epithelial putative stem/progenitor cell populations, a finding also suggestive of cellular reprogramming. Taken together, the results reported here establish the critical importance of

Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20.

Science.gov (United States)

Jin, Yuesheng; Zhang, Hao; Tsao, Sai Wah; Jin, Charlotte; Lv, Mei; Strömbeck, Bodil; Wiegant, Joop; Wan, Thomas Shek Kong; Yuen, Po Wing; Kwong, Yok-Lam

2004-01-01

This study aimed at identifying the genetic events involved in immortalization of ovarian epithelial cells, which might be important steps in ovarian carcinogenesis. The genetic profiles of five human ovarian surface epithelial (HOSE) cell lines immortalized by retroviral transfection of the human papillomavirus (HPV) E6/E7 genes were thoroughly characterized by chromosome banding and fluorescence in situ hybridization (FISH), at various passages pre- and post-crisis. In pre-crisis, most cells had simple, non-clonal karyotypic changes. Telomere association was the commonest aberration, suggesting that tolermase dysfunction might be an important genetic event leading to cellular crisis. After immortalization post-crisis, however, the karyotypic patterns were non-random. Loss of genetic materials was a characteristic feature. The commonest numerical aberrations were -13, -14, -16, -17, -18, and +5. Among them, loss of chromosome 13 was common change observed in all lines. The only recurrent structural aberration was homogeneously staining regions (hsr) observed in three lines. FISH and combined binary ratio labeling (COBRA)-FISH showed in two cases that the hsrs were derived from chromosome 20. Clonal evolution was observed in four of the lines. In one line, hsr was the only change shared by all subclones, suggesting that it might be a primary event in cell immortalization. The results of the present study suggested that loss of chromosome 13 and the amplification of chromosome 20 might be early genetic events involved in ovarian cell immortalization, and might be useful targets for the study of genomic aberrations in ovarian carcinogenesis.

Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization.

Science.gov (United States)

Kamada, Mizuna; Kumazaki, Tsutomu; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

2012-06-01

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

Estradiol-mediated hepatocyte growth factor is involved in the implantation of endometriotic cells via the mesothelial-to-mesenchymal transition in the peritoneum.

Science.gov (United States)

Ono, Yoshihiro J; Hayashi, Masami; Tanabe, Akiko; Hayashi, Atsushi; Kanemura, Masanori; Terai, Yoshito; Ohmichi, Masahide

2015-06-01

The pathogenesis of endometriosis, a chronic painful gynecological disease characterized by the presence of endometrial tissue located outside of the uterus and often adhering to the peritoneum, is known to be estrogen dependent. However, the precise pathophysiology of endometriosis remains elusive. Recent studies indicate that the epithelial-to-mesenchymal transition (EMT) of human endometrial cells is important for the progression of endometriosis, and another previous study has implicated hepatocyte growth factor (HGF) in endometriosis progression. The aim of the present study was to examine the role of estradiol in the regulation of HGF production and progression of peritoneal endometriosis, focusing on the interactions between the peritoneum and endometriotic cells. Consequently, estradiol was found to promote the proliferation, invasion, and migration of immortalized human endometrial epithelial cells (hEECs) via HGF upregulation, and the estradiol-induced direct binding of estrogen receptor-α to the HGF promoter was confirmed on a chromatin immunoprecipitation (ChIP) assay. Estradiol also induced the EMT in hEECs by promoting HGF production. Furthermore, human mesothelial cells underwent the mesothelial-to-mesenchymal transition (MMT) during culture with estradiol-stimulated hEEC conditioned medium. Importantly, estradiol itself did not induce the MMT, and the estradiol-stimulated hEEC-conditioned medium in the presence of HGF antibodies reversed the MMT process. These results, which were obtained using immortalized hEECs, indicate that estradiol-induced HGF production may play a crucial role in the peritoneal implantation of human endometriotic cells by exerting proliferative and invasive effects via the EMT in hEECs and promoting the MMT in mesothelial cells. Copyright © 2015 the American Physiological Society.

Research on human placenta-derived mesenchymal stem cells ...

African Journals Online (AJOL)

Research on human placenta-derived mesenchymal stem cells transfected with pIRES2-EGFP-VEGF165 using liposome. ... African Journal of Biotechnology. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue ...

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

Science.gov (United States)

Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

2016-06-01

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

[The concept of cellular immortality, a myth or a reality. Example of "immortalized" articular chondrocytes].

Science.gov (United States)

Adolphe, M; Thenet, S

1990-01-01

The concept of cellular immortality, which arose from the historical studies of A. Carrel, is getting a new start with the progress of virology. However, the definition of cell immortalization is still ambiguous. Although scientists agree that cells regarded as immortal have acquired an infinite growth capacity, the relationship of this change with the first stages of transformation is difficult to clearly define. Immortalized cell lines have already been obtained from numerous cell types by using viral infection or transfection with viral and cellular genes. Immortalization of cells is interesting for three main reasons: it permits study of the steps in progression to transformation, allows establishment of cell lines for producing biological products, and permits various cell types to maintain a part of their differentiated functions. For example, hypothalamic neurosecretory cells, macrophages, astrocytes and intestinal epithelial cells have been immortalized and these lines can be used for understanding the balance between division and differentiation, and also for pharmacotoxicological studies. In our laboratory, we immortalized rabbit articular chondrocytes by transfection with SV40 large T and little t encoding genes. At the 9th subculture, when the control culture was senescent, clones of polygonal cells appeared in the transfected cell cultures. Three clones have been selected and have been maintained in culture for two years. Growth curves of normal and SV40-transfected chondrocytes were compared and displayed similar doubling times (approximately 20 hours). The exponential phase of growth was longer for immortalized cells resulting in a 2-fold higher saturation density. These cells appear to be not fully transformed and maintain some properties of differentiated chondrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional activity of telomerase complex in CD34- stem cells of cord blood in dependence of preparation time.

Directory of Open Access Journals (Sweden)

M Bojdys-Szyndlar

2009-12-01

Full Text Available The aim of the study was to determine whether the expression of telomerase subunits encoding genes changes during the process of cord blood preparation. It should establish if the commonly accepted 24 hours time interval in stem cells kriopreservation procedure significantly influences their immortalization and so decreases the "quality" of cord blood stem cells. Investigation includes 69 women. Spontaneous labour was the inclusion condition. The material was collected at birth after clamping of umbilical cord by direct vasopuncture. CD34- cells were extracted from cord blood (MACS, Miltenyi Biotec; Bisley, Surrey, UK. The expression profile of telomerase activators and inhibitors encoding genes was determined using HG_U133A oligonucleotide microarray (Affymetrix. We used a real-time quantitative RT-PCR assay to quantify the telomerase TERT, hTR and TP1 subunits mRNA copy numbers in CD34- cells in 0, 6, 12 and 24 hours after cord blood collection. We observed significant decrease of numbers of copies of TERTA+B mRNA within the successive hours of observation. Significant decrease of numbers of TERTA mRNA copies was confirmed after 24 hours. However, we observed significant increase of numbers of copies of TERTB mRNA after 6 hours of observation. Similar level was maintained during another 6h. The significantly lower number of copies of TERTB mRNA was observed after 24h. We also observed significant increase of number of copies of TERT mRNA after 6 hours. Number of copies of TERT mRNA significantly decreased after another 6h, remaining, however, on a higher then initial one. The significant lower number of copies of TERT mRNA was observed 24h after delivery. The possible explanation of those results is discussed in the paper.

Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

Science.gov (United States)

Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

2017-04-01

The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

Imaging gene expression in human mesenchymal stem cells: from small to large animals

DEFF Research Database (Denmark)

Willmann, Jürgen K; Paulmurugan, Ramasamy; Rodriguez-Porcel, Martin

2009-01-01

To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning.......To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning....

Tooth engineering: searching for dental mesenchymal cells sources.

Directory of Open Access Journals (Sweden)

Laetitia eKeller

2011-03-01

Full Text Available The implantation of cultured re-associations between embryonic dental mesenchymal cells and epithelial cells from mouse molars at ED14 allowed making full teeth with crown, root, periodontal ligament fibers and bone. Although representing valuable tools to set up methodologies embryonic cells are not easily available. This work thus aimed to replace the embryonic cells by dental mesenchymal cell lines or cultured expanded embryonic cells, and to test their ability to mediate tooth development in vitro when re-associated with a competent dental epithelium. Histology, immunostaining and RT-PCR allowed getting complementary sets of results. Two different immortalized cell lines from ED18 dental mesenchyme failed in mediating tooth formation. The potentialities of embryonic dental mesenchymal cells decreased from ED14 to ED16 and were lost at ED18. This is likely related to a change in the mesenchymal cell phenotype and/or populations during development. Attempts to cultivate ED14 or ED16 embryonic dental mesenchymal cells prior to re-association led to the loss of their ability to support tooth development. This was accompanied by a down-regulation of Fgf3 transcription. Supplementation of the culture medium with FGF2 allowed restoring Fgf3 expression, but not the ability of mesenchymal cells to engage in tooth formation. Altogether, these observations suggest that a competent cell population exists in the dental mesenchyme at ED14, progressively decreases during development, and cannot as such be maintained in vitro. This study evidenced the need for specific conditions to maintain the ability of dental mesenchymal cells to initiate whole tooth formation, when re-associated with an odontogenic epithelium. Efforts to improve the culture conditions will have to be combined with attempts to characterize the competent cells within the dental mesenchyme.

Association of telomerase activity with radio- and chemosensitivity of neuroblastomas

Directory of Open Access Journals (Sweden)

Willich Normann

2010-07-01

Full Text Available Abstract Background Telomerase activity compensates shortening of telomeres during cell division and enables cancer cells to escape senescent processes. It is also supposed, that telomerase is associated with radio- and chemoresistance. In the here described study we systematically investigated the influence of telomerase activity (TA and telomere length on the outcome of radio- and chemotherapy in neuroblastoma. Methods We studied the effects on dominant negative (DN mutant, wild type (WT of the telomerase catalytic unit (hTERT using neuroblastoma cell lines. The cells were irradiated with 60Co and treated with doxorubicin, etoposide, cisplatin and ifosfamide, respectively. Viability was determined by MTS/MTT-test and the GI50 was calculated. Telomere length was measured by southernblot analysis and TA by Trap-Assay. Results Compared to the hTERT expressing cells the dominant negative cells showed increased radiosensitivity with decreased telomere length. Independent of telomere length, telomerase negative cells are significantly more sensitive to irradiation. The effect of TA knock-down or overexpression on chemosensitivity were dependent on TA, the anticancer drug, and the chemosensitivity of the maternal cell line. Conclusions Our results supported the concept of telomerase inhibition as an antiproliferative treatment approach in neuroblastomas. Telomerase inhibition increases the outcome of radiotherapy while in combination with chemotherapy the outcome depends on drug- and cell line and can be additive/synergistic or antagonistic. High telomerase activity is one distinct cancer stem cell feature and the here described cellular constructs in combination with stem cell markers like CD133, Aldehyddehydrogenase-1 (ALDH-1 or Side population (SP may help to investigate the impact of telomerase activity on cancer stem cell survival under therapy.

Acute myocardial infarction: 'telomerasing' for cardioprotection

OpenAIRE

Sanchís-Gomar, Fabián; Lucía Mulas, Alejandro

2015-01-01

Reactivating the telomerase gene through gene therapy after acute myocardial infarction (AMI) has been recently reported to improve survival in mice. Given that regular physical exercise also activates this gene, therapeutic and lifestyle interventions targeting telomerase need to be explored as possible additions to the current armamentarium for myocardial regeneration. 9.292 JCR (2015) Q1, 17/289 Biochemistry & mollecular biology, 17/187 Cell biology, 8/124 Medicine, research & experimen...

Telomerase and drug resistance in cancer

OpenAIRE

Lipinska, Natalia; Romaniuk, Aleksandra; Paszel-Jaworska, Anna; Toton, Ewa; Kopczynski, Przemyslaw; Rubis, Blazej

2017-01-01

It is well known that a decreased expression or inhibited activity of telomerase in cancer cells is accompanied by an increased sensitivity to some drugs (e.g., doxorubicin, cisplatin, or 5-fluorouracil). However, the mechanism of the resistance resulting from telomerase alteration remains elusive. There are theories claiming that it might be associated with telomere shortening, genome instability, hTERT translocation, mitochondria functioning modulation, or even alterations in ABC family gen...

Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

Science.gov (United States)

Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

2011-02-01

Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly

Energy Technology Data Exchange (ETDEWEB)

Her, Joonyoung [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of); Chung, In Kwon, E-mail: topoviro@yonsei.ac.kr [Departments of Biology and Integrated Omics for Biomedical Science, Yonsei University, Seoul 120-749 (Korea, Republic of)

2012-01-20

Highlights: Black-Right-Pointing-Pointer Identification of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. Black-Right-Pointing-Pointer NVL2 associates with catalytically active telomerase via an interaction with hTERT. Black-Right-Pointing-Pointer NVL2 is a telomerase component essential for holoenzyme assembly. Black-Right-Pointing-Pointer ATP-binding activity of NVL2 is required for hTERT binding and telomerase assembly. -- Abstract: Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.

Immortality versus resurrection in the Christian tradition.

Science.gov (United States)

Murphy, Nancey

2011-10-01

For those in contemporary society who believe in an afterlife, there are a number of views available. The most common may be based on belief in an immortal soul. However, the early Christian account was, instead, bodily resurrection. As Christianity moved throughout the Mediterranean world, apologists and theologians adapted their teaching on human nature and the afterlife to Greek and Roman philosophies. By the time of Augustine (d. 430), the doctrines of body-soul dualism and immortality of the soul were firmly entrenched in Christian teaching. The incorporation of the concept of an immortal soul into Christian accounts of life after death produced a hybrid account. The body dies, the soul (at least of those who were to be saved) travels to heaven. At the end of history, there would be a general resurrection, and the souls would be reunited with their bodies, although the bodies would be in a transformed, indestructible state. This hybrid account of life after death went largely uncontested until the twentieth century. In this essay, I describe this history and argue for a return to the early Christian view of humans as a unity, not a duality, and for belief in resurrection of the body as the appropriate expectation for eternal life. This would not only be truer to Christian sources, but, valuable, I believe, in focusing Christian attention on the need to care for the environment. © 2011 New York Academy of Sciences.

Comparison of the biological features between human fetal hepatocyte and immortalized L-02 hepatocyte in vitro

International Nuclear Information System (INIS)

Kong Weiwei; Teng Gaojun

2004-01-01

Objective: To evaluate the feasibilities of the potential donors in liver cell transplantation using the human fetal hepatocytes and immortalized L-02 hepatocytes by comparing their biological features. Methods: Human fetal hepatocytes were isolated from aborted fetal livers (gestational ages from 14 w to 24 w) by an improved two-stage perfusion method and cultured in a conditioned medium without any growth factors. α-fetal protein (AFP) and albumin (ALB) were detected by radioimmunoassay (RIA) and cytokeratin-19 (CK-19 ) was identified by cellular immunochemistry study. Immortalized L-02 hepatocytes were cultured in the same condition and the characteristic proteins were detected by the same methods. Results: The viability of human fetal hepatocytes was approximately 95% using the perfusion method, and the maximum survival time of the cultured hepatocytes was 3 weeks. The expression of AFP, ALB, and CK19 was detected at the same time, especially during Day 3 to Day 7 in the culture. By comparison, the proliferation ability of L-02 hepatocyte was greater, although with a lower level of ALB secretion. The expression of AFP and CK19 was not detected. Furthermore, during the long culture, L-02 hepatocytes may undergo a morphologic change and fail to express ALB. Conclusion: Human fetal hepatocyte may be a practical donor for hepatocyte transplantation with its high-level protein expression and potential bi-differentiation ability. In view of the absent expression of ALB and the morphologic change in culture, although with better proliferation, L-02 hepatocyte seems not useful for hepatocyte transplantation

Nanoscale Mechanical Stimulation of Human Mesenchymal Stem Cells

Directory of Open Access Journals (Sweden)

H Nikukar

2014-05-01

We observed significant responses after 1 and 2-week stimulations in cell number, cell shapes and phenotypical markers. Microarray was performed for all groups. Cell count showed normal cell growth with stimulation. However, cell surface area, cell perimeter, and arboration after 1-week stimulation showed significant increases. Immunofluorescent studies have showed significant increase in osteocalcin production after stimulation. Conclusions: Nanoscale mechanical vibration showed significant changes in human mesenchymal stem cell behaviours. Cell morphology changed to become more polygonal and increased expression of the osteoblast markers were noted. These findings with gene regulation changes suggesting nanoscale mechanostimulation has stimulated osteoblastogenesis.  Keywords:  Mesenchymal, Nanoscale, Stem Cells.

Dietary restriction ameliorates haematopoietic ageing independent of telomerase, whilst lack of telomerase and short telomeres exacerbates the ageing phenotype.

Science.gov (United States)

Al-Ajmi, Nouf; Saretzki, Gabriele; Miles, Colin; Spyridopoulos, Ioakim

2014-10-01

Ageing is associated with an overall decline in the functional capacity of tissues and stem cells, including haematopoietic stem and progenitor cells (HSPCs), as well as telomere dysfunction. Dietary restriction (DR) is a recognised anti-ageing intervention that extends lifespan and improves health in several organisms. To investigate the role of telomeres and telomerase in haematopoietic ageing, we compared the HSPC profile and clonogenic capacity of bone marrow cells from wild type with telomerase-deficient mice and the effect of DR on these parameters. Compared with young mice, aged wild type mice demonstrated a significant accumulation of HSPCs (1.3% vs 0.2%, P=0.002) and elevated numbers of granulocyte/macrophage colony forming units (CFU-GM, 26.4 vs 17.3, P=0.0037) consistent with myeloid "skewing" of haematopoiesis. DR was able to restrict the increase in HSPC number as well as the myeloid "skewing" in aged wild type mice. In order to analyse the influence of short telomeres on the ageing phenotype we examined mice lacking the RNA template for telomerase, TERC(-/-). Telomere shortening resulted in a similar bone marrow phenotype to that seen in aged mice, with significantly increased HSPC numbers and an increased formation of all myeloid colony types but at a younger age than wild type mice. However, an additional increase in erythroid colonies (BFU-E) was also evident. Mice lacking telomerase reverse transcriptase without shortened telomeres, TERT(-/-), also presented with augmented haematopoietic ageing which was ameliorated by DR, demonstrating that the effect of DR was not dependent on the presence of telomerase in HSPCs. We conclude that whilst shortened telomeres mimic some aspects of haematopoietic ageing, both shortened telomeres and the lack of telomerase produce specific phenotypes, some of which can be prevented by dietary restriction. Copyright © 2014 Elsevier Inc. All rights reserved.

Clinical Outcomes of Lung Transplantation in Patients with Telomerase Mutations

Science.gov (United States)

Tokman, Sofya; Singer, Jonathan P.; Devine, Megan S.; Westall, Glen P.; Aubert, John-David; Tamm, Michael; Snell, Gregory I.; Lee, Joyce S.; Goldberg, Hilary J.; Kukreja, Jasleen; Golden, Jeffrey A.; Leard, Lorriana E.; Garcia, Christine K.; Hays, Steven R.

