High Levels Of Telomerase Activity Research Articles

Novel protective effect of the FOXO3 longevity genotype on mechanisms of cellular aging in Okinawans

The genetic association of FOXO3 genotypes with human longevity is well established, although the mechanism is not fully understood. We now report on the relationship of the FOXO3 longevity variant rs2802292 with telomere length, telomerase activity, FOXO3 expression, and inflammatory cytokine levels in men and women. In agreement with earlier work, the FOXO3 longevity variant conferred protection against telomere shortening of peripheral blood mononuclear cells from adults aged 55 years and older. This was accompanied by higher levels of telomerase activity in mononuclear cells for carriers of the longevity-associated FOXO3 G-allele of SNP rs2802292 (P = 0.015). FOXO3 mRNA expression increased slightly with age in both young (P = 0.02) and old (P = 0.08) G-allele carriers. Older female G-allele carriers displayed a modest decline in levels of pro-inflammatory cytokine IL-6 with age (P = 0.07). In contrast, older male G-allele carriers displayed an age-dependent increase in levels of anti-inflammatory cytokine IL-10 with age (P = 0.04). Thus, FOXO3 may act through several different pro-longevity mechanisms, which may differ by age and sex.

THE FOXO3 LONGEVITY GENOTYPE PROTECTS AGAINST MECHANISMS OF CELLULAR AGING IN OKINAWANS

Abstract The genetic association of FOXO3 genotypes with human longevity is well established, although the mechanism is not fully understood. We now report on the relationship of the FOXO3 longevity variant rs2802292 with telomere length, telomerase activity, FOXO3 expression and inflammatory cytokine levels in men and women. In agreement with earlier work, the FOXO3 longevity variant conferred protection against telomere shortening of peripheral blood mononuclear cells from adults aged 55 and older. This was accompanied by higher levels of telomerase activity in carriers of the longevity-associated FOXO3 G-allele (P=0.015). FOXO3 mRNA expression increased slightly with age in both young (P=0.02) and old (P=0.08) G-allele carriers. Older female G-allele carriers displayed a modest decline in levels of pro-inflammatory cytokine IL-6 with age (P=0.07). In contrast, older male G-allele carriers displayed an age-dependent increase in levels of anti-inflammatory cytokine IL-10 with age (P=0.04). Thus, FOXO3 may act through several different pro-longevity mechanisms, which may differ by age and sex.

Discovery of potent telomerase activators: Unfolding new therapeutic and anti-aging perspectives.

Telomere length, a marker of cellular aging, decreases with age and it has been associated with aging-related diseases. Environmental factors, including diet and lifestyle factors, affect the rate of telomere shortening which can be reversed by telomerase. Telomerase activation by natural molecules has been suggested to be an anti-aging modulator that can play a role in the treatment of aging-related diseases. This study aimed to investigate the effect of natural compounds on telomerase activity in human peripheral blood mononuclear cells (PBMCs). The tested compounds included Centella asiatica extract formulation (08AGTLF), Astragalus extract formulation (Nutrient 4), TA-65 (containing Astragalus membranaceus extract), oleanolic acid (OA), maslinic acid (MA), and 3 multi-nutrient formulas (Nutrients 1, 2 and 3) at various concentrations. The mean absorbance values of telomerase activity measured following treatment with some of the above-mentioned formulations were statistically significantly higher compared to those of the untreated cells. In particular, in order of importance with respect to telomerase activation from highest to lowest, 08AGTLF, OA, Nutrient 4, TA-65, MA, Nutrient 3 and Nutrient 2, triggered statistically significant increase in telomerase activity compared to the untreated cells. 08AGTLF reached the highest levels of telomerase activity reported to date, at least to our knowledge, increasing telomerase activity by 8.8 folds compared to untreated cells, while Nutrient 4 and OA were also potent activators (4.3-fold and 5.9-fold increase, respectively). On the whole, this study indicates that the synergistic effect of nutrients and natural compounds can activate telomerase and produce more potent formulations. Human clinical studies using these formulations are required to evaluate their mode of action. This would reveal the health benefits of telomerase activation through natural molecules and would shed new light onto the treatment of aging-related diseases.

