Ectopic hTERT expression facilitates reprograming of fibroblasts derived from patients with Werner syndrome as a WS cellular model
Shuyan WangZhongfeng LiuYanxia YeBingnan LiTiantian LiuWeiqi ZhangGuang‐Hui LiuY. Alex ZhangJing QuDawei XuZhiguo Chen
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Abstract The induced pluripotent stem cell (iPSC) technology has provided a unique opportunity to develop disease-specific models and personalized treatment for genetic disorders, and is well suitable for the study of Werner syndrome (WS), an autosomal recessive disease with adult onset of premature aging caused by mutations in the RecQ like helicase (WRN) gene. WS-derived fibroblasts were previously shown to be able to generate iPSCs; however, it remains elusive how WS-derived iPSCs behave and whether they are able to mimic the disease-specific phenotype. The present study was designed to address these issues. Unexpectedly, we found that a specific WS fibroblast line of homozygous truncation mutation was difficult to be reprogrammed by using the Yamanaka factors even under hypoxic conditions due to their defect in induction of hTERT, the catalytic unit of telomerase. Ectopic expression of hTERT restores the ability of this WS fibroblast line to form iPSCs, although with a low efficiency. To examine the phenotype of WRN-deficient pluripotent stem cells, we also generated WRN knockout human embryonic stem (ES) cells by using the CRISPR/Cas9 method. The iPSCs derived from WS-hTERT cells and WRN-/- ESCs are fully pluripotent, express pluripotent markers and can differentiate into three germ layer cells; however, WS-iPSCs and WRN-/- ESCs show S phase defect in cell cycle progression. Moreover, WS-iPSCs and WRN-/- ESCs, like WS patient-derived fibroblasts, remain hypersensitive to topoisomerase inhibitors. Collectively, WS-derived iPSCs and WRN-/- ESCs mimic the intrinsic disease phenotype, which may serve as a suitable disease model, whereas not be good for a therapeutic purpose without gene correction.Keywords:
Ectopic expression
Progeria
Background: Telomere length maintenance is essential for tumorigenesis. Most human tumours stabilise their chromosome ends by telomerase, a specialised reverse transcriptase that adds telomeric repeats (TTAGGG) to these ends. The main components of this telomerase complex are a reverse transcriptase (hTERT) and an integral RNA component (hTR). Most typical meningiomas, however, do not have active telomerase, although some express the hTERT component. The aim of this study was to evaluate telomerase activity and its reverse transcriptase for 33 (30 typical and three atypical) meningiomas in vivo and in vitro. Materials and Methods: Telomerase activity was examined by the telomeric repeat amplification protocol (TRAP) assay. The protein, telomerase reverse transcriptase, was visualised by immunohistochemistry. Results: In vivo, telomerase activity was detectable in one out of 30 typical meningiomas and in two out of three atypical meningiomas. hTERT protein expression in vivo was positive in 14 out of 33 (42%) cases. The mean percentage of positive nuclei was 12.9% (SD=21.0). In vitro, 22 out of 33 (66%) meningiomas were positive for hTERT, with a mean percentage of positive nuclei of 31.8% (SD=37.1). Only four expressed telomerase activity in vitro, from which three had expressed telomerase activity in vivo. A significant association was found for telomerase activity (p<0.001) and hTERT expression (p<0.001) in vivo versus in vitro; a significant association was found for hTERT expression and telomerase activity in vivo (p<0.05) and in vitro (p<0.05). Conclusion: The results of our study suggest that hTERT expression is an early event in carcinogenesis in contrast to telomerase activity. Fast-proliferating hTERT-positive tumour cells may overgrow in vitro by clonal selection. Telomeres are nucleoprotein complexes at the ends of
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This study investigated the relationship between the levels of human telomerase reverse transcriptase (hTERT) mRNA and that of telomerase activity in hepatocellular carcinoma (HCC). A significant correlation between hTERT mRNA expression and telomerase activity by transfecting the gene encoding hTERT into telomerase-negative human fibroblast cells has clearly been demonstrated. However, the relationship between levels of telomerase activity and that of hTERT mRNA has yet to be elucidated. In this study, the levels of hTERT mRNA were analyzed in 24 HCC patients by real-time PCR. And the intensity of telomerase activity was analyzed by fluorescence-based TRAP method. The difference of hTERT mRNA level was highly significant between tumor tissues and non-cancerous liver tissues. And there were significant correlations between the levels of hTERT mRNA and that of telomerase activity (r=0.751) in tumor tissues. We observed a strong correlation between levels of hTERT mRNA and that of telomerase activity in HCC. Our results suggest that the levels of hTERT mRNA would be useful in genetic diagnostic tests, instead of telomerase activity, to screen at-risk patients of HCC in human liver tissues.