2017-01-01

Background Successful lung transplantation (LT) for patients with pulmonary fibrosis from telomerase mutations is limited by systemic complications of telomerase dysfunction including myelosuppression, cirrhosis, and malignancy. We describe clinical outcomes among 14 LT recipients with telomerase mutations. Methods Subjects underwent LT between February 2005 and April 2014 at 5 LT centers. We abstracted data from medical records, focusing on outcomes reflecting post-LT treatment effects likely to be complicated by telomerase mutations. Results The median age of subjects was 60.5 years (IQR 52.0–62.0), 64.3% were male, and the mean post-LT observation time was 3.2 years (SD ±2.9). Eleven subjects had a mutation in telomerase reverse transcriptase, 2 in telomerase RNA component, and 1 had an uncharacterized mutation. Ten subjects were leukopenic post-LT; leukopenia prompted cessation of mycophenolate mofetil in 5 and treatment with filgrastim in 4. Six subjects had recurrent lower respiratory tract infections (LRTI), 7 had acute cellular rejection (ACR) (A1), and 4 developed chronic lung allograft dysfunction (CLAD). Ten LT recipients developed chronic renal insufficiency and 8 experienced acute, reversible renal failure. Three developed cancer, none had cirrhosis. Thirteen subjects were alive at data censorship. Conclusions The clinical course for LT recipients with telomerase mutations is complicated by renal disease, leukopenia prompting a change in the immunosuppressive regimen, and recurrent LTRI. In contrast, cirrhosis was absent, ACR was mild, and development of CLAD was comparable to other LT populations. While posing challenges, lung transplantation may be feasible for patients with pulmonary fibrosis due to telomerase mutations. PMID:26169663

Human Papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

Science.gov (United States)

Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

2012-01-01

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone. PMID:22284893

Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

Directory of Open Access Journals (Sweden)

Lindolfo da Silva Meirelles

2016-03-01

Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15.

Directory of Open Access Journals (Sweden)

Artur Brandt

Full Text Available We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT in conjunction with a novel thermolabile mutant (U19dl89-97tsA58 of SV40 large T antigen (LT. This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15

Science.gov (United States)

Brandt, Artur; Löhers, Katharina; Beier, Manfred; Leube, Barbara; de Torres, Carmen; Mora, Jaume; Arora, Parineeta; Jat, Parmjit S.; Royer-Pokora, Brigitte

2016-01-01

We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells. PMID:27213811

Comparison of Inhibitory Effect of Curcumin Nanoparticles and Free Curcumin in Human Telomerase Reverse Transcriptase Gene Expression in Breast Cancer

Directory of Open Access Journals (Sweden)

Nosratollah Zarghami

2013-02-01

Full Text Available Purpose: Telomerase is expressed in most cancers, including breast cancer. Curcumin, a polyphenolic compound that obtained from the herb of Curcuma longa, has many anticancer effects. But, its effect is low due to poor water solubility. In order to improve its solubility and drug delivery, we have utilized a β-cyclodextrin-curcumin inclusion complex. Methods: To evaluate cytotoxic effects of cyclodextrin-curcumin and free curcumin, MTT assay was done. Cells were treated with equal concentration of cyclodextrin-curcumin and free curcumin. Telomerase gene expression level in two groups was compared by Real-time PCR. Results: MTT assay demonstrated that β-cyclodextrin-curcumin enhanced curcumin delivery in T47D breast cancer cells. The level of telomerase gene expression in cells treated with cyclodextrin-curcumin was lower than that of cells treated with free curcumin (P=0.001. Conclusion: Results are suggesting that cyclodextrin-curcumin complex can be more effective than free curcumin in inhibition of telomerase expression.

Telomeres and Telomerase in Hematopoietic Dysfunction: Prognostic Implications and Pharmacological Interventions

Directory of Open Access Journals (Sweden)

Theresa Vasko

2017-10-01

Full Text Available Leukocyte telomere length (TL has been suggested as a marker of biological age in healthy individuals, but can also reflect inherited and acquired hematopoietic dysfunctions or indicate an increased turnover of the hematopoietic stem and progenitor cell compartment. In addition, TL is able to predict the response rate of tyrosine kinase inhibitor therapy in chronic myeloid leukemia (CML, indicates clinical outcomes in chronic lymphocytic leukemia (CLL, and can be used as screening tool for genetic sequencing of selected genes in patients with inherited bone marrow failure syndromes (BMFS. In tumor cells and clonal hematopoietic disorders, telomeres are continuously stabilized by reactivation of telomerase, which can selectively be targeted by telomerase-specific therapy. The use of the telomerase inhibitor Imetelstat in patients with essential thrombocythmia or myelofibrosis as well as the use of dendritic cell-based telomerase vaccination in AML patients with complete remissions are promising examples for anti-telomerase targeted strategies in hematologic malignancies. In contrast, the elevation in telomerase levels through treatment with androgens has become an exciting clinical intervention for patients with BMFS. Here, we review recent developments, which highlight the impact of telomeres and telomerase targeted therapies in hematologic dysfunctions.

Human stromal (mesenchymal) stem cells

DEFF Research Database (Denmark)

Aldahmash, Abdullah; Zaher, Walid; Al-Nbaheen, May

2012-01-01

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self......-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number...

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells.

Science.gov (United States)

Yoshioka, Masahiro; Ohashi, Shinya; Ida, Tomomi; Nakai, Yukie; Kikuchi, Osamu; Amanuma, Yusuke; Matsubara, Junichi; Yamada, Atsushi; Miyamoto, Shin'ichi; Natsuizaka, Mitsuteru; Nakagawa, Hiroshi; Chiba, Tsutomu; Seno, Hiroshi; Muto, Manabu

2017-08-01

Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells. Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated. Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR

Effective control of acute myeloid leukaemia and acute lymphoblastic leukaemia progression by telomerase specific adoptive T-cell therapy.

Science.gov (United States)

Sandri, Sara; De Sanctis, Francesco; Lamolinara, Alessia; Boschi, Federico; Poffe, Ornella; Trovato, Rosalinda; Fiore, Alessandra; Sartori, Sara; Sbarbati, Andrea; Bondanza, Attilio; Cesaro, Simone; Krampera, Mauro; Scupoli, Maria T; Nishimura, Michael I; Iezzi, Manuela; Sartoris, Silvia; Bronte, Vincenzo; Ugel, Stefano

2017-10-20

Telomerase (TERT) is a ribonucleoprotein enzyme that preserves the molecular organization at the ends of eukaryotic chromosomes. Since TERT deregulation is a common step in leukaemia, treatments targeting telomerase might be useful for the therapy of hematologic malignancies. Despite a large spectrum of potential drugs, their bench-to-bedside translation is quite limited, with only a therapeutic vaccine in the clinic and a telomerase inhibitor at late stage of preclinical validation. We recently demonstrated that the adoptive transfer of T cell transduced with an HLA-A2-restricted T-cell receptor (TCR), which recognize human TERT with high avidity, controls human B-cell chronic lymphocytic leukaemia (B-CLL) progression without severe side-effects in humanized mice. In the present report, we show the ability of our approach to limit the progression of more aggressive leukemic pathologies, such as acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). Together, our findings demonstrate that TERT-based adoptive cell therapy is a concrete platform of T cell-mediated immunotherapy for leukaemia treatment.

Data on isolating mesenchymal stromal cells from human adipose tissue using a collagenase-free method

Directory of Open Access Journals (Sweden)

Wassim Shebaby

2016-03-01

Full Text Available The present dataset describes a detailed protocol to isolate mesenchymal cells from human fat without the use of collagenase. Human fat specimen, surgically cleaned from non-fat tissues (e.g., blood vessels and reduced into smaller fat pieces of around 1–3 mm size, is incubated in complete culture media for five to seven days. Then, cells started to spread out from the fat explants and to grow in cultures according to an exponential pattern. Our data showed that primary mesenchymal cells presenting heterogeneous morphology start to acquire more homogenous fibroblastic-like shape when cultured for longer duration or when subcultured into new flasks. Cell isolation efficiency as well as cell doubling time were also calculated throughout the culturing experimentations and illustrated in a separate figure thereafter. This paper contains data previously considered as an alternative protocol to isolate adipose-derived mesenchymal stem cell published in “Proliferation and differentiation of human adipose-derived mesenchymal stem cells (ASCs into osteoblastic lineage are passage dependent” [1]. Keywords: Adipose tissue, mesenchymal stromal cell, cell culture, doubling time

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

International Nuclear Information System (INIS)

Cogan, Nicola; Baird, Duncan M.; Phillips, Ryan; Crompton, Lucy A.; Caldwell, Maeve A.; Rubio, Miguel A.; Newson, Roger; Lyng, Fiona; Case, C. Patrick

2010-01-01

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

Energy Technology Data Exchange (ETDEWEB)

Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

2010-01-05

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells.

Science.gov (United States)

Kinoshita, Yusuke; Kamitani, Hideki; Mamun, Mahabub Hasan; Wasita, Brian; Kazuki, Yasuhiro; Hiratsuka, Masaharu; Oshimura, Mitsuo; Watanabe, Takashi

2010-05-01

Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

Periodicity and Immortality in Reversible Computing

OpenAIRE

Kari , Jarkko; Ollinger , Nicolas

2008-01-01

Additional material available on the web at http://www.lif.univ-mrs.fr/~nollinge/rec/gnirut/; We investigate the decidability of the periodicity and the immortality problems in three models of reversible computation: reversible counter machines, reversible Turing machines and reversible one-dimensional cellular automata. Immortality and periodicity are properties that describe the behavior of the model starting from arbitrary initial configurations: immortality is the property of having at le...

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe.

Science.gov (United States)

Margalef, Pol; Kotsantis, Panagiotis; Borel, Valerie; Bellelli, Roberto; Panier, Stephanie; Boulton, Simon J

2018-01-25

Telomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1 -/- cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

Science.gov (United States)

Lemieux, Bruno; Laterreur, Nancy; Perederina, Anna; Noël, Jean-François; Dubois, Marie-Line; Krasilnikov, Andrey S; Wellinger, Raymund J

2016-05-19

Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts.

Science.gov (United States)

Luciano, Pierre; Coulon, Stéphane; Faure, Virginie; Corda, Yves; Bos, Julia; Brill, Steven J; Gilson, Eric; Simon, Marie-Noelle; Géli, Vincent

2012-04-18

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

Antioxidant therapy attenuates myocardial telomerase activity reduction in superoxide dismutase-deficient mice.

Science.gov (United States)

Makino, Naoki; Maeda, Toyoki; Oyama, Jun-ichi; Sasaki, Makoto; Higuchi, Yoshihiro; Mimori, Koji; Shimizu, Takahiko

2011-04-01

Oxidative stress plays a pathological role in the development of heart failure. This study examined telomere biology in heart/muscle-specific manganese superoxide dismutase-deficient mice (H/M-SOD2(-/-)), which develop progressive congestive heart failure and exhibit pathology typical of dilated cardiomyopathy. EUK-8 (25mg/kg/day), a superoxide dismutase and catalase mimetic, was administered to H/M-SOD2(-/-) mice for four weeks beginning at 8 weeks of age. Telomere length, telomerase activity, telomere-associated proteins, and cell death signals were assessed in hearts from control wild-type mice (H/M-Sod2 (lox/ lox)) and H/M-SOD2(-/-) mice either treated or untreated with EUK-8. While cardiac function was unchanged in these experimental mice, the end-diastolic dimension in H/M-SOD2(-/-) mice was notably dilated and could be significantly reduced by EUK-8 treatment. At the end of the study, no shortening of telomere length was observed in heart tissues from all mice tested, but telomerase activity was decreased in heart tissue from H/M-SOD2(-/-) mice compared to control mice. Protein expression for telomerase reverse transcriptase and telomere repeat binding factor 2 was also downregulated in H/M-SOD2(-/-) heart tissue as was expression of phospho-Akt, insulin-like growth factor, and endothelial nitric oxide synthase. Expression levels of Sirt1, a lifespan modulator, were enhanced while FoxO3a was depressed in H/M-SOD2(-/-) hearts. All of the changes seen in H/M-SOD2(-/-) heart tissue could be inhibited by EUK-8 treatment. Taken together, the results suggest that oxidant stress might affect myocardial telomerase activity and telomere-associated proteins. Telomerase may therefore play a pivotal role in antioxidant defense mechanisms, and may be useful as a novel therapeutic tool for treating human heart failure. Copyright © 2010 Elsevier Ltd. All rights reserved.

Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.

Science.gov (United States)

Lavranos, T C; Mathis, J M; Latham, S E; Kalionis, B; Shay, J W; Rodgers, R J

1999-08-01

We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells.

The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

International Nuclear Information System (INIS)

Ganzenmueller, Tina; Matthaei, Markus; Muench, Peter; Scheible, Michael; Iftner, Angelika; Hiller, Thomas; Leiprecht, Natalie; Probst, Sonja; Stubenrauch, Frank; Iftner, Thomas

2008-01-01

Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19 ARF induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53

Telomeres, telomerase and premature ovarian failure

Directory of Open Access Journals (Sweden)

Renata Košir Pogačnik

2011-11-01

Full Text Available Telomeres are specialized structures at the ends of chromosomes, consisting of six repeated nucleotides in TTAGGG sequence. Genome stability is partly maintained by the architecture of telomeres and is gradually lost as telomeres progressively shorten with each cell replication. Critically shortened telomeres are recognized by DNA repair mechanisms as DNA damage and the cell replication cycle stops. The cell eventually dies or undergoes cell apoptosis. Telomere represents a cellular marker of biological age and are therefore also called cell mitotic clock. The enzyme that counteracts telomere shortening by adding nucleotides to the 3’ end of DNA strand is called telomerase. It is composed of the RNA subunit (TR, which is special type of messenger RNA (mRNA, the catalytic protein subunit (TERT, which works as a reverse transcriptase and numerous additional proteins. Telomerase is active in some germline, epithelial and haemopoietic cells, but in most somatic cells the activity is undetectable. In literature, the length of telomeres is closely connected with premature ovarian failure (POF. POF is generally defined as the onset of menopause before the age of 40. The causes of disease are genetical, autoimmune, iatrogenic or if we cannot establish the cause – idiopathic. A lot of studies examined correlation between idiopathic POF, length of telomeres and telomerase activity. The studies mostly show that women with POF have shortened telomeres and decreased activity of telomerase as compared to healthy women.

Serum telomerase levels in smokers and smokeless tobacco users as Maras powder.

Science.gov (United States)

Bozkuş, Fulsen; Atilla, Nurhan; Şimşek, Seçil; Kurutaş, Ergül; Samur, Anıl; Arpağ, Hüseyin; Kahraman, Hasan

2017-09-01

To the best of our knowledge, no previous study regarding the serum telomerase levels in Maras powder users (MPUs) has been founded. The aim of the current study was to investigate serum telomerase levels in smokers and MPUs. The study was carried out with 98 patients (36 MPUs, 32 smokers and 30 non-smokers). Blood samples were collected, and after having measured the serum telomerase and malondialdehyde (MDA) levels of the patients, comparison were made between the groups. It has been observed that the serum telomerase and MDA levels of smokers (pnon-smoker control subjects. In addition, the levels of serum telomerase and MDA were observed to be higher in the MPU group compared to those of the smoker group (psmokers. In this context, it may be useful to further measure and assess telomerase activity in such patients in order to better determine the harmful effects associated with these habits.

Human amnion mesenchymal stem cells promote proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells.

Science.gov (United States)

Wang, Yuli; Yin, Ying; Jiang, Fei; Chen, Ning

2015-02-01

Human amnion mesenchymal stem cells (HAMSCs) can be obtained from human amniotic membrane, a highly abundant and readily available tissue. HAMSC sources present fewer ethical issues, have low immunogenicity, anti-inflammatory properties, considerable advantageous characteristics, and are considered an attractive potential treatment material in the field of regenerative medicine. We used a co-culture system to determine whether HAMSCs could promote osteogenesis in human bone marrow mesenchymal stem cells (HBMSCs). We isolated HAMSCs from discarded amnion samples and collected them using pancreatin/collagenase digestion. We cultured HAMSCs and HBMSCSs in basal medium. Activity of alkaline phosphatase (ALP), an early osteogenesis marker, was increased in the co-culture system compared to the control single cultures, which we also confirmed by ALP staining. We used immunofluorescence testing to investigate the effects of co-culturing with HAMSCs on HBMSC proliferation, which revealed that the co-culturing enhanced EdU expression in HBMSCs. Western blotting and quantitative real-time PCR indicated that co-culturing promoted osteogenesis in HBMSCs. Furthermore, Alizarin red S staining revealed that extracellular matrix calcium levels in mineralized nodule formation produced by the co-cultures were higher than that in the controls. Using the same co-culture system, we further observed the effects of HAMSCs on osteogenic differentiation in primary osteoblasts by Western blotting, which better addressed the mechanism for HAMSCs in bone regeneration. The results showed HAMSCs are osteogenic and not only play a role in promoting HBMSC proliferation and osteogenic differentiation but also in osteoblasts, laying the foundation for new regenerative medicine methods.

Telomerase Activity in Chicken EmbryoFibroblast Cell Cultures Infected withMarek's Disease Virus

Directory of Open Access Journals (Sweden)

Gregory A. Tannock

2010-07-01

Full Text Available Background:Telomerase is a ribonucleoprotein, which adds telomeric repeats onto the 3’end of existing telomers at the end of chromosomes ineukaryotes. One hypothesis states that telomere length may function as a mitoticclock, therefore expression of telomerase activity in cancer cells may be a necessary and essential step for tumor development and progression.Methods:The detectability of telomerase activity in chicken embryofibroblast (CEF cells infected with different passages of Marek's disease virus(MDV was tested with the TRAPEZE® telomerase detection kit at passages14 (P14, P80/1 and P120 for the Woodland strain, and passage 9 (P9 for theMPF57 strain. Results:The results showed increased telomerase activity in MDV Woodlands strain at P14 and MPF57 strain at P9. Conclusion:Our results suggest that MDV-transformed cells at low passage are a suitable system for the study of telomerases in tumor developmentand for testing telomerase-inhibiting drugs.

Analysis of telomerase target gene expression effects from murine models in patient cohorts by homology translation and random survival forest modeling

Directory of Open Access Journals (Sweden)

Frederik Otzen Bagger

2016-03-01

Full Text Available Acute myeloid leukemia (AML is an aggressive and rapidly fatal blood cancer that affects patients of any age group. Despite an initial response to standard chemotherapy, most patients relapse and this relapse is mediated by leukemia stem cell (LSC populations. We identified a functional requirement for telomerase in sustaining LSC populations in murine models of AML and validated this requirement using an inhibitor of telomerase in human AML. Here, we describe in detail the contents, quality control and methods of the gene expression analysis used in the published study (Gene Expression Omnibus GSE63242. Additionally, we provide annotated gene lists of telomerase regulated genes in AML and R code snippets to access and analyze the data used in the original manuscript. Keywords: AML, Leukemia, Stem cells, Telomere, Telomerase

Human mesenchymal stromal cells : biological characterization and clinical application

NARCIS (Netherlands)

Bernardo, Maria Ester

2010-01-01

This thesis focuses on the characterization of the biological and functional properties of human mesenchymal stromal cells (MSCs), isolated from different tissue sources. The differentiation capacity of MSCs from fetal and adult tissues has been tested and compared. Umbilical cord blood (UCB) has

Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

Directory of Open Access Journals (Sweden)

Denise Salzig

2016-01-01

Full Text Available The manufacture of human mesenchymal stem cells (hMSCs for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT as well as primary cells derived from bone marrow (bm-hMSCs and adipose tissue (ad-hMSCs. We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells. The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types.