Telomeres and Telomerase in Cutaneous Squamous Cell Carcinoma.

The role of telomere biology and telomerase activation in skin cancers has been investigated in melanoma and basal cell carcinoma but limited evidence is available for cutaneous squamous cell carcinoma (cSCC). We will review the current knowledge on the role of telomere and telomerase pathway in cSCC pathogenesis. At the somatic level, both long and short telomere lengths have been described in cSCC. This telomere dichotomy is probably related to two different mechanisms of tumour initiation which determines two tumour subtypes. Telomere shortening is observed during the invasive progression from in situ forms of cSCC, such as Bowen’s disease (BD) and actinic keratosis (AK), to invasive cSCC. At the germline level, controversial results have been reported on the association between constitutive telomere length and risk of cSCC. Approximately 75–85% of cSCC tumours are characterized by a high level of telomerase activity. Telomerase activation has been also reported in AKs and BD and in sun-damaged skin, thus supporting the hypothesis that UV modulates telomerase activity in the skin. Activating TERT promoter mutations have been identified in 32–70% of cSCCs, with the majority showing the UV-signature. No significant correlation was observed between TERT promoter mutations and cSCC clinico-pathological features. However, TERT promoter mutations have been recently suggested to be independent predictors of an adverse outcome. The attention on telomere biology and telomerase activity in cSCC is increasing for the potential implications in the development of effective tools for prognostic assessment and of therapeutic strategies in patients with cutaneous cSCC.

Cancer stem cell‐like characteristics and telomerase activity of the nasopharyngeal carcinoma radioresistant cell line CNE‐2R

The radioresistance of nasopharyngeal carcinoma (NPC) may be related to cancer stem cells (CSCs), and the characteristics of CSCs may be maintained by telomerase activity. In this study, we explored the CSC‐like characteristics and telomerase activity of the NPC radioresistant cell line CNE‐2R. This work provides a foundation for future studies on stem cell‐targeted therapies by targeting the radioresistance of NPC. The expression of stem cell‐related genes/proteins and the hTERT gene/protein in CNE‐2R and its parent radiosensitive cell line CNE‐2 were detected using qPCR/Western Blot. Label‐retaining cells (LRCs) were detected through immunocytochemistry, and telomerase activity was detected using a PCR‐ELISA kit. CD133 expression was detected with flow cytometry. CNE‐2R‐CD133+ and CNE‐2R‐CD133− cells were separated with magnetic‐activated cell sorting. The proliferation and tumorigenesis capacities of CNE‐2R‐CD133+, CNE‐2R‐CD133−, and CNE‐2R cells were compared with a CCK‐8 assay, sphere formation assay, and an in vivo experiment. Our results showed that the expression of stem cell‐related genes and the hTERT gene in CNE‐2R cells was higher than those in CNE‐2 cells. Similarly, the expression of stem cell‐related proteins and the hTERT protein in CNE‐2R cells was markedly higher than those in CNE‐2 cells. The proportion of LRCs in CNE‐2R and CNE‐2 cells was (3.10 ± 0.63%) vs (0.40 ± 0.35%; P < 0.001), respectively. Telomerase activity in CNE‐2R cells was remarkably higher than that in CNE‐2 cells. Flow cytometry suggested that the CD133 positive rates in CNE‐2R and CNE‐2 cells were (2.49 ± 0.56%) vs (0.76 ± 0.25%; P = 0.008), respectively. Meanwhile, the proliferation capacity, tumorigenesis capacity, and telomerase activity of CNE‐2R‐CD133+ cells were notably higher than those of CNE‐2R‐CD133− and CNE‐2R cells. Collectively, CNE‐2R displayed CSC‐like characteristics; our results also showed that CNE‐2R cells, especially the sorted CSCs, had high telomerase activity levels.

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

Background: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. Materials and Methods: Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. Results: Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30−/CD15− cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival (p < 10−3), event-free survival (p < 10−4), and freedom from progression (p < 10−3) and the presence of an ALT profile in lymph nodes of EBV+ patients. Conclusion: The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.

