Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.     

Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes.

We have measured telomere length and telomerase activity throughout the life span of clones of human B lymphocytes transformed by Epstein-Barr virus. Shortening of telomeres occurred at similar rates in all populations and persisted until chromosomes had little telomeric DNA remaining. At this stage, some of the clones entered a proliferative crisis and died. Only clones in which telomeres were stabilized, apparently by activation of telomerase, continued to proliferate indefinitely, i.e., became immortal. Since loss of telomeres impairs chromosome function, and may thus affect cell survival, we propose that telomerase activity is required for immortality. We have now detected this enzyme in a variety of immortal human cells transformed by different viruses, indicating that telomerase activation may be a common step in immortalization.     

Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.     

Effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts

Myostatin (GDF-8) inhibits the activation, proliferation, and differentiation of myogenic satellite cells. The relative importance of this growth factor is demonstrated in myostatin-null mice and cattle possessing defective myostatin genes. These defects result in greatly enhanced musculature. In the present study, we examined the effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts. Compared with controls (P<0.05), proliferation of both turkey embryonic myoblasts and satellite cells was inhibited between 26 and 45% in serum-free medium containing 20 ng/mL myostatin. While individual turkey satellite cell clones differed in their responsiveness to myostatin, there were no significant differences in the responsiveness of fast and slow growing cells as groups (P>0.05). A slow growing clone that exhibited the greatest response to myostatin also exhibited the greatest depression of differentiation with this growth factor (P<0.05). All other turkey satellite cell clones exhibited similar responses to the differentiation depressing effects of myostatin (P>0.05). However, myostatin had no effect on differentiation of turkey embryonic myoblasts (P>0.05). When exposed to myostatin, 4 of 6 proliferating clones and all differentiating clones increased their expression of decorin, a growth inhibitor (P<0.05). The present study demonstrates that myostatin inhibits the proliferation and differentiation of satellite cells and suggests a role for decorin in myostatin action in muscle development.     

Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.     

端粒与端粒酶研究进展

细胞分裂中染色体因其末端（端粒）的DNA不能完全复制而短缩,使细胞逐渐失去增殖能力而衰老．端粒酶可延长染色体末端DNA,端粒酶的活化使细胞无限增殖．85％左右的恶性肿瘤端粒酶表达阳性,生殖细胞和无限繁殖的细胞系中端粒酶表达也呈阳性．文章综述了端粒的构成和功能、端粒酶在端粒合成中的作用,介绍了端粒酶活性的测定方法、细胞恶变与端粒酶激活的关系,并论及通过抑制端粒酶活性来治疗癌症的可能性．     

Cancer cell dynamics in presence of telomerase inhibitors: analysis of in vitro data

The inhibition of telomerase activity in actively dividing cells leads to suppression of cell growth after a time delay (inhibitory delay) required to reach a threshold telomeric DNA size. We developed a mathematical model of the dynamics of telomere size distribution and cell growth in the presence of telomere inhibitors that allowed quantification of the inhibitory delay. The model based on the solution of a system of differential equations described quantitatively recent experimental data on dynamics of cultured cells in presence of telomerase inhibitors. The analysis of the data by this model suggested the existence of at least two distinct subpopulations of cells with different proliferative activity. Size distribution of telomeres, fraction of proliferating cells, and tumor doubling times are of critical importance for the dynamics of cancer cells growth in presence of telomerase inhibitors. Rapidly growing cells with large telomeric DNA heterogeneity and small proliferating fractions as well as those with very short homogeneous telomeres would be the most sensitive to telomerase inhibitors.     

Satellite cells of growing turkeys: influence of donor age and sex on proliferation and differentiation in vitro

Proliferation and differentiation (fusion) of myogenic satellite cells isolated from Nicholas tom and hen turkeys at 3, 9, and 15 weeks of age and the response of a satellite cell clone to serum from these birds were examined. Responsiveness of satellite cells to culture conditions was proportional to donor age, and sex of bird had no effect on satellite cell proliferation. Fusion percentages were similar in all cultures. When a turkey satellite cell clone was exposed to serum from tom and hen turkeys at each age, an age-related decline in proliferation occurred with serum from hen but not from tom turkeys. Minimal cell fusion occurred in turkey serum and neither proliferation nor fusion was correlated with serum IGF-I levels. Cells derived from pectoralis major and anterior latissimus dorsi muscles of 15-week-old tom turkeys possessed similar proliferation and fusion properties in vitro. It appears that satellite cells retain a high proliferative activity throughout growth in turkeys, and the mitogenic properties of turkey serum may be evaluated using turkey satellite cells in culture.     

Changes in Telomerase Activity and Telomere Length during Human T Lymphocyte Senescence

It has been proposed that telomeres shorten with every cell cycle because the normal mechanism of DNA replication cannot replicate the end sequences of the lagging DNA strand. Telomerase, a ribonucleoprotein enzyme that synthesizes telomeric DNA repeats at the DNA 3′ ends of eukaryotic chromosomes, can compensate for such shortening, by extending the template of the lagging strand. Telomerase activity has been identified in human germline cells and in neoplastic immortal somatic cells, but not in most normal somatic cells, which senesce after a certain number of cell divisions. We and others have found that telomerase activity is present in normal human lymphocytes and is upregulated when the cells are activated. But, unlike the immortal cell lines, presence of telomerase activity is not sufficient to make T cells immortal and telomeres from these cells shorten continuously duringin vitroculture. After senescence, telomerase activity, as detected by the TRAP technique, was downregulated. A cytotoxic T lymphocyte (CTL) cell line that was established in the laboratory has very short terminal restriction fragments (TRFs). Telomerase activity in this cell line is induced during activation and this activity is tightly correlated with cell proliferation. The level of telomerase activity in activated peripheral blood T cells, the CTL cell line, and two leukemia cell lines does not correlate with the average TRF length, suggesting that other factors besides telomerase activity are involved in the regulation of telomere length.     