Detection of telomerase activity in Plasmodium falciparum using a nonradioactive method

Directory of Open Access Journals (Sweden)

Rubiano Claudia C

2003-01-01

Full Text Available A simple, quick and sensitive method was used to detect telomerase activity in Plasmodium falciparum. The telomeric repeat amplification protocol (TRAP assay was modified using electrophoresis and staining with SYBR-green I to detect telomerase activity in a range of 10² to 10(7 parasites. This might be a useful way to ascertain telomerase activity in different types of nontumor cells.

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane

Science.gov (United States)

Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja

Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.

Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability.

Science.gov (United States)

Robart, Aaron R; O'Connor, Catherine M; Collins, Kathleen

2010-03-01

Telomerase adds simple-sequence repeats to chromosome 3' ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

Generation of insulin-producing human mesenchymal stem cells using recombinant adeno-associated virus.

Science.gov (United States)

Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

2007-02-28

The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet beta-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced with AAV which is containing furin-cleavable human preproinsulin gene to generate insulin-producing cells as surrogate beta-cells for the type 1 diabetes therapy. In the rAAV production procedure, rAAV was generated by transfection of AD293 cells. Human mesenchymal stems cells were transduced using rAAV with a various multiplicity of infection. Transduction of recombinant AAV was also tested using beta-galactosidse expression. Cell viability was determined by using MTT assay to evaluate the toxicity of the transduction procedure. Expression and production of Insulin were tested using reverse transcriptase-polymerase chain reaction and immunocytochemistry. Secretion of human insulin and C-peptide from the cells was assayed using enzyme-linked immunosorbent assay. Production of insulin and C-peptide from the test group represented a higher increase compared to the control group. In this study, we examined generation of insulin-producing cells from mesenchymal stem cells by genetic engineering for diabetes therapy. This work might be valuable to the field of tissue engineering for diabetes treatment.

Genetic effect of low dose rate radiation on human cells immortalized with the hTERT gene

International Nuclear Information System (INIS)

Nakamura, Hideaki; Fukami, Hiroko; Hayashi, Yuko; Kiyono, Tohru; Ishizaki, Kanji; Tachibana, Akira; Nakatsugawa, Shigekazu; Hamaguchi, Michinari

2003-01-01

We established immortal human cells by introducing the hTERT gene into skin fibroblast cells obtained from normal (SuSa) and ataxia telangiectasia (AT: AT1OS) individuals of Japanese origin. These immortalized cells showed the same characteristics as the original cells except expanded life span. We irradiated SuSa/T-n and AT1OS/T-n cells with low-dose-rate (LDR; 0.3 mGy/min) irradiation at confluent state in low-serum medium. Then, survival rate and micronucleus frequency of each cell line were analyzed. In SuSa/T-n cells, frequency of HPRT mutation induction was also determined by 6TG selection. In SuSa/T-n cells, survival rate and micronucleus frequency showed higher resistance after irradiation with LDR than high-dose-rate (HDR; 2 Gy/min) irradiation. In contrast, no significant difference was observed in survival and micronucleus induction in AT1OS/T-n cells between HDR and LDR irradiation, suggesting that AT1OS/T-n cells may have some defect in DNA repair activity. In SuSa/T-n cells, the frequency of HPRT mutation after LDR irradiation decreased to approximately one eighth that after HDR irradiation. (author)

Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

Science.gov (United States)

Salamon, Achim; van Vlierberghe, Sandra; van Nieuwenhove, Ine; Baudisch, Frank; Graulus, Geert-Jan; Benecke, Verena; Alberti, Kristin; Neumann, Hans-Georg; Rychly, Joachim; Martins, José C.; Dubruel, Peter; Peters, Kirsten

2014-01-01

Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies. PMID:28788517

The influence of the telomere-telomerase system on diabetes mellitus and its vascular complications.

Science.gov (United States)

Qi Nan, Wu; Ling, Zhang; Bing, Chen

2015-06-01

The telomere-telomerase system plays an important role in the pathogenesis and disease progression of diabetes mellitus as well as in its vascular complications. Recent studies suggest that telomere shortening and abnormal telomerase activity occur in patients with diabetes mellitus, and targeting the telomere-telomerase system has become a prospective treatment for diabetes mellitus and its vascular complications. This review highlights the significance of the telomere-telomerase system and supports its role as a possible therapeutic target for patients with diabetes mellitus and its vascular complications Areas covered: This review covers the advances in understanding the telomere-telomerase system over the last 30 years and its significance in diabetes mellitus. In addition, it provides knowledge regarding the significance of the telomere-telomerase system in diabetes mellitus and its vascular complications as well as its role and mechanisms in oxidative stress, cell therapy and antioxidant activity Expert opinion: The telomere-telomerase system may be a potential therapeutic target that can protect against DNA damage and apoptosis in patients with diabetes mellitus and its vascular complications. DNA damage and apoptosis are associated with oxidative stress and are involved in the dysfunction of pancreatic β cells, insulin resistance, and its vascular complications. Abnormalities in the telomere-telomerase system may be associated with diabetes mellitus and its vascular complications. Therapies targeting telomere-telomerase system, telomerase reverse transcriptase transfection and alterative telomere lengthening must be identified before gene therapy can commence.

Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

Science.gov (United States)

Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

2011-01-01

We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro

Directory of Open Access Journals (Sweden)

Chao Yang

2014-01-01

Full Text Available Human mesenchymal stem cells (MSCs have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs and umbilical cord-derived mesenchymal stem cells (UC-MSCs. We found (1 MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2 MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3 real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4 furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

International Nuclear Information System (INIS)

Rashid-Kolvear, Fariborz; Taboski, Michael AS; Nguyen, Johnny; Wang, Dong-Yu; Harrington, Lea A; Done, Susan J

2010-01-01

Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated. In this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference. We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's t test. To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined

Generation of a transplantable erythropoietin-producer derived from human mesenchymal stem cells.

Science.gov (United States)

Yokoo, Takashi; Fukui, Akira; Matsumoto, Kei; Ohashi, Toya; Sado, Yoshikazu; Suzuki, Hideaki; Kawamura, Tetsuya; Okabe, Masataka; Hosoya, Tatsuo; Kobayashi, Eiji

2008-06-15

Differentiation of autologous stem cells into functional transplantable tissue for organ regeneration is a promising regenerative therapeutic approach for cancer, diabetes, and many human diseases. Yet to be established, however, is differentiation into tissue capable of producing erythropoietin (EPO), which has a critical function in anemia. We report a novel EPO-producing organ-like structure (organoid) derived from human mesenchymal stem cells. Using our previously established relay culture system, a human mesenchymal stem cell-derived, human EPO-competent organoid was established in rat omentum. The organoid-derived levels of human EPO increased in response to anemia induced by rapid blood withdrawal. In addition, the presence of an organoid in rats suppressed for native (rat) EPO production enhanced recovery from anemia when compared with control animals lacking the organoid. Together these results confirmed the generation of a stem cell-derived organoid that is capable of producing EPO and sensitive to physiological regulation.

Correlation between telomerase activity and matrix metalloproteinases 2 expression in gastric cancer.

Science.gov (United States)

Wang, Gang; Wang, Wenling; Zhou, Jianjiang; Yang, Xiaofeng

2013-01-01

To investigate the relationship between telomerase activity (TA) and matrix metallo proteinases 2 (MMP-2) on malignant behavior and prognosis predictable value in gastric cancer. Telomerase activity and MMP-2 protein expressions were tested in 40 gastric surgical resected cancer samples and the clinicopathological data of enrolled patients were obtained to get correlation analysis results. The expression of telomerase was up-regulated with infiltrating depth, lymph node metastasis and stage (P correlated with infiltrating depth (P < 0.05). Combined detections of telomerase activity and MMP2 protein could identify patients at high risk in disease recurrence and prognosis more efficiently.

Quantitative and qualitative analysis of telomerase activity in benign and malignant thyroid tissues

International Nuclear Information System (INIS)

Zheng Rongxiu; Fang Peihua; Tan Jian; Lu Mei; Li Yigong

2002-01-01

Objective: To study the status of telomerase activity during the development of thyroid tumors, and to determine whether telomerase activity can be used clinically as a molecular marker in the differential diagnosis of thyroid cancer. Methods: Telomerase activity was measured in 37 thyroid carcinomas, 33 benign thyroid lesions and 30 normal thyroid tissue samples by means of a modified TRAP-PCR. The assay was also applied to 15 fine needle aspirates (FNAs) of thyroid carcinomas to test its sensitivity. Results: Thirty-one of 37 thyroid carcinomas (83.8%), 7 of 33 benign thyroid lesions (21.2%), and 4 of 30 adjacent normal thyroid tissue samples expressed telomerase activity, 15 FNAs also had positive telomerase activity, just as their corresponding tissue specimens. The quantitative analysis showed that the telomerase activity was significantly higher in thyroid carcinomas than that in benign thyroid tissue samples. And medullary carcinomas and anaplastic carcinomas had higher levels of telomerase activity than papillary carcinomas. Conclusions: Telomerase activity is a good marker for thyroid carcinomas. The quantitative TRAP-PCR might have more potential application in the differential diagnosis of tumors and the estimation of tumor progression and prognosis. And this sensitive assay could become a useful new modality for supplementing microscopic cytopathology in the detection of cancer cells in small tissue samples and FNAs

Label-free electrochemiluminescence biosensor for ultrasensitive detection of telomerase activity in HeLa cells based on extension reaction and intercalation of Ru(phen)3 (2.).

Science.gov (United States)

Lin, Yue; Yang, Linlin; Yue, Guiyin; Chen, Lifen; Qiu, Bin; Guo, Longhua; Lin, Zhenyu; Chen, Guonan

2016-10-01

Telomerase is one of the most common markers of human malignant tumors, such as uterine, stomach, esophageal, breast, colorectal, laryngeal squamous cell, thyroid, bladder, and so on. It is necessary to develop some sensitive but convenient detection methods for telomerase activity determination. In this study, a label-free and ultrasensitive electrochemiluminescence (ECL) biosensor has been fabricated to detect the activity of telomerase extracted from HeLa cells. Thiolated telomerase substrate (TS) primer was immobilized on the gold electrode surface through gold-sulfur (Au-S) interaction and then elongated by telomerase specifically. Then, it was hybridized with complementary DNA to form double-stranded DNA (dsDNA) fragments on the electrode surface, and Ru(phen)3 (2+) has been intercalated into the dsDNA grooves to act as the ECL probe. The enhanced ECL intensity has a linear relationship with the number of HeLa cells in the range of 5∼5000 and with a detection limit of 2 HeLa cells. The proposed ECL biosensor has high specificity to telomerase in the presence of common interferents. The relative standard deviations (RSDs) were HeLa cells. The proposed method provides a convenient approach for telomerase-related cancer screening or diagnosis.

RAD51 and RTEL1 compensate telomere loss in the absence of telomerase.

Science.gov (United States)

Olivier, Margaux; Charbonnel, Cyril; Amiard, Simon; White, Charles I; Gallego, Maria E

2018-03-16

Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.

Generation of functional islets from human umbilical cord and placenta derived mesenchymal stem cells.

Science.gov (United States)

Kadam, Sachin; Govindasamy, Vijayendran; Bhonde, Ramesh

2012-01-01

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been used for allogeneic application in tissue engineering but have certain drawbacks. Therefore, mesenchymal stem cells (MSCs) derived from other adult tissue sources have been considered as an alternative. The human umbilical cord and placenta are easily available noncontroversial sources of human tissue, which are often discarded as biological waste, and their collection is noninvasive. These sources of MSCs are not subjected to ethical constraints, as in the case of embryonic stem cells. MSCs derived from umbilical cord and placenta are multipotent and have the ability to differentiate into various cell types crossing the lineage boundary towards endodermal lineage. The aim of this chapter is to provide a detailed reproducible cookbook protocol for the isolation, propagation, characterization, and differentiation of MSCs derived from human umbilical cord and placenta with special reference to harnessing their potential towards pancreatic/islet lineage for utilization as a cell therapy product. We show here that mesenchymal stromal cells can be extensively expanded from umbilical cord and placenta of human origin retaining their multilineage differentiation potential in vitro. Our report indicates that postnatal tissues obtained as delivery waste represent a rich source of mesenchymal stromal cells, which can be differentiated into functional islets employing three-stage protocol developed by our group. These islets could be used as novel in vitro model for screening hypoglycemics/insulin secretagogues, thus reducing animal experimentation for this purpose and for the future human islet transplantation programs to treat diabetes.

Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA

NARCIS (Netherlands)

Fontijn, R.; Hop, C.; Brinkman, H. J.; Slater, R.; Westerveld, A.; van Mourik, J. A.; Pannekoek, H.

1995-01-01

Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more

Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

Directory of Open Access Journals (Sweden)

Achim Salamon

2014-02-01

Full Text Available Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.

Calcium phosphate thin films enhance the response of human mesenchymal stem cells to nanostructured titanium surfaces

Directory of Open Access Journals (Sweden)

Mura M McCafferty

2014-05-01

Full Text Available The development of biomaterial surfaces possessing the topographical cues that can promote mesenchymal stem cell recruitment and, in particular, those capable of subsequently directing osteogenic differentiation is of increasing importance for the advancement of tissue engineering. While it is accepted that it is the interaction with specific nanoscale topography that induces mesenchymal stem cell differentiation, the potential for an attendant bioactive chemistry working in tandem with such nanoscale features to enhance this effect has not been considered to any great extent. This article presents a study of mesenchymal stem cell response to conformal bioactive calcium phosphate thin films sputter deposited onto a polycrystalline titanium nanostructured surface with proven capability to directly induce osteogenic differentiation in human bone marrow–derived mesenchymal stem cells. The sputter deposited surfaces supported high levels of human bone marrow–derived mesenchymal stem cell adherence and proliferation, as determined by DNA quantification. Furthermore, they were also found to be capable of directly promoting significant levels of osteogenic differentiation. Specifically, alkaline phosphatase activity, gene expression and immunocytochemical localisation of key osteogenic markers revealed that the nanostructured titanium surfaces and the bioactive calcium phosphate coatings could direct the differentiation towards an osteogenic lineage. Moreover, the addition of the calcium phosphate chemistry to the topographical profile of the titanium was found to induce increased human bone marrow–derived mesenchymal stem cell differentiation compared to that observed for either the titanium or calcium phosphate coating without an underlying nanostructure. Hence, the results presented here highlight that a clear benefit can be achieved from a surface engineering strategy that combines a defined surface topography with an attendant, conformal

Titanium phosphate glass microcarriers induce enhanced osteogenic cell proliferation and human mesenchymal stem cell protein expression

Directory of Open Access Journals (Sweden)

Nilay J Lakhkar

2015-11-01

Full Text Available In this study, we have developed 50- to 100-µm-sized titanium phosphate glass microcarriers (denoted as Ti5 that show enhanced proliferation of human mesenchymal stem cells and MG63 osteosarcoma cells, as well as enhanced human mesenchymal stem cell expression of bone differentiation markers, in comparison with commercially available glass microspheres at all time points. We also demonstrate that these microcarriers provide superior human mesenchymal stem cell proliferation with conventional Dulbecco’s Modified Eagle medium than with a specially developed commercial stem cell medium. The microcarrier proliferative capacity is revealed by a 24-fold increase in MG63 cell numbers in spinner flask bioreactor studies performed over a 7-day period, versus only a 6-fold increase in control microspheres under the same conditions; the corresponding values of Ti5 and control microspheres under static culture are 8-fold and 7-fold, respectively. The capability of guided osteogenic differentiation is confirmed by ELISAs for bone morphogenetic protein-2 and osteopontin, which reveal significantly greater expression of these markers, especially osteopontin, by human mesenchymal stem cells on the Ti5 microspheres than on the control. Scanning electron microscopy and confocal laser scanning microscopy images reveal favorable MG63 and human mesenchymal stem cell adhesion on the Ti5 microsphere surfaces. Thus, the results demonstrate the suitability of the developed microspheres for use as microcarriers in bone tissue engineering applications.

Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

NARCIS (Netherlands)

Fernandes, Hugo; Mentink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells

NARCIS (Netherlands)

Fernandes, H.A.M.; Mentink-Leusink, Anouk; Bank, Ruud; Stoop, Reinout; van Blitterswijk, Clemens; de Boer, Jan

2010-01-01

Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

NARCIS (Netherlands)

Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

2010-01-01

Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix

The Golden Bough: Literature and the Idea of Immortality.

Science.gov (United States)

Klotz, Kenneth

1979-01-01

All works of literature reflect man's obsession with death and his creative attempts to evade or postpone it. Art cannot solve the problem, but within the limits of human capabilities, we come to art, whether as artists or audience, for our own small share in the "intimations of immortality." (Author/SJL)

Towards Maturity in 1 Peter: Freedom, Holiness, Immortality

Directory of Open Access Journals (Sweden)

Paul B. Decock

2016-11-01

Full Text Available Growth towards maturity is dependent on the presence of freedom, holiness and immortality. These are presented as divine qualities that are utterly lacking in human beings. However, while human beings are ignorant and weak, sinful and mortal the addressees of 1 Peter1 are reminded that they have also been begotten anew by the imperishable seed of God’s Word, the Good News of Jesus Christ in order to share immortal life. This article looks first at human beings who as God’s creatures are ‘flesh’, but are also enabled to acknowledge their ‘fleshly’ state, to appreciate (‘desire’ and ‘taste’ (2:2–3 the Gospel and to submit to God. The second part considers the saving role of Christ as the powerful yet rejected ‘stone’ placed and offered by God as the model and means to transcend the ‘flesh’ in the flesh (4:1–2. A final part focuses on the new birth and the growth process in which the fleshly desires and ways of living give way to a manner of life, which is a witness to God’s saving power.

Telomerase lost?

Czech Academy of Sciences Publication Activity Database

Mason, J. M.; Randall, T. A.; Čapková Frydrychová, Radmila

2016-01-01

Roč. 125, č. 1 (2016), s. 65-73 ISSN 0009-5915 R&D Projects: GA ČR GA14-07172S Grant - others:GA JU(CZ) 052/2013/P; GA JU(CZ) 038/2014/P; European Union Seventh Framework Programme(CZ) 316304 Program:FP7 Institutional support: RVO:60077344 Keywords : telomerase * DNA sequences * Bombyx mori Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.414, year: 2016 http://link.springer.com/article/10.1007%2Fs00412-015-0528-7

The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

International Nuclear Information System (INIS)

William Petersen, Ole; Lind Nielsen, Helga; Gudjonsson, Thorarinn; Villadsen, René; Rønnov-Jessen, Lone; Bissell, Mina J

2001-01-01

The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression

The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

Energy Technology Data Exchange (ETDEWEB)

Petersen, Ole William; Nielsen, Helga Lind; Gudjonsson, Thorarinn; Villadsen, Ren& #233; ; Ronnov-Jessen, Lone; Bissell, Mina J.

2001-05-12

The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may indeed have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression.

Universal mortality law and immortality

Science.gov (United States)

Azbel', Mark Ya.

