[Show abstract][Hide abstract] ABSTRACT: Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.
[Show abstract][Hide abstract] ABSTRACT: Infections with high-risk human papillomaviruses (hrHPV) contribute to cervical carcinoma. The cdk inhibitor and tumor suppressor p16(INK4A) is consistently upregulated in cervical carcinoma cells for reasons that are poorly understood. We report here that downregulation of p16(INK4A)gene expression in three different cervical carcinoma cell lines reduced expression of the E7 oncogene, suggesting a positive feedback loop involving E7 and p16(INK4A). p16(INK4A) depletion induced cellular senescence in HeLa but not CaSki and MS-751 cervical carcinoma cells.
This study demonstrates that the cdk inhibitor p16(INK4A), frequently used as surrogate marker for transforming infections by human papillomaviruses of the high-risk group, is required for high level expression of the E7 oncoproteins of HPV-16, HPV-18 and HPV-45 in cervical carcinoma cells. It is also demonstrated that depletion of p16(INK4A) induces senescence in HeLa but not CaSki or MS-751 cervical carcinoma cells.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts. RESULTS: We observed a parallel activation of the p53/p21WAF1 and p16INK4a/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101. CONCLUSION: We performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a network of miRNA-mRNA interactions mediating UVB-induced senescence. In addition, miR-101 and Ezh2 were identified as key players in UVB-induced senescence of HDF.
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) generated by NADPH oxidases play an important role in cellular signal transduction regulating cell proliferation, survival and differentiation. NADPH oxidase 4 (Nox4) induces cellular senescence in human endothelial cells; however, intracellular targets for Nox4 remained elusive. Here we show that Nox4 induces mitochondrial dysfunction in human endothelial cells. Nox4 depletion induced alterations in mitochondrial morphology, stabilized mitochondrial membrane potential, and decreased production of hydrogen peroxide in mitochondria. High-resolution respirometry in permeabilized cells combined with native polyacrylamide gel electrophoresis demonstrated that Nox4 specifically inhibits the activity of mitochondrial electron transport chain complex I, and this was associated with a decreased concentration of complex I subunits. These data suggest a new pathway by which sustained Nox4 activity decreases mitochondrial function.
[Show abstract][Hide abstract] ABSTRACT: NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition.
[Show abstract][Hide abstract] ABSTRACT: Availability of methionine is known to modulate the rate of aging in model organisms, best illustrated by the observation that dietary methionine restriction extends the lifespan of rodents. However, the underlying mechanisms are incompletely understood. In eukaryotic cells, methionine can be converted to cysteine through the reverse transsulfuration pathway thereby modulating intracellular methionine availability. Whereas previous results obtained in yeast and fruit flies suggest that alterations in the reverse transsulfuration pathway modulate the rate of aging, it is not known whether this function is conserved in evolution. Here we show that depletion of cystathionine beta synthase (CBS), a rate limiting enzyme in the reverse transsulfuration pathway, induces premature senescence in human endothelial cells. We found that CBS depletion induces mild mitochondrial dysfunction and increases the sensitivity of endothelial cells to homocysteine, a known inducer of endothelial cell senescence and an established risk factor for vascular disease. Our finding that CBS deficiency induces endothelial cell senescencein vitro, involving both mitochondrial dysfunction and increased susceptibility of the cells to homocysteine, suggests a new mechanism linking CBS deficiency to vascular aging and disease.
[Show abstract][Hide abstract] ABSTRACT: Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-deficient point-mutant, we found that S149 phosphorylation is necessary for mediating the resistance to apoptosis upon oxidative stress and is partially necessary for elongating the cellular life span. Therefore, ATM dependent phosphorylation of SNEVhPrp19/hPso4 upon DNA damage or oxidative stress might represent a novel axis capable of modulating cellular life span.
[Show abstract][Hide abstract] ABSTRACT: Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins (1, 2). Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response (3), cell cycle regulation and cellular differentiation (4) or in immune system response (5). The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases (4, 6). Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging (7, 8, 9, 10). Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)-dgn fusion protein (GFP-dgn, unstable) and a variant carrying a frameshift mutation (GFP-dgnFS, stable (11)) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP-dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.
