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Skin aging is a complex process influenced by intrinsic factors and environmental stressors, including ultraviolet (UV) radiation and air pollution, among others. In this study, we investigated the effects of UVA and UVB radiation, combined with urban particulate matter (UPM), on human dermal fibroblasts (HDF). We show here that treatment of HDF with a subcytotoxic dose of UVA/UVB results in a series of events leading to mitochondrial dysfunction, increased ROS levels, and DNA damage. These effects are known to trigger either cellular senescence or cell death, depending on the cells' ability to clear damage by activating autophagy. Whereas UPM treatment in isolation did not affect proliferation or survival of HDF, of note, simultaneous UPM treatment of UV‐irradiated cells selectively inhibited autophagic flux, thereby changing cell fate of a fraction of the cell population from senescence to apoptotic cell death. Our findings highlight the synergistic effects of UV radiation and UPM on skin aging, emphasizing the need to consider these factors in assessing the impact of environmental stressors on human health and opening opportunities for developing comprehensive approaches to protect and preserve skin integrity in the face of growing environmental challenges. [ABSTRACT FROM AUTHOR]

The human skin is exposed daily to different environmental factors such as air pollutants and ultraviolet (UV) light. Air pollution is considered a harmful environmental risk to human skin and is known to promote aging and inflammation of this tissue, leading to the onset of skin disorders and to the appearance of wrinkles and pigmentation issues. Besides this, components of air pollution can interact synergistically with ultraviolet light and increase the impact of damage to the skin. However, little is known about the modulation of air pollution on cellular senescence in skin cells and how this can contribute to skin aging. In this review, we are summarizing the current state of knowledge about air pollution components, their involvement in the processes of cellular senescence and skin aging, as well as the current therapeutic and cosmetic interventions proposed to prevent or mitigate the effects of air pollution in the skin. [ABSTRACT FROM AUTHOR]

Organismal aging is normally accompanied by an increase in the number of senescent cells, growth-arrested metabolic active cells that affect normal tissue function. These cells present a series of characteristics that have been studied over the last few decades. The damage in cellular organelles disbalances the cellular homeostatic processes, altering the behavior of these cells. Lysosomal dysfunction is emerging as an important factor that could regulate the production of inflammatory molecules, metabolic cellular state, or mitochondrial function. [ABSTRACT FROM AUTHOR]

Due to its ability to cross the epidermis and reach the upper dermis where it causes cumulative DNA damage and increased oxidative stress, UVB is considered the most harmful component of sunlight to the skin. The consequences of chronic exposition to UVB are related to photoaging and photocarcinogenesis. There are limitations to the study of human skin aging and for this reason the use of models is required. Human dermal fibroblasts submitted to mild and repeated doses of UVB are considered a versatile model to study UVB effects in the process of skin photoaging, which depends on the accumulation of senescent cells, in particular in the dermis. Here we provide updated information about the current model of UVB-induced senescence with special emphasis on the process of protein quality control. [ABSTRACT FROM AUTHOR]

Transformation by the ras oncogene can result in promotion of metastasis as well as induction of senescence via increased tissue remodeling, for example, by matrix metalloproteases. Increased production of mitochondrial reactive oxygen species (mtROS) via NADPH oxidase 4 (NOX4) is implicated in this process. Hydrogen peroxide‐inducible clone‐5 (HIC‐5) is postulated to sense both matrix detachment of transformed cells and intracellular ROS and can inhibit ras signaling via inhibition of NOX4. [ABSTRACT FROM AUTHOR]

This review consolidates a decade of research on fumarylacetoacetate hydrolase domain containing protein 1 (FAHD1), a mitochondrial oxaloacetate tautomerase and decarboxylase with profound implications in cellular metabolism. Despite its critical role as a regulator in mitochondrial metabolism, FAHD1 has remained an often‐overlooked enzyme in broader discussions of mitochondrial function. After more than 12 years of research, it is increasingly clear that FAHD1's contributions to cellular metabolism, oxidative stress regulation, and disease processes such as cancer and aging warrant recognition in both textbooks and comprehensive reviews. The review delves into the broader implications of FAHD1 in mitochondrial function, emphasizing its roles in mitigating reactive oxygen species (ROS) levels and regulating complex II activity, particularly in cancer cells. This enzyme's significance is further highlighted in the context of aging, where FAHD1's activity has been shown to influence cellular senescence, mitochondrial quality control, and the aging process. Moreover, FAHD1's involvement in glutamine metabolism and its impact on cancer cell proliferation, particularly in aggressive breast cancer subtypes, underscores its potential as a therapeutic target. In addition to providing a comprehensive account of FAHD1's biochemical properties and structural insights, the review integrates emerging hypotheses regarding its role in metabolic reprogramming, immune regulation, and mitochondrial dynamics. By establishing a detailed understanding of FAHD1's physiological roles and therapeutic potential, this work advocates for FAHD1's recognition in foundational texts and resources, marking a pivotal step in its integration into mainstream metabolic research and clinical applications in treating metabolic disorders, cancer, and age‐related diseases. [ABSTRACT FROM AUTHOR]