2004-10-01

Well-protected human and laboratory animal populations with abundant resources are evolutionarily unprecedented. Physical approach, which takes advantage of their extensively quantified mortality, establishes that its dominant fraction yields the exact law, which is universal for all animals from yeast to humans. Singularities of the law demonstrate new kinds of stepwise adaptation. The law proves that universal mortality is an evolutionary by-product, which at any given age is reversible, independent of previous life history, and disposable. Life expectancy may be extended, arguably to immortality, by minor biological amendments in the animals. Indeed, in nematodes with a small number of perturbed genes and tissues it increased 6-fold (to 430 years in human terms), with no apparent loss in health and vitality. The law relates universal mortality to specific processes in cells and their genetic regulation.

The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

International Nuclear Information System (INIS)

Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.; Zhang, J.; Oreffo, R.O.C.

2006-01-01

RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPARγ) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPARγ-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negative siRNA). The inhibitory effect of PPARγ-siRNA was supported by testing human PPARγ mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP 3 ) expression, an adipocyte-specific marker. The current studies indicate that PPARγ-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells

Krása a nesmrteľnosť. O plodení u Platóna a Shakespeara (Beauty and Immortality. On Procreation in Plato and Shakespeare

Directory of Open Access Journals (Sweden)

Anton Vydra

2007-03-01

Full Text Available There are two moments referred in this essay: (1 A human being, which desires for immortality, desires for to be alive in his child or in an artwork. (2 Full human being’s desire is related for beauty. Is a beauty the same thing as immortality? How looks the relation between them? Shakespeare’s Sonnets begin with the challenge to his friend to procreate the offspring, because this is a way for immortality of the friend’s beauty. And if it is not a child, than verse refused him before the death or the Lethe. Similarly, Plato’s Socrates says in Symposium about his meeting with Diotima of Mantineia. She told him the oration on real Beauty without accidents. To see real Beauty means to be immortal, likewise the gods. Thus, could be a human being immortal?

Can widely used cell type markers predict the suitability of immortalized or primary mammary epithelial cell models?

Directory of Open Access Journals (Sweden)

Edgar Corneille Ontsouka

Full Text Available BACKGROUND: Mammary cell cultures are convenient tools for in vitro studies of mammary gland biology. However, the heterogeneity of mammary cell types, e.g., glandular milk secretory epithelial or myoepithelial cells, often complicates the interpretation of cell-based data. The present study was undertaken to determine the relevance of bovine primary mammary epithelial cells isolated from American Holstein (bMEC US or Swiss Holstein-Friesian (bMEC CH cows, and of primary bovine mammary alveolar epithelial cells stably transfected with simian virus-40 (SV-40 large T-antigen (MAC-T for in vitro analyses. This was evaluated by testing their expression pattern of cytokeratin (CK 7, 18, 19, vimentin, and α-smooth muscle actin (α-SMA. RESULTS: The expression of the listed markers was assessed using real-time quantitative PCR, flow cytometry and immunofluorescence microscopy. Characteristic markers of the mesenchymal (vimentin, myoepithelial (α-SMA and glandular secretory cells (CKs showed differential expression among the studied cell cultures, partly depending on the analytical method used. The relative mRNA expression of vimentin, CK7 and CK19, respectively, was lower (P < 0.05 in immortalized than in primary mammary cell cultures. The stain index (based on flow cytometry of CK7 and CK19 protein was lower (P < 0.05 in MAC-T than in bMECs, while the expression of α-SMA and CK18 showed an inverse pattern. Immunofluorescence microscopy analysis mostly confirmed the mRNA data, while partly disagreed with flow cytometry data (e.g., vimentin level in MAC-T. The differential expression of CK7 and CK19 allowed discriminating between immortal and primary mammary cultures. CONCLUSIONS: The expression of the selected widely used cell type markers in primary and immortalized MEC cells did not allow a clear preference between these two cell models for in vitro analyses studying aspects of milk composition. All tested cell models exhibited to a variable

Carvacrol promotes angiogenic paracrine potential and endothelial differentiation of human mesenchymal stem cells at low concentrations.

Science.gov (United States)

Matluobi, Danial; Araghi, Atefeh; Maragheh, Behnaz Faramarzian Azimi; Rezabakhsh, Aysa; Soltani, Sina; Khaksar, Majid; Siavashi, Vahid; Feyzi, Adel; Bagheri, Hesam Saghaei; Rahbarghazi, Reza; Montazersaheb, Soheila

2018-01-01

Phenolic monoterpene compound, named Carvacrol, has been found to exert different biological outcomes. It has been accepted that the angiogenic activity of human mesenchymal stem cells was crucial in the pursuit of appropriate regeneration. In the current experiment, we investigated the contribution of Carvacrol on the angiogenic behavior of primary human mesenchymal stem cells. Mesenchymal stem cells were exposed to Carvacrol in a dose ranging from 25 to 200μM for 48h. We measured cell survival rate by MTT assay and migration rate by a scratch test. The oxidative status was monitored by measuring SOD, GPx activity. The endothelial differentiation was studied by evaluating the level of VE-cadherin and vWF by real-time PCR and ELISA analyses. The content of VEGF and tubulogenesis behavior was monitored in vitro. We also conducted Matrigel plug in vivo CAM assay to assess the angiogenic potential of conditioned media from human mesenchymal stem cells after exposure to Carvacrol. Carvacrol was able to increase mesenchymal stem cell survival and migration rate (pcells by detecting vWF and VE-cadherin expression (pmesenchymal stem cells conditioned media improved angiogenesis tube formation in vitro (pmesenchymal stem cells by modulating cell differentiation and paracrine angiogenic response. Copyright © 2017 Elsevier Inc. All rights reserved.

Long telomeres produced by telomerase-resistant recombination are established from a single source and are subject to extreme sequence scrambling.

Directory of Open Access Journals (Sweden)

Jianing Xu

Full Text Available Considerable evidence now supports the idea that the moderate telomere lengthening produced by recombinational telomere elongation (RTE in a Kluyveromyces lactis telomerase deletion mutant occurs through a roll-and-spread mechanism. However, it is unclear whether this mechanism can account for other forms of RTE that produce much longer telomeres such as are seen in human alternative lengthening of telomere (ALT cells or in the telomerase-resistant type IIR "runaway" RTE such as occurs in the K. lactis stn1-M1 mutant. In this study we have used mutationally tagged telomeres to examine the mechanism of RTE in an stn1-M1 mutant both with and without telomerase. Our results suggest that the establishment stage of the mutant state in newly generated stn1-M1 ter1-Δ mutants surprisingly involves a first stage of sudden telomere shortening. Our data also show that, as predicted by the roll-and-spread mechanism, all lengthened telomeres in a newly established mutant cell commonly emerge from a single telomere source. However, in sharp contrast to the RTE of telomerase deletion survivors, we show that the RTE of stn1-M1 ter1-Δ cells produces telomeres whose sequences undergo continuous intense scrambling via recombination. While telomerase was not necessary for the long telomeres in stn1-M1 cells, its presence during their establishment was seen to interfere with the amplification of repeats via recombination, a result consistent with telomerase retaining its ability to add repeats during active RTE. Finally, we observed that the presence of active mismatch repair or telomerase had important influences on telomeric amplification and/or instability.

Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

International Nuclear Information System (INIS)

Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun; Juhn, Kyoung-Mi; Woo, Seon Rang; Kim, Hee-Young; Han, Young-Hoon; Hwang, Sang-Gu; Hong, Sung-Hee; Kang, Chang-Mo; Yoo, Young-Do; Park, Won-Bong; Cho, Myung-Haing; Park, Gil Hong; Lee, Kee-Ho

2010-01-01

Research highlights: → In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. → The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. → The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. → P53 status is not associated with the occurrence of unsensitized clone. → Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeutic regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC -/- cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC -/- clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.

The inhibitory effect of Curcuma longa extract on telomerase activity ...

African Journals Online (AJOL)

STORAGESEVER

2010-02-08

Feb 8, 2010 ... curcumin, could have important effect on treatment of lung cancer. Curcumin ... study inhibitory effect of C. longa total extract on telomerase in A549 lung cancer cell line as in vitro model of ..... If A > 2× (OD of negative control), then, telomerase activity ... radiation, chemotherapy, laser therapy, photodynamic.

Telomere-independent functions of telomerase in nuclei, cytoplasm, and mitochondria

Energy Technology Data Exchange (ETDEWEB)

Chiodi, Ilaria; Mondello, Chiara, E-mail: mondello@igm.cnr.it [Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia (Italy)

2012-09-28

Telomerase canonical activity at telomeres prevents telomere shortening, allowing chromosome stability and cellular proliferation. To perform this task, the catalytic subunit (telomerase reverse transcriptase, TERT) of the enzyme works as a reverse transcriptase together with the telomerase RNA component (TERC), adding telomeric repeats to DNA molecule ends. Growing evidence indicates that, besides the telomeric-DNA synthesis activity, TERT has additional functions in tumor development and is involved in many different biological processes, among which cellular proliferation, gene expression regulation, and mitochondrial functionality. TERT has been shown to act independently of TERC in the Wnt-β-catenin signaling pathway, regulating the expression of Wnt target genes, which play a role in development and tumorigenesis. Moreover, TERT RNA-dependent RNA polymerase activity has been found, leading to the genesis of double-stranded RNAs that act as precursor of silencing RNAs. In mitochondria, a TERT TERC-independent reverse transcriptase activity has been described that could play a role in the protection of mitochondrial integrity. In this review, we will discuss some of the extra-telomeric functions of telomerase.

A supporting role of Chinese National Immortalized Cell Bank in life science research.

Science.gov (United States)

Xu, Chong-feng; Duan, Zi-yuan

2017-01-20

A biorepository of human samples is essential to support the research of life science. Lymphoblastoid B cell line (LCL), which is easy to be prepared and can reproduce indefinitely, is a convenient form of sample preservation. LCLs are established from human B cells transformed by Epstein-Barr virus (EBV). Chinese National Immortalized Cell Bank has preserved human LCLs from different ethnic groups in China. As there are many studies on the nature of LCLs and public available resources with genome-wide data for LCLs, they have been widely applied in genetics, immunology, pharmacogenetics/genomics, regenerative medicine, cancer pathogenesis and immunotherapy, screening and generation of fully human neutralizing monoclonal antibodies and study on EBV pathogenesis. Here, we review the characteristics of LCLs and their contributions to scientific research, and introduce preserved samples in Chinese National Immortalized Cell Bank to the scientific community. We hope this bank can support more areas in the scientific research.

MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors

NARCIS (Netherlands)

Georgi, Nicole; Taipaleenmaeki, H.; Raiss, C.C.; Groen, N.; Portalska, K.K.; van Blitterswijk, Clemens; de Boer, Jan; Post, Janine Nicole; van Wijnen, A.; Karperien, Hermanus Bernardus Johannes

2015-01-01

The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Because the differentiation potential of hMSCs differs between donors, it is necessary to establish

Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

Directory of Open Access Journals (Sweden)

Roberta Fedriga

2001-01-01

Full Text Available Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU. Telomerase activity in urine ranged from 0 to 106 AEU (median 0 in healthy donors and from 0 to 282 AEU (median 87 in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21 of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

A Highly Sensitive Telomerase Activity Assay that Eliminates False-Negative Results Caused by PCR Inhibitors

Directory of Open Access Journals (Sweden)

Hidenobu Yaku

2013-09-01

Full Text Available An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR on magnetic beads (MBs and subsequent application of cycling probe technology (CPT is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGGn-3' of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.

Urine Telomerase for Diagnosis and Surveillance of Bladder Cancer

Directory of Open Access Journals (Sweden)

Angela Lamarca

2012-01-01

Full Text Available Bladder cancer has increased incidence during last decades. For those patients with nonmuscle involved tumors, noninvasive diagnosis test and surveillance methods must be designed to avoid current cystoscopies that nowadays are done regularly in a lot of patients. Novel urine biomarkers have been developed during last years. Telomerase is important in cancer biology, improving the division capacity of cancer cells. Even urinary telomerase could be a potentially useful urinary tumor marker; its use for diagnosis of asymptomatic and symptomatic patients or its impact during surveillance is still unknown. Moreover, there will need to be uniformity and standardization in the assays before it can become useful in clinical practice. It does not seem to exist a real difference between the most classical assays for the detection of urine telomerase (TRAP and hTERT. However, the new detection methods with modified TeloTAGGG telomerase or with gold nanoparticles must also be taken into consideration for the correct development of this diagnosis method. Maybe the target population would be the high-risk groups within screening programs. To date there is no enough evidence to use it alone and to eliminate cystoscopies from the diagnosis and surveillance of these patients. The combination with cytology or FISH is still preferred.

Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

DEFF Research Database (Denmark)

Foster, Leonard J; Zeemann, Patricia A; Li, Chen

2005-01-01

in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...

Immortality in view of Maimonides and Spinoza

Directory of Open Access Journals (Sweden)

Morteza Shajari

2014-12-01

Full Text Available Desire for immortality can be seen as the essential natural impulse. Therefore, different religions and thinkers have attempted to see the issue from different viewpoints. The great Jewish philosopher. Maimonides, due to deep fixation to Judaism, has tried to express their issues to be consistent with the Bible and his own community believes. He, in his discussion of resurrection, believed to three basic steps: The Messiah, the resurrection, and the world hereafter. His standpoint of eternity is dedicated to the hereafter. And we can be immortalized only by acting and teachings in accordance with the Bible and righteousness. Like Maimonides, Spinoza – the other Jewish philosopher - considered the immortality as Ultimate bliss through which the “immutable and eternal love of God" can be achieved. In his opinion, a person reaches this stage, when the lusts and emotions can reasonably be overcome, and also, when the power and anger and contempt and disregard others will respond with love and dignity. Thus, a man can be reached its proper perfection and immortality is reached. The difference between these two philosophers is that Maimonides believes through "actual intellect" -that is Emanation of the active intellect- can be immortalized but, for Spinoza, eternity can be reached through the adequate Ideas.

Detection of telomerase on upconversion nanoparticle modified cellulose paper.

Science.gov (United States)

Wang, Faming; Li, Wen; Wang, Jiasi; Ren, Jinsong; Qu, Xiaogang

2015-07-25

Herein we report a convenient and sensitive method for the detection of telomerase activity based on upconversion nanoparticle (UCNP) modified cellulose paper. Compared with many solution-phase systems, this paper chip is more stable and easily stores the test results. What's more, the low background fluorescence of the UCNPs increases the sensitivity of this method, and the low telomerase levels in different cell lines can clearly be discriminated by the naked eye.

The Origin of Human Mesenchymal Stromal Cells Dictates Their Reparative Properties

DEFF Research Database (Denmark)

Naftali-Shani, Nili; Itzhaki-Alfia, Ayelet; Landa-Rouben, Natalie

2013-01-01

Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair....

Ibn Qayyim Al-Jawziyyah and Allameh Tabataba’i on Immortality in Hell

Directory of Open Access Journals (Sweden)

Janan Izadi

2016-09-01

Full Text Available The Immortality of the people of hell and their eternal torment is one of the most important and complex debates, preoccupying religious scholars of different religions and sects. Each of them has taken a different way based on their intellectual principles of belief to solve this problem and the questions thereof, including how the immortality of the inhabitants of hell hellions and their eternal torment is consistent with the mercy and justice of God. How is it reasonable to endure infinite torment for limited and finite sins? Does a Merciful God, born a sinful slave forever in the fire of the hereafter? Or  is this not the case and He punishes the sinful people for a limited period of time, whether it is short or long, and then releases them from  torture and provides them with  comfort. There is a difference of opinions on these problems in the works of Ibn Qayyim Al- Jawziyyah Al-Ash͑ari and Allameh Tabataba’i, the Shiite philosopher and commentator.Ibn Qayyim addresses widely and systematically the issue of the immortality of the inhabitants of hell. In fact, he interprets immortality (khulūd as a long time, so that the long fire and scourge annihilate the evil from the souls that evil has mixed in their being.  In his viewpoint God has created human monotheist. If the monotheistic nature of the person is changed by vices, these vices and the corrupted nature can be changed by torment and fire. He quotes in his works the ideas of the believers in immortality in torment and criticizes and rebuts them. Stating so many arguments, Ibn Qayyim Al-Jawziyya tries to deny the immortality and eternality in torment. Interpreting the verses of The Holy Quran on immortality of the inhabitants of hell in torment, Allameh Tabataba’i strongly asserts the immortality principle. He relates the happiness and misery, and good and evil among human beings to the development and appearance of the carnal states and habits they gained in the earthly

NAC selectively inhibit cancer telomerase activity: A higher redox homeostasis threshold exists in cancer cells

Directory of Open Access Journals (Sweden)

Pengying Li

2016-08-01

Full Text Available Telomerase activity controls telomere length, and this plays an important role in stem cells, aging and tumors. Antioxidant was shown to protect telomerase activity in normal cells but inhibit that in cancer cells, but the underlying mechanism is elusive. Here we found that 7721 hepatoma cells held a higher redox homeostasis threshold than L02 normal liver cells which caused 7721 cells to have a higher demand for ROS; MnSOD over-expression in 7721 decreased endogenous reactive oxygen species (ROS and inhibited telomerase activity; Akt phosphorylation inhibitor and NAC both inhibited 7721 telomerase activity. The over-elimination of ROS by NAC resulted in the inhibition of Akt pathway. Our results suggest that ROS is involved in the regulation of cancer telomerase activity through Akt pathway. The different intracellular redox homeostasis and antioxidant system in normal cells and tumor cells may be the cause of the opposite effect on telomerase activity in response to NAC treatment. Our results provide a theoretical base of using antioxidants selectively inhibit cancer telomerase activity. Findings of the present study may provide insights into novel approaches for cancer treatment.

Glucosamine-Based Supramolecular Nanotubes for Human Mesenchymal Cell Therapy.

Science.gov (United States)

Talloj, Satish Kumar; Cheng, Bill; Weng, Jen-Po; Lin, Hsin-Chieh

2018-04-23

Herein, we demonstrate an example of glucosamine-based supramolecular hydrogels that can be used for human mesenchymal cell therapy. We designed and synthesized a series of amino acid derivatives based on a strategy of capping d-glucosamine moiety at the C-terminus and fluorinated benzyl group at the N-terminus. From a systematic study on chemical structures, we discovered that the glucosamine-based supramolecular hydrogel [pentafluorobenzyl (PFB)-F-Glu] self-assembled with one-dimensional nanotubular structures at physiological pH. The self-assembly of a newly discovered PFB-F-Glu motif is attributed to the synergistic effect of π-π stacking and extensive intermolecular hydrogen bonding network in aqueous medium. Notably, PFB-F-Glu nanotubes are proven to be nontoxic to human mesenchymal stem cells (hMSCs) and have been shown to enhance hMSC proliferation while maintaining their pluripotency. Retaining of pluripotency capabilities provides potentially unlimited source of undifferentiated cells for the treatment of future cell therapies. Furthermore, hMSCs cultured on PFB-F-Glu are able to secrete paracrine factors that downregulate profibrotic gene expression in lipopolysaccharide-treated human skin fibroblasts, which demonstrates that PFB-F-Glu nanotubes have the potential to be used for wound healing applications. Overall, this article addresses the importance of chemical design to generate supramolecular biomaterials for stem cell therapy.

