Using the telobox to search for plant telomere binding proteins

Telobox is a Myb-related DNA-binding domain which is present in a number of yeast, plant and animal proteins. Its capacity to bind preferentially double-stranded telomeric DNA has been used in numerous studies to search for candidate telomeric proteins in various organisms, including plants. Here we provide an overview of these studies with a special emphasis on plants, where a specific subfamily of the proteins possessing the N-terminally positioned telobox is present in addition to more common C-terminal telobox proteins. We further demonstrate the presence of a telobox protein (CpTBP1) in Cestrum parqui, a plant lacking typical telomeres and telomerase. The protein shows nuclear localisation and association with chromatin. The role of this protein in ancestral and current telomere structure is discussed in the evolutionary context. Altogether, the present overview shows the importance of the telobox domain in a search for candidate telomere proteins but at the same time warns against oversimplified identification of any telobox protein with telomere structure without appropriate evidence of its telomeric localisation and function.     

Telomere and Telomerase as Targets for Cancer Therapy

Telomere maintenance and telomerase reactivation is essential for the transformation of most human cancer cells. Telomere shortening to the threshold length, mutations of the telomere-associated proteins, and/or telomerase RNA lead to telomeric dysfunction and therefore genomic instability. Telomerase up-regulation in 85% of human cancer cells has become a hallmark of cancers, hence a promising target for anticancer therapy. In this review, we discuss the mechanism of cancer due to telomere dysfunction and the resulting biological effects, the control of telomerase activity, and the new developments in cancer therapies targeting telomere and telomerase.     

端粒酶活性在氯化镧诱导MDCC-MSB1细胞凋亡中的作用

探讨端粒酶活性在LaCl_3诱导MDCC-MSB1细胞凋亡中细胞中的作用.肿瘤细胞常规培养于RPMI1640培养液中,加入终浓度为3 mmol·L~(-1)的LaCl_3,继续培养12,24,36,48 h后,应用琼脂糖凝胶电泳和AO/EB双荧光染色检测细胞凋亡,以FITC-(C_3TA_2)_3 PNA为荧光探针检测细胞内端粒长度.以TRAP-PCR银染法检测端粒酶活性.LaCl_3浓度为3 mmol·L~(-1),作用0～48 h,细胞的琼脂精凝胶电泳和AO/EB双荧光染色法均观察到明显的细胞凋亡变化,细胞内端粒长度缩短,端粒酶活性下降,并呈时间-效应关系.LaCl_3可通过抑制MDCC-MSB1细胞端粒酶活性而诱导其发生凋亡.     

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells

Transformation with viral oncogene extends the lifespan of normal cells beyond replicative senescence called M1, but most of them eventually succumb to second crisis called M2 when telomeres become critically short. To acquire an infinite growth capacity, these cells have to overcome M2 crisis, which is known to follow telomerase activation. We have investigated if telomerase expression is required for virus-transformed pre-M2 cells to avert M2 crisis. Human retinal pigment epithelial (RPE) cells were transformed with simian virus 40 large T antigen and a VR3 clone in pre-M2 stage was obtained. Then, VR3 cells were transfected with a telomerase-containing vector and two cell lines that expressed telomerase temporarily or continuously were cloned and designated as ST1 and ST2, respectively. Normal RPE cells went into senescence after 36 population doublings. Although the lifespan was extended in the VR3 clone about 20 times more, it eventually underwent second crisis. The telomere length of VR3 decreased compared to that of normal RPE cells and the decrease continued during subculture. However, the ST1 and ST2 clones that expressed both T antigen and telomerase could avert this crisis. The initial telomere length of ST1 and ST2 was longer than that of normal cells. The ST1 underwent growth arrest again as telomerase expression faded out and elongated telomere was shortened, but the ST2 that maintained telomerase activity and telomere length proliferated continuously. In conclusion, telomerase activation is definitely required to overcome M2 crisis and acquire an infinite lifespan in human somatic epithelial cells and this mechanism is independent from M1 crisis escape in cell immortalization.     

Recombination proteins and telomere stability in plants

Repair of DNA damage is essential for the maintenance of the integrity and transmission of the genome in development and reproduction. Telomeres are nucleoprotein structures which protect the ends of (linear) eukaryotic chromosomes. Telomere dysfunction results in loss of this protection and the telomeres being recognised as DNA damage by the cellular DNA Damage Repair and Response (DDR) machinery, leading to senescence or cell death. Telomeric homeostasis is thus tightly controlled and many specific and non-specific proteins are involved in its regulation. Among these, DNA damage and Repair proteins contribute both to the recognition of telomere dysfunction and more surprisingly, are directly implicated in telomere homeostasis itself. Plants offer a great opportunity to study these mechanisms due to the fact that many key DNA repair and recombination proteins are non-essential in plants, in contrast to vertebrates. In the following text, after a brief summary of the current state of knowledge on telomere-specific proteins in plants, we review the DDR processes and the related proteins implicated in plant telomere stability. We focus specifically on telomere signalling and on recombination events induced by unprotected telomeres, at the origin of genome rearrangements and instability when telomere function is affected.     