Influence of Inflammation in the Process of T Lymphocyte Differentiation: Proliferative, Metabolic, and Oxidative Changes.

T lymphocytes, from their first encounter with their specific antigen as naïve cell until the last stages of their differentiation, in a replicative state of senescence, go through a series of phases. In several of these stages, T lymphocytes are subjected to exponential growth in successive encounters with the same antigen. This entire process occurs throughout the life of a human individual and, earlier, in patients with chronic infections/pathologies through inflammatory mediators, first acutely and later in a chronic form. This process plays a fundamental role in amplifying the activating signals on T lymphocytes and directing their clonal proliferation. The mechanisms that control cell growth are high levels of telomerase activity and maintenance of telomeric length that are far superior to other cell types, as well as metabolic adaptation and redox control. Large numbers of highly differentiated memory cells are accumulated in the immunological niches where they will contribute in a significant way to increase the levels of inflammatory mediators that will perpetuate the new state at the systemic level. These levels of inflammation greatly influence the process of T lymphocyte differentiation from naïve T lymphocyte, even before, until the arrival of exhaustion or cell death. The changes observed during lymphocyte differentiation are correlated with changes in cellular metabolism and these in turn are influenced by the inflammatory state of the environment where the cell is located. Reactive oxygen species (ROS) exert a dual action in the population of T lymphocytes. Exposure to high levels of ROS decreases the capacity of activation and T lymphocyte proliferation; however, intermediate levels of oxidation are necessary for the lymphocyte activation, differentiation, and effector functions. In conclusion, we can affirm that the inflammatory levels in the environment greatly influence the differentiation and activity of T lymphocyte populations. However, little is known about the mechanisms involved in these processes. The elucidation of these mechanisms would be of great help in the advance of improvements in pathologies with a large inflammatory base such as rheumatoid arthritis, intestinal inflammatory diseases, several infectious diseases and even, cancerous processes.

Mechanisms of increased lifespan in hypoxia

Genetic variation accounts for a relatively small amount of the variation in lifespan (generally 15–30%), while environmental stressors are very strong predictors (Finch and Kirkwood 2000). Hypoxia is an environmental stress that increases longevity in some contexts, but the mechanisms remain poorly understood. Our long‐term goal is to understand the mechanisms by which hypoxia extends lifespan. In the bee Megachile rotundata, lifespan doubles upon rearing in hypoxia (Abdelrahman et al. 2014). We hypothesize that that hypoxia increases longevity by limiting telomere loss from reactive oxygen species (ROS). In this study, prepupal bees were maintained in 10, 21 or 24% oxygen for 9 months. Each month, two subsets of bees were removed. One group was immediately frozen for processing. A second subset of bees was reared to adulthood and then frozen for processing. Similar to our previous findings, all bees had extended lifespans compared to bees in the wild, but bees exposed to hypoxia had high survival after 9 months in 10% oxygen. At nine months, bees reared in 24% oxygen had 14% survival, while those in normoxia had 41% survival. Because they survived better than bees in normoxia or hyperoxia, we predicted that bees in hypoxic conditions would have reduced telomere loss rates, lower levels of ROS, and/or higher levels of telomerase activity than bees exposed to normoxia. We measured total antioxidant activity and found no differences among treatments from either prepupal or adult bees, indicating that oxidative stress is not regulated at the level of antioxidants. Because antioxidants are invariant, we predict that increased oxidative damage to proteins will be observed under normoxic and hyperoxic conditions. We expect bees reared in hypoxia to have longer telomeres than bees reared in normoxia or hyperoxia. In addition, we expect bees reared in hypoxia to have lower levels of oxidative damage than bees reared in normoxia or hyperoxia.Support or Funding InformationNIH COBRE to NDSU, USDA‐ARS