Herpesvirus telomerase RNA (vTR with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

Directory of Open Access Journals (Sweden)

Benedikt B Kaufer

2011-10-01

Full Text Available Telomerase reverse transcriptase (TERT and telomerase RNA (TR represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5 by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1 that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2 that this strategy could be used to generate novel vaccine candidates

TWIST1 promotes invasion through mesenchymal change in human glioblastoma

Directory of Open Access Journals (Sweden)

Wakimoto Hiroaki

2010-07-01

Full Text Available Abstract Background Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro. Results To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells. Conclusions Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is

Irradiation-induced telomerase activity and gastric cancer risk: a case-control analysis in a Chinese Han population

International Nuclear Information System (INIS)

He, Xianli; Qiao, Qing; Ge, Naijian; Nan, Jing; Shen, Shuqun; Wang, Zizhong; Yang, Yefa; Bao, Guoqiang

2010-01-01

Telomerase expression is one of the characteristics of gastric cancer (GC) cells and telomerase activity is frequently up-regulated by a variety of mechanisms during GC development. Therefore, we hypothesized that elevated levels of activated telomerase might enhance GC risk due to increased propagation of cells with DNA damage, such as induced by γ-radiation. To explore this hypothesis, 246 GC cases and 246 matched controls were recruited in our case-control study. TRAP-ELISA was used to assess the levels of telomerase activity at baseline and after γ-radiation and the γ-radiation-induced telomerase activity (defined as after γ-irradiation/baseline) in cultured peripheral blood lymphocytes (PBLs). Our data showed that there was no significant difference for the baseline telomerase activity between GC cases and controls (10.17 ± 7.21 vs. 11.02 ± 8.03, p = 0.168). However, after γ-radiation treatment, γ-radiation-induced telomerase activity was significantly higher in the cases than in the controls (1.51 ± 0.93 vs. 1.22 ± 0.66, p < 0.001). Using the median value of γ-radiation-induced telomerase activity in the controls as a cutoff point, we observed that high γ-radiation-induced telomerase activity was associated with a significantly increased GC risk (adjusted odds ratio, 2.45; 95% confidence interval, 1.83-3.18). Moreover, a dose response association was noted between γ-radiation-induced telomerase activity and GC risk. Age, but not sex, smoking and drinking status seem to have a modulating effect on the γ-radiation-induced telomerase activities in both cases and controls. Overall, our findings for the first time suggest that the increased γ-radiation-induced telomerase activity in PBLs might be associated with elevated GC risk. Further confirmation of this association using a prospective study design is warranted

Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

International Nuclear Information System (INIS)

Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu

2011-01-01

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of β-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.

Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents.

Science.gov (United States)

Harrington, Dean J; Cemeli, Eduardo; Carder, Joanna; Fearnley, Jamie; Estdale, Sian; Perry, Philip J; Jenkins, Terence C; Anderson, Diana

2003-01-01

Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. Copyright 2003 Wiley-Liss, Inc.

Exploring the motifs of death and immortality | Maina | Journal of ...

African Journals Online (AJOL)

felt threatened by the eventuality of death, inculcating in them a fear so great that all possible strategies are engaged in the search for an avenue that would prepare them for this eventuality. A careful exploration of human activities surrounding the issues of death and immortality reveals an obsession with the expression of ...

Epigenetic modulation of cancer-germline antigen gene expression in tumorigenic human mesenchymal stem cells: implications for cancer therapy

DEFF Research Database (Denmark)

Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole

2009-01-01

Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found...... of cancer-germline antigens in hMSC-TERT20 cells, while their expression levels in primary human mesenchymal stem cells remained unaffected. The expression pattern of cancer-germline antigens in tumorigenic mesenchymal stem cells and sarcomas, plus their susceptibility to enhancement by epigenetic...

Differentiation of human mesenchymal stem cell spheroids under microgravity conditions

Directory of Open Access Journals (Sweden)

Wolfgang H Cerwinka

2012-01-01

Full Text Available To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 106 cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

Telomerase and Tel1p Preferentially Associate with Short Telomeres in S. cerevisiae

Science.gov (United States)

Sabourin, Michelle; Tuzon, Creighton T.; Zakian, Virginia A.

2009-01-01

SUMMARY In diverse organisms, telomerase preferentially elongates short telomeres. We generated a single short telomere in otherwise wild-type (WT) S. cerevisiae cells. The binding of the positive regulators Ku and Cdc13p was similar at short and WT-length telomeres. The negative regulators Rif1p and Rif2p were present at the short telomere, although Rif2p levels were reduced. Two telomerase holoenzyme components, Est1p and Est2p, were preferentially enriched at short telomeres in late S/G2 phase, the time of telomerase action. Tel1p, the yeast ATM-like checkpoint kinase, was highly enriched at short telomeres from early S through G2 phase and even into the next cell cycle. Nonetheless, induction of a single short telomere did not elicit a cell-cycle arrest. Tel1p binding was dependent on Xrs2p and required for preferential binding of telomerase to short telomeres. These data suggest that Tel1p targets telomerase to the DNA ends most in need of extension. PMID:17656141

Interaction of human mesenchymal stem cells with osteopontin coated hydroxyapatite surfaces

DEFF Research Database (Denmark)

Jensen, Thomas; Dolatshahi-Pirouz, Alireza; Foss, Morten

2010-01-01

In vitro studies of the initial attachment, spreading and motility of human bone mesenchymal stem cells have been carried out on bovine osteopontin (OPN) coated hydroxyapatite (HA) and gold (Au) model surfaces. The adsorption of OPN extracted from bovine milk was monitored by the quartz crystal...

A novel telomerase activator suppresses lung damage in a murine model of idiopathic pulmonary fibrosis.

Science.gov (United States)

Le Saux, Claude Jourdan; Davy, Philip; Brampton, Christopher; Ahuja, Seema S; Fauce, Steven; Shivshankar, Pooja; Nguyen, Hieu; Ramaseshan, Mahesh; Tressler, Robert; Pirot, Zhu; Harley, Calvin B; Allsopp, Richard

2013-01-01

The emergence of diseases associated with telomere dysfunction, including AIDS, aplastic anemia and pulmonary fibrosis, has bolstered interest in telomerase activators. We report identification of a new small molecule activator, GRN510, with activity ex vivo and in vivo. Using a novel mouse model, we tested the potential of GRN510 to limit fibrosis induced by bleomycin in mTERT heterozygous mice. Treatment with GRN510 at 10 mg/kg/day activated telomerase 2-4 fold both in hematopoietic progenitors ex vivo and in bone marrow and lung tissue in vivo, respectively. Telomerase activation was countered by co-treatment with Imetelstat (GRN163L), a potent telomerase inhibitor. In this model of bleomycin-induced fibrosis, treatment with GRN510 suppressed the development of fibrosis and accumulation of senescent cells in the lung via a mechanism dependent upon telomerase activation. Treatment of small airway epithelial cells (SAEC) or lung fibroblasts ex vivo with GRN510 revealed telomerase activating and replicative lifespan promoting effects only in the SAEC, suggesting that the mechanism accounting for the protective effects of GRN510 against induced lung fibrosis involves specific types of lung cells. Together, these results support the use of small molecule activators of telomerase in therapies to treat idiopathic pulmonary fibrosis.

Oxygen Tension Regulates Human Mesenchymal Stem Cell Paracrine Functions

OpenAIRE

Paquet, Joseph; Deschepper, Mickael; Moya, Adrien; Logeart-Avramoglou, Delphine; Boisson-Vidal, Catherine; Petite, Hervé

2015-01-01

This study examined the shift of the human mesenchymal stem cell (hMSC) cytokine signature induced by oxygen tension. Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These results elucidate important aspects of using MSCs in regenerative medicine, contribute to improving the efficacy of such therapies, and highlight the interest in using c...

Telomerase Inhibition by Everolimus Suppresses Smooth Muscle Cell Proliferation and Neointima Formation Through Epigenetic Gene Silencing.

Science.gov (United States)

Aono, Jun; Ruiz-Rodriguez, Ernesto; Qing, Hua; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

2016-01-01

The present study sought to investigate the mechanisms underlying the mitogenic function of telomerase and to test the hypothesis that everolimus, commonly used on drug-eluting stents, suppresses smooth muscle cells (SMC) proliferation by targeting telomerase. Proliferation of SMC during neointima formation is prevented by drug-eluting stents. Although the replicative capacity of mammalian cells is enhanced by telomerase expression, the contribution of telomerase to the proliferative response underlying neointima formation and its potential role as a pharmacological target remain to be investigated. We first employed constitutive expression of telomerase reverse transcriptase (TERT) in cell systems to study transcriptional mechanisms by which telomerase activates a mitogenic program. Second, overexpression of telomerase in mice provided a model to study the role of telomerase as a drug target for the antiproliferative efficacy of everolimus. Inhibition of neointima formation by everolimus is lost in mice overexpressing TERT, indicating that repression of telomerase confers the antiproliferative efficacy of everolimus. Everolimus reduces TERT expression in SMC through an Ets-1-dependent inhibition of promoter activation. The inhibition of TERT-dependent SMC proliferation by everolimus occurred in the absence of telomere shortening but rather as a result of a G1→S phase arrest. Although everolimus failed to inhibit phosphorylation of the retinoblastoma protein as the gatekeeper of S-phase entry, it potently repressed downstream target genes. Using chromatin immunoprecipitation assays, we finally demonstrate that TERT induces E2F binding to S-phase gene promoters and supports histone acetylation, effects that are inhibited by everolimus and mediate its antiproliferative activity. These results characterize telomerase as a previously unrecognized target for the antiproliferative activity of everolimus. Our studies further identify a novel mitogenic pathway in SMC

Encapsulated glucagon-like peptide-1-producing mesenchymal stem cells have a beneficial effect on failing pig hearts

DEFF Research Database (Denmark)

Wright, Elizabeth J; Farrell, Kelly A; Malik, Nadim

2012-01-01

-life in vivo. The effects of prolonged GLP-1 delivery from stromal cells post-MI were evaluated in a porcine model. Human mesenchymal stem cells immortalized and engineered to produce a GLP-1 fusion protein were encapsulated in alginate (bead-GLP-1 MSC) and delivered to coronary artery branches. Control groups...... were cell-free beads and beads containing unmodified MSCs (bead-MSC), n = 4-5 per group. Echocardiography confirmed left ventricular (LV) dysfunction at time of delivery in all groups. Four weeks after intervention, only the bead-GLP-1 MSC group demonstrated LV function improvement toward baseline...... and showed decreased infarction area compared with controls. Histological analysis showed reduced inflammation and a trend toward reduced apoptosis in the infarct zone. Increased collagen but fewer myofibroblasts were observed in infarcts of the bead-GLP-1 MSC and bead-MSC groups, and significantly more...

Mechanism of Surface-Enhanced Raman Scattering Based on 3D Graphene-TiO2 Nanocomposites and Application to Real-Time Monitoring of Telomerase Activity in Differentiation of Stem Cells.

Science.gov (United States)

Zheng, Tingting; Feng, Enduo; Wang, Zhiqiang; Gong, Xueqing; Tian, Yang

2017-10-25

With a burst development of new nanomaterials for plasmon-free surface-enhanced Raman scattering (SERS), the understanding of chemical mechanism (CM) and further applications have become more and more attractive. Herein, a novel SERS platform was specially designed through electrochemical deposition of graphene onto TiO 2 nanoarrays (EG-TiO 2 ). The developed EG-TiO 2 nanocomposite SERS platform possessed remarkable Raman activity using copper phthalocyanine (CuPc) as a probe molecule. X-ray photoelectron spectroscopy measurement revealed that the chemical bond Ti-O-C was formed at the interface between graphene and TiO 2 in EG-TiO 2 nanocomposites. Both experimental and theoretical results demonstrated that the obvious Raman enhancement was attributed to TiO 2 -induced Fermi level shift of graphene, resulting in effective charge transfer between EG-TiO 2 nanocomposites and molecules. Taking advantage of a marked Raman response of the CuPc molecule on the EG-TiO 2 nanocomposite surface as well as specific recognition of CuPc toward multiple telomeric G-quadruplex, EG-TiO 2 nanocomposites were tactfully employed as the SERS substrate for selective and ultrasensitive determination of telomerase activity, with a low detection limit down to 2.07 × 10 -16 IU. Interestingly, the self-cleaning characteristic of EG-TiO 2 nanocomposites under visible light irradiation successfully provided a recycling ability for this plasmon-free EG-TiO 2 substrate. The present SERS biosensor with high analytical performance, such as high selectivity and sensitivity, has been further explored to determine telomerase activity in stem cells as well as to count the cell numbers. More importantly, using this useful tool, it was discovered that telomerase activity plays an important role in the proliferation and differentiation from human mesenchymal stem cells to neural stem cells. This work has not only established an approach for gaining fundamental insights into the chemical mechanism (CM

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit

Directory of Open Access Journals (Sweden)

O. G. Lyublinskaya

2015-01-01

Full Text Available The present study focuses on the involvement of reactive oxygen species (ROS in the process of mesenchymal stem cells “waking up” and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs, we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol blocked G1–S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

DEFF Research Database (Denmark)

Petersen, Ole William; Nielsen, Helga Lind; Gudjonsson, Thorarinn

2001-01-01

The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neopl...

Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

International Nuclear Information System (INIS)

Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.

2015-01-01

Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling

Does telomerase activity have an effect on infertility in patients with endometriosis?

Science.gov (United States)

Sofiyeva, Nigar; Ekizoglu, Seda; Gezer, Altay; Yilmaz, Handan; Kolomuc Gayretli, Tugba; Buyru, Nur; Oral, Engin

2017-06-01

This study aimed to investigate the role of telomerase activity in the development of endometriosis-related infertility by evaluation of the serum telomerase in eutopic and ectopic endometrial tissue. Eutopic endometrium, cystic wall/ovarian cortex, and venous blood were assessed in forty-seven patients. The following groups of patients were identified: females with endometriosis requiring surgical intervention and healthy control females. Patients with histopathologically confirmed endometriosis were further subdivided in the infertile (n=14) and fertile (n=17) groups. Patients who underwent hysterectomy and oophorectomy for benign gynecological conditions were enrolled in the healthy control group (n=16). Telomerase activity was evaluated with three-group, endometriosis-based and fertility-based designs. Analyses were performed regardless the menstrual cycle phase (Phase G), in proliferative (Phase P) (n=22) and secretory phases (Phase S) (n=25). Telomeric Repeat Amplification Protocol PCR was applied for telomerase activity assessment. All statistical analyses were performed with STATA 14.2, GraphPad Prisma 7.01. In analyses of the eutopic endometrium, with three-group design, a significant difference was not found in Phase G and P (p=0.58 and p=0.33, respectively). However, a statistical difference was shown in Phase S (p=0.008). A significant difference was not established in Phase G, P and S of endometriosis-based design (p=0.35, p=1.0, p=0.13, respectively). No difference was detected in Phase G and P of fertility-based design (p=0.66 and p=0.14, respectively), whereas in secretory phase difference was approved (p=0,049). Telomerase activity was not established in ectopic endometrium and in serum assessment. Telomerase activity is useless as a biomarker in peripheric blood analysis. The absence of activity in cystic wall approves the high differentiation of endometriosis tissue, what is the possible reason of low malignancy risk. The high rate of telomerase

Telomerase activity promotes osteoblast differentiation by modulating IGF-signaling pathway

DEFF Research Database (Denmark)

Saeed, Hamid; Qiu, Weimin; Li, Chen

2015-01-01

-regulation of several components of insulin-like growth factor (IGF) signaling. Specifically, a significant increase in IGF-induced AKT phosphorylation and alkaline phosphatase (ALP) activity were observed in hMSC-TERT. Enhanced ALP activity was reduced in presence of IGF1 receptor inhibitor: picropodophyllin....... In addition, telomerase deficiency caused significant reduction in IGF signaling proteins in osteoblastic cells cultured from telomerase deficient mice (Terc (-/-)). The low bone mass exhibited by Terc (-/-) mice was associated with significant reduction in serum levels of IGF1 and IGFBP3 as well as reduced...... skeletal mRNA expression of Igf1, Igf2, Igf2r, Igfbp5 and Igfbp6. IGF1-induced osteoblast differentiation was also impaired in Terc (-/-) MSC. In conclusion, our data demonstrate that impaired IGF/AKT signaling contributes to the observed decreased bone mass and bone formation exhibited by telomerase...

Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome.

Science.gov (United States)

Jeyapalan, Jennie N; Varley, Helen; Foxon, Jenny L; Pollock, Raphael E; Jeffreys, Alec J; Henson, Jeremy D; Reddel, Roger R; Royle, Nicola J

2005-07-01

Immortal human cells maintain telomere length by the expression of telomerase or through the alternative lengthening of telomeres (ALT). The ALT mechanism involves a recombination-like process that allows the rapid elongation of shortened telomeres. However, it is not known whether activation of the ALT pathway affects other sequences in the genome. To address this we have investigated, in ALT-expressing cell lines and tumours, the stability of tandem repeat sequences known to mutate via homologous recombination in the human germline. We have shown extraordinary somatic instability in the human minisatellite MS32 (D1S8) in ALT-expressing (ALT+) but not in normal or telomerase-expressing cell lines. The MS32 mutation frequency varied across 15 ALT+ cell lines and was on average 55-fold greater than in ALT- cell lines. The MS32 minisatellite was also highly unstable in three of eight ALT+ soft tissue sarcomas, indicating that somatic destabilization occurs in vivo. The MS32 mutation rates estimated for two ALT+ cell lines were similar to that seen in the germline. However, the internal structures of ALT and germline mutant alleles are very different, indicating differences in the underlying mutation mechanisms. Five other hypervariable minisatellites did not show elevated instability in ALT-expressing cell lines, indicating that minisatellite destabilization is not universal. The elevation of MS32 instability upon activation of the ALT pathway and telomere length maintenance suggests there is overlap between the underlying processes that may be tractable through analysis of the D1S8 locus.

Krása a nesmrteľnosť. O plodení u Platóna a Shakespeara (Beauty and Immortality. On Procreation in Plato and Shakespeare)

OpenAIRE

Anton Vydra

2007-01-01

There are two moments referred in this essay: (1) A human being, which desires for immortality, desires for to be alive in his child or in an artwork. (2) Full human being’s desire is related for beauty. Is a beauty the same thing as immortality? How looks the relation between them? Shakespeare’s Sonnets begin with the challenge to his friend to procreate the offspring, because this is a way for immortality of the friend’s beauty. And if it is not a child, than verse refused him before the de...

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

International Nuclear Information System (INIS)

Uziel, Orit; Kanfer, Gil; Beery, Einat; Yelin, Dana; Shepshelovich, Daniel; Bakhanashvili, Mary; Nordenberg, Jardena; Lahav, Meir

2014-01-01

Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway

The effects of erythropoietin signaling on telomerase regulation in non-erythroid malignant and non-malignant cells

Energy Technology Data Exchange (ETDEWEB)

Uziel, Orit, E-mail: Oritu@clalit.org.il [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Kanfer, Gil [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Beery, Einat [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Yelin, Dana; Shepshelovich, Daniel [Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Bakhanashvili, Mary [Unit of Infectious Diseases, Sheba Medical Center, Tel-Hashomer (Israel); Nordenberg, Jardena [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Dep. of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Endocrinology Laboratory, Beilinson Medical Center, Petah-Tikva (Israel); Lahav, Meir [Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel); Medicine A, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv (Israel)

2014-07-18

Highlights: • We assumed that some of erythropoietin adverse effects may be mediated by telomerase activity. • EPO administration increased telomerase activity, cells proliferation and migration. • The inhibition of telomerase modestly repressed the proliferative effect of erythropoietin. • Telomere shortening caused by long term inhibition of the enzyme totally abolished that effect. • This effect was mediated via the Lyn–AKT axis and not by the canonical JAK2–STAT pathway. - Abstract: Treatment with erythropoietin (EPO) in several cancers is associated with decreased survival due to cancer progression. Due to the major importance of telomerase in cancer biology we hypothesized that some of these effects may be mediated through EPO effect on telomerase. For this aim we explored the possible effects of EPO on telomerase regulation, cell migration and chemosensitivity in non-erythroid malignant and non-malignant cells. Cell proliferation, telomerase activity (TA) and cell migration increased in response to EPO. EPO had no effect on cancer cells sensitivity to cisplatinum and on the cell cycle status. The inhibition of telomerase modestly repressed the proliferative effect of EPO. Telomere shortening caused by long term inhibition of the enzyme abolished the effect of EPO, suggesting that EPO effects on cancer cells are related to telomere dynamics. TA was correlated with the levels of Epo-R. The increase in TA was mediated post-translationally through the Lyn-Src and not the canonical JAK2 pathway.

