Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.     

端粒和端粒酶与癌症

端粒为真核细胞染色体末端的特殊结构,即染色体末端ＤＮＡ多个重复序列。人们曾经认为染色体末端ＤＮＡ稳定不变,实际上绝大多数生物体内染色体末端的端粒均处于不断地缩短或缩短和延长的变化之中。过去的１５年间,生物学界有许多意想不到的发现,特别是发现了一种不寻...     

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

Chondrocyte ex vivo expansion currently performed to replace damaged articular surfaces is associated with a loss of telomeric repeats similar to decades of aging in vivo. This might affect the incidence or time of onset of age-related disorders within transplanted cells or tissues. This study examined whether more immature progenitor cells, such as mesenchymal stem cells (MSC), which can be expanded and subsequently differentiated into chondrocytes is advantageous regarding telomere-length related limitations of expansion protocols. Primary chondrocytes and bone-marrow-derived MSC were isolated from 12 donors, expanded separately to 4 x 10(6) cells, and (re-)differentiated as three-dimensional chondrogenic spheroids. Cells were collected during expansion, after three-dimensional culturing and chondrogenic differentiation, and sequential analyses of telomere length and telomerase activity were performed. Surprisingly, telomeres of expanded MSC were significantly shorter than those from expanded chondrocytes from the same donor (11.4+/-2.5 vs. 13.4+/-2.2 kb) and tended to remain shorter after differentiation in chondrogenic spheroids (11.9+/-1.8 vs. 13.0+/- kb). While telomere lengths in native chondrocytes and MSC were not related to the age of the donor, significant negative correlations with age were observed in expanded (136 bp/year), three-dimensionally reconstituted (188 bp/year), and redifferentiated (229 bp/year) chondrocytes. Low levels of telomerase activity were found in MSC and chondrocytes during expansion and after (re-)differentiation to chondrogenic spheroids. In terms of replicative potential, as determined by telomere length, ex vivo expansion followed by chondrogenic differentiation of MSC did not provide a benefit compared to the expansion of adult chondrocytes. However, accelerated telomere shortening with age during expansion and redifferentiation argues for an "age phenotype" in chondrocytes as opposed to MSC and suggests an advantage for the use of MSC especially in older individuals and protocols requiring extensive expansion     

端粒-端粒酶与诺贝尔奖

2009年10月5日，北京时间17时30分，瑞典皇家科学院诺贝尔奖委员会在瑞典卡罗林斯卡医学院宣布，将2009年度生理学或医学奖授予美国加利福尼亚旧金山大学的伊丽莎白．布莱克本（Elizabeth Blackburn）、美国巴尔的摩约翰一霍普金斯大学医学院的卡罗尔-格雷德（Carol Greider）和美国哈佛医学院的杰克．绍斯塔克（Jack Szostak），     

Telomere shortening and apoptosis in telomerase-inhibited human tumor cells

Despite a strong correlation between telomerase activity and malignancy, the outcome of telomerase inhibition in human tumor cells has not been examined. Here, we have addressed the role of telomerase activity in the proliferation of human tumor and immortal cells by inhibiting TERT function. Inducible dominant-negative mutants of hTERT dramatically reduced the level of endogenous telomerase activity in tumor cell lines. Clones with short telomeres continued to divide, then exhibited an increase in abnormal mitoses followed by massive apoptosis leading to the loss of the entire population. This cell death was telomere-length dependent, as cells with long telomeres were viable but exhibited telomere shortening at a rate similar to that of mortal cells. It appears that telomerase inhibition in cells with short telomeres lead to chromosomal damage, which in turn trigger apoptotic cell death. These results provide the first direct evidence that telomerase is required for the maintenance of human tumor and immortal cell viability, and suggest that tumors with short telomeres may be effectively and rapidly killed following telomerase inhibition.     

Genetic and epigenetic changes in human epithelial cells immortalized by telomerase

Exogenous expression of hTERT, the catalytic component of telomerase, is sufficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammary epithelial cells (HMECs). These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expression of hTERT and loss of p16(INK4a) expression, indicating that the retinoblastoma (Rb) pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we further characterize hTERT-immortalized HFKs and human adenoid epithelial cells (HAKs) for genotypic and phenotypic alterations that are associated with immortalization. Of five hTERT-immortalized HFK and HAK cell lines examined, four exhibited repression of p16(INK4a) expression by promoter methylation or specific large-scale deletion of chromosome 9p, the location of p16(INK4a). Interestingly, one cell line exhibited complete down-regulation of expression of p14(ARF), with only slight down-regulation of expression of p16(INK4a). Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of the five cell lines. All of the cell lines retained a growth block response with the expression of mutant ras. When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on terminal differentiation. Our results indicate that immortalization of epithelial cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation.     