Contribution of telomere G-quadruplex stabilization to the inhibition of telomerase-mediated telomere extension by chemical ligands

Inhibition of telomerase activity through stabilizing telomere G-quadruplex with small chemical ligands is emerging as a novel strategy for cancer therapy. For the large number of ligands that have been reported to inhibit telomerase activity, it is difficult to validate the contribution of G-quadruplex stabilization to the overall inhibition. Using a modified telomere repeat amplification protocol (TRAP) method to differentiate the telomere G-quadruplex independent effect from dependent ones, we analyzed several ligands that have high affinity and/or selectivity to telomere G-quadruplex. Our results show that these ligands effectively inhibited telomerase activity in the absence of telomere G-quadruplex. The expected G-quadruplex-dependent inhibition was only obvious for the cationic ligands at low K(+) concentration, but it dramatically decreased at physiological concentration of K(+). These observations demonstrate that the ligands are much more than G-quadruplex stabilizers with a strong G-quadruplex-irrelevant off-target effect. They inhibit telomerase via multiple pathways in which stabilization of telomere G-quadruplex may only make a minor or neglectable contribution under physiologically relevant conditions depending on the stability of telomere G-quadruplex under ligand-free conditions.     

端粒酶检测新方法研究进展

端粒酶是一种核蛋白逆转录酶,能以自身的RNA为模板添加重复序列(TTAGGG)n至染色体末端,稳定端粒长度。端粒酶在85%～90%的癌细胞中处于激活状态,已经引起了人们的广泛关注,成为肿瘤诊断和临床治疗的重要突破口。因此,准确、高效地测定端粒酶活性具有重要意义。近年来,许多灵敏、准确的体外或原位检测技术在端粒酶活性检测方面得到了发展。该文综述了近年来端粒酶活性检测的最新进展,并对其发展趋势进行了展望。     

Cellular delivery of peptide nucleic acids and inhibition of human telomerase.

BACKGROUND: Human telomerase has an essential RNA component and is an ideal target for developing rules correlating oligonucleotide chemistry with disruption of biological function. Similarly, peptide nucleic acids (PNAs), DNA analogs that bind complementary sequences with high affinity, are outstanding candidates for inducing phenotypic changes through hybridization. RESULTS: We identify PNAs directed to nontemplate regions of the telomerase RNA that can overcome RNA secondary structure and inhibit telomerase by intercepting the RNA component prior to holoenzyme assembly. Relative potencies of inhibition delineate putative structural domains. We describe a novel protocol for introducing PNAs into eukaryotic cells and report efficient inhibition of cellular telomerase by PNAs. CONCLUSIONS: PNAs directed to nontemplate regions are a new class of telomerase inhibitor and may contribute to the development of novel antiproliferative agents. The dependence of inhibition by nontemplate-directed PNAs on target sequence suggests that PNAs have great potential for mapping nucleic acid structure and predictably regulating biological processes. Our simple method for introducing PNAs into cells will not only be useful for probing the complex biology surrounding telomere length maintenance but can be broadly applied for controlling gene expression and functional genomics.     

Telomerase inhibitors identified by a forward chemical genetics approach using a yeast strain with shortened telomere length

Telomerase has been proposed as a selective target for cancer chemotherapy. We established a forward chemical genetics approach using a yeast strain with shortened telomere length. Since this strain rapidly enters cell senescence in the absence of active telomerase, compounds that induce selective growth defects against telomere-shortened yeast could be candidates for drugs acting on telomeres and telomerase. We screened our microbial products library and identified three structurally unrelated antibiotics, chrolactomycin, UCS1025A, and radicicol, as active compounds. Detailed analysis showed that chrolactomycin inhibited human telomerase in a cell-free assay as well as in a cellular assay. Long-term culture of cancer cells with chrolactomycin revealed population-doubling-dependent antiproliferative activity accompanied by telomere shortening. These results suggest that chrolactomycin is a telomerase inhibitor, and that the yeast-based assay is useful for discovering the small molecules acting on human telomerase.     