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo " (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo. This primer introduces the "minimal information on cellular senescence experimentation in vivo " (MICSE) guidelines, which aim to address the challenges in identifying and characterizing senescent cells. It provides an overview of senescence markers in various systems and offers a practical toolset to enhance our understanding of cellular senescence in vivo. [ABSTRACT FROM AUTHOR]

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress‐induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX‐dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB‐irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB‐irradiated NIX‐depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB‐induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF. [ABSTRACT FROM AUTHOR]

Growth differentiation factor 15 (GDF15) is a stress‐responsive cytokine also known as a mitokine; however, its role in mitochondrial homeostasis and cellular senescence remained elusive. We show here that knocking down GDF15 expression in human dermal fibroblasts induced mitochondrial dysfunction and premature senescence, associated with a distinct senescence‐associated secretory phenotype. Fibroblast‐specific loss of GDF15 expression in a model of 3D reconstructed human skin induced epidermal thinning, a hallmark of skin aging. Our results suggest GDF15 to play a so far undisclosed role in mitochondrial homeostasis to delay both the onset of cellular senescence and the appearance of age‐related changes in a 3D human skin model. [ABSTRACT FROM AUTHOR]

Premature aging of the skin is a prominent side effect of psoralen photoactivation, a therapy widely and successfully used for different skin disorders. Recently, we demonstrated that treatment of fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in growth arrest with morphological and functional changes reminiscent of replicative senescence. In this minireview we will focus on the similarities between intrinsic and extrinsic aging and PUVA-induced senescence-like growth arrest both resulting in the loss of the structural integrity of the dermal connective tissue as a hallmark of intrinsic aging and photoaging (extrinsic aging) of the skin, and we will discuss the important role of oxidative stress related telomere attrition in the PUVA-induced phenotype of dermal fibroblasts.With the PUVA-induced growth arrest of fibroblasts a new model has been added to the growing number of in vitro models with longterm growth arrest upon exposure to sublethal stressors (i.e. hyperoxia, hydrogen peroxide, ethanol), which are characterized by morphological and functional changes common for cellular senescence. This model may be particularly suited for further studies addressing mechanisms of stress-induced senescence-like growth arrest in vitro and in vivo, since many dermatological patients are treated with PUVA allowing the analysis of putative stress-induced premature senescence in vivo. [Copyright &y& Elsevier]

Humans and animals are structurally designed as a compromise to guarantee optimal survival until the time of reproduction based on natural selection that is effective until that age. Modern conditions of life including improvement of hygiene, preventive and curative medicine as well as socio-economic and political developments have led to an increase of the mean life expectancy that allows ever larger proportions of the population to reach an age that is far beyond that of the reproductive phase. The concept of a Darwinian-evolutionary basis for the development of age-related diseases in principle postulates that genetic traits that are beneficial in younger years to allow for successful reproduction may become deleterious in the elderly, i.e. when selective pressure does not seem to be effective anymore. Examples for this mechanism of pleiotropic antagonism taken from the work of the Institute for Biomedical Aging Research in Innsbruck, Austria, include atherosclerosis, benign and malignant prostate hypertrophy, Alzheimer''s disease and the reciprocal relationship between cellular senescence and cancer. [Copyright &y& Elsevier]