[Show abstract][Hide abstract] ABSTRACT: Persistent infections by high-risk human papillomaviruses (HPVs) are the main etiological factor for cervical cancer, and expression of HPV E7 oncoproteins was suggested to be a potential marker for tumor progression. The objective of this study was to generate new reagents for the detection of the HPV18 E7 oncoprotein in cervical smears. Rabbit monoclonal antibodies against recombinant E7 protein of HPV type 18 (HPV18) were generated and characterized using Western blotting, epitope mapping, indirect immunofluorescence, and immunohistochemistry. One clone specifically recognizing HPV18 E7 was used for the development of a sandwich enzyme-linked immunosorbent assay (ELISA). The assay was validated using recombinant E7 proteins of various HPV types and lysates from E7-positive cervical carcinoma cells. A total of 14 HPV18 DNA-positive cervical swab specimens and 24 HPV DNA-negative-control specimens were used for the determination of E7 protein levels by the newly established sandwich ELISA. On the basis of the average absorbance values obtained from all 24 negative controls, a cutoff above which a clinical sample can be judged E7 positive was established. Significant E7 signals 6- to 30-fold over background were found in 7 out of 14 abnormal HPV18 DNA-positive cervical smear specimens. This feasibility study demonstrates for the first time that HPV18 E7 oncoprotein can be detected in cervical smears.
Journal of clinical microbiology 11/2011; 50(2):246-57. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21(Cip1) and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21(Cip1) and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21(Cip1) expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21(Cip1) expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21(Cip1) core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21(Cip1) promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21(Cip1) expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21(Cip1) expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21(Cip1) gene expression.
[Show abstract][Hide abstract] ABSTRACT: The human fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is part of the FAH protein superfamily, but its enzymatic function is unknown. In the quest for a putative enzymatic function of FAHD1, we found that FAHD1 exhibits acylpyruvase activity, demonstrated by the hydrolysis of acetylpyruvate and fumarylpyruvate in vitro, whereas several structurally related compounds were not hydrolyzed as efficiently. Conserved amino acids Asp-102 and Arg-106 of FAHD1 were found important for its catalytic activity, and Mg(2+) was required for maximal enzyme activity. FAHD1 was found expressed in all tested murine tissues, with highest expression in liver and kidney. FAHD1 was also found in several human cell lines, where it localized to mitochondria. In summary, the current work identified mammalian FAHD1 as a novel mitochondrial enzyme with acylpyruvate hydrolase activity.
Journal of Biological Chemistry 08/2011; 286(42):36500-8. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) regulates cell proliferation and survival by extracellular interaction and inactivation of the growth factor IGF-I. Beyond that, IGF-independent actions mediated by intracellular IGFBP-3 including nuclear-IGFBP-3, have also been described. We here show, using both confocal and electron microscopy and cell fractionation, that the extracellular addition of IGFBP-3 to living cells results in rapid uptake and nuclear delivery of IGFBP-3, by yet partly unknown mechanisms. IGFBP-3 is internalized through a dynamin-dependent pathway, traffics through endocytic compartments and is finally delivered into the nucleus. We observed docking of IGFBP-3 containing structures to the nuclear envelope and found IGFBP-3 containing dot-like structures to permeate the nuclear envelope. In summary, our findings establish the pathway by which this tumor suppressor protein is delivered from extracellular space to the nucleus.
International Journal of Cancer 08/2011; 130(7):1544 - 1557. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular senescence can be induced by a variety of mechanisms, and recent data suggest a key role for cytokine networks to maintain the senescent state. Here, we have used a proteomic LC-MS/MS approach to identify new extracellular regulators of senescence in human fibroblasts. We identified 26 extracellular proteins with significantly different abundance in conditioned media from young and senescent fibroblasts. Among these was insulin-like growth factor binding protein-6 (IGFBP-6), which was chosen for further analysis. When IGFBP-6 gene expression was downregulated, cell proliferation was inhibited and apoptotic cell death was increased. Furthermore, downregulation of IGFBP-6 led to premature entry into cellular senescence. Since IGFBP-6 overexpression increased cellular lifespan, the data suggest that IGFBP-6, in contrast to other IGF binding proteins, is a negative regulator of cellular senescence in human fibroblasts.