Ruxolitinib therapy and telomere length in myelofibrosis

Telomeres are specialized structures of repetitive nucleotide sequences that cap the ends of human chromosomes. Their main purpose is to maintain genome stability and integrity, and to protect the cell from progressive DNA shortening during repeated division. Human enzyme telomerase complex maintains the length of telomere repeats.1 Despite the high levels of telomerase activity in cancer cells, telomere shortening can still occur. Some recent reports describe reduced telomere length in myelofibrosis (MF) regardless of hydroxycarbamide therapy, suggesting a possible prognostic relevance for this biomarker.2, 3, 4 As yet, no studies have investigated telomere dynamics following treatment with ruxolitinib, a JAK1/2 inhibitor approved for the treatment of intermediate-2 and high risk MF, primary or post-polycythemia vera (PV) and essential thrombocytemia (ET).5

The germline/soma dichotomy: implications for aging and degenerative disease.

Human somatic cells are mortal due in large part to telomere shortening associated with cell division. Limited proliferative capacity may, in turn, limit response to injury and may play an important role in the etiology of age-related pathology. Pluripotent stem cells cultured in vitro appear to maintain long telomere length through relatively high levels of telomerase activity. We propose that the induced reversal of cell aging by transcriptional reprogramming, or alternatively, human embryonic stem cells engineered to escape immune surveillance, are effective platforms for the industrial-scale manufacture of young cells for the treatment of age-related pathologies. Such cell-based regenerative therapies will require newer manufacturing and delivery technologies to insure highly pure, identified and potent pluripotency-based therapeutic formulations.

Activity of telomerase and telomeric length in Apis mellifera.

Telomerase is an enzyme that adds repeats of DNA sequences to the ends of chromosomes, thereby preventing their shortening. Telomerase activity is associated with proliferative status of cells, organismal development, and aging. We report an analysis of telomerase activity and telomere length in the honeybee, Apis mellifera. Telomerase activity was found to be regulated in a development and caste-specific manner. During the development of somatic tissues of larval drones and workers, telomerase activity declined to 10% of its level in embryos and remained low during pupal and adult stages but was upregulated in testes of late pupae, where it reached 70% of the embryo level. Upregulation of telomerase activity was observed in the ovaries of late pupal queens, reaching 160% of the level in embryos. Compared to workers and drones, queens displayed higher levels of telomerase activity. In the third larval instar of queens, telomerase activity reached the embryo level, and an enormous increase was observed in adult brains of queens, showing a 70-fold increase compared to a brain of an adult worker. Southern hybridization of terminal TTAGG fragments revealed a high variability of telomeric length between different individuals, although the same pattern of hybridization signals was observed in different tissues of each individual.

Telomerase Activity and Telomere Length in Human Benign Prostatic Hyperplasia Stem-like Cells and Their Progeny Implies the Existence of Distinct Basal and Luminal Cell Lineages

Benign prostatic hyperplasia (BPH) treatments have changed little over many years and do not directly address the underlying cause. Because BPH is characterised by uncontrolled cell growth, the chromosomal telomeres should be eroded in the reported absence or low levels of telomerase activity, but this is not observed. We investigated the telomere biology of cell subpopulations from BPH patients undergoing transurethral resection of prostate (TURP). Measurement of TERC, TERT, and telomerase activity revealed that only the epithelial stem-like and progenitor fractions expressed high levels of telomerase activity (p<0.01) and individual enzyme components (p<0.01). Telomerase activity and TERT expression were not detected in stromal cells. Telomere length measurements reflected this activity, although the average telomere length of (telomerase-negative) luminal cells was equivalent to that of telomerase-expressing stem/progenitor cells. Immunohistochemical analysis of patient-derived BPH arrays identified distinct areas of luminal hyperproliferation, basal hyperproliferation, and basal–luminal hyperproliferation, suggesting that basal and luminal cells can proliferate independently of each other. We propose a separate lineage for the luminal and basal cell components in BPH. Patient summaryWe unexpectedly found an enzyme called telomerase in the cells that maintain benign prostatic hyperplasia (BPH), suggesting that telomerase inhibitors could be used to alleviate BPH symptoms.

Widespread telomere instability in prostatic lesions.

