Quantitative determination of telomerase activity in breast cancer and benign breast diseases.

Telomerase plays an important role in maintaining the stability of chromosomes. This ribonucleoprotein prevents chromosome ends (telomeres) from gradual loss with each cell division. It enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Telomerase activity has been detected in the majority of tumor and germ cells and in immortalized cell lines. Quantitative telomerase PCR-ELISA (TeloTAGGG Telomerase PCR ELISA(PLUS)) was evaluated for distinguishing benign and malignant breast tissue. Activity of telomerase was determined in 27 samples of fibrocystic and dysplastic tissues, 28 fibroadenomas and phylloid tumors, and 154 breast cancer tissues; 59 specimens were analyzed retrospectively. Analytical precision and linearity of the assay was tested using breast carcinoma cell line ZR-75-1 and breast tumor tissue extracts. About 4% of tumor samples were excluded from analysis due to interferences in the PCR reaction. Relative telomerase activity differed significantly in the groups of dysplastic tissues, fibroadenomas and carcinomas. The highest activity was found in breast cancer tissue. This method can identify breast cancer tissue with 73% clinical sensitivity and 93% specificity as compared to benign breast tumors. We did not find a correlation between telomerase activity and the tissue levels of estrogen and progesterone receptors, HER-2/neu oncoprotein concentration, tumor size, and lymph node positivity. Probability of disease-free survival was significantly lower for patients with telomerase activity higher than median value. As the assay for telomerase activity has very high analytical sensitivity and high specificity for cancer cells, this routinely used method may prove useful for distinguishing malignant phenotype of breast tissues.     

Telomere dysfunction alters the chemotherapeutic profile of transformed cells

Telomerase inhibition has been touted as a novel cancer-selective therapeutic goal based on the observation of high telomerase levels in most cancers and the importance of telomere maintenance in long-term cellular growth and survival. Here, the impact of telomere dysfunction on chemotherapeutic responses was assessed in normal and neoplastic cells derived from telomerase RNA null (mTERC(-/-)) mice. Telomere dysfunction, rather than telomerase per se, was found to be the principal determinant governing chemosensitivity specifically to agents that induced double-stranded DNA breaks (DSB). Enhanced chemosensitivity in telomere dysfunctional cells was linked to therapy-induced fragmentation and multichromosomal fusions, whereas telomerase reconstitution restored genomic integrity and chemoresistance. Loss of p53 function muted the cytotoxic effects of DSB-inducing agents in cells with telomere dysfunction. Together, these results point to the combined use of DSB-inducing agents and telomere maintenance inhibition as an effective anticancer therapeutic approach particularly in cells with intact p53-dependent checkpoint responses.     

Oxidative stress causes telomere damage in Fanconi anaemia cells - a possible predisposition for malignant transformation

Fanconi anaemia (FA) is an autosomal recessive and X-linked disease characterized by severe genetic instability and increased incidence of cancer. One explanation for this instability may be the cellular hypersensitivity to oxidative stress leading to chromosomal breaks. This study explored the possible oxidative damage to telomeres of FA lymphocyte cell line, HSC536/N, and its possible effect on telomere function. We postulated that combination of oxidative damage with overexpression of telomerase may provide a possible model for malignant transformation in FA. The cells were grown in the presence of telomerase inhibitor and exposed for 1 month to H₂O₂ combined with various antioxidants. This exposure caused shortening of telomere length and damage to the telomere single stranded overhang, which was prevented by several oxidants. This shortening was associated with development of severe telomere dysfunction. Control cells did not exhibit this sensitivity to H₂O₂. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal P53 upregulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxidative damage. These results suggest a scenario in which oxidative stress causes telomere shortening and ensuing telomere dysfunction may form the basis for malignant transformation in FA cells. Upregulation of telomerase activity in sporadic FA cells may perpetuate that process, thus explaining the malignant character of FA cells in vivo .     

