Levels Of Telomerase Activity Research Articles

Detection of telomerase activity in gastric cancer

Telomerase activity (TA) was examined in gastric adenocarcinomas and gastric lymphoma using a modified TRAP assay. TA was present in 16 of 18 (89%) gastric adenocarcinomas and in gastric lymphoma, whereas no TA was detected in normal tissue. Almost all samples had "high" and "very high" TA levels. Telomerase is undoubtedly associated with the process of malignant transformation and therefore can be an important marker for diagnostics of gastric cancer.

The Telomerase Antagonist, Imetelstat, Efficiently Targets Glioblastoma Tumor-Initiating Cells Leading to Decreased Proliferation and Tumor Growth

Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel human telomerase antagonist, imetelstat, on primary human glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo. Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro. The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels, telomere length, proliferation, clonogenicity, and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts were used to assess the in vivo efficacy of imetelstat. Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC(50) 0.45 micromol/L). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation, and eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood-brain barrier in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic administration of imetelstat (30 mg/kg every day for three days), showed approximately 70% inhibition of telomerase activity. Chronic systemic treatment produced a marked decrease in the rate of xenograft subcutaneous tumor growth. This preclinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination with standard therapies.

Nuclear translocation of telomerase reverse transcriptase: a critical process in chemical induced hepatocellular carcinogenesis

Telomerase, a ribonuclearprotein complex, functions not only in cancer development but also in apoptosis, and senescence. As a catalytic subunit of telomerase, telomerase reverse transcriptase (TERT) has been confirmed to regulate telomerase activity in a rate-limiting manner. Although a lot of work has been done, the dynamic state of TERT protein and the relationship with telomerase have not been delineated systematically in cancer development. The purpose of this study was to do such an exploration. To investigate the role of TERT in the in vivo carcinogenesis, we performed immunofluorescence and Western blot analysis, respectively, to detect the alteration of TERT status as well as telomeric repeat amplification protocol (TRAP) assay to detect telomerase activity in diethyl nitrosoamine (DENA) induced rat hepatocellular carcinoma (HCC). The course of cancer development was divided into three main stages, which were inflammation ( <12 weeks), hepatocirrhosis (15 weeks), and hepatocarcinoma (18-21 weeks). In normal liver and its early inflammatory stage, concomitant with a weak positive TERT signal, which was detected exclusively in the cytoplasm, telomerase activity was very low at this stage. However, in late hepatocirrhosis and particularly cancer stage, high levels of TERT expression (P = 0.044 and P = 0.001, respectively) and telomerase activity (P = 0.02 and P = 0.01, respectively) were observed following TERT nuclear translocation. Our data suggest that TERT protein might regulate telomerase activity. TERT translocation from cytoplasm to the nucleus might be a turning point in cancer development. Therefore, TERT translocation might be more useful than TERT expression level and telomerase activity in predicting the progression of HCC.

Effect of aromatase inhibitor (fadrozole) on proliferation, estradiol production and telomerase activity in pig granulosa cells in vitro

The objective of the present work was to study the effect of a nonsteroidal aromatase inhibitor (fadrozole) on proliferation, estradiol production, aromatase expression and telomerase activity (TA) in pig granulosa cells (GC) from small (1–2 mm) and large (5–7 mm) follicles. The cells were treated with fadrozole for 48 h and 72 h in basal and FSH-stimulated conditions. Fadrozole caused a decrease (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) of 3H-thymidine incorporation in granulosa cells derived from small (1–2 mm) and large follicles (5–7 mm). The proliferative potential of small-follicle GC was significantly higher (&lt;i&gt;P&lt;/i&gt; &lt; 0.01) under all culture conditions. Estradiol production was suppressed (&lt;i&gt;P&lt;/i&gt; &lt; 0.01) in both granulosa cell populations cultured in the presence of fadrozole for 48 and 72 h. Fadrozole caused a decrease (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) of aromatase gene expression in small-follicle granulosa cell incubated for 72 h and in large-follicle GC after 48 h of culture. Large-follicle GC were characterized by a higher (&lt;i&gt;P&lt;/i&gt; &lt; 0.01) level of estradiol production and aromatase gene expression. Telomerase activity decreased (&lt;i&gt;P&lt;/i&gt; &lt; 0.05) in large-follicle granulosa cells incubated in the presence of an aromatase inhibitor for 72 h. The TA level in large-follicle granulosa cells was higher (&lt;i&gt;P&lt;/i&gt; &lt; 0.01) in comparison to small-follicle GC in all culture conditions after 72 h of incubation. The results of the present study suggest the important role of telomerase in the process of follicular growth and development.