The metabolism of tumor cells (tumor metabolome) is characterized by a high concentration of glycolytic enzymes including pyruvate kinase isoenzyme type M2 (M2-PK), a high glutaminolytic capacity, high fructose 1,6-bisphosphate (FBP) levels and a low (ATP+GTP):(CTP+UTP) ratio. The sequence of events required for the establishment of the tumor metabolome is presently unknown. In non-transformed rat kidney (NRK) cells we observed a high glutaminolytic flux rate and a low (ATP+GTP):(CTP+UTP) ratio, whereas FBP levels and M2-PK activity are still extremely low. After stable expression of oncogenic ras in NRK cells a strong upregulation of FBP levels and of M2-PK activity was observed. Elevated FBP levels induce a tetramerization of M2-PK and its migration into the glycolytic enzyme complex. AMP levels increase whereas UTP and CTP levels strongly decrease. Thus, ras expression completes the glycolytic part of tumor metabolism leading to the inhibition of nucleic acid synthesis and cell proliferation. The HPV-16 E7 oncoprotein, which cooperates with ras in cell transformation, directly binds to M2-PK, induces its dimerization and restores nucleic acid synthesis as well as cell proliferation. Apparently, the combination of the different metabolic effects of ras and E7 constructs the perfect tumor metabolome as generally found in tumor cells. Oncogene (2001) 20, 6891–6898. [ABSTRACT FROM AUTHOR]

Background: In the nematode Caenorhabditis elegans, longevity in response to germline ablation, but not in response to reduced insulin/IGF1-like signaling, is strongly dependent on the conserved protein kinase minibrain-related kinase 1 (MBK-1). In humans, the MBK-1 ortholog DYRK1A is associated with a variety of disorders, most prominently with neurological defects observed in Down syndrome. To better understand mbk-1's physiological roles and their dependence on genetic background, we analyzed the influence of mbk-1 loss on the transcriptomes of wildtype and long-lived, germline-deficient or insulin-receptor defective, C. elegans strains by RNA-sequencing. Results: mbk-1 loss elicited global changes in transcription that were less pronounced in insulin-receptor mutant than in germline-deficient or wildtype C. elegans. Irrespective of genetic background, mbk-1 regulated genes were enriched for functions in biological processes related to organic acid metabolism and pathogen defense. qPCR-studies confirmed mbk-1 dependent induction of all three C. elegans Δ9-fatty acid desaturases, fat-5, fat-6 and fat-7, in wildtype, germline-deficient and insulin-receptor mutant strains. Conversely, mbk-1 dependent expression patterns of selected pathogen resistance genes, including asp-12, dod-24 and drd-50, differed across the genetic backgrounds examined. Finally, cth-1 and cysl-2, two genes which connect pathogen resistance to the metabolism of the gaseous messenger and lifespan regulator hydrogen sulfide (H2S), were commonly suppressed by mbk-1 loss only in wildtype and germline-deficient, but not in insulin-receptor mutant C. elegans. Conclusion: Our work reveals previously unknown roles of C. elegans mbk-1 in the regulation of fatty acid desaturase- and H2S metabolic-genes. These roles are only partially dependent on genetic background. Considering the particular importance of fatty acid desaturation and H2S for longevity of germline-deficient C. elegans, we propose that these processes at least in part account for the previous observation that mbk-1 preferentially regulates lifespan in these worms. [ABSTRACT FROM AUTHOR]

Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., 'senolytics') or inactivating/switching damage‐inducing properties of senescent cells (i.e., 'senostatics/senomorphics'), such as the senescence‐associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging. [ABSTRACT FROM AUTHOR]

FAH domain containing protein 1 (FAHD1) is a mammalian mitochondrial protein, displaying bifunctionality as acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. We report the crystal structure of mouse FAHD1 and structural mapping of the active site of mouse FAHD1. Despite high structural similarity with human FAHD1, a rabbit monoclonal antibody (RabMab) could be produced that is able to recognize mouse FAHD1, but not the human form, whereas a polyclonal antibody recognized both proteins. Epitope mapping in combination with our deposited crystal structures revealed that the epitope overlaps with a reported SIRT3 deacetylation site in mouse FAHD1. [ABSTRACT FROM AUTHOR]

Human fumarylacetoacetate hydrolase (FAH) domain containing protein 1 (FAHD1) is a mitochondrial oxalocatate decarboxylase, the first of its kind identified in eukaryotes. The physiological role of FAHD1 in other eukaryotes is still poorly understood. In C. elegans loss of the FAHD1 ortholog FAHD-1 was reported to impair mitochondrial function, locomotion and egg-laying behavior, yet the underlying mechanisms remained unclear. Using tissue-specific rescue of fahd-1(-) worms, we find that these phenotypic abnormalities are at least in part due to fahd-1’s function in neurons. Moreover, we show that egg-laying defects in fahd-1(-) worms can be fully rescued by external dopamine administration and that depletion of fahd-1 expression induces expression of several enzymes involved in serotonin biosynthesis. Together, our results support a role for fahd-1 in modulating serotonin levels and suggest this protein as a novel link between metabolism and neurotransmitter signaling in the nervous system. Finally, we propose a model to explain how a metabolic defect could ultimately lead to marked changes in neuronal signaling. [ABSTRACT FROM AUTHOR]