Potato type I and II proteinase inhibitors: modulating plant physiology and host resistance

Serine protease inhibitors (PIs) are a large and complex group of plant proteins. Members of the potato type I (Pin1) and II (Pin2) proteinase inhibitor families are among the first and most extensively characterized plant PIs. Many insects and phytopathogenic microorganisms use intracellular and extracellular serine proteases playing important roles in pathogenesis. Plants, however, are able to fight these pathogens through the activation of an intricate defence system that leads to the accumulation of various PIs, including Pin1 and Pin2. Several transgenic plants over-expressing members of the Pin1 and Pin2 families have been obtained in the last twenty years and their enhanced defensive capabilities demonstrated against insects, fungi and bacteria. Furthermore, Pin1 and Pin2 genetically engineered plants showed altered regulation of different plant physiological processes (e.g., dehydratation response, programmed cell death, plant growth, trichome density and branching), supporting an endogenous role in various plant species in addition to the well established defensive one. This review summarizes the current knowledge about Pin1 and Pin2 structure, the role of these proteins in plant defence and physiology, and their potential exploitation in biotechnology.     

Magnetic beads-based electrochemiluminescence assay for rapid and sensitive detection of telomerase activity

Telomerase is a potential cancer marker. We developed a new and robust telomerase activity assay which combines the modified telomere repeat amplification protocol (TRAP) with magnetic beads-based electrochemiluminescence (ECL) detection. The high performance of this assay is related to the determination of telomerase activity from single cell levels, and ECL intensity is linear over the range of 1–1000 HeLa cell equivalents. The proposed telomerase assay offers a highly cost- and time-effective alternative to presently available telomerase assays, which are limited by tedious and complicated post-PCR detection.     

Current literature highlights - April 2002

Telomerase Inhibitors: Telomerase is the enzyme responsible for maintaining telomere length and it has activity not observed in normal somatic cells. In contrast, high expression of telomerase is observed in around 85-90% of human tumour cells and therefore telomerase is regarded as a specific target for development of cancer chemotherapeutic agents. There are several types of inhibitor known. For example antisense oligodeoxynucleotides and related compounds which exhibit potent inhibition of telomerase in the picomolar range. In spite of this research there have been no clinical trials of inhibitors to date, and discovery of novel inhibitors will contribute to evaluation of telomerase inhibitors for cancer chemotherapy. Recent developments have highlighted new telomerase inhibitors based on the bisindole unit (i) (S. Sasaki et. al., Bioorg. Med. Chem. Lett., 11, (2001), 583).     

Cyclic Naphthalene Diimide Dimer with a Strengthened Ability to Stabilize Dimeric G-Quadruplex

A new type of dimeric cyclic naphthalene diimide derivatives (cNDI-dimers) carrying varied linker length were designed and synthesized to recognize dimeric G-quadruplex structures. All of the cNDI-dimers exhibited a high preference for recognizing G-quadruplex structures, and significantly enhanced the thermal stability of the dimeric G-quadruplex structure over the cNDI monomer by increasing the melting temperature by more than 23 °C, which indicated the strengthened ability of cNDI dimers for stabilizing dimeric G-quadruplex. cNDI dimers also showed a stronger ability to inhibit telomerase activity and stop telomere DNA elongation than cNDI monomer, which showed an improved anticancer potentiality for further therapeutic application.     

Assays for human telomerase activity: progress and prospects

Human telomerase is a ribonucleoprotein complex that functions as a telomere terminal transferase by adding multiple TTAGGG hexamer repeats using its integral RNA as the template. There is a very strong association between telomerase activity and malignancy in nearly all types of cancer, suggesting that telomerase could be used not only as a diagnostic and prognostic marker but also as a therapeutic target for managing cancer. The significant progress in biomedical telomerase research has necessitated the development of new bioanalytical methods for the rapid, sensitive, and reliable detection of telomerase activity in a particular cell or clinical tissue and body fluids. In this review, we highlight some of the latest methods for identifying telomerase activity and inhibition and discuss some of the challenges for designing innovative telomerase assays. We also summarise the current technologies and speculate on future directions for telomerase testing.     

Convergence of The Nobel Fields of Telomere Biology and DNA Repair

The fields of telomere biology and DNA repair have enjoyed a great deal of cross‐fertilization and convergence in recent years. Telomeres function at chromosome ends to prevent them from being falsely recognized as chromosome breaks by the DNA damage response and repair machineries. Conversely, both canonical and nonconical functions of numerous DNA repair proteins have been found to be critical for preserving telomere structure and function. In 2009, Elizabeth Blackburn, Carol Greider and Jack Szostak were awarded the Nobel prize in Physiology or Medicine for the discovery of telomeres and telomerase. Four years later, pioneers in the field of DNA repair, Aziz Sancar, Tomas Lindahl and Paul Modrich were recognized for their seminal contributions by being awarded the Nobel Prize in Chemistry. This review is part of a special issue meant to celebrate this amazing achievement, and will focus in particular on the convergence of nucleotide excision repair and telomere biology, and will discuss the profound implications for human health.     