Telomerase Activity in Breast Tumor Tissues and its Possible use for Detection of Circulating Carcinoma Cells

Czech Academy of Sciences Publication Activity Database

Šimíčková, M.; Nekulová, M.; Pecen, Ladislav; Vagundová, M.; Maláska, J.; Obermannová, R.; Lauerová, L.

2002-01-01

Roč. 5, - (2002), s. 98 ISSN 1211-8869. [Central European Conference on Human Tumor Markers /4./. 13.02.2003-16.02.2003, Karlovy Vary] Institutional research plan: CEZ:AV0Z1030915 Keywords : telomerase activity * early detection of distant metastases * cancer reccurence Subject RIV: BB - Applied Statistics, Operational Research

Designed modulation of sex steroid signaling inhibits telomerase activity and proliferation of human prostate cancer cells

International Nuclear Information System (INIS)

Verma, Vikas; Sharma, Vikas; Singh, Vishal; Sharma, Siddharth; Bishnoi, Ajay Kumar; Chandra, Vishal; Maikhuri, J.P.; Dwivedi, Anila; Kumar, Atul; Gupta, Gopal

2014-01-01

The predominant estrogen-receptor (ER)-β signaling in normal prostate is countered by increased ER-α signaling in prostate cancer (CaP), which in association with androgen-receptor (AR) signaling results in pathogenesis of the disease. However CaP treatments mostly target AR signaling which is initially effective but eventually leads to androgen resistance, hence simultaneous targeting of ERs has been proposed. A novel series of molecules were designed with multiple sex-steroid receptor modulating capabilities by coalescing the pharmacophores of known anti-CaP molecules that act via modulation of ER(α/β) and/or AR, viz. 3,3′diindolylmethane (DIM), mifepristone, toremifene, tamoxifen and raloxifene. N,N-diethyl-4-((2-(4-methoxyphenyl)-1H-indol-3-yl)methyl) aniline (DIMA) was identified as the most promising structure of this new series. DIMA increased annexin-V labelling, cell-cycle arrest and caspase-3 activity, and decreased expression of AR and prostate specific antigen in LNCaP cells, in vitro. Concurrently, DIMA increased ER-β, p21 and p27 protein levels in LNCaP cells and exhibited ∼ 5 times more selective binding for ER-β than ER-α, in comparison to raloxifene. DIMA exhibited a dose-dependent ER-β agonism and ER-α antagonism in classical gene reporter assay and decreased hTERT (catalytic subunit of telomerase) transcript levels in LNCaP at 3.0 μM (P < 0.05). DIMA also dose-dependently decreased telomerase enzyme activity in prostate cancer cells. It is thus concluded that DIMA acts as a multi-steroid receptor modulator and effectively inhibits proliferation of prostate cancer cells through ER-β mediated telomerase inhibition, by countering actions of ER-α and AR. Its unique molecular design can serve as a lead structure for generation of potent agents against endocrine malignancies like the CaP

Designed modulation of sex steroid signaling inhibits telomerase activity and proliferation of human prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Verma, Vikas; Sharma, Vikas; Singh, Vishal [Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Sharma, Siddharth; Bishnoi, Ajay Kumar [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Chandra, Vishal; Maikhuri, J.P.; Dwivedi, Anila [Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Kumar, Atul [Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow 226 031 (India); Gupta, Gopal, E-mail: g_gupta@cdri.res.in [Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow 226 031 (India)

2014-10-15

The predominant estrogen-receptor (ER)-β signaling in normal prostate is countered by increased ER-α signaling in prostate cancer (CaP), which in association with androgen-receptor (AR) signaling results in pathogenesis of the disease. However CaP treatments mostly target AR signaling which is initially effective but eventually leads to androgen resistance, hence simultaneous targeting of ERs has been proposed. A novel series of molecules were designed with multiple sex-steroid receptor modulating capabilities by coalescing the pharmacophores of known anti-CaP molecules that act via modulation of ER(α/β) and/or AR, viz. 3,3′diindolylmethane (DIM), mifepristone, toremifene, tamoxifen and raloxifene. N,N-diethyl-4-((2-(4-methoxyphenyl)-1H-indol-3-yl)methyl) aniline (DIMA) was identified as the most promising structure of this new series. DIMA increased annexin-V labelling, cell-cycle arrest and caspase-3 activity, and decreased expression of AR and prostate specific antigen in LNCaP cells, in vitro. Concurrently, DIMA increased ER-β, p21 and p27 protein levels in LNCaP cells and exhibited ∼ 5 times more selective binding for ER-β than ER-α, in comparison to raloxifene. DIMA exhibited a dose-dependent ER-β agonism and ER-α antagonism in classical gene reporter assay and decreased hTERT (catalytic subunit of telomerase) transcript levels in LNCaP at 3.0 μM (P < 0.05). DIMA also dose-dependently decreased telomerase enzyme activity in prostate cancer cells. It is thus concluded that DIMA acts as a multi-steroid receptor modulator and effectively inhibits proliferation of prostate cancer cells through ER-β mediated telomerase inhibition, by countering actions of ER-α and AR. Its unique molecular design can serve as a lead structure for generation of potent agents against endocrine malignancies like the CaP.

Biomimetic alginate/polyacrylamide porous scaffold supports human mesenchymal stem cell proliferation and chondrogenesis

Energy Technology Data Exchange (ETDEWEB)

Guo, Peng [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China); Yuan, Yasheng, E-mail: yuanyasheng@163.com [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China); Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 (United States); Chi, Fanglu [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China)

2014-09-01

We describe the development of alginate/polyacrylamide (ALG/PAAm) porous hydrogels based on interpenetrating polymer network structure for human mesenchymal stem cell proliferation and chondrogenesis. Three ALG/PAAm hydrogels at molar ratios of 10/90, 20/80, and 30/70 were prepared and characterized with enhanced elastic and rubbery mechanical properties, which are similar to native human cartilage tissues. Their elasticity and swelling properties were also studied under different physiological pH conditions. Finally, in vitro tests demonstrated that human mesenchymal stem cells could proliferate on the as-synthesized hydrogels with improved alkaline phosphatase activities. These results suggest that ALG/PAAm hydrogels may be a promising biomaterial for cartilage tissue engineering. - Highlights: • ALG/PAAm hydrogels were prepared at different molar ratios for cartilage tissue engineering. • ALG/PAAm hydrogels feature an interpenetrating polymer network structure. • ALG/PAAm hydrogels demonstrate strengthened elastic and rubbery mechanical properties. • hMSCs could be cultured on the ALG/PAAm hydrogels for proliferation and chondrogenesis.

Biomimetic alginate/polyacrylamide porous scaffold supports human mesenchymal stem cell proliferation and chondrogenesis

International Nuclear Information System (INIS)

Guo, Peng; Yuan, Yasheng; Chi, Fanglu

2014-01-01

We describe the development of alginate/polyacrylamide (ALG/PAAm) porous hydrogels based on interpenetrating polymer network structure for human mesenchymal stem cell proliferation and chondrogenesis. Three ALG/PAAm hydrogels at molar ratios of 10/90, 20/80, and 30/70 were prepared and characterized with enhanced elastic and rubbery mechanical properties, which are similar to native human cartilage tissues. Their elasticity and swelling properties were also studied under different physiological pH conditions. Finally, in vitro tests demonstrated that human mesenchymal stem cells could proliferate on the as-synthesized hydrogels with improved alkaline phosphatase activities. These results suggest that ALG/PAAm hydrogels may be a promising biomaterial for cartilage tissue engineering. - Highlights: • ALG/PAAm hydrogels were prepared at different molar ratios for cartilage tissue engineering. • ALG/PAAm hydrogels feature an interpenetrating polymer network structure. • ALG/PAAm hydrogels demonstrate strengthened elastic and rubbery mechanical properties. • hMSCs could be cultured on the ALG/PAAm hydrogels for proliferation and chondrogenesis

IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells.

Directory of Open Access Journals (Sweden)

Masahiro Kondo

Full Text Available OBJECTIVE: Mesenchymal stem cells (MSCs can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs. METHODS: Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression. RESULTS: Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA, which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis. CONCLUSIONS: IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

Osteo-/odontogenic differentiation of induced mesenchymal stem cells generated through epithelial-mesenchyme transition of cultured human keratinocytes.

Science.gov (United States)

Yi, Jin-Kyu; Mehrazarin, Shebli; Oh, Ju-Eun; Bhalla, Anu; Oo, Jenessa; Chen, Wei; Lee, Min; Kim, Reuben H; Shin, Ki-Hyuk; Park, No-Hee; Kang, Mo K

2014-11-01

Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT). We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities. Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation. The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Mesenchymal stem cells from human umbilical cord ameliorate testicular dysfunction in a male rat hypogonadism model

Directory of Open Access Journals (Sweden)

Zhi-Yuan Zhang

2017-01-01

Full Text Available Androgen deficiency is a physical disorder that not only affects adults but can also jeopardize children′s health. Because there are many disadvantages to using traditional androgen replacement therapy, we have herein attempted to explore the use of human umbilical cord mesenchymal stem cells for the treatment of androgen deficiency. We transplanted CM-Dil-labeled human umbilical cord mesenchymal stem cells into the testes of an ethane dimethanesulfonate (EDS-induced male rat hypogonadism model. Twenty-one days after transplantation, we found that blood testosterone levels in the therapy group were higher than that of the control group (P = 0.037, and using immunohistochemistry and flow cytometry, we observed that some of the CM-Dil-labeled cells expressed Leydig cell markers for cytochrome P450, family 11, subfamily A, polypeptide 1, and 3-β-hydroxysteroid dehydrogenase. We then recovered these cells and observed that they were still able to proliferate in vitro. The present study shows that mesenchymal stem cells from human umbilical cord may constitute a promising therapeutic modality for the treatment of male hypogonadism patients.

Colorimetry and SERS dual-mode detection of telomerase activity: combining rapid screening with high sensitivity.

Science.gov (United States)

Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Hu, Guohua; Liu, Min; Chen, Peng; Cui, Yiping

2014-01-01

As an important biomarker and therapeutic target, telomerase has attracted considerable attention concerning its detection and monitoring. Here, we present a colorimetry and surface enhanced Raman scattering (SERS) dual-mode telomerase activity detection method, which has several distinctive advantages. First, colorimetric functionality allows rapid preliminary discrimination of telomerase activity by the naked eye. Second, the employment of SERS technique results in greatly improved detection sensitivity. Third, the combination of colorimetry and SERS into one detection system can ensure highly efficacious and sensitive screening of numerous samples. Besides, the avoidance of polymerase chain reaction (PCR) procedures further guarantees fine reliability and simplicity. Generally, the presented method is realized by an "elongate and capture" procedure. To be specific, gold nanoparticles modified with Raman molecules and telomeric repeat complementary oligonucleotide are employed as the colorimetric-SERS bifunctional reporting nanotag, while magnetic nanoparticles functionalized with telomerase substrate oligonucleotide are used as the capturing substrate. Telomerase can synthesize and elongate telomeric repeats onto the capturing substrate. The elongated telomeric repeats subsequently facilitate capturing of the reporting nanotag via hybridization between telomeric repeat and its complementary strand. The captured nanotags can cause a significant difference in the color and SERS intensity of the magnetically separated sediments. Thus both the color and SERS can be used as indicators of the telomerase activity. With fast screening ability and outstanding sensitivity, we anticipate that this method would greatly promote practical application of telomerase-based early-stage cancer diagnosis.

P. berghei telomerase subunit TERT is essential for parasite survival.

Directory of Open Access Journals (Sweden)

Agnieszka A Religa

Full Text Available Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA, though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT, is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further

Telomeres and telomerase as therapeutic targets to prevent and treat age-related diseases [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Christian Bär

2016-01-01

Full Text Available Telomeres, the protective ends of linear chromosomes, shorten throughout an individual’s lifetime. Telomere shortening is a hallmark of molecular aging and is associated with premature appearance of diseases associated with aging. Here, we discuss the role of telomere shortening as a direct cause for aging and age-related diseases. In particular, we draw attention to the fact that telomere length influences longevity. Furthermore, we discuss intrinsic and environmental factors that can impact on human telomere erosion. Finally, we highlight recent advances in telomerase-based therapeutic strategies for the treatment of diseases associated with extremely short telomeres owing to mutations in telomerase, as well as age-related diseases, and ultimately aging itself.

Binding of the sphingolipid S1P to hTERT stabilizes telomerase at the nuclear periphery by allosterically mimicking protein phosphorylation†

Science.gov (United States)

Selvam, Shanmugam P.; De Palma, Ryan M.; Oaks, Joshua J.; Oleinik, Natalia; Peterson, Yuri K.; Stahelin, Robert V.; Skordalakes, Emmanuel; Ponnusamy, Suriyan; Garrett-Mayer, Elizabeth; Smith, Charles D.; Ogretmen, Besim

2015-01-01

During DNA replication, the enzyme telomerase maintains the ends of chromosomes, called telomeres. Shortened telomeres trigger cell senescence, and cancer cells often have increased telomerase activity to promote their ability to proliferate indefinitely. The catalytic subunit, human telomerase reverse transcriptase (hTERT), is stabilized by phosphorylation. Here, we found that the lysophospholipid sphingosine 1-phosphate (S1P), generated by sphingosine kinase 2 (SK2), bound hTERT at the nuclear periphery in human and mouse fibroblasts. Docking predictions and mutational analyses revealed that binding occurred between a hydroxyl group (C′3-OH) in S1P and Asp684 in hTERT. Inhibiting or depleting SK2 or mutating the S1P binding site decreased the stability of hTERT in cultured cells and promoted senescence and loss of telomere integrity. S1P binding inhibited the interaction of hTERT with MKRN1, an E3 ubiquitin ligase that tags hTERT for degradation. Murine Lewis lung carcinoma (LLC) cells formed smaller tumors in mice lacking SK2 than in wild-type mice, and knocking down SK2 in LLC cells before implantation into mice suppressed their growth. Pharmacologically inhibiting SK2 decreased the growth of subcutaneous A549 lung cancer cell-derived xenografts in mice, and expression of wild-type hTERT, but not an S1P-binding mutant, restored tumor growth. Thus, our data suggest that S1P binding to hTERT allosterically mimicks phosphorylation, promoting telomerase stability and hence telomere maintenance, cell proliferation, and tumor growth PMID:26082434

Effect of Mifepristone on the Telomerase Activity in Chorion and Decidua during Early Pregnancy

Institute of Scientific and Technical Information of China (English)

Ge-qing XIA; Ya-li XIONG; Yong-hong SUN

2004-01-01

Objective To investigate telomerase activity in chorion and decidua from abortion induced by mifepristone incorporated with misoprostol at early pregnancy Methods TRAP-SYBR Green assay was used to detect the expression of telomerase. Forty specimen were obtained from medicinal abortion (experiment group) and forty were from normal induced abortion (control group).Results Positive expression, of chorion telomerase was significantly different between the experimental group (28％, 11/40) and the control group (73％, 29/40) (P＜0. 05).While in decidua, the positive rate was 28％ (11/40) in the experimental group and 20％ (9/40) in the control group, there was no significant difference (P＞0. 05).Conclusion It is suggested that miferistone may significantly decrease the telomerase activity in chorion but not in decidua.

Clonal nature of spontaneously immortalized 3T3 cells.

Science.gov (United States)

Rittling, S R

1996-11-25

Mouse embryo fibroblasts (MEFs), when plated at appropriate densities, proliferate vigorously for several passages, and then the growth rate of the culture slows considerably. If the cells are plated at a high enough density and continuously passed, the cultures will eventually overcome this "crisis" period and resume rapid growth. Here, we have addressed the question of what the changes are that cells undergo in overcoming the growth restraints of crisis. Primary MEF cells were infected with a retrovirus which confers G418 resistance and selected in G418. The resultant pre-crisis population comprised cells which each contained a retrovirus integrated at a unique genomic location. These cells were then passed according to the 3T3 protocol until immortal, rapidly growing cells emerged. The integration pattern of the retrovirus in the immortal population was examined. In two independent experiments, the immortal population of cells grown in the presence of G418 comprised two independent clones of cells, with additional clones undetectable at the level of detection of the assays used. The integration pattern was also examined in parallel infected cultures grown in the absence of selection. In one experiment the unselected immortal population contained the same labeled clone that appeared in the sister infected culture, indicating that an immortal precursor was present in the precrisis population. These results are consistent with the idea that a mutation is responsible for the immortal phenotype.

Biocompatibility of quantum dots (CdSe/ZnS ) in human amniotic membrane-derived mesenchymal stem cells in vitro.

Science.gov (United States)

Wang, Gongping; Zeng, Guangwei; Wang, Caie; Wang, Huasheng; Yang, Bo; Guan, Fangxia; Li, Dongpeng; Feng, Xiaoshan

2015-06-01

Amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) are a potential source of mesenchymal stem cells which could be used to repair skin damage. The use of mesenchymal stem cells to repair skin damage requires safe, effective and biocompatible agents to evaluate the effectiveness of the result. Quantum dots (QDs) composed of CdSe/ZnS are semiconductor nanocrystals with broad excitation and narrow emission spectra, which have been considered as a new chemical and fluorescent substance for non-invasively labeling different cells in vitro and in vivo. This study investigated the cytotoxic effects of QDs on hAM-dMSCs at different times following labeling. Using 0.75, 1.5 and 3.0 μL between quantum dots, labeled human amniotic mesenchymal stem cells were collected on days 1, 2 and 4 and observed morphological changes, performed an MTT cell growth assay and flow cytometry for mesenchymal stem cells molecular markers. Quantum dot concentration 0.75 μg/mL labeled under a fluorescence microscope, cell morphology was observed, The MTT assay showed cells in the proliferative phase. Flow cytometry expression CD29, CD31, CD34, CD44, CD90, CD105 and CD106. Within a certain range of concentrations between quantum dots labeled human amniotic mesenchymal stem cells has good biocompatibility.

Alterations of telomerase activity and terminal restriction fragment in gastric cancer and its premalignant lesions.