线粒体自噬和内质网应激调控血管内皮细胞功能研究进展

血管内皮细胞在调节人体血液循环功能、维持人体心血管系统稳定及促进血管结构重塑中发挥着重要作用.近年来的研究成果证实,线粒体自噬与人体血管壁、内皮组织细胞氧化稳态和细胞氧化应激机制关系密切.内质网是一种维持人体细胞内部结构和生理功能的重要亚特性细胞器,参与人体细胞的众多自然生理化学活动.多种细胞刺激化学因素作用造成的内质...     

端粒酶hTR反义寡核苷酸对K562细胞端粒酶活性的影响

目的：探讨端粒酶RNA反义寡核苷酸（antisense oligodeoxynucleotide,ASODN)对人白血病K562细胞端粒酶活性及细胞生长的影响。方法：应用与人端粒酶RNA组分模板区互补的硫代ASODN处理K562细胞，用端粒酶重复扩增分析（telomere repeat amplification protocol,TRAP)-PCR-ELISA检测法观察端粒酶活性的变化；用Annexin V分析法及流式细胞术检测凋亡细胞。结果：经ASODN作用后，细胞端粒酶活性明显受到抑制，并与ASODN的浓度和处理时间相关。作用5d后，凋亡细胞明显增加。结论：端粒酶RNA模板区ASODN可明显抑制白血病K562细胞端粒酶活性，抑制细胞生长和增殖，诱导细胞凋亡，可能成为白血病基因治疗新靶点。     

Lack of cell cycle regulation of telomerase activity in human cells

Conflicting reports have appeared concerning the cell cycle regulation of telomerase activity and its possible repression during quiescence and cell differentiation. We have reexamined these issues in an attempt to uncover the basis for the discrepancies. Variations in extracted telomerase activity during the cell cycle are not observed in cells sorted on the basis of DNA content. Variations are observed in cells synchronized using some biochemical cell cycle inhibitors, but only with those agents where cellular toxicity is evident. A progressive decline in telomerase activity is observed in cells whose growth rate is reduced from seven to eight population doublings per week to one to two doublings per week. Telomerase is largely absent in cells that truly exit the cell cycle and do not divide over the 7-day period. Although it is not necessary for all cell types to regulate telomerase in the same way, we conclude that in the immortal cultured cell lines examined, extracted telomerase activity does not change significantly during progression through the stages of the cell cycle. Telomerase activity generally correlates with growth rate and is repressed in cells that exit the cell cycle and become quiescent.     

Alcohol and mitochondria: a dysfunctional relationship

Mitochondria are intimately involved in the generation of and defense against reactive oxygen species (ROS). Mitochondria are themselves targets of oxidative stress and also contribute to mechanisms by which oxidative stress-related signals control cell fate. Ethanol promotes oxidative stress, both by increasing ROS formation and by decreasing cellular defense mechanisms. These effects of ethanol are prominent in the liver, the major site of ethanol metabolism in the body. The question remains to what extent this contributes to ethanol-dependent tissue damage or the susceptibility of cells to other stressors. In this review, we consider how mitochondrial actions of ethanol influence oxidative stress management of liver cells. Mitochondrial electron transport constitutes the major intracellular source of ROS, and ethanol treatment imposes conditions that promote ROS formation by mitochondria, the effects of which may be enhanced by a decrease in mitochondrial oxidative stress defenses. A significant target of ethanol-related increases in oxidative stress is mitochondrial DNA. Ethanol-induced damage to mitochondrial DNA, if not adequately repaired, impairs mitochondrial function, which further increases oxidative stress in the cell, leading to a vicious cycle of accumulating cell damage that is more apparent with advancing age. Uncontrolled mitochondrial formation of ROS promotes the inappropriate activation of the mitochondrial permeability transition, increasing the sensitivity of cells to other pro-apoptotic or damage signals. In combination with ethanol-induced defects in mitochondrial function, these alterations may promote both apoptotic and necrotic cell death in response to otherwise benign or beneficial challenges and contribute to the onset or progression of alcohol-induced liver diseases.     