基于一维序列的三维染色质相分离：驱动力、过程与功能

染色质高级结构在基因调控中起到不可忽视的作用,染色质结构的形成与调控机制受到广泛关注。"相分离"理论近年来受到较多关注,异染色质与转录因子在其中的作用引人瞩目。但是,目前的相分离模型更关注结合因子与表观遗传性质,对DNA序列自身的作用理解尚较不充分。许多物种基因组的序列分布均具有多尺度的不均一性,仅基于Cp G岛(Cp G island,CGI)密度差异这一序列性质,就可以划分出基因、表观遗传、结构和转录性质都截然不同的高CGI密度"森林"和低CGI密度"草原"两种序列区域,体现了基因组自身的马赛克性。本文聚焦染色质结构的序列依赖性,讨论了染色质结构模型的研究进展,关注在序列几乎相同的不同细胞类型中的序列–结构关系及其功能调控,对发育、分化、衰老、疾病等多种过程的染色质结构变化进行了系统分析。针对基于序列的染色质相分离模型,对其物理驱动力进行了讨论,并在该模型的框架下基于相分离的物理特性,对温度、序列不均一性等物理因素对染色质结构可能造成的影响进行了探讨。     

Autoacetylation induced specific structural changes in histone acetyltransferase domain of p300: probed by surface enhanced Raman spectroscopy

Reversible acetylation of histone and non-histone proteins plays an important role in the regulation of gene expression and cellular homeostasis. A balance between acetylation and deacetylation of these proteins are maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Among different HATs, p300/CBP is the most widely studied chromatin modifying enzymes. p300 is involved in several physiological processes like cell growth, regulation of gene expression, development, and tumor suppressor, and therefore its dysfunction causes different diseases. The autoacetylation of p300 is one of the key regulators of its catalytic activity. Mechanistically, autoacetylation induced structural changes in the p300 HAT domain acts as a master switch. In this report, we have shown that the natural HAT inhibitor garcinol could potently inhibit the autoacetylation activity. Furthermore, for the first time, we demonstrate that indeed autoacetylation induces structural changes in p300 HAT domain, as probed by surface-enhanced Raman scattering. Presumably, SERS will be a very useful tool to find out the structural changes in the other self-modifying enzymes like kinases and methyltransferases.     

Spectroscopic studies of dactinomycin and vinorelbine binding to deoxyribonucleic acid and chromatin

Dactinomycin and vinorelbine are anticancer drugs, widely used as chemotherapeutic agents for the treatment of various cancers. This study represents an attempt to compare the effect of these drugs on DNA and soluble chromatin employing UV/vis, fluorescence and circular dichroism spectroscopy techniques. The results demonstrated that the absorbance at 260 and 210 nm was decreased and quenching of drugs with DNA and chromatin chromospheres induced reduction of fluorescence emission intensity. Circular dichroism profiles showed that the binding of drugs induced structural changes in both positive and negative extremes of DNA and chromatin revealing release or displacement of histone proteins from chromatin upon dactinomycin binding whereas vinorelbine preceded the chromatin into compaction. The results suggest that dactinomycin shows higher affinity to DNA compared to chromatin, whereas, vinorelbine recognizes the chromatin structure with higher affinity than free DNA. Also chromatin proteins play a fundamental role in drug-chromatin complex process.     

Plant cell walls: supramolecular assembly, signalling and stress

The structure of the primary cell wall in non-graminaceous plants is briefly reviewed and its role in providing mechanical strength to the plant and protecting it from microbial infection are described. A variety of signalling mechanisms involve oligosaccharides released by glycanase enzymes from microbial pathogens, and some of the mechanisms may be implicated in the regulation of metabolism in ripening fruits. There is some evidence that cell walls are able to sense damage or loss of integrity and that signals can accordingly be passed back to the cytoplasm. Primary cell walls must combine the mechanical and other functions with the capacity to grow in a controlled way. A modification of the ‘Molecular Velcro’ model developed originally to describe deformation of wood is used to predict load-deformation curves like those described by the Lockhart equation for the relationship between turgor stress and growth. Predicting a stress threshold for growth does not require the assumption of enzyme activity, although in fact enzyme activity is indeed required to permit growth at the rates normally observed.