Dynamics of telomerase activity in response to acute psychological stress

Telomerase activity plays an essential role in cell survival, by lengthening telomeres and promoting cell growth and longevity. It is now possible to quantify the low levels of telomerase activity in human leukocytes. Low basal telomerase activity has been related to chronic stress in people and to chronic glucocorticoid exposure in vitro. Here we test whether leukocyte telomerase activity changes under acute psychological stress. We exposed 44 elderly women, including 22 high stress dementia caregivers and 22 matched low stress controls, to a brief laboratory psychological stressor, while examining changes in telomerase activity of peripheral blood mononuclear cells (PBMCs). At baseline, caregivers had lower telomerase activity levels than controls, but during stress telomerase activity increased similarly in both groups. Across the entire sample, subsequent telomerase activity increased by 18% one hour after the end of the stressor ( p < 0.01). The increase in telomerase activity was independent of changes in numbers or percentages of monocytes, lymphocytes, and specific T cell types, although we cannot fully rule out some potential contribution from immune cell redistribution in the change in telomerase activity. Telomerase activity increases were associated with greater cortisol increases in response to the stressor. Lastly, psychological response to the tasks (greater threat perception) was also related to greater telomerase activity increases in controls. These findings uncover novel relationships of dynamic telomerase activity with exposure to an acute stressor, and with two classic aspects of the stress response – perceived psychological stress and neuroendocrine (cortisol) responses to the stressor.

Ets2 transcription factor, telomerase activity and breast cancer

1. The enzyme telomerase maintains telomeres (ends of chromosomes) by synthesizing telomeric DNA at each end of the chromosomes. Its association with telomeres has implicated telomerase in cell immortalization. 2. Numerous studies have shown significant levels of telomerase activity in 85% of various types of cancer. Transcriptional control of the catalytic subunit, telomerase reverse transcriptase (TERT), dominates regulation of telomerase. Although several major factors have been identified in regulating TERT, they cannot explain all the transcriptional activity of the hTERT gene. 3. The Ets transcription factor (TF) family is becoming a regular feature in tumourigenesis, particularly in breast cancer. However, the roles and mechanisms of different Ets TFs are largely unknown. 4. The present minireview discusses the research that identified Ets as a regulator of telomerase required for breast cancer cell survival and proliferation, highlighting the discoveries central to understanding the molecular acts used by Ets TFs to mediate TERT gene transcription.

Leukemic T-PLL Cells Exhibit Short and Narrowly Distributed Telomere Lengths and High Telomerase Activities, Associated with Rapid and Dose-Dependent Cell Death Following Telomerase Inhibition by Imetelstat Sodium.