An impaired mitochondrial electron transport chain increases retention of the hypoxia imaging agent diacetylbis(4-methylthiosemicarbazonato)copperII

Radiolabeled diacetylbis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] is an effective positron-emission tomography imaging agent for myocardial ischemia, hypoxic tumors, and brain disorders with regionalized oxidative stress, such as mitochondrial myopathy, encephalopathy, and lactic acidosis with stroke-like episodes (MELAS) and Parkinson's disease. An excessively elevated reductive state is common to these conditions and has been proposed as an important mechanism affecting cellular retention of Cu from Cu(II)(atsm). However, data from whole-cell models to demonstrate this mechanism have not yet been provided. The present study used a unique cell culture model, mitochondrial xenocybrids, to provide whole-cell mechanistic data on cellular retention of Cu from Cu(II)(atsm). Genetic incompatibility between nuclear and mitochondrial encoded subunits of the mitochondrial electron transport chain (ETC) in xenocybrid cells compromises normal function of the ETC. As a consequence of this impairment to the ETC we show xenocybrid cells upregulate glycolytic ATP production and accumulate NADH. Compared to control cells the xenocybrid cells retained more Cu after being treated with Cu(II)(atsm). By transfecting the cells with a metal-responsive element reporter construct the increase in Cu retention was shown to involve a Cu(II)(atsm)-induced increase in intracellular bioavailable Cu specifically within the xenocybrid cells. Parallel experiments using cells grown under hypoxic conditions confirmed that a compromised ETC and elevated NADH levels contribute to increased cellular retention of Cu from Cu(II)(atsm). Using these cell culture models our data demonstrate that compromised ETC function, due to the absence of O(2) as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu(II)(atsm) that increases cellular retention of the Cu.     

Metformin and the ATM DNA damage response (DDR): accelerating the onset of stress-induced senescence to boost protection against cancer

By activating the ataxia telangiectasia mutated (ATM)-mediated DNA Damage Response (DDR), the AMPK agonist metformin might sensitize cells against further damage, thus mimicking the precancerous stimulus that induces an intrinsic barrier against carcinogenesis. Herein, we present the new hypothesis that metformin might function as a tissue sweeper of pre-malignant cells before they gain stem cell/tumor initiating properties. Because enhanced glycolysis (the Warburg effect) plays a causal role in the gain of stem-like properties of tumor-initiating cells by protecting them from the pro-senescent effects of mitochondrial respiration-induced oxidative stress, metformin's ability to disrupt the glycolytic metabotype may generate a cellular phenotype that is metabolically protected against immortalization. The bioenergetic crisis imposed by metformin, which may involve enhanced mitochondrial biogenesis and oxidative stress, can lower the threshold for cellular senescence by pre-activating an ATM-dependent pseudo-DDR. This allows an accelerated onset of cellular senescence in response to additional oncogenic stresses. By pushing cancer cells to use oxidative phosphorylation instead of glycolysis, metformin can rescue cell surface major histocompatibility complex class I (MHC-I) expression that is downregulated by oncogenic transformation, a crucial adaptation of tumor cells to avoid the adaptive immune response by cytotoxic T-lymphocytes (CTLs). Aside from restoration of tumor immunosurveillance at the cell-autonomous level, metformin can activate a senescence-associated secretory phenotype (SASP) to reinforce senescence growth arrest, which might trigger an immune-mediated clearance of the senescent cells in a non-cell-autonomous manner. By diminishing the probability of escape from the senescence anti-tumor barrier, the net effect of metformin should be a significant decrease in the accumulation of dysfunctional, pre-malignant cells in tissues, including those with the ability to initiate tumors. As life-long or late-life removal of senescent cells has been shown to prevent or delay the onset or progression of age-related disorders, the tissue sweeper function of metformin may inhibit the malignant/metastatic progression of pre-malignant/senescent tumor cells and increase the human lifespan.     