Whereas enzymes in the fumarylacetoacetate hydrolase (FAH) superfamily catalyze several distinct chemical reactions, the structural basis for their multi-functionality remains elusive. As a well-studied example, human FAH domain-containing protein 1 (FAHD1) is a mitochondrial protein displaying both acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. As mitochondrial ODx, FAHD1 acts antagonistically to pyruvate carboxylase, a key metabolic enzyme. Despite its importance for mitochondrial function, very little is known about the catalytic mechanisms underlying FAHD1 enzymatic activities, and the architecture of its ligated active site is currently ill defined. We present crystallographic data of human FAHD1 that provide new insights into the structure of the catalytic center at high resolution, featuring a flexible 'lid'-like helical region which folds into a helical structure upon binding of the ODx inhibitor oxalate. The oxalate-driven structural transition results in the generation of a potential catalytic triad consisting of E33, H30 and an associated water molecule. In silico docking studies indicate that the substrate is further stabilized by a complex hydrogen-bond network, involving amino acids Q109 and K123, identified herein as potential key residues for FAHD1 catalytic activity. Mutation of amino acids H30, E33 and K123 each had discernible influence on the ApH and/or ODx activity of FAHD1, suggesting distinct catalytic mechanisms for both activities. The structural analysis presented here provides a defined structural map of the active site of FAHD1 and contributes to a better understanding of the FAH superfamily of enzymes. [ABSTRACT FROM AUTHOR]

Prokaryotic and eukaryotic fumarylacetoacetate hydrolase (FAH) superfamily members, sharing conserved regions that form the so-called FAH-domain, catalyze a remarkable variety of reactions. These enzymes are essential in the metabolic pathways to degrade aromatic compounds in prokaryotes and eukaryotes. It appears that prokaryotic FAH superfamily members evolved mainly to allow microbes to generate energy and useful metabolites from complex carbon sources. We review recent findings, indicating that both prokaryotic and eukaryotic members of the FAH superfamily also display oxaloacetate decarboxylase (ODx) activity. The identification of human FAH domain-containing protein 1 as mitochondrial ODx regulating mitochondrial function supports the new concept that, during evolution, eukaryotic FAH superfamily members have acquired important regulatory functions beyond catabolism of complex carbon sources. Molecular studies on the evolution and function of FAH superfamily members are expected to provide new mechanistic insights in their physiological roles. [ABSTRACT FROM AUTHOR]

In this study we report the identification of FAH domain containing protein 1 (FAHD1), a recently described member of the fumarylacetoacetate hydrolase (FAH) superfamily of metabolic enzymes, as a novel player in the regulation of cellular senescence. FAHD1 was found in a proteomic screen searching for mitochondrial proteins, which are differentially regulated in mitochondria from young and senescent human endothelial cells, and subsequently identified as oxaloacetate decarboxylase. We report here that depletion of FAHD1 from human endothelial cells inhibited mitochondrial energy metabolism and subsequently induced premature senescence. Whereas senescence induced by FAHD1 depletion was not associated with DNA damage, we noted a reduction of mitochondrial ATP-coupled respiration associated with upregulation of the cdk inhibitor p21. These results indicate that FAHD1 is required for mitochondrial function in human cells and provide additional support to the growing evidence that mitochondrial dysfunction can induce cellular senescence by metabolic alterations independent of the DNA damage response pathway. [ABSTRACT FROM AUTHOR]

In the current study, we have extended previous findings aiming at a better understanding of molecular mechanisms underlying UVB-induced senescence of diploid human dermal fibroblasts (HDFs), an experimental model to study the process of photoaging in the skin. We provide evidence that the inhibition of proteasomal degradation of damaged proteins and the activation of autophagosome formation are early events in UVB-induced senescence of HDFs, dependent on UVB-induced accumulation of reactive oxygen species. Our data suggest that autophagy is required for the establishment of the senescent phenotype in UVB-treated HDFs and that inhibition of autophagy is sufficient to change the cell fate from senescence to cell death by apoptosis. Studies in reconstructed skin equivalents revealed that UVB irradiation triggers hallmarks of autophagy induction in the dermal layer. These findings have potential implications for fundamental as well as translational research into skin aging, in particular photoaging. [ABSTRACT FROM AUTHOR]