Science.gov (United States)

Yang, S M; Fang, D C; Luo, Y H; Lu, R; Battle, P D; Liu, W W

2001-08-01

In order to explore the role of alterations of telomerase activity and terminal restriction fragment (TRF) length in the development and progression of gastric cancer. Telomerase activity was detected in 176 specimens of gastric mucosa obtained through an operation or endoscopical biopsy by using the telomeric repeat amplification protocol (TRAP) assay. Meanwhile, the mean length of TRF was measured with the use of a Southern blot in part of those samples. Telomerase activity was detected in 14 of 57 (24.6%) chronic atrophy gastritis patients, six of 18 (33.3%) intestinal metaplasia patients, three of eight (37.5%) dysplasia patients and 60 of 65 (92.3%) gastric cancer patients, respectively. Normal gastric mucosa revealed no telomerase activity. No association was found between telomerase activity and any clinicopathological parameters. The mean TRF length was decreased gradually with age in normal mucosa and in gastric cancer tissue. Regression analysis demonstrated that the reduction rate in these tissues was 41 +/- 12 base pairs/year. Among 35 gastric cancers, TRF length was shown to be shorter in 20 cases (57.1%), similar in 12 cases (34.3%) and elongated in three cases (7.6%), compared to the corresponding adjacent tissues. The mean TRF length tended to decrease as the mucosa underwent chronic atrophy gastritis, intestinal metaplasia, dysplasia and into gastric cancer. The mean TRF length in gastric cancer was not statistically correlated with clinicopathological parameters and telomerase activity. Our results suggest that telomerase is expressed during the early stage of gastric carcinogenesis, and that the clinical significance of TRF length appears to be limited in gastric cancer.

Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

DEFF Research Database (Denmark)

Mortensen, O V; Thomassen, M; Larsen, M B

1999-01-01

were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A...

Recovery of important physiological functions in 3D culture of immortal hepatocytes

DEFF Research Database (Denmark)

Wrzesinski, Krzysztof; Fey, S. J.

2011-01-01

to grow human liver cells in ‘3 dimensional’ cultures so that they behave very similar to the liver in our bodies. By growing the immortal hepatocytes in specially designed bioreactors they form small pieces of ‘pseudotissue’ which exhibit several of the functions seen in the adult liver. We have grown...

UV-activated 7-dehydrocholesterol-coated titanium implants promote differentiation of human umbilical cord mesenchymal stem cells into osteoblasts.

Science.gov (United States)

Satué, María; Ramis, Joana M; Monjo, Marta

2016-01-01

Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants. © The Author(s) 2015.

Locus of Control and Level of Conflict as Correlates of Immortality Orientation.

Science.gov (United States)

O'Dowd, William

1985-01-01

Assessed the orientation of 14 male professors toward immortality as a psychological motive. Results showed a generally low conscious concern with immortality issues; however, respondents who have accepted some sort of immortality show a more internal locus of control and better adjustment. (JAC)

3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

International Nuclear Information System (INIS)

Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

2009-01-01

Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPARα) signaling. Furthermore, using PPARα agonists and antagonists, we also analyzed the effect of PPARα signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.

Direct Genesis of Functional Rodent and Human Schwann Cells from Skin Mesenchymal Precursors

Directory of Open Access Journals (Sweden)

Matthew P. Krause

2014-07-01

Full Text Available Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs, a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

Telomerase Inhibition by Everolimus Suppresses Smooth Muscle Cell Proliferation and Neointima Formation Through Epigenetic Gene Silencing

Directory of Open Access Journals (Sweden)

Jun Aono, MD, PhD

2016-01-01

Full Text Available Proliferation of smooth muscle cells (SMCs during neointima formation is prevented by drug-eluting stents. The replicative capacity of mammalian cells is enhanced by telomerase expression; however, the contribution of telomerase to the proliferative response underlying neointima formation and its potential role as a pharmacological target are unknown. The present study investigated the mechanisms underlying the mitogenic function of telomerase, and tested the hypothesis that everolimus, which is commonly used on drug-eluting stents, suppresses SMC proliferation by targeting telomerase. Inhibition of neointima formation by everolimus was lost in mice overexpressing telomerase reverse transcriptase (TERT, indicating that repression of telomerase confers the anti-proliferative efficacy of everolimus. Everolimus reduced TERT expression in SMC through an Ets-1-dependent inhibition of promoter activation. The inhibition of TERT-dependent SMC proliferation by everolimus occurred in the absence of telomere shortening but rather as a result of a G1→S-phase arrest. Although everolimus failed to inhibit phosphorylation of the retinoblastoma protein as the gatekeeper of S-phase entry, it potently repressed downstream target genes. Chromatin immunoprecipitation assays demonstrated that TERT induced E2F binding to S-phase gene promoters and supported histone acetylation. These effects were sensitive to inhibition by everolimus. These results characterize telomerase as a previously unrecognized target for the antiproliferative activity of everolimus, and further identify a novel mitogenic pathway in SMC that depends on the epigenetic activation of S-phase gene promoters by TERT.

Ameloblastin Peptides Modulates the Osteogenic Capacity of Human Mesenchymal Stem Cells

Czech Academy of Sciences Publication Activity Database

Stakkestad, O.; Lyngstadaas, S. P.; Vondrášek, Jiří; Gordeladze, J. O.; Reseland, J. E.

2017-01-01

Roč. 8, Feb 7 (2017), č. článku 58. ISSN 1664-042X Institutional support: RVO:61388963 Keywords : ameloblastin * biomineralization * bone growth * exon 5 * human mesenchymal stem cells * osteogenesis * proliferation Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 4.134, year: 2016 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293776/pdf/fphys-08-00058.pdf

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

Science.gov (United States)

Jin, Hye Jin; Kwon, Ji Hye; Kim, Miyeon; Bae, Yun Kyung; Choi, Soo Jin; Oh, Wonil; Yang, Yoon Sun; Jeon, Hong Bae

2016-04-01

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required

Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

Directory of Open Access Journals (Sweden)

Irit Meivar-Levy

2011-01-01

Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells

Directory of Open Access Journals (Sweden)

O Schätti

2011-10-01

Full Text Available ere is great interest in how bone marrow derived stem cells make fate decisions. Numerous studies have investigated the role of individual growth factors on mesenchymal stem cell differentiation, leading to protocols for cartilage, bone and adipose tissue. However, these protocols overlook the role of biomechanics on stem cell differentiation. There have been various studies that have applied mechanical stimulation to constructs containing mesenchymal stem cells, with varying degrees of success. One critical fate decision is that between cartilage and bone. Articular motion is a combination of compressive, tensile and shear deformations; therefore, one can presume that compression alone is unlikely to be a sufficient mechanical signal to generate a cartilage-like tissue in vitro. Within this study, we aimed to determine the role of shear on the fate of stem cell differentiation. Specifically, we investigated the potential enhancing effect of surface shear, superimposed on cyclic axial compression, on chondrogenic differentiation of human bone marrow-derived stem cells. Using a custom built loading device we applied compression, shear or a combination of both stimuli onto fibrin/polyurethane composites in which human mesenchymal stem cells were embedded, while no exogenous growth-factors were added to the culture medium. Both compression or shear alone was insufficient for the chondrogenic induction of human mesenchymal stem cells. However, the application of shear superimposed upon dynamic compression led to significant increases in chondrogenic gene expression. Histological analysis detected sulphated glycosaminoglycan and collagen II only in the compression and shear group. The results obtained may provide insight into post-operative care after cell therapy involving mesenchymal stromal cells.

Low LET radiation-induced telomerase catalytic subunit promoter activation is mediated by nuclear factor Kappa B

International Nuclear Information System (INIS)

Natarajan, M.; Hong, F.A.; Mohan, S.; Herman, T.S.

2003-01-01

Full text: The objective of this study is to understand whether low doses of low LET radiation induces survival advantage in normal cells. As an increase in telomerase activity is associated with longevity and cell proliferation, we examined the telomerase response following gamma-irradiation in normal aortic endothelial cells. Telomeric Repeat Amplification Protocol assay following low LET radiation showed an increase in telomerase enzyme activity as early as 8 h post irradiation and reaches its maximum at 24 h. Subsequent analysis revealed that the increased telomerse enzyme activity is due to increased synthesis resulting from an increased transcription. Examination of transcriptional activation of telomerase reverse transcriptase (TERT) promoter regulation showed an enhanced transcription of the telomerse gene following gamma-irradiation. In our previous reports we documented an increase in NF-kB DNA-binding property following low LET radiation (3). Therefore, to determine whether the activation of NF-kB-signaling is responsible for induced TERT promoter activation, cells transiently transfected with minimal promoter region of TERT containing wild type or mutant NF-kB binding site were examined following low LET radiation. TERT promoter activation was induced in wild type transfected cells whereas, in mutant kB binding site, the activation remained at the basal level similar to that of un-irradiated cells. More significantly, the gamma-ray mediated promoter activation of telomerase gene as well as induce telomerase enzyme activity was abrogated by ectopically expressing the IkBa mutant (IkBa (S32A/S36A)), which blocks NF-kB activation. The results thus suggest that exposure to low LET radiation could induce telomerase activity and the activation is at least, in part, mediated by the transcription factor NF-kB. Sustained activation of telomerase in these cells after low LET radiation may impart extended life span

Effects of exogenous ATM gene on mRNA expression of human telomerase reverse transcriptase in AT cells induced by irradiation

International Nuclear Information System (INIS)

Sheng Fangjun; Cao Jianping; Luo Jialin; Zhu Wei; Liu Fenju; Feng Shuang; Song Jianyuan; Li Chong

2005-01-01

The study is to observe effects of exogenous ATM gene on mRNA expression of hTERT (human telomerase reverse transcriptase) in fibroblast cells (AT5BIVA cells) from skin of Ataxia-telangiectasia (AT) patients and to study the regulation of ATM to hTERT. Using reverse transcription polymerase chain reaction (RT-PCR), mRNA expression of hTERT in AT, PEBS7-AT, ATM + -AT and GM cells irradiated with 0 and 3 Gy of 60 Co γ-rays were examined respectively. The difference of the mRNA expression of hTERT among AT, PEBS7-AT, ATM + -AT and GM cells were analyzed. Difference of the mRNA expression of hTERT between 0 Gy and 3 Gy groups was analyzed, too. The results showed that the mRNA expression of hTERT in GM cells was negative, but positive mRNA expression of hTERT in AT cells. The mRNA expression of hTERT in ATM + -AT cells decreased significantly (p 60 Co γ-rays, the mRNA expression of hTERT in GM cells was positive, and that in AT, PEBS7-AT, ATM + -AT cells was increased (p + -AT cells was lower than that in AT and PEBS7-AT cells respectively (p<0.05). It is postulated that exogenous ATM is able to downregulate the mRNA expression of hTERT in AT cells, ionizing radiation can induce the mRNA expression of hTERT in cells and telomerase anticipates the repair of damaged DNA. (authors)

Cell cycle-dependent transcription factors control the expression of yeast telomerase RNA.

Science.gov (United States)

Dionne, Isabelle; Larose, Stéphanie; Dandjinou, Alain T; Abou Elela, Sherif; Wellinger, Raymund J

2013-07-01

Telomerase is a specialized ribonucleoprotein that adds repeated DNA sequences to the ends of eukaryotic chromosomes to preserve genome integrity. Some secondary structure features of the telomerase RNA are very well conserved, and it serves as a central scaffold for the binding of associated proteins. The Saccharomyces cerevisiae telomerase RNA, TLC1, is found in very low copy number in the cell and is the limiting component of the known telomerase holoenzyme constituents. The reasons for this low abundance are unclear, but given that the RNA is very stable, transcriptional control mechanisms must be extremely important. Here we define the sequences forming the TLC1 promoter and identify the elements required for its low expression level, including enhancer and repressor elements. Within an enhancer element, we found consensus sites for Mbp1/Swi4 association, and chromatin immunoprecipitation (ChIP) assays confirmed the binding of Mbp1 and Swi4 to these sites of the TLC1 promoter. Furthermore, the enhancer element conferred cell cycle-dependent regulation to a reporter gene, and mutations in the Mbp1/Swi4 binding sites affected the levels of telomerase RNA and telomere length. Finally, ChIP experiments using a TLC1 RNA-binding protein as target showed cell cycle-dependent transcription of the TLC1 gene. These results indicate that the budding yeast TLC1 RNA is transcribed in a cell cycle-dependent fashion late in G1 and may be part of the S phase-regulated group of genes involved in DNA replication.

Cancer stem cell overexpression of nicotinamide N-methyltransferase enhances cellular radiation resistance

DEFF Research Database (Denmark)

D’Andrea, Filippo P.; Safwat, Akmal; Kassem, Moustapha

2011-01-01

BackgroundCancer stem cells are thought to be a radioresistant population and may be the seeds for recurrence after radiotherapy. Using tumorigenic clones of retroviral immortalized human mesenchymal stem cell with small differences in their phenotype, we investigated possible genetic expression...

The telomerase reverse transcriptase subunit from the dimorphic fungus Ustilago maydis.

Directory of Open Access Journals (Sweden)

Dolores Bautista-España

Full Text Available In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1 contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

DEFF Research Database (Denmark)

Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed

2012-01-01

) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence......Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA...... results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells....

Nonstimulated human uncommitted mesenchymal stem cells express cell markers of mesenchymal and neural lineages.

Science.gov (United States)

Minguell, José J; Fierro, Fernando A; Epuñan, María J; Erices, Alejandro A; Sierralta, Walter D

2005-08-01

Ex vivo cultures of human bone marrow-derived mesenchymal stem cells (MSCs) contain subsets of progenitors exhibiting dissimilar properties. One of these subsets comprises uncommitted progenitors displaying distinctive features, such as morphology, a quiescent condition, growth factor production, and restricted tissue biodistribution after transplantation. In this study, we assessed the competence of these cells to express, in the absence of differentiation stimuli, markers of mesoderm and ectodermic (neural) cell lineages. Fluorescence microscopy analysis showed a unique pattern of expression of osteogenic, chondrogenic, muscle, and neural markers. The depicted "molecular signature" of these early uncommitted progenitors, in the absence of differentiation stimuli, is consistent with their multipotentiality and plasticity as suggested by several in vitro and in vivo studies.

Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma Cells

Energy Technology Data Exchange (ETDEWEB)

Bu, Xinxin; Jia, Fengqi; Wang, Weifeng; Guo, Xianling; Wu, Mengchao; Wei, Lixin [Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Hospital, Second Military Medical Universisty, 225 Changhai Road, Shanghai 200438 (China)

2007-11-12

Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation. This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively. Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells. These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.

Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma Cells

International Nuclear Information System (INIS)

Bu, Xinxin; Jia, Fengqi; Wang, Weifeng; Guo, Xianling; Wu, Mengchao; Wei, Lixin

2007-01-01

Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation. This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively. Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells. These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

International Nuclear Information System (INIS)

Tsuno, Hiroaki; Yoshida, Toshiko; Nogami, Makiko; Koike, Chika; Okabe, Motonori; Noto, Zenko; Arai, Naoya; Noguchi, Makoto; Nikaido, Toshio

2012-01-01

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy. - Highlights: ► Human amniotic mesenchymal cells include cells (HAMα cells) that have the properties of MSCs. ► HAMα cells have excellent osteogenic differentiation potential. ► Osteogenic differentiation ability of HAMα was amplified by calcium phosphate scaffolds. ► HAMα cells can be applicable to allogeneic cell transplantation in bone regenerative therapy.

MicroRNA Regulation of Telomerase Reverse Transcriptase (TERT: Micro Machines Pull Strings of Papier-Mâché Puppets

Directory of Open Access Journals (Sweden)

Ammad Ahmad Farooqi

2018-04-01

Full Text Available Substantial fraction of high-quality information is continuously being added into the existing pool of knowledge related to the biology of telomeres. Based on the insights gleaned from decades of research, it is clear that chromosomal stability needs a highly controlled and dynamic balance of DNA gain and loss in each terminal tract of telomeric repeats. Telomeres are formed by tandem repeats of TTAGGG sequences, which are gradually lost with each round of division of the cells. Targeted inhibition of telomerase to effectively induce apoptosis in cancer cells has attracted tremendous attention and overwhelmingly increasingly list of telomerase inhibitors truthfully advocates pharmacological significance of telomerase. Telomerase reverse transcriptase (TERT is a multi-talented and catalytically active component of the telomerase-associated protein machinery. Different proteins of telomerase-associated machinery work in a synchronized and orchestrated manner to ensure proper maintenance of telomeric length of chromosomes. Rapidly emerging scientific findings about regulation of TERT by microRNAs has revolutionized our understanding related to the biology of telomeres and telomerase. In this review, we have comprehensively discussed how different miRNAs regulate TERT in different cancers. Use of miRNA-based therapeutics against TERT in different cancers needs detailed research in preclinical models for effective translation of laboratory findings to clinically effective therapeutics.

Mesenchymal stem cells in human placental chorionic villi reside in a vascular Niche

NARCIS (Netherlands)

Castrechini, N. M.; Murthi, P.; Gude, N. M.; Erwich, J. J. H. M.; Gronthos, S.; Zannettino, A.; Brennecke, S. R.; Kalionis, B.; Brennecke, S.P.

The chorionic villi of human term placentae are a rich source of mesenchymal stem cells (PMSCs) The stem cell "niche" within the chorionic villi regulates how PMSCs participate in placental tissue generation, maintenance and repair, but the anatomic location of the niche has not been defined A

CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

Directory of Open Access Journals (Sweden)

Yu. В. Basok

2018-01-01

Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

Telomere Maintenance Mechanisms in Cancer

OpenAIRE

Tiago Bordeira Gaspar; Ana Sá; José Manuel Lopes; Manuel Sobrinho-Simões; Paula Soares; João Vinagre

2018-01-01

Tumour cells can adopt telomere maintenance mechanisms (TMMs) to avoid telomere shortening, an inevitable process due to successive cell divisions. In most tumour cells, telomere length (TL) is maintained by reactivation of telomerase, while a small part acquires immortality through the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. In the last years, a great amount of data was generated, and different TMMs were reported and explained in detail, benefiting from g...

The (not so) immortal strand hypothesis.

Science.gov (United States)

Tomasetti, Cristian; Bozic, Ivana

2015-03-01

Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an "immortal" DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. Copyright © 2015. Published by Elsevier B.V.

DNA methylation patterns of imprinting centers for H19, SNRPN, and KCNQ1OT1 in single-cell clones of human amniotic fluid mesenchymal stem cell

Directory of Open Access Journals (Sweden)

Hsiu-Huei Peng

2012-09-01

Conclusion: In conclusion, human amniotic fluid mesenchymal stem cells contain a unique epigenetic signature during in vitro cell culture. H19 and KCNQ1OT1 possessed a substantial degree of hypermethylation status, and variable DNA methylation patterns of SNRPN was observed during in vitro cell culture of human amniotic fluid mesenchymal stem cells. Our results urge further understanding of epigenetic status of human amniotic fluid mesenchymal stem cells before it is applied in cell replacement therapy.

Effect of Human Adipose Tissue Mesenchymal Stem Cells on the Regeneration of Ovine Articular Cartilage.