Telomeres and telomerase activity in pig tissues

The current state of the art concerning telomeres and telomerase stems almost exclusively from the analysis of protozoa, yeast, and a small number of mammals. In the present study, we confirm that the pig telomeric sequence is indeed T(2)AG(3), as previously suggested. By making use of sequence analysis of pig telomeric DNA variant telomeric repeats in the medial region of the telomeres, interspersed with canonical T(2)AG(3) repeats, were identified. This telomere organization is similar to the one present in humans. Analysis of terminal restriction fragments showed that the majority of telomeres from different pig tissues are longer than in humans but shorter than in Mus musculus. Telomeres from spermatozoa were found to be longer, ranging in size between 13 and 44 kb. Most of the somatic pig tissues expressed significant levels of telomerase activity, a situation more similar to mouse and that contrasts with the one in humans and dog. Moreover, the analysis of sperm cells from different epididymal compartments of an adult animal showed that telomerase activity is absent in maturing spermatozoa, suggesting that sperm telomere elongation is restricted during spermatogenesis.     

Human Ku70/80 associates physically with telomerase through interaction with hTERT

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.     

The assembly of large BACs by in vivo recombination

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre-lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of alpha satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene. The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E. coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA. This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences.     

多形胶质母细胞瘤细胞系端粒酶活性的测定

端粒缩短见于星形细胞瘤发展过程中,但其长度在胶质母细胞瘤／细胞系相对稳定,提示胶质瘤细胞内存在端粒修复机制的可能性．为证实此点,利用端粒重复片段扩增技术（ＴＲＡＰ）,对８株人／大鼠多形胶质母细胞系的蛋白提取液中端粒酶活性加以测定．结果显示：８例胶质瘤样本的反应液均可见端粒ＰＣＲ扩增片段;用无ＤＮａｓｅ的ＲＮａｓｅ事先处理蛋白提取液,可明显降低或消除ＰＣＲ产物的出现,说明ＴＲＡＰ反应中的ＰＣＲ扩增是在端粒酶的介导下进行而非ＤＮＡ污染或其它端粒修复因子所致．从而不但建立起检测人癌细胞内端粒酶活性的可靠方法,也为针对端粒酶的胶质母细胞瘤生物／药物治疗提供了实验依据．     

Inter-telomeric recombination is present in telomerase-positive human cells

Immortal cells require a mechanism of telomere length control in order to divide infinitely. One mechanism is telomerase, an enzyme that compensates the loss of telomeric DNA. The second mechanism is the alternative lengthening of telomeres (ALT) pathway. In ALT pathway cells, homologous recombination between telomeric DNA is the mechanism by which telomere homeostasis is achieved. We developed a novel homologous recombination reporter system that is able to measure inter-telomeric recombination in a sensitive manner. We asked the fundamental question if homologous recombination between different telomeres is present in telomerase-positive cells. In this in vitro study, we showed that homologous recombination between telomeres is detectable in ALT cells with the same frequency as in cells that utilize the telomerase pathway. We further described an ALT cell clone that showed peaks of recombination which were not detected in telomerase-positive clones. In telomerase-positive cells the frequency of inter-telomeric recombination was not increased by shortened telomeres or by a fragile telomere phenotype induced with aphidicolin. ALT cells, in contrast, responded to aphidicolin with an increase in the frequency of recombination. Our results indicate that inter-telomeric recombination is present in both pathways of telomere length control, but the factors that increase recombination are different in ALT and telomerase-positive cells.     

Dynamics of telomeric DNA turnover in yeast.

Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.     

Phospho-MAPK as a marker of myogenic satellite cell responsiveness to growth factors

To determine if differential response to growth factor stimuli between subpopulations of satellite cells was due to variation in the levels of activated intracellular signaling proteins, the levels of phospho-MAPK (phospho-ERK 1/2) were determined in clonal populations of turkey (Meleagris gallopavo) satellite cells. Relative levels of phospho-ERK 1/2 between clones were determined by Western blotting of extracts from satellite cells exposed to growth factor stimuli. Initial measurements using serum mitogenic stimuli showed differences in phospho-MAPK levels between the clonal subpopulations, but the responses did not correlate with proliferation rates of the individual clones (P>0.05). IGF-I alone did not increase phospho-MAPK levels compared to unstimulated controls (P>0.05), whereas FGF-2 did increase levels (P<0.05). A synergistic response was seen in satellite cells as well as embryonic myoblasts administered both IGF-I and FGF-2. When administered FGF-2 and IGF-I, 2 of the slow growing satellite cell clones exhibited lowest levels of phospho-MAPK (P<0.05). One of the slow growing clones had levels of phospho-MAPK similar to the three fast growing clones (P>0.05). The results suggest that variation in responsiveness to growth factor stimuli among satellite cell populations within muscles may be due to several different reasons. Some differences in cell responsiveness appear to be due to variation in phospho-MAPK generation.