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Telomerase extends the repetitive DNA at the ends of linear chromosomes, and it is normally active in stem cells. When expressed in somatic diploid cells, it can lead to cellular immortalization. Human papillomaviruses (HPVs) are associated with and high-risk for cancer activate telomerase through the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). The expression of hTERT is affected by both high-risk HPVs, E6 and E7. Seminal studies over the last two decades have identified the transcriptional, epigenetic, and post-transcriptional roles high-risk E6 and E7 have in telomerase induction. This review will summarize these findings during infection and highlight the importance of telomerase activation as an oncogenic pathway in HPV-associated cancer development and progression.
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Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase‐associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down‐ or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate‐limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activi`ty. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.
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在 telomerestelomerase 和年龄相关的疾病之间有一种靠近的关系。人的 telomerase 颠倒 transcriptase (hTERT ) 两个都是人的 telomerase 和 telomerase 活动的限制率的决定因素的催化部件。Itstranscriptional 规定是 telomerase 活动的控制的主要模式。为探索 hTERT 表示和 telomerase 活动的规章的机制发现蛋白质 interactingwith hTERT 是批评的。在这研究， theyeast 二混血儿的系统被用来屏蔽 hTERT 的潜在的交互蛋白质。六蛋白质被获得， amongwhich T 星， LOXL3， HKR3，和 Par-4 被 co-immunoprecipitation.Then 进一步作为 hTERT 的交往的蛋白质证实感觉和 antisense 基因包含这四基因的真核细胞的表示向量被构造， transfectedinto 肿瘤房间排队。在这四蛋白质， hTERT 的表示水平，和 telomerase 活动的表示层次之中的关联被分析。结果证明 T 星表示和 down-regulationof HKR3 表示的起来规定导致了 hTERT 表示和 telomerase 活动的增加，当 LOXL3and Par-4 表情的起来规定和下面规定没有明显的效果时。当 HKR3 可以是一个否定管理者时，我们的结果建议 T 星与 telomeraseactivity 有积极关联。这个结论是重要的进一步探索人的 telomerase 活动的影响因素 orregulation 小径，它可能为潜在的临床的申请是很重要的。
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There is a close relationship between telomeres-telomerase and age-related disease. Human telomerase reverse transcriptase (hTERT) is both the catalytic component of human telomerase and the rate-limiting determinant of telomerase activity. Its transcriptional regulation is the primary mode of control of telomerase activity. It is critical to find the proteins interacting with hTERT for exploring the regulatory mechanisms of the hTERT expression and the telomerase activity. In this study, the yeast two-hybrid system was used to screen the potential interactive proteins of hTERT. Six proteins were obtained, among which T-STAR, LOXL3, HKR3, and Par-4 were further confirmed as the interacting proteins of hTERT by co-immunoprecipitation. Then the sense and antisense gene eukaryotic expression vectors containing these four genes were constructed and transfected into tumor cell lines. The correlations among the expression levels of these four proteins, the expression level of hTERT, and the telomerase activity were analyzed. Results showed that the up-regulation of T-STAR expression and down-regulation of HKR3 expression led to the increase of hTERT expression and telomerase activity, while the up- and down-regulation of LOXL3 and Par-4 expressions had no obvious effect. Our results suggested that T-STAR has a positive correlation with the telomerase activity while HKR3 may be a negative regulator. This conclusion is important to further explore the influencing factors or regulation pathways of human telomerase activity, which may be of great importance for the potential clinical application.
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Telomerase is a reverse transcriptase that adds telomeric repeats to chromosomal ends. In most normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to limited proliferative life-span. Telomerase reactivation is associated with cellular immortalization and is a frequent event during tumorigenesis. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein subunit [human telomerase reverse transcriptase (hTERT)] and a template RNA (hTR). hTR is constitutively expressed, while hTERT is almost universally absent in telomerase-negative cells. Although repression of telomerase is transcriptional in telomerase-negative cells, post-transcriptional and assembly processes are likely to play important roles in regulating telomerase activity in those that are telomerase-positive. The telomerase transcript can also be alternatively spliced into a variety of non-functional forms. To establish the quantitative relationships between telomerase activity and its various components, we determined the numbers of molecules of hTR and hTERT mRNA, and the levels of alternatively spliced hTERT mRNA variants in normal, in vitro immortalized and cancer cell lines. We report here that there is surprisingly little variation in the proportion of alternatively spliced forms of hTERT in different cell lines. The only variation observed occurred when a change in splicing to non-functional forms appeared in response to conditions that repress telomerase activity in IDH4 cells. We also found that most telomerase-positive cell lines only contain a few molecules of potentially functional hTERT mRNA, and there is a correlation between telomerase activity and the levels of both hTR and hTERT +α+β mRNA.
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