A critical function of the telomere is to disguise chromosome ends from cellular recognition as double strand breaks, thereby preventing aberrant chromosome fusion events. Such chromosome end-to-end fusions are known to initiate genomic instability via breakage-fusion-bridge cycles. Telomere dysfunction and other forms of genomic assault likely result in misregulation of genes involved in growth control, cell death, and senescence pathways, lowering the threshold to malignancy and likely drive disease progression. Shortened telomeres and anaphase bridges have been reported in a wide variety of early precursor and malignant cancer lesions including those of the prostate. These findings are being extended using methods for the analysis of telomere fusions (decisive genetic markers for telomere dysfunction) specifically within human tissue DNA. Here we report that benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (PIN), and prostate cancer (PCa) prostate lesions all contain similarly high frequencies of telomere fusions and anaphase bridges. Tumor-adjacent, histologically normal prostate tissue generally did not contain telomere fusions or anaphase bridges as compared to matched PCa tissues. However, we found relatively high levels of telomerase activity in this histologically normal tumor-adjacent tissue that was reduced but closely correlated with telomerase levels in corresponding PCa samples. Thus, we present evidence of high levels of telomere dysfunction in BPH, an established early precursor (PIN) and prostate cancer lesions but not generally in tumor adjacent normal tissue. Our results suggest that telomere dysfunction may be a common gateway event leading to genomic instability in prostate tumorigenesis. .

Predicting clinical outcome in feline oral squamous cell carcinoma: tumour initiating cells, telomeres and telomerase.

Feline oral squamous cell carcinoma (SCC) has very poor prognosis. Here, a retrospective pilot study was conducted on 20 feline oral SCC patients who underwent stereotactic radiation therapy (SRT), to evaluate: (1) the value of putative tumour initiating cell (TIC) markers of human head and neck SCC (CD44, Bmi-1); (2) telomere length (TL) specifically in putative TICs; and (3) tumour relative telomerase activity (TA). Significant inverse correlations were found between treatment outcomes and Bmi-1 expression, supporting the predictive value of Bmi-1 as a negative prognostic indicator. While TL exhibited a wide range of variability, particularly in very short fractions, many tumours possessed high levels of TA, which correlated with high levels of Bmi-1, Ki67 and EGFR. Taken together, our results imply that Bmi-1 and telomerase may represent novel therapeutic targets in feline oral SCC, as their inhibition-in combination with SRT-would be expected to have beneficial treatment outcome.

Effect of a qigong intervention program on telomerase activity and psychological stress in abused Chinese women: a randomized, wait-list controlled trial

BackgroundAbused women, who suffer from chronic psychological stress, have been shown to have shorter telomeres than never abused women. Telomere shortening is associated with increased risk of cell death, and it is believed that adopting health-promoting behaviors can help to increase the activity of telomerase, an enzyme that counters telomere shortening. Qigong is an ancient Chinese mind-body integration, health-oriented practice designed to enhance the function of qi, an energy that sustains well-being. Therefore, an assessor-blind, randomized, wait-list controlled trial was developed to evaluate the effect of a qigong intervention on telomerase activity (primary objective) and proinflammatory cytokines, perceived stress, perceived coping, and depressive symptoms (secondary objectives) in abused Chinese women.Methods/DesignA total of 240 Chinese women, aged ≥18 years, who have been abused by an intimate partner within the past three years will be recruited from a community setting in Hong Kong and randomized to receive either a qigong intervention or wait-list control condition as follows: the qigong intervention will comprise (i) a 2-hour group qigong training session twice a week for 6 weeks, (ii) a 1-hour follow-up group qigong exercise session once a week for 4 months, and (iii) a 30-minute self-practice qigong exercise session once a day for 5.5 months. The wait-list control group will receive qigong training after the intervention group completes the program. Upon completion of the qigong intervention program, it is expected that abused Chinese women in the intervention group will have higher levels of telomerase activity and perceived coping and lower levels of proinflammatory cytokines, perceived stress, and depressive symptoms than will abused Chinese women in the wait-list control group.DiscussionThis study will provide information about the effect of qigong exercise on telomerase activity and chronic psychological stress in abused Chinese women. The findings will inform the design of interventions to relieve the effects of IPV-related psychological stress on health. Also, the concept that health-promoting behaviors could slow down cellular aging might even motivate abused women to change their lifestyles.Trial registrationCurrent Controlled Trials NCT02060123. Registered February 6, 2014.