Objective: Human papilloma virus (HPV) induced head and neck squamous cell carcinoma (HNSCC) represents a distinct tumor subset. We questioned how accurately a brushing from the tumor surface detects HPV in patients with HNSCC.Materials and Methods: Brushings from the tumor surface were compared with HPV DNA isolation from formalin-fixed and paraffin-embedded (FFPE) tumor biopsies, which served as the reference standard. In both matrices, HPV DNA was detected using a commercially available test kit. In addition, p16 was assessed in tumor biopsies by immunohistochemistry (IHC). The tumors were considered p16 positive if 70% or more of cancer cells expressed p16.Results: 93 patients with HNSCC were included. Sensitivity and specificity of the brush test were 83% (95%CI: 67-92%) and 85% (95%CI: 72-93%). Results of p16 IHC were concordant with FFPE samples DNA determinations in 73/93 patients. In 53 patients (57%) the tumor was located in the oropharynx and in 40 patients (43%) the tumor was located in the non-oropharynx region. Sensitivity and specificity of the brush test in patients with oropharyngeal cancer was higher with 86% (95%CI: 70-95%) and 89% (95%CI: 65-99%).Conclusion: Superficial brushes from the tumor surface may be used to identify HPV positive HNSCC. [ABSTRACT FROM AUTHOR]

Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor. [ABSTRACT FROM AUTHOR]

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin-/- mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a group of small non-coding executor RNAs. Their function as key modulators of cellular senescence has been widely recognized recently. By cross-comparing several human aging models we previously identified dozens of miRNAs being differentially regulated during aging. Here the functions of two miRNAs, mir-24 and mir-424, were investigated in an oxidative stress-induced fibroblast premature senescence model. Using pre-miRNA precursors, miRNAs were overexpressed in cells undergoing premature senescence induced by oxidative stress. More senescent cells were observed in mir-24 transfected cells. p53 was upregulated in mir-24 overexpressing cells, but downregulated in mir-424 overexpressing cells. DNA topoisomerase I (TOP1), an enzyme controlling DNA topology, was identified as a target of mir-24, whose expression was induced by oxidative stress. Knocking down TOP1 induced cellular senescence. These results suggest that mir-24 activity propagates stress-induced senescence by down regulating TOP1. [ABSTRACT FROM AUTHOR]

The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of fahd-1, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of fahd-1 resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the fahd-1(tm5005) mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of fahd-1(tm5005) animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of fahd-1. Together these data establish a role of fahd-1 to maintain mitochondrial function and consequently physical activity in nematodes. [ABSTRACT FROM AUTHOR]

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins occur in both prokaryotes and eukaryotes, where they carry out diverse enzymatic reactions, probably related to structural differences in their respective FAH domains; however, the precise relationship between structure of the FAH domain and the associated enzyme function remains elusive. In mammals, three FAH domain-containing proteins, FAHD1, FAHD2A, and FAHD2B, are known; however, their enzymatic function, if any, remains to be demonstrated. In bacteria, oxaloacetate is subject to enzymatic decarboxylation; however, oxaloacetate decarboxylases (ODx) were so far not identified in eukaryotes. Based on molecular modeling and subsequent biochemical investigations, we identified FAHD1 as a eukaryotic ODx enzyme. The results presented here indicate that dedicated oxaloacetate decarboxylases exist in eukaryotes. [ABSTRACT FROM AUTHOR]

Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts ( HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mt DNA-encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly. [ABSTRACT FROM AUTHOR]

Infections with high-risk human papillomaviruses (hrHPV) contribute to cervical carcinoma. The cdk inhibitor and tumor suppressor p16INK4A is consistently upregulated in cervical carcinoma cells for reasons that are poorly understood. We report here that downregulation of p16INK4A gene expression in three different cervical carcinoma cell lines reduced expression of the E7 oncogene, suggesting a positive feedback loop involving E7 and p16INK4A. p16INK4A depletion induced cellular senescence in HeLa but not CaSki and MS-751 cervical carcinoma cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ROS (reactive oxygen species) generated by NADPH oxidases play an important role in cellular signal transduction regulating cell proliferation, survival and differentiation. Nox4 (NADPH oxidase 4) induces cellular senescence in human endothelial cells; however, intracellular targets for Nox4 remained elusive. In the present study, we show that Nox4 induces mitochondrial dysfunction in human endothelial cells. Nox4 depletion induced alterations in mitochondrialmorphology, stabilized mitochondrial membrane potential and decreased production of H2O2 in mitochondria. High-resolution respirometry in permeabilized cells combined with native PAGE 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. [ABSTRACT FROM AUTHOR]