Mechanisms of ageing and development 07/2011; 132(10):468-79. · 4.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor binding proteins (IGFBPs) are key regulators of insulin-like growth factor (IGF) mediated signal transduction and thereby can profoundly influence cellular phenotypes and cell fate. Whereas IGFBPs are extracellular proteins, intracellular activities were described for several IGFBP family members, such as IGFBP-3, which can be reinternalized by endocytosis and reaches the nucleus through routes that remain to be fully established. Within the family of IGFBPs, IGFBP-6 is unique for its specific binding to IGF-II. IGFBP-6 was described to possess additional IGF-independent activities, which have in part been attributed to its translocation to the nucleus; however, cellular uptake of IGFBP-6 was not described. To further explore IGFBP-6 functions, we developed a new method for the purification of native human IGFBP-6 from cell culture supernatants, involving a four-step affinity purification procedure, which yields highly enriched IGFBP-6. Whereas protein purified in this way retained the capacity to interact with IGF-II and modulate IGF-dependent signal transduction, our data suggest that, unlike IGFBP-3, human IGFBP-6 is not readily internalized by human tumor cells. To summarize, this work describes a novel and efficient method for the purification of native human insulin-like growth factor binding protein 6 (IGFBP-6) from human cell culture supernatants, applying a four-step chromatography procedure. Intactness of purified IGFBP-6 was confirmed by IGF ligand Western blot and ability to modulate IGF-dependent signal transduction. Cellular uptake studies were performed to further characterize the purified protein, showing no short-term uptake of IGFBP-6, in contrast to IGFBP-3.
Journal of Cell Communication and Signaling 03/2011; 5(4):277-89.
[Show abstract][Hide abstract] ABSTRACT: According to the mitochondrial theory of aging, reactive oxygen species (ROS) derived primarily from mitochondria cause cumulative oxidative damage to various cellular molecules and thereby contribute to the aging process. On the other hand, a pivotal role of the proteasome, as a main proteolytic system implicated in the degradation of oxidized proteins during aging, is suggested. In this study, we analyzed mitochondrial function in dermal fibroblasts derived from biopsies obtained from healthy young, middle-aged, and old donors. We also determined proteasome activity in these cells, using a degron-destabilized green fluorescent protein (GFP)-based reporter protein. We found a significant decrease in mitochondrial membrane potential in samples from aged donors, accompanied by a significant increase in ROS levels. Respiratory activity was not significantly altered with donor age, probably reflecting genetic variation. Proteasome activity was significantly decreased in fibroblasts from middle-aged donors compared with young donors; fibroblasts derived from the oldest donors displayed a high heterogeneity in this assay. We also found intraindividual coregulation of mitochondrial and proteasomal activities in all human fibroblast strains tested, suggesting that both systems are interdependent. Accordingly, pharmacological inhibition of the proteasome led to decreased mitochondrial function, whereas inhibition of mitochondrial function in turn reduced proteasome activity.
Journal of Investigative Dermatology 03/2011; 131(3):594-603. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heterologous prime-boost immunization strategies in general establish higher frequencies of antigen-specific T lymphocytes than homologous prime-boost protocols or single immunizations. We developed virosomes and recombinant Semliki Forest virus (rSFV) as antigen delivery systems, each capable of inducing strong CTL responses in homologous prime-boost protocols. Here, we demonstrate that a heterologous prime-boost with recombinant Semliki Forest virus (rSFV) encoding a fusion protein of E6 and E7 of human papillomavirus (HPV) type 16 and virosomes containing the HPV16 E7 protein resulted in higher numbers of antigen-specific CTL in mice than homologous protocols. Evasion of vector-specific immunity appeared to play a role in establishing these high frequencies, as coinduction of vector-specific responses during the prime immunization reduced the frequency of antigen-specific CTL after a heterologous booster. However, the high numbers of CTL initially primed by the heterologous protocols did not correlate with enhanced responsiveness to in vitro antigenic stimulation, nor in improved cytolytic activity or antitumor responses in vivo compared to a homologous protocol with rSFV. This lack of correlation could not be explained by changes in numbers of regulatory T cells. However, we observed differences in the frequencies of T cell subsets within the E7-specific CD8(+) T cell population, e.g. higher frequencies of central memory T cells upon homologous immunizations compared to heterologous immunizations. The induction of central memory T cells is crucial for a cancer vaccine as these cells are known to rapidly expand upon recall stimulation. This study demonstrates that the strongly increased number of antigen-specific CTL as induced by heterologous prime-boost immunizations, often used as a proof for the enhanced efficacy of such regimes, does not necessarily equal superior functional antitumor responses.