Abstract 2647Poster Board II-623 Background/Aims:Reflecting their proliferative history, malignant cells generally have shorter telomeres compared to their normal counterparts. We have reported that leukemic cells from patients with T-prolymphocytic leukaemia (T-PLL) have exceptionally short telomeres (telomere lengths around 1 kb) compared to other malignancies, and high levels of telomerase activity (Roeth et al., Leukemia, 2007). We have also previously presented (Roeth et al., ASCO 2008) cytotoxic effects of the telomerase inhibitory agent imetelstat sodium (GRN163L), which is currently in clinical trials. The objective of this study was to further characterize the relationship between mean telomere length (TL), the single chromosome telomeric size distributions (single TL), telomerase levels and imetelstat-induced telomerase inhibition and cytotoxicity in T-PLL, B-CLL, and normal cells. Methods:Cells were obtained from peripheral blood of normal donors and patients with T-PLL and B-CLL and cryopreserved. TL was measured by flow-FISH (which yields a multicellular average measurement across all chromosomes) and the distribution of individual telomere lengths by single telomere length analysis (STELA, XpYp specific). Telomerase activity (TA) was assessed by TRAP. Effects of imetelstat upon cell survival, apoptosis and TA were studied in short-term cell cultures. Apoptosis was measured by Annexin V-PI double staining. Results:Average (±SD) TL of the T-PLL cells was 1.8 ± 0.73 kb (n=13) which was shorter than for B-CLL cells (ZAP-70+/CD38+ CLL: 2.46 ± 1.08 kb (n=30); ZAP-70neg/CD38neg CLL: 5.06 ± 1.76 kb (n=29), Roeth et al, Br. J. Haem., 2008). T-PLL cells were found to have a narrow range of single chromosome telomere lengths, while both subtypes of B-CLL cells demonstrated a broader distribution of telomere lengths. No attrition of mean telomere length was observed over time in two patients with T-PLL who were followed over time, despite continued clinical progression. In contrast, we have observed that most patients with B-CLL do have decreases in telomere length over the course of their disease.Leukemic T-PLL cells exhibited high levels of telomerase activity, whereas there was little or no detectable telomerase activity in normal, unstimulated T-cells and most B-CLL cells. Imetelstat (GRN163L, Geron Corporation), a potent inhibitor of telomerase, induced significant and dose-dependent inhibition of telomerase activity in T-PLL cells after 2 hours of culture at 1μM and 10μM. Imetelstat significantly reduced cell viability of T-PLL cells in vitro after 7-10 days of culture (viability as % of control (mean ± SD) with 1, 3, 10 μM imetelstat: 69.9 ± 27.3, 51.7 ± 30.2, 37.1 ± 27.6). No significant cytotoxicity of imetelstat was observed in CLL samples, or in unstimulated and stimulated T-cells from 5 normal donors. Leukemic cells from one T-PLL patient early in the course of the disease did not exhibit loss of viability when exposed to imetelstat at levels up to 10 μM; however, when cells from this patient were tested later during the progression of the disease significant cytotoxicity was observed. Conclusions:In summary, leukemic T-PLL cells exhibit extremely short telomere lengths, narrower ranges of single chromosome telomere size distributions, and higher telomerase activity compared to B-CLL cells and normal T cells. These observations may explain why inhibition of telomerase activity in T-PLL cells by imetelstat is associated with rapid and dose-dependent cell death. Disclosures:Baerlocher:Geron Corporation: Research Funding. Bassett:Geron Corporation: Employment, Equity Ownership. Elias:Geron Corporation: Employment, Equity Ownership. Go:Geron Corporation: Employment, Equity Ownership. Roeth:Geron Corporation: Research Funding.

TRANSLATIONAL IMPACT

Dyskeratosis congenita (DKC) is a collection of autosomal dominant diseases that result in abnormalities in the skin, nails, bone marrow and lungs. DKC arises from hereditary mutations in factors that replenish or cap telomeres, which are at the ends of the chromosomes. When the level of telomere-replenishing factors dips, telomeres erode and chromosomes rearrange. The affected cells either die or become unable to divide. Patients with DKC often die before the age of 30, usually from infection because of bone marrow dysfunction, or as a result of their increased predisposition to cancer.The authors use a mouse model that resembles DKC in humans to detemine the potential for adaptive extension of telomeres. They use mTert mice, which express one functional allele and one disrupted allele of the telomere-replenishing factor, telomerase reverse transcriptase (Tert). The initial generations of mTert heterozygotes exhibit the same telomere erosion as seen in DKC patients. The authors show that, after several generations, telomeres in mTert mice are restored to nearly the same lengths as those found in normal animals. This restoration depends on the inheritance of Tert, which actively supports lengthening of the telomeres. The telomere length restoration that is observed prevents cell death, and the mice do not exhibit phenotypes that are normally associated with loss of telomere function in the intestine, testes and the bone marrow. This result is similar to findings in yeast but shows that telomerase can correct telomere length over time in a mammal.The ability to maintain a functional telomere cap appears to be more important than the actual length of the telomere itself. In yeast cells, long telomeres are bound by factors that inhibit telomere extension by telomerase. Short telomeres are not bound by as many inhibitory factors and telomerase can gain access and extend the telomere. The authors speculate that such an equilibration process might protect normal human stem cells. Furthermore, activation of the process in DKC-affected cells might reduce symptoms of the disease that result from lost telomere function. The capacity to lengthen telomeres in normal tissues with low levels of telomerase activity might also protect against the inhibition of telomerase during cancer therapy, and improve its efficacy.