Biomarkers of oxidative stress and cataract. Novel drug delivery therapeutic strategies targeting telomere reduction and the expression of telomerase activity in the lens epithelial cells with N-acetylcarnosine lubricant eye drops: anti-cataract which helps to prevent and treat cataracts in the eyes of dogs and other animals.

Cataracts in small animals are shown to be at least partially caused by oxidative damage to lens epithelial cells (LECs) and the internal lens; biomarkers of oxidative stress in the lens are considered as general biomarkers for life expectancy in the canine and other animals. Telomeres lengths and expressed telomerase activity in canine LECs may serve as important monitors of oxidative damage in normal LECs with documented higher levels of telomerase activity in cataractous LECs during cells' lifespan. Loss of functional telomere length below a critical threshold in LECs of canines during the effect of UV and chronic oxidative stress or metabolic failure, can activate programs leading to LEC senescence or death. Telomerase is induced in LECs of canines at critical stages of cataractogenesis initiation and exposure to oxidative stress through the involvement of catalytically active prooxidant transition metal (iron) ions. This work documents that transition metal ions (such as, ferrous ions- catalytic oxidants) might induce premature senescence in LECs of canines, telomere shortening with increased telomerase activity as adaptive response to UV light, oxidative and metabolic stresses. The therapeutic treatment with 1% N-acetylcarnosine (NAC) prodrug delivery is beneficial for prevention and dissolution of ripe cataracts in canines. This biological activity is based on the findings of ferroxidase activity pertinent to the dipeptide carnosine released ophthalmically from NAC prodrug of L-carnosine, stabilizing properties of carnosine on biological membranes based on the ability of the imidazole-containing dipeptides to interact with lipid peroxidation products and reactive oxygen species (ROS), to prevent membrane damage and delute the associated with membrane fragements protein aggregates. The advent of therapeutic treatment of cataracts in canines with N-acetylcarnosine lubricant eye drops through targeting the prevention of loss of functional telomere length below a critical threshold and "flirting" with an indirect effect with telomerase expression in LECs of canines during the effects of UV, chronic oxidative stress increases the successful rate of cataract management challenges in home veterinary care.

Telomere Length Plays No Role in Clinical Unit Dominance After Double-Unit Cord Blood Transplantation

Abstract 4661 Background:Umbilical cord blood (CB) is an alternative source of allogeneic hematopoietic stem cells for transplantation and has the advantage of a reduced stringency of the required human leukocyte antigen-match. It is limited, however, by low total nucleated cell (TNC) dose and a low progenitor number in single CB units, thus restricting the use of CB transplantation (CBT) in larger children and adults. One strategy to augment engraftment is to combine 2 units from 2 different donors in a double-unit graft. We have previously shown that in most cases, one unit emerges as the sole source of hematopoiesis long-term, but as yet the mechanism of unit dominance remains unknown (Blood, 2010, 116(19):3999–4006). CB units are known to have an inherent biological variation in telomere length, the repeat sequence capping the ends of chromosomes. Telomere length variation and progressive shortening of telomere could later hematopoietic potential. Methods:We evaluated if telomere length has a role in unit dominance and how telomere length progresses over time post-transplant. We purified mononuclear cells from small aliquots of each unit of double-unit grafts and post-transplant peripheral blood (days +28, 100, 180 and 1 year) in 12 adult double-unit CBT recipients transplanted for hematologic malignancies at MSKCC. Average telomere length was measured using the TeloTTAGGG Telomere Length Assay (Roche) which utilizes Southern analysis of terminal restriction fragments (TRF) that are obtained by the digestion of isolated genomic DNA. Results:All 12 patients engrafted with one unit showing predominance. While there was a range of telomere length (5.57–12.09 kb) in the 24 units evaluated on the day of transplant, when comparing the telomere length of engrafting to non-engrafting units, there was no association between telomere length at day 0 and subsequent unit dominance. In 5 patients the engrafting unit had longer telomeres and in 7 patients the non-engrafting unit had longer telomeres. In serial assays, 7 of 12 patients demonstrated telomere length stabilization post-transplant in the dominant unit with a mean percentage loss of telomere length of 7.15% ± 2.33%. A second group (n = 5) demonstrated a decrease in telomere length with a mean percentage loss of 29.48% ± 5.12%. While this difference is significant (p = 0.0015) the clinical significance of this finding is uncertain. We are currently following these patients in order to correlate telomere length (stabilization versus decrease) with clinical outcomes. Conclusions:This data suggests that unit dominance is not influenced by telomere length. It is likely, based on emerging data from our laboratory as well as others, that unit dominance is immune mediated. However, the influence of telomere length on the quality of engraftment is of interest and analysis of this is ongoing with larger numbers of patients required to also consider the infused cell dose. It is notable that 7/12 patients had telomere length stabilization which correlates with the high levels of telomerase activity previously reported in the in vitro expansion of CB (Blood, 2004, 103(12):4440-8). Disclosures:No relevant conflicts of interest to declare.