[Show abstract][Hide abstract] ABSTRACT: Stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, is a hallmark of benign prostatic hyperplasia (BPH) and solid tumors, including prostate cancer (PCa). Increased local production of TGFβ1 is considered the inducing stimulus. Given that stromal remodeling actively promotes BPH/PCa development, there is considerable interest in developing stromal-targeted therapies. Microarray and quantitative PCR analysis of primary human prostatic stromal cells induced to undergo fibroblast-to-myofibroblast differentiation with TGFβ1 revealed up-regulation of the reactive oxygen species (ROS) producer reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and down-regulation of the selenium-containing ROS-scavenging enzymes glutathione peroxidase 3, thioredoxin reductase 1 (TXNRD1), and the selenium transporter selenoprotein P plasma 1. Consistently, NOX4 expression correlated specifically with the myofibroblast phenotype in vivo, and loss of selenoprotein P plasma 1 was observed in tumor-associated stroma of human PCa biopsies. Using lentiviral NOX4 short hairpin RNA-mediated knockdown, pharmacological inhibitors, antioxidants, and selenium, we demonstrate that TGFβ1 induction of NOX4-derived ROS is required for TGFβ1-mediated phosphorylation of c-jun N-terminal kinase, which in turn is essential for subsequent downstream cytoskeletal remodeling. Significantly, selenium supplementation inhibited differentiation by increasing ROS-scavenging selenoenzyme biosynthesis because glutathione peroxidase 3 and TXNRD1 expression and TXNRD1 enzyme activity were restored. Consistently, selenium depleted ROS levels downstream of NOX4 induction. Collectively, this work demonstrates that dysregulated redox homeostasis driven by elevated NOX4-derived ROS signaling underlies fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma. Further, these data indicate the potential clinical value of selenium and/or NOX4 inhibitors in preventing the functional pathogenic changes of stromal cells in BPH and PCa.
[Show abstract][Hide abstract] ABSTRACT: Age-related gene expression patterns of Homo sapiens as well as of model organisms such as Mus musculus, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster are a basis for understanding the genetic mechanisms of ageing. For an effective analysis and interpretation of expression profiles it is necessary to store and manage huge amounts of data in an organized way, so that these data can be accessed and processed easily.
GiSAO.db (Genes involved in senescence, apoptosis and oxidative stress database) is a web-based database system for storing and retrieving ageing-related experimental data. Expression data of genes and miRNAs, annotation data like gene identifiers and GO terms, orthologs data and data of follow-up experiments are stored in the database. A user-friendly web application provides access to the stored data. KEGG pathways were incorporated and links to external databases augment the information in GiSAO.db. Search functions facilitate retrieval of data which can also be exported for further processing.
We have developed a centralized database that is very well suited for the management of data for ageing research. The database can be accessed at https://gisao.genome.tugraz.at and all the stored data can be viewed with a guest account.
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor-like cytokine 1A (TL1A) is expressed in endothelial cells and contributes to T-cell activation, via an extracellular fragment TL1A(L72-L251), generated by ectodomain shedding. Fragments of TL1A, referred to as vascular endothelial growth inhibitor, were found to induce growth arrest and apoptosis in endothelial cells; however, the underlying mechanisms remained obscure. Here, we show that full-length TL1A is the major detectable gene product in both human umbilical vein endothelial cells and circulating endothelial progenitor cells. TL1A expression was significantly enhanced in senescent circulating endothelial progenitor cells, and knockdown of TL1A partially reverted senescence. TL1A overexpression induced premature senescence in both circulating endothelial progenitor cells and human umbilical vein endothelial cells. We also identified a novel extracellular fragment of TL1A, TL1A(V84-L251), resulting from differential ectodomain shedding, which induced growth arrest and apoptosis in human umbilical vein endothelial cells. These findings suggest that TL1A is involved in the regulation of endothelial cell senescence, via a novel fragment produced by differential ectodomain shedding.
The Journals of Gerontology Series A Biological Sciences and Medical Sciences 11/2010; 65(11):1165-80. · 4.31 Impact Factor