Analyses and comparisons of telomerase activity and telomere length in human T and B cells: Insights for epidemiology of telomere maintenance

Telomeres are the DNA–protein complexes that protect the ends of eukaryotic chromosomes. The cellular enzyme telomerase counteracts telomere shortening by adding telomeric DNA. A growing body of literature links shorter telomere length and lower telomerase activity with various age-related diseases and earlier mortality. Thus, leukocyte telomere length (LTL) and telomerase activity are emerging both as biomarkers and contributing factors for age-related diseases. However, no clinical study has directly examined telomerase activity and telomere length in different lymphocyte subtypes isolated from the same donors, which could offer insight into the summary measure of leukocyte telomere maintenance. We report the first quantitative data in humans examining both levels of telomerase activity and telomere length in four lymphocyte subpopulations from the same donors—CD4+, CD8+CD28+ and CD8+CD28− T cells and B cells, as well as total PBMCs—in a cohort of healthy women. We found that B cells had the highest telomerase activity and longest telomere length; CD4+ T cells had slightly higher telomerase activity than CD8+CD28+ T cells, and similar telomere length. Consistent with earlier reports that CD8+CD28− T cells are replicatively senescent cells, they had the lowest telomerase activity and shortest telomere length. In addition, a higher percentage of CD8+CD28− T cells correlated with shorter total PBMC TL ( r = − 0.26, p = 0.05). Interestingly, telomerase activities of CD4+ and CD8+CD28+ T cells from the same individual were strongly correlated ( r = 0.55, r < 0.001), indicating possible common mechanisms for telomerase activity regulation in these two cell subtypes. These data will facilitate the understanding of leukocyte aging and its relationship to human health.

Cellular senescence and longevity of osteophyte‐derived mesenchymal stem cells compared to patient‐matched bone marrow stromal cells

This study aimed to determine the cellular aging of osteophyte-derived mesenchymal cells (oMSCs) in comparison to patient-matched bone marrow stromal cells (bMSCs). Extensive expansion of the cell cultures was performed and early and late passage cells (passages 4 and 9, respectively) were used to study signs of cellular aging, telomere length, telomerase activity, and cell-cycle-related gene expression. Our results showed that cellular aging was more prominent in bMSCs than in oMSCs, and that oMSCs had longer telomere length in late passages compared with bMSCs, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSCs and not in bMSCs. In osteophyte tissues telomerase-positive cells were found to be located perivascularly and were Stro-1 positive. Fifteen cell-cycle regulator genes were investigated and only three genes (APC, CCND2, and BMP2) were differentially expressed between bMSC and oMSC. Our results indicate that oMSCs retain a level of telomerase activity in vitro, which may account for the relatively greater longevity of these cells, compared with bMSCs, by preventing replicative senescence.

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomere Dysfunction and DNA Damage Checkpoints in Diseases and Cancer of the Gastrointestinal Tract

Telomerase activity in benign and malignant adrenal tumors.

Histological analysis of surgically removed adrenal masses often fails to differentiate between benign and malignant tumors. In normal cells, the telomeric ends of the chromosomes are shortened with each cell division, leading to chromosome destabilization and cellular senescence after a critical number of cell cycles. In tumor cells, telomere shortening is prevented by a specific DNA polymerase, called telomerase. In an effort to clarify the role of telomerase in the pathogenesis of adrenal tumors, and to test whether its activity could serve as marker of malignancy, we measured telomerase activity in 41 human adrenal tissue samples that were classified both by the clinical course and by histological examination. Telomerase activity was determined by TRAP ELISA and expressed as high (>50% of positive control telomerase activity), medium (31-50%), low (11-30%), very low (< or = 10%), or absent (0%). The 8 normal adrenal tissue samples showed very low levels of telomerase activity. Mean telomerase activity also very low in 3/3 incidentalomas, 6/6 Cushing adenomas, 6/6 Conn adenomas, 7/7 adrenocortical carcinomas, 8/8 benign pheochromocytomas, and 2/3 malignant pheochromocytomas. In contrast, one malignant pheochromocytoma showed high telomerase activity. These data indicate that telomerase activity may not be a suitable marker for malignancy in the adrenal gland. Our results also challenge the current dogma of close correlation between cell dedifferentiation and telomerase activity.