Science.gov (United States)

Zorzi, Alessandro R; Amstalden, Eliane M I; Plepis, Ana Maria G; Martins, Virginia C A; Ferretti, Mario; Antonioli, Eliane; Duarte, Adriana S S; Luzo, Angela C M; Miranda, João B

2015-11-09

Cell therapy is a promising approach to improve cartilage healing. Adipose tissue is an abundant and readily accessible cell source. Previous studies have demonstrated good cartilage repair results with adipose tissue mesenchymal stem cells in small animal experiments. This study aimed to examine these cells in a large animal model. Thirty knees of adult sheep were randomly allocated to three treatment groups: CELLS (scaffold seeded with human adipose tissue mesenchymal stem cells), SCAFFOLD (scaffold without cells), or EMPTY (untreated lesions). A partial thickness defect was created in the medial femoral condyle. After six months, the knees were examined according to an adaptation of the International Cartilage Repair Society (ICRS 1) score, in addition to a new Partial Thickness Model scale and the ICRS macroscopic score. All of the animals completed the follow-up period. The CELLS group presented with the highest ICRS 1 score (8.3 ± 3.1), followed by the SCAFFOLD group (5.6 ± 2.2) and the EMPTY group (5.2 ± 2.4) (p = 0.033). Other scores were not significantly different. These results suggest that human adipose tissue mesenchymal stem cells promoted satisfactory cartilage repair in the ovine model.

Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.

Directory of Open Access Journals (Sweden)

Aparna Raval

Full Text Available Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC, aplastic anemia (AA and myelodysplastic syndromes (MDS. However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/- mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

Nanocurcumin-Mediated Down-Regulation of Telomerase Via Stimulating TGFβ1 Signaling Pathway in Hepatocellular Carcinoma Cells

Science.gov (United States)

Shariati, Molood; Hajigholami, Samira; Veisi Malekshahi, Ziba; Entezari, Maliheh; Bodaghabadi, Narges; Sadeghizadeh, Majid

2017-10-10

Curcumin, extracted from turmeric, represents enormous potential to serve as an anticancer agent. Telomerase is viewed as a prominent molecular target of curcumin, and Transforming growth factor-β1 (TGFβ1) has proven to be a major inhibitory signaling pathway for telomerase activity. In the current study, we aimed to explore suppressive effects of nanocurcumin on telomerase expression through TGFβ1 pathway in a hepatocellular carcinoma cell line (Huh7). MTT assay was used to determine the effect of nonocurcumin on viability of Huh7 cells. RT-PCR was used to analyze the gene expression patterns. MTT assay revealed that nanocurcumin acts in a dose- and time-dependent manner to diminish the cell viability. RT-PCR analysis indicated that nanocurcumin results in augmentation of TGFβ1 72 hours post treatment and leads to the reduction of telomerase expression 48 and 72 hours post exposure. Also, up-regulation of Smad3 and E2F1 and down-regulation of Smad7 confirmed the effect of nanocurcumin on intermediate components of TGFβ1 pathway. Furthermore, transfection of the proximal promoter of telomerase triggered a significant reduction in luciferase activity. The data from the present study lead us to develop a deeper understanding of the mechanisms underlying nanocurcumin-mediated regulation of telomerase expression, thereby presenting a new perspective to the landscape of using nanocurcumin as a cancer-oriented therapeutic agent.

Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma Cells

Directory of Open Access Journals (Sweden)

Wu Mengchao

2007-11-01

Full Text Available Abstract Background Hepatocellular carcinoma (HCC is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation. Methods This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu. We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively. Results Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells. Conclusion These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

Science.gov (United States)

Muiños-López, Emma; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; de Toro-Santos, Javier; Blanco, Francisco Javier; Díaz-Prado, Silvia María

2017-09-01

Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better

Life Beyond the Physical Body: The Possibilities of Digital Immortality

OpenAIRE

Galvão, Vinícius F.; Maciél, Cristiano; Garcia, Ana Cristina B.; Viterbo, José

2017-01-01

We are on the verge of a major shift in the way we perceive digital life, what may cause a significant impact to the real world. Gradually, through increasing knowledge in the areas of artificial intelligence, big data and machine learning, computers have been emulating deceased human beings and, symbolically, with the aid of technology, have been managing to conquer death. This article seeks to understand and problematize the ways in which digital immortality has manifested itself, particula...

Postirradiation expression of lethal mutations in an immortalized human keratinocyte cell line

International Nuclear Information System (INIS)

O'Reilly, S.; Mothersill, C.; Seymour, C.B.

1994-01-01

The quantification of the extent of delayed cell death and the rate and pattern of its occurrence in relation to the cell division cycle is important in radiotherapy and also in radiation transformation studies related to protection and dose limits. Here the numbers of lethal mutations occurring over 45 population doublings (clonal expansion to about 10 13 cells per cell originally surviving irradiation) was measured in an HPV 16 immortalized human keratinocyte cell lines used for transformation studies. The results showed that when postirradiation (dose range 1-6 Gy) growth curves were constructed, the difference in slopes could be accounted for entirely by correcting for the non-clonogenic fraction in the cell count, excluding a longer cell generation time as an explanation. When the cell loss was examined over the entire growth period of 6 weeks (about 45 doublings of the cell population), it was found to be dose dependent for the first two passages, but then to become more independent of dose. The results allow a time/cell generation dependent factor to be derived for the cell line and used in survival curve equations where effects of radiation are being measured at times distant from the original exposure. (author)

Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review.

Science.gov (United States)

Panchalingam, Krishna M; Jung, Sunghoon; Rosenberg, Lawrence; Behie, Leo A

2015-11-23

Human mesenchymal stem cells (hMSCs), also called mesenchymal stromal cells, have been of great interest in regenerative medicine applications because of not only their differentiation potential but also their ability to secrete bioactive factors that can modulate the immune system and promote tissue repair. This potential has initiated many early-phase clinical studies for the treatment of various diseases, disorders, and injuries by using either hMSCs themselves or their secreted products. Currently, hMSCs for clinical use are generated through conventional static adherent cultures in the presence of fetal bovine serum or human-sourced supplements. However, these methods suffer from variable culture conditions (i.e., ill-defined medium components and heterogeneous culture environment) and thus are not ideal procedures to meet the expected future demand of quality-assured hMSCs for human therapeutic use. Optimizing a bioprocess to generate hMSCs or their secreted products (or both) promises to improve the efficacy as well as safety of this stem cell therapy. In this review, current media and methods for hMSC culture are outlined and bioprocess development strategies discussed.

β-Cyclodextrin-curcumin complex inhibit telomerase gene ...

African Journals Online (AJOL)

Yomi

2011-12-21

Dec 21, 2011 ... have various applications in cancer therapy. But, its low water solubility and bioavailability is possible for poor drug delivery of curcumin. In this study, we prepared β-cyclodextrin-curcumin complex to determine the inhibitory effect of this drug on telomerase gene expression. Curcumin was encapsulated.

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Energy Technology Data Exchange (ETDEWEB)

Tsuno, Hiroaki [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Yoshida, Toshiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nogami, Makiko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Orthopedic Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Koike, Chika; Okabe, Motonori [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Noto, Zenko [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Arai, Naoya; Noguchi, Makoto [Department of Oral and Maxillofacial Surgery, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan); Nikaido, Toshio, E-mail: tnikaido@med.u-toyama.ac.jp [Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences for Research, University of Toyama, 2630 Sugitani Toyama, Toyama 930-0194 (Japan)

2012-12-01

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAM{alpha} cells and induced to osteogenic status-their in vivo osteogenesis was subsequently investigated in rats. It was found that HAM{alpha} cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAM{alpha} cells. The expression of osteocalcin mRNA was increased in HAM{alpha} cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAM{alpha} cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy. - Highlights: Black-Right-Pointing-Pointer Human amniotic mesenchymal cells include cells (HAM{alpha} cells) that have the properties of MSCs. Black-Right-Pointing-Pointer HAM{alpha} cells have excellent osteogenic differentiation potential. Black-Right-Pointing-Pointer Osteogenic differentiation ability of HAM{alpha} was amplified by calcium phosphate scaffolds. Black-Right-Pointing-Pointer HAM{alpha} cells can be applicable to allogeneic cell transplantation in bone regenerative therapy.

Niclosamide inhibits epithelial-mesenchymal transition and tumor growth in lapatinib-resistant human epidermal growth factor receptor 2-positive breast cancer.

Science.gov (United States)

Liu, Junjun; Chen, Xiaosong; Ward, Toby; Mao, Yan; Bockhorn, Jessica; Liu, Xiaofei; Wang, Gen; Pegram, Mark; Shen, Kunwei

2016-02-01

Acquired resistance to lapatinib, a human epidermal growth factor receptor 2 kinase inhibitor, remains a clinical problem for women with human epidermal growth factor receptor 2-positive advanced breast cancer, as metastasis is commonly observed in these patients. Niclosamide, an anti-helminthic agent, has recently been shown to exhibit cytotoxicity to tumor cells with stem-like characteristics. This study was designed to identify the mechanisms underlying lapatinib resistance and to determine whether niclosamide inhibits lapatinib resistance by reversing epithelial-mesenchymal transition. Here, two human epidermal growth factor receptor 2-positive breast cancer cell lines, SKBR3 and BT474, were exposed to increasing concentrations of lapatinib to establish lapatinib-resistant cultures. Lapatinib-resistant SKBR3 and BT474 cells exhibited up-regulation of the phenotypic epithelial-mesenchymal transition markers Snail, vimentin and α-smooth muscle actin, accompanied by activation of nuclear factor-кB and Src and a concomitant increase in stem cell marker expression (CD44(high)/CD24(low)), compared to naive lapatinib-sensitive SKBR3 and BT474 cells, respectively. Interestingly, niclosamide reversed epithelial-mesenchymal transition, induced apoptosis and inhibited cell growth by perturbing aberrant signaling pathway activation in lapatinib-resistant human epidermal growth factor receptor 2-positive cells. The ability of niclosamide to alleviate stem-like phenotype development and invasion was confirmed. Collectively, our results demonstrate that lapatinib resistance correlates with epithelial-mesenchymal transition and that niclosamide inhibits lapatinib-resistant cell viability and epithelial-mesenchymal transition. These findings suggest a role of niclosamide or derivatives optimized for more favorable bioavailability not only in reversing lapatinib resistance but also in reducing metastatic potential during the treatment of human epidermal growth factor receptor

Fetal Mesenchymal Stromal Cells Differentiating towards Chondrocytes Acquire a Gene Expression Profile Resembling Human Growth Plate Cartilage

NARCIS (Netherlands)

van Gool, S.A.; Emons, J.A.M.; Leijten, Jeroen Christianus Hermanus; Decker, E.; Sticht, C.; van Houwelingen, J.C.; Goeman, J.J.; Kleijburg, C.; Scherjon, S.; Gretz, N.; Wit, J.M.; Rappold, G.; Post, Janine Nicole; Karperien, Hermanus Bernardus Johannes

2012-01-01

Abstract We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether

Primary mesenchymal stem cells in human transplanted lungs are CD90/CD105 perivascularly located tissue-resident cells

DEFF Research Database (Denmark)

Rolandsson, Sara; Andersson Sjöland, Annika; Brune, Jan C

2014-01-01

BACKGROUND: Mesenchymal stem cells (MSC) have not only been implicated in the development of lung diseases, but they have also been proposed as a future cell-based therapy for lung diseases. However, the cellular identity of the primary MSC in human lung tissues has not yet been reported. This st......BACKGROUND: Mesenchymal stem cells (MSC) have not only been implicated in the development of lung diseases, but they have also been proposed as a future cell-based therapy for lung diseases. However, the cellular identity of the primary MSC in human lung tissues has not yet been reported...

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.

Science.gov (United States)

Eigenmann, Daniela Elisabeth; Jähne, Evelyn Andrea; Smieško, Martin; Hamburger, Matthias; Oufir, Mouhssin

2016-03-01

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.

Developmentally inspired programming of adult human mesenchymal stromal cells toward stable chondrogenesis.

Science.gov (United States)

Occhetta, Paola; Pigeot, Sebastien; Rasponi, Marco; Dasen, Boris; Mehrkens, Arne; Ullrich, Thomas; Kramer, Ina; Guth-Gundel, Sabine; Barbero, Andrea; Martin, Ivan

2018-05-01

It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.

Establishment and characterization of a spontaneously immortalized trophoblast cell line (HPT-8) and its hepatitis B virus-expressing clone.

Science.gov (United States)

Zhang, Lei; Zhang, Weilu; Shao, Chen; Zhang, Jingxia; Men, Ke; Shao, Zhongjun; Yan, Yongping; Xu, Dezhong

2011-08-01

Most trophoblast cell lines currently available to study vertical transmission of hepatitis B virus (HBV) are immortalized by viral transformation. Our goal was to establish and characterize a spontaneously immortalized human first-trimester trophoblast cell line and its HBV-expressing clone. Chorionic villi of Asian human first-trimester placentae were digested with trypsin and collagenase I to obtain the primary trophoblast cell culture. A spontaneously immortalized trophoblast cell line (HPT-8) was analyzed by scanning and transmission electron microscopy, cell cycle analysis, immunohistochemistry and immunofluorescence. HPT-8 cells were stably transfected with the adr subtype of HBV (HPT-8-HBV) and characterized by PCR and enzyme-linked immunosorbent assay. We obtained a clonal derivative of a spontaneously immortalized primary cell clone (HPT-8). HPT-8 cells were epithelioid and polygonal, and formed multinucleate, giant cells. They exhibited microvilli, distinct desmosomes between adjacent cells, abundant endoplasm, lipid inclusions and glycogen granules, which are all characteristic of cytotrophoblasts. HPT-8 cells expressed cytokeratin 7, cytokeratin 18, vimentin, cluster of differentiation antigen 9, epidermal growth factor receptor, stromal cell-derived factor 1 and placental alkaline phosphatase. They secreted prolactin, estradiol, progesterone and hCG, and were positive for HLA-G, a marker of extravillous trophoblasts. HPT-8-HBV cells were positive for HBV relaxed-circular, covalently closed circular DNA and pre-S sequence. HPT-8-HBV cells also produced and secreted HBV surface antigen and HBV e antigen. We established a trophoblast cell line, HPT-8 and its HBV-expressing clone which could be valuable in exploring the mechanism of HBV viral integration in human trophoblasts during intrauterine infection.

Triterpenoids from Ganoderma lucidum inhibit the activation of EBV antigens as telomerase inhibitors.

Science.gov (United States)

Zheng, Dong-Shu; Chen, Liang-Shu

2017-10-01

Nasopharyngeal carcinoma (NPC) is a malignant disease that threatens the health of humans. To find effective agents for the inhibition of Epstein-Barr virus (EBV) infection, which is associated with NPC, a phytochemical investigation of Ganoderma lucidum was carried out in the present study. Five triterpenoids were identified, including ganoderic acid A (compound 1), ganoderic acid B (compound 2), ganoderol B (compound 3), ganodermanontriol (compound 4), and ganodermanondiol (compound 5), on the basis of spectroscopic analysis. An inhibition of EBV antigens activation assay was implemented to elucidate the triterpenoids from G. lucidum and potentially prevent NPC. All the triterpenoids showed significant inhibitory effects on both EBV EA and CA activation at 16 nmol. At 3.2 nmol, all the compounds moderately inhibited the activation of the two antigens. The activity of telomerase was inhibited by these triterpenoids at 10 µM. Molecular docking demonstrated that compound 1 was able to inhibit telomerase as a ligand. In addition, the physicochemical properties of these compounds were calculated to elucidate their drug-like properties. These results provided evidence for the application of these triterpenoids and whole G. lucidum in the treatment of NPC.

Inhibition of cell proliferation and induction of apoptosis by oleanane triterpenoid (CDDO-Me) in pancreatic cancer cells is associated with the suppression of hTERT gene expression and its telomerase activity

International Nuclear Information System (INIS)

Deeb, Dorrah; Gao, Xiaohua; Liu, Yongbo; Kim, Sahn-Ho; Pindolia, Kirit R.; Arbab, Ali S.; Gautam, Subhash C.

2012-01-01

Highlights: ► CDDO-Me inhibits hTERT gene expression. ► CDDO-Me inhibits hTERT protein expression. ► CDDO-Me inhibits hTERT telomerase activity. ► CDDO-Me inhibits hTERT regulatory proteins. -- Abstract: Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a multifunctional oleanane synthetic triterpenoid with potent anti-inflammatory and antitumorigenic properties. The mechanisms of the antisurvival and apoptosis-inducing activities of CDDO-Me and related derivatives of oleanolic acid have been defined; however, to date, no study has been carried out on the effect of CDDOs on human telomerase reverse transcriptase (hTERT) gene or telomerase activity. Here we report for the first time that inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cell lines is associated with the inhibition of hTERT gene expression, hTERT telomerase activity and a number of proteins that regulate hTERT expression and activity. Furthermore, abrogation or overexpression of hTERT protein altered the susceptibility of tumor cells to CDDO-Me. These findings suggest that telomerase (hTERT) is a relevant target of CDDO-Me in pancreatic cancer cells.

Changes in stress, eating, and metabolic factors are related to changes in telomerase activity in a randomized mindfulness intervention pilot study.

Science.gov (United States)

Daubenmier, Jennifer; Lin, Jue; Blackburn, Elizabeth; Hecht, Frederick M; Kristeller, Jean; Maninger, Nicole; Kuwata, Margaret; Bacchetti, Peter; Havel, Peter J; Epel, Elissa

2012-07-01

Psychological distress and metabolic dysregulation are associated with markers of accelerated cellular aging, including reduced telomerase activity and shortened telomere length. We examined whether participation in a mindfulness-based intervention, and, secondarily, improvements in psychological distress, eating behavior, and metabolic factors are associated with increases in telomerase activity in peripheral blood mononuclear cells (PBMCs). We enrolled 47 overweight/obese women in a randomized waitlist-controlled pilot trial (n=47) of a mindfulness-based intervention for stress eating and examined changes in telomerase activity from pre- to post-intervention. In secondary analyses, changes in telomerase activity across the sample were examined in relation to pre- to post-intervention changes in psychological distress, eating behavior, and metabolic factors (weight, serum cortisol, fasting glucose and insulin, and insulin resistance). Both groups increased in mean telomerase activity over 4 months in intent-to-treat and treatment efficacy analyses (peating behavior, and metabolic health and increases in telomerase activity. These findings suggest that telomerase activity may be in part regulated by levels of both psychological and metabolic stress. Published by Elsevier Ltd.

Effects of Curcuma longa Extract on Telomerase Activity in Lung and Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Nosratollah Zarghami

2014-10-01

Full Text Available Background: The purpose of this study is to evaluate the effect of Curcuma longa extract on the telomerase gene expression in QU-DB lung cancer and T47D breast cancer cell lines. Materials and Methods: The present study is an experimental research. Using 3 different phases n-hexane, dichloromethane and methanol, total extract of Curcuma longa in a serial dilution was prepared and three phases was analyzed for determining which phase has more curcuminoids. Then the extract cytotoxicity effect was tested on breast cancer cell line (T47D, and lung cancer cell line (QU-DB by 24, 48 and 72 h MTT (Dimethyl thiazolyl diphenyl tetrazolium assay. Then, the cells were treated with serial concentrations of the extract. Finally, total protein was extracted from the control and test groups, its quantity was determined and telomeric repeat amplification protocol (TRAP assay was performed for measurement of possible inhibition of the telomerase activity. Results: Cell viability and MTT-based cytotoxicity assay show that the total extract of Curcuma longa has cytotoxic effect with different IC50s in breast and lung cancer cell lines. Analysis of TRAP assay also shows a significant reduction in telomerase activity on both cancer cells with different levels. Conclusion: Curcuma longa extract has anti-proliferation and telomerase inhibitory effects on QU-DB lung cancer and T47D breast cancer cells with differences in levels of telomerase inhibition.

Human multipotent mesenchymal stromal cells in the treatment of postoperative temporal bone defect: an animal model