Mitochondria at the interface between danger signaling and metabolism: role of unfolded protein responses in chronic inflammation

Inflammatory bowel diseases (IBDs), like many other chronic diseases, feature multiple cellular stress responses including endoplasmic reticulum (ER) unfolded protein response (UPR). Maintaining protein homeostasis is indispensable for cell survival and, consequently, distinct signaling pathways have evolved to transmit organelle stress. While the ER UPR, aiming to restore ER homeostasis after challenges to ER function, has been extensively studied in the context of chronic diseases, only recently the related mitochondrial UPR (mtUPR), induced by disturbances of mitochondrial proteostasis, has drawn some attention. ER and mitochondria are in close contact and interact physically and functionally. Accumulating data have placed mitochondria at the center of diverse cellular functions and suggest mitochondria as integrators of signaling pathways such as autophagy and inflammation. Consequently, it is likely that mitochondrial stress and ER stress cannot be regarded separately and that mitochondrial stress, as well as ER stress, participates in the pathology of IBD. Protein homeostasis is particularly sensitive toward infections, oxidative stress, and energy deficiency. Thus, environmental disturbances impacting organelle function lead to the concerted activation of distinct UPRs. The metabolic status might therefore serve as an innate mechanism to sense the epithelial environment, including luminal-derived and host-derived factors. This review highlights mtUPR and its interrelation with ER UPR, focuses on recent studies identifying mitochondria as integrators of cellular danger signaling, and, furthermore, illustrates the importance ER UPR and mitochondrial dysfunction in IBD.     

线粒体途径保护心肌缺血再灌注损伤的研究进展

线粒体是细胞能量代谢和细胞内信号传导过程的关键细胞器,参与多种复杂信号介导的细胞生存和死亡。线粒体功能障碍及由此产生的氧化应激与心肌缺血再灌注损伤密切相关,保护线粒体功能将有助于减缓心肌损伤的严重程度或进展。最近,线粒体生物学进展启发人们研制作用于线粒体的选择性靶向药物,保护心肌缺血再灌注损伤。本文就此做一综述。     

Telomerase activity in Malaysian patients with central nervous system tumors

Telomerase, the enzyme that stabilizes telomere length is reactivated with almost all cancer types, and may be a useful diagnostic marker for malignancy. Telomerase activity has been detected in germ line cells and most cancer cells, whereas most normal somatic cells have no clearly detectable telomerase activity. In our study, we aim to detect telomerase activity in 20 human central nervous system tumors from Malaysian patients. Telomerase activity was detected based on a highly sensitive procedure consisting of a CHAPS detergent-based extraction from frozen tissues and a PCR-based telomeric repeat amplification protocol (TRAP) using a TRAPEZE Telomerase Detection Kit (Intergen, Co). Telomerase activity was considered positive when a ladder of products was observed starting at 50bp, with 6bp increments. The activity was detected in 30% of the samples analysed, included glioblastoma multiforme, meduloblastoma, paraganglioma and oligodendroglioma. The result of Fisher's exact test indicated that there was a significant association between telomerase activity status with tumor grade (p=0.003). These results suggest that telomerase activity may be an important marker for tumor malignancy.     

A continuous correlation between oxidative stress and telomere shortening in fibroblasts

Telomere shortening in cells with low intrinsic telomerase activity like fibroblasts is governed by various mechanisms including the so-called end-replication problem, end processing and oxidative DNA damage. To assess the impact of oxidative stress on telomere shortening rates, we compared telomere shortening rates measured in fibroblasts from two different donor species (human and sheep) under both pro- and antioxidative culture regimes. Over an almost 50-fold change in peroxide indicator dye fluorescence intensity, we found a continuous, exponential correlation between cellular oxidative stress levels and telomere shortening rates, which was independent of donor species and cell strain. This correlation suggests stress-mediated telomere DNA damage as an important determinant of telomere shortening.     

Percolation and criticality in a mitochondrial network

Synchronization of mitochondrial function is an important determinant of cell physiology and survival, yet little is known about the mechanism of interorganellar communication. We have recently observed that coordinated cell-wide oscillations in the mitochondrial energy state of heart cells can be induced by a highly localized perturbation of a few elements of the mitochondrial network, indicating that mitochondria represent a complex, self-organized system. Here, we apply percolation theory to explain the mechanism of intermitochondrial signal propagation in response to oxidative stress. A global phase transition (mitochondrial depolarization) is shown to occur when a critical density of mitochondria accumulate reactive oxygen species above a threshold to form an extended spanning cluster. The scaling and fractal properties of the mitochondrial network at the edge of instability agree remarkably well with the idea that mitochondria are organized as a percolation matrix, with reactive oxygen species as a key messenger.