Establishment of a human neonatal hepatocyte cell line

Hepatocytes are routinely used to generate and identify drug metabolites and hepatic toxicity. Primary cultures of human hepatocytes are the model cell of choice for most of these pharmacological and toxicological studies. However, major problems are encountered with primary liver cell cultures: the dwindling availability of viable livers, hepatocytes having a limited life span, the loss of liver-specific functions in culture, and the donor to donor variability. These limitations have created a significant need for an in vitro hepatocyte system, which has both the potential for use in toxicological and pharmaceutical studies as well as clinical applications. Ectopic expression of human telomerase reverse transcriptase (hTERT) is one of the major strategies used to develop immortalized cells. Immortalization of primary cells using hTERT allows retention of the original cellular characteristics and functions and avoids some of the genetic and phenotypic instabilities associated with using known oncogenes. In the present study, we developed a cell line from human neonatal hepatocytes by transduction with a recombinant retrovirus expressing the hTERT gene. Induction of stable expression of hTERT in the neonatal cells led to immortalization of these cells. The cell line was cultured continuously for more than 25 passages, equivalent to >25 population doublings, whereas the parental cells senesced within five passages. Analysis of telomerase activity as measured by telomeric repeat amplification protocol assay indicated elevated levels of telomerase activity in immortalized cells compared to the parental cells. These immortalized human hepatocytes cells maintained a normal diploid karyotype as well as the gene expression profile similar to that of human normal neonatal hepatocytes. The data suggest that these immortalized cells preserved some of the biological characteristics of hepatic progenitor cells and might be useful as an in vitro model for pharmacological and toxicity studies.

Isolation and characterization of stem cells from the human parathyroid gland

Somatic stem cells can be obtained from a variety of adult human tissues. However, it was not clear whether human parathyroid glands, which secrete parathyroid hormones and are essential in maintaining homeostasis levels of calcium ions in the circulation, contained stem cells. We aimed to investigate the possibility of isolating such parathyroid-derived stem cells (PDSC). Surgically removed parathyroid glands were obtained with informed consent. Cell cytogenetics was used to observe chromosomal abnormalities. Surface phenotypes were characterized by flow cytometry. Telomerase repeat amplification protocol (TRAP) assay was performed to observe the telomerase activity. RT-PCR and real-time PCR was was used to detect gene expressions. Real-time calcium uptake imaging was performed for extent of calcium uptake and transmission electron microscopy and immunofluorecent staining for smooth muscle actin. After enzymatic digestion and primary culture, plastic-adherent, fibroblast-like cells appeared in culture and a morphologically homogeneous population was derived from subsequent limiting dilution and clonal expansion. Karyotyping was normal and doubling time of clonal cell growth was estimated to be 70.7 +/- 14.5 h (mean +/- standard deviation). The surface phenotype of the cells was positive for CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, and MHC class I, and negative for CD34, CD133, CD117, CD114, CD31, CD62P, EGF-R, ICAM-3, CD26, CXCR4, CD106, CD90 and MHC class II, similar to mesenchymal stem cells (MSC). Detectable levels of telomerase activity along with pluripotency Sall4 gene expression were observed from the isolated PDSCs. Expression of calcium-sensing receptor gene along with alpha-smooth muscle actin was induced and cellular uptake of extracellular calcium ions was observed. Furthermore, PDSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials. Our results reveal that PDSCs were similar phenotypically to MSCs and further studies are needed to formulate induction conditions to differentiate PDSCs into parathyroid hormone-secreting chief cells.