Loss of the Malignant Phenotype of Human Neuroblastoma Cells by a Catalytically Inactive Dominant-Negative hTERT Mutant

Telomerase, a ribonucleoprotein complex mainly composed of the reverse transcriptase catalytic subunit (human telomerase reverse transcriptase, hTERT) and the RNA component (hTR), is a key enzyme of cancer progression. That aggressive stage 4-neuroblastoma expressed high levels of telomerase activity, whereas favorable tumors had no or little telomerase expression and activity, prompted us to investigate the role of this enzyme in this tumor model of altered proliferation, neuronal differentiation, and apoptosis. A human MYCN-amplified neuroblastoma cell line (IGR-N-91) was engineered to stably express either the normal hTERT protein (WT-hTERT) or a catalytically inactive dominant-negative mutant of this protein (DN-hTERT). We showed that DN-hTERT expression inhibited the endogenous hTERT in the malignant neuroblasts without telomere shortening nor loss of in vitro proliferative capacity. Importantly, DN-hTERT expression induced major changes in cell morphology of neuroblasts that switched them from a neuronal to a substrate adherent phenotype, which was more prone to apoptosis and lost their tumorigenic properties in nude mice. These biologic effects arose from modifications in the expression of genes involved in both apoptosis and neuroblastoma biology. Taken together these results highlighted the functional relevance of noncanonical functions of hTERT in the determination of neuroblast cell fate. Therefore, our results envision new therapeutic strategies for metastatic neuroblastoma therapeutic management.

Establishment of mesenchymal cell line derived from human developing odontoma

An odontoma, which shows proliferating odontogenic epithelium and mesenchymal tissue, is one of the most common odontogenic tumors encountered. These are commonly found in tooth-bearing regions, although the etiology remains unknown. There are no previous reports of an established line of immortalized human odontoma cells. Using odontoma fragments obtained from a girl treated at our department, we established an immortalized human odontoma cell line and investigated cell morphology, dynamic proliferation, the presence of contamination, and karyotype. Moreover, cell characterization was examined using osteogenic and odontogenic markers. We successfully established a mesenchymal odontoma cell (mOd cells). The cells were found to be fibroblastic and had a high level of telomerase activity. Cell growth was confirmed after more than 200 population doublings without significant growth retardation. mOd cells expressed mRNA for differentiation markers, including collagen type I (COLI), alkaline phosphatase, bone sialoprotein, osteopontin, osteocalcin, cementum-derived protein (CP-23), dentin sialophosphoprotein (DSPP), and distal-less homeobox 3 (DLX3), as well as bone morphogenetic proteins (BMPs). In addition, they showed a high level of calcified nodule formation activity in vitro. We successfully established a cell line that may be useful for investigating the mechanisms of normal odontogenesis as well as characteristics of odontoma tumors.

High Efficiency of Reprogramming CD34+ Cells Derived from Human Amniotic Fluid into Induced Pluripotent Stem Cells with Oct4

Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34⁺ cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34⁺ HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.