Telomere length in subpopulations of human hematopoietic cells

In order to test the hypothesis that the telomere length in human hematopoietic cells correlates with their proliferative potential, we analyzed the telomere length in highly purified subpopulations of bone marrow cells. Cells were sorted on the basis of CD34 and CD38 cell surface markers, and two samples were additionally sorted on the basis of Hoechst 33342 dye efflux allowing isolation of side population (SP) cells. The telomere length in limiting numbers of sorted cells was analyzed using a newly developed fluorescence in situ hybridization (flow-FISH) method in which hybridization of telomere probe in cells of interest is measured relative to control cells in the same tube. In all seven bone marrow samples analyzed, the telomere length in CD34(+)CD38(-) cells was longer than in CD34(+)CD38(+) cells from the same donor (p < 0.02). Results with sorted SP cells were less clear: the telomere fluorescence in these cells was very heterogeneous, and a reproducible difference in telomere length relative to CD34(+)CD38(-) cells could not be observed. We conclude that the telomere length in subpopulations of hematopoietic cells does appear to be correlated with the known proliferative potential of such cells and that further characterization of cells on the basis of telomere length is warranted for enrichment of very rare precursors of hematopoietic and other tissues.     

乳腺良恶性病变组织端粒长度和端粒酶活性检测

目的　比较乳腺良恶性病变端粒长度改变及其在肿瘤发生发展中的意义 ;探讨端粒酶活性与临床病理参数的关系及其在乳腺癌诊断中的价值。方法　Southern印迹杂交检测TRF长度 ,端粒重复扩增分析 (TRAP)方法检测端粒酶活性。结果　乳腺癌组织平均TRF为 (5 2± 2 8)kb ,与正常组织比较明显缩短 (P <0 0 0 1) ,从正常乳腺组织到乳腺良性病变、乳腺原位癌及浸润性癌平均TRF呈递减趋势。 5 8例乳腺癌中 4 9例端粒酶阳性 (84 7% ) ,端粒酶活性与临床病理参数无相关性 ;癌旁组织端粒酶为阴性 ,而 7例乳腺增生症和 6例乳腺纤维腺瘤中分别有 1例端粒酶阳性 ,与乳腺癌比其差异有显著性 (P <0 0 0 1)。结论　端粒长度在肿瘤发生发展过程中渐进性缩短 ,并最终触发端粒酶的激活 ;端粒酶活性检测有望成为乳腺癌诊断的可靠标记物     

Telomere structure, function and maintenance in Arabidopsis

The stability of eukaryotic genomes is provided in part by the integrity of telomeres, the nucleoprotein caps on the ends of chromosome. Recent studies reveal that proper telomere architecture is required for long-term proliferation capacity. Here we describe molecular mechanisms that protect and maintain chromosome ends and discuss why Arabidopsis is emerging as a powerful new model for elucidating fundamental aspects of telomere biology.     

端粒、端粒酶与细胞衰老及永生化

正常细胞体外培养时,表现为有限生长特性,经一定的细胞倍增次数后,失去了对促分裂因子刺激的反应,不可逆地失去增殖能力而停止分裂,细胞开始衰老的历程.目前认为染色体末端(端粒)的缺失会使细胞逐渐失去增殖能力,导致细胞的衰老和死亡.人端粒酶催化亚单位(hTERT)可以激活端粒酶的活性,延长染色体末端DNA,维持基因组的稳定.端粒、端粒酶、hTERT的发现为细胞衰老的研究提供了新的思路,同时也应用于永生化细胞系的建立,特别是在组织工程种子细胞生物性能研究和细胞库的建立中将发挥重要作用.     

Acetylcholinesterase and somatostatin-immunoreactivity coexist in human neocortex

Immunohistochemistry was combined with enzyme histochemistry to examine the localization of somatostatin (SOM) and acetylcholinesterase (AChE) in the human neocortex. Many of the SOM-immunoreactive cortical neurons were found to display specific AChE activity. Similar coexistence was seen in the rat cortex. In contrast, AChE and SOM appear to be present in distinct cell groups in the human caudate nucleus.     