Attenuated expression of SECIS binding protein 2 causes loss of telomeric reserve without affecting telomerase

The family of selenoproteins have a broad range of functions, including protection against oxidative damage. Previous studies have shown that elevated levels of oxidative damage can induce accelerated loss of telomeric DNA during proliferation of mammalian cells. The incorporation of selenocysteine (Sec) into proteins in mammalian cells requires the Sec insertion sequence (SECIS) binding protein 2 (SBP2). Thus in the present study we have assessed the effect of knocking down the expression of SBP2 on telomere length. Following knock-down of SBP2 expression in two different human cell lines, the MSTO mesothelioma cell line (∼5 Kb average telomere length) and SY5Y neuroblastoma cell line (∼4.2 Kb average telomere length), we observed a significant reduction (−0.6 to −1.1 Kb; P ⩽ 0.01) in telomere length as compared to control cells. This reduction in telomere length was independent of affects on telomerase, since both telomerase activity levels and Tert mRNA expression levels were not altered by knock-down of SBP2 expression. Furthermore, telomeres were particularly sensitive to S1 nuclease digestion following SBP2 knock-down, indicating an increased frequency of oxidative damage-induced lesions in the telomeric DNA in these cells. Together, these observations imply that selenoproteins may help protect telomeric reserve in mammalian cells.

Generation of Pig Induced Pluripotent Stem Cells with a Drug-Inducible System

Domesticated ungulate pluripotent embryonic stem (ES) cell lines would be useful for generating precise gene-modified animals. To date, many efforts have been made to establish domesticated ungulate pluripotent ES cells from early embryos without success. Here, we report the generation of porcine-induced pluripotent stem (iPS) cells using drug-inducible expression of defined factors. We showed that porcine iPS cells expressed alkaline phosphatase, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Oct3/4, Nanog, Sox2, Rex1 and CDH1. Pig iPS cells expressed high levels of telomerase activity and showed normal karyotypes. These cells could differentiate into cell types of all three germ layers in vitro and in teratomas. Our study reveals properties of porcine pluripotent stem cells that may facilitate the eventual establishment of porcine ES cells. Moreover, the porcine iPS cells produced may be directly useful for the generation of precise gene-modified pigs.

Novel Isolation and Biochemical Characterization of Immortalized Fibroblasts for Tissue Engineering Vocal Fold Lamina Propria

Tissue regeneration of the vocal fold lamina propria extracellular matrix (ECM) will be facilitated by the use of suitable vocal fold fibroblast (VFF) cell lines in appropriate model systems. Primary human VFFs (hVFFs) were steadily transduced by a retroviral vector containing human telomerase reverse transcriptase (hTERT) gene; immortalized cells grew and divided vigorously for more than 120 days. Biochemical characterization of the six transduced lines included, at different time points, expression of hTERT, telomerase activity, telomere lengths, and transcript levels of ECM constituents. Telomere lengths of the transfected lines were elongated and stable. Gene expression levels of collagen Ialpha1, collagen Ialpha2, collagen VIalpha3, elastin, and fibronectin were measured between the transduced cell clones and the primary hVFFs to verify transcription. Absence of inter- and intraspecies contamination was confirmed with DNA fingerprinting and karyotype analysis. Cell morphology, growth, and transcription expression were examined on 2D scaffolds-collagen, fibronectin, and hyaluronic acid. Immortalized hVFFs demonstrated normal attachment and spread on 2D scaffolds. Collagen Ialpha1, collagen Ialpha2, collagen VIalpha3, elastin, and fibronectin transcript expression was measured from immortalized hVFFs, for all surfaces. This is the first report of immortalization and biochemical characterization of hVFFs, providing a novel and invaluable tool for tissue regeneration applications in the larynx.

Effect of c-MYC and E2F1 Gene Silencing and of 5-Azacytidine Treatment on Telomerase Activity in Pancreatic Cancer-Derived Cell Lines

Background:The gene promoter region of human telomerase reverse transcriptase (hTERT) contains binding sites for c-myc and E2F1 as well as CpG islands, suggesting regulation by genetic factors and epigenetically by methylation. Hence, the effect of the demethylating agent 5-azacytidine and silencing of c-MYC and E2F1 genes on its expression and consequently on telomerase activity were studied in pancreatic cancer-derived cell lines. Methods: MlaPaCa-2 and PANC-1 cell lines were transfected with SiRNA against E2F1 and c-MYC genes separately as well as along with 5-azacytidine treatment. The hTERT gene methylation status was determined by methylation-specific PCR and telomerase activity quantitated by TRAP-PCR-ELISA. Results: Demethylation by 5-azacytidine resulted in hTERTinhibition with a reduction in telomerase activity to 37–49% of controls. Silencing of E2F-1 or c-MYC also decreased the hTERT transcript and telomerase activity with a more pronounced effect with respect to c-MYC silencing. There was a synergistic effect of demethylation and gene silencing on the inhibition of hTERT mRNA expression which resulted in undetectable levels of telomerase activity. Conclusion: Telomerase activity, which is necessary for cellular immortalization, can be shut down by a combined approach using SiRNA-mediated gene silencing and demethylating agents, which has therapeutic implications.

Targeting prostate cancer progenitor cells with telomerase interference: A novel therapeutic strategy

e22029 Background: We investigated whether telomerase, which is critical for benign stem cell activation, also plays a role in prostate cancer progenitor cells (PCPCs), which are thought to mediate therapy resistance and cancer progression, and we tested whether telomerase interference can effectively inhibit PCPC proliferation. Methods: A putative PCPC population was isolated from human prostatectomy specimens via collagen attachment and FACS selection for integrin α2β1 and CD44. PCPCs were characterized for gene expression (RT-PCR), clonogenicity (colony formation), invasiveness (matrigel chamber), and telomerase activity (qPCR-TRAP). PCPC telomerase interference was accomplished by lentiviral expression of 2 constructs: telomerase RNA with an altered template region (MT-Ter) and siRNA targeting wild-type telomerase RNA (anti-Ter siRNA). The effects of these constructs were assessed by measuring PCPC viability (MTS) and apoptosis (TUNEL assay). Results: An integrin α2β1+CD44+ putative PCPC population was isolated from 6 human prostate tumors. This population expressed high levels of “progenitor phenotype” genes (ABCG2, β-catenin, NANOG, Oct3/4) and low levels of “differentiated phenotype” genes (AR and PSA). PCPCs yielded &gt;50 colonies per 1000 cells seeded on collagen after 3 weeks vs. none from FACS- cells, and matrigel chamber assay showed 10% of the PCPC population invading over 24 hours vs. none of the FACS- population. Most importantly, PCPCs possessed at least 20- fold greater telomerase activity than FACS- cells, and induction of telomerase interference in PCPCs via MT-hTer and anti- hTer siRNA expression elicited a brisk apoptotic response (TUNEL) by day 3 in &gt;90% of cells, with concomitant near-complete growth inhibition (MTS). Conclusions: We have shown that human prostate tumors contain a subpopulation of prostate cancer progenitor cells (PCPCs) marked by an undifferentiated gene expression profile, vigorous clonogenicity and invasiveness, and high levels of telomerase activity that can be successfully exploited to neutralize these cells. Ongoing studies are investigating the in vivo effects of telomerase interference on PCPC tumorigenicity in mouse models. No significant financial relationships to disclose.

Fetal Growth Restriction is Associated with Accelerated Telomere Shortening and Increased Expression of Cell Senescence Markers in the Placenta

A hallmark of fetal growth restriction (FGR) is restricted placental development and insufficient nutrient supply to the fetus. It has previously been shown that activity levels of telomerase, the enzyme responsible for completing replication of telomeric DNA during cell division, is suppressed in FGR placenta samples as compared to control placenta samples from donors of the same gestational age. Here we examine whether telomere length maintenance is also compromised in FGR placenta samples. Southern analysis of telomere length for placenta and cord blood samples from 32 FGR and 36 control donors, ranging in gestational age from 37 to 40 weeks, revealed significantly shorter telomeres (P≤0.001) in FGR placenta samples, but not cord blood samples. Furthermore, analysis of telomerase extracts, RNA and DNA placental samples from donors with and without idiopathic FGR confirmed a direct association between suppression of telomerase activity and reduced telomere length in FGR placenta. In addition, expression levels of markers of telomere-induced senescence, p21, p16 and EF-1α, were significantly elevated in FGR placenta samples (P≤0.01). These observations support a direct affect of reduced telomerase activity levels on the placental pathology associated with FGR.