Heterologous mitochondrial DNA recombination in human cells

Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants. Nevertheless, its occurrence in human cells is still controversial. To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers. In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes. Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches. First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker. Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events. Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes. This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells. These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.     

Telomere dysfunction drives chromosomal instability in human mammary epithelial cells

The development of genomic instability is an important step toward generating the multiple genetic changes required for cancer. Telomere dysfunction is one of the factors that contribute to tumorigenesis. Telomeres shorten with each cell division in the absence of telomerase. Human mammary epithelial cells (HMECs) obtained from normal human tissue demonstrate two growth phases. After an initial phase of active growth, HMECs exhibit a growth plateau termed selection. However, some cells can emerge from this growth plateau by spontaneously losing expression of the p16(INK4a) protein. These post-selection HMECs are capable of undergoing an additional 20-50 population doublings in culture. Continued proliferation of these post-selection HMECs leads to further telomere erosion, loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter bridge-fusion-breakage (BFB) cycles, generating massive chromosomal instability before terminating in a population growth plateau termed agonescence. We have found that the chromosome arms carrying the shortest telomeres are those involved in telomere-telomere type rearrangements. In addition, we found that the risk of a particular chromosome being unstable differs between individuals. Most importantly, we identified sister chromatid fusion as a first event in generating genomic instability in HMECs. During post-selection HMEC growth, double strand breaks are generated by both fused chromosomes as well as individual chromosomes with fused chromatids entering BFB cycles. These broken chromosome extremities are able to join other broken ends or eroded telomeres, producing massive chromosomal instability at the later passages of the cell culture. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Telomere length, telomerase activity, and expression of human telomerase reverse transcriptase mRNA in growth plate of epiphyseal articular cartilage in femoral head during normal human development and in thanatophoric dysplasia

Telomeres are important in chromosome structure and function, protecting against their degradation. However, few studies have examined telomeres in growth plates within articular cartilage during normal development. We investigated frozen sections that were obtained from 57 reference autopsy cases (aged from 16 weeks of gestation to 91 years) and from 2 patients with thanatophoric dysplasia. In the reference cases, telomere length was significantly longer in growth plates obtained from the 10 cases that were aged from 16 weeks of gestation to 10 years than in those from 47 of the adult cases (aged 20 to 91 years). In fetal, neonatal, and child cases, telomerase activity was significantly higher in the hypertrophied zone (HZ) in growth plates than in the other 3 zones. The hTERT mRNA staining intensity (staining area) was stronger (larger) in HZ and the proliferating zone than in the calcified zone and resting zone. In thanatophoric dysplasia, telomere length and telomerase activity were short and low, respectively, compared with those of normal growth plates at an equivalent age, and expression of hTERT mRNA was negative or weakly positive in all 4 zones within growth plates. These results suggest that telomere length and telomerase activity have significant effects in the growth plates of articular cartilage, particularly at developmental ages from fetus to child. We speculate that short telomere length and low telomerase activity may be important for chondrocyte differentiation in rhizomeric shortening of the limbs in thanatophoric dysplasia.     

Effects of telomerase modulation in human hematopoietic progenitor cells

Loss of telomeric repeats has been causally linked to replicative senescence and aging in human cells. In contrast to normal somatic cells, which are telomerase-negative, hematopoietic stem cells have low levels of telomerase, which can be transiently upregulated upon cytokine stimulation. To examine whether ectopic expression of telomerase can overcome telomere erosion in hematopoietic progenitor cells, we overexpressed telomerase in CD34+ and AC133+ cord blood (CB) cells using retroviral vectors containing hTERT, the catalytic component of telomerase. Although the hTERT-transduced CB cells exhibited significantly elevated telomerase activity (approximately 10-fold), the mean telomere length was only increased up to 600 bp, which was in contrast to hTERT-transduced fibroblast cells gaining more than 2-kb telomeric repeats. Moreover, ectopic telomerase activity did not prevent overall telomere shortening, which was in the range of 1.3 kb in serum-free expansion culture. We also blocked endogenous telomerase activity by ectopic expression of dominant-negative hTERT. Whereas CB cells with absent telomerase activity showed reduced absolute numbers of colony-forming cells, we observed increased rates only for burst-forming units erythroid when the enzyme was overexpressed. These results suggest that telomere shortening in human hematopoietic progenitor cells cannot be compensated by increased levels of telomerase alone and is likely to be dependent on other factors, such as telomere binding proteins. Furthermore, telomerase function seems to be directly associated with the proliferative capacity of stem cells and may exert an additional role in lineage differentiation.     

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.