Abstract: We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21Cip1 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 p21Cip1 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 p21Cip1 expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21Cip1 expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21Cip1 core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21Cip1 promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21Cip1 expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21Cip1 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 p21Cip1 gene expression. [Copyright &y& Elsevier]

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 Mg2+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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors. [ABSTRACT FROM AUTHOR]

Background: 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. Description: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor–like cytokine 1A (TL1A) is expressed in endothelial cells and contributes to T-cell activation, via an extracellular fragment TL1AL72-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, TL1AV84-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. [ABSTRACT FROM PUBLISHER]

Abstract: Mitotic cells in culture show a limited replicative potential and after extended subculturing undergo a terminal growth arrest termed cellular senescence. When cells reach the senescent phenotype, this is accompanied by a significant change in the cellular phenotype and massive changes in gene expression, including the upregulation of secreted factors. In human fibroblasts, senescent cells also acquire resistance to apoptosis. In contrary, in human endothelial cells, both replicative and stress-induced premature senescence is accompanied by increased cell death; however mechanisms of cell death are poorly explored. In this communication, we addressed the role of endonuclease G (EndoG), a mitochondrial mediator of caspase-independent cell death, in senescence-associated cell death of human endothelial cells. Using immunofluorescence microscopy, we found, that EndoG is localized in the mitochondria in young cells, but relocalizes to the nucleus upon senescence. When EndoG gene expression was downregulated by lentiviral shRNA vectors, we found a significant reduction in the replicative life span and a corresponding increase in cell death. We also observed a slight shift in the cell death phenotype from necrosis to apoptosis. Together these observations suggest an important role of EndoG in the senescence program of human endothelial cells. [Copyright &y& Elsevier]

Abstract: The accumulation of oxidative damage in mitochondrial proteins, membranes and DNA during ageing is supposed to lead to mitochondrial inactivation, downstream molecular impairments and subsequent decline of biological systems. In a quantitative study investigating the age-related changes of mitochondrial proteins on the level of oxidative posttranslational modifications, we previously found a set of conserved biomarkers across ageing models in five species with consistent oxidative break-up of tryptophan residues and formation of N-formyl kynurenine. In an additional proteomic profiling of a long-living Drosophila mutant overexpressing mitochondrial Hsp22 and controls, we found age-related redundant isoforms of voltage-dependent anion channel 1 (VDAC-1). A re-examination of data from human umbilical vein endothelial cells (with normal and chemically accelerated in vitro ageing), revealed similar age-dependent alterations of voltage-dependent anion channel isoforms. Building on these results, we examined the expression of VDAC-1 in an in vitro model of excitotoxicity. We show that glutamate-induced calcium toxicity in neurons induces changes of voltage-dependent anion channel 1 related to downstream events of mitochondrial apoptosis like poly-ADP-ribosylation. [Copyright &y& Elsevier]

Abstract: We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also been described to possess additional IGF-independent activities, which rely, at least in part, on their nuclear localization. However, the mechanisms of IGF-independent biological activities of IGFBP-3 are not well understood. For the study of these functions, recombinant IGFBP-3 is used. However, it has traditionally been difficult to obtain recombinant protein in sufficient quality and quantity. Here we describe a new procedure for the purification of recombinant IGFBP-3 from cell culture supernatants involving a two-step affinity purification procedure. Using this new protocol, we obtained pure IGFBP-3 free of any visible contaminants. We also provide evidence that the protein purified in this way retains biological activity, to bind IGF and modulate IGF-dependent signal transduction. We also show that the purified protein produced by the new procedure is readily internalized by human fibroblasts, suggesting that this protein is also suitable to study intracellular trafficking of IGFBP-3. [Copyright &y& Elsevier]

Abstract: High-risk human papillomaviruses (HPV) cause cervical cancer. The biological properties of HPV-45, the third most prevalent high-risk HPV-genotype, are unknown. We demonstrate here that the HPV-45 E7 protein transforms immortalized NIH3T3 fibroblasts, while mutations in either the conserved LXCXE sequence (C28G) or the carboxyl-terminus (Δ87LQQLF91) significantly abolish this activity. To address the mechanisms underlying cell transformation by HPV-45 E7, we investigated its impact on the cell cycle. We show that HPV-45 E7 associates with the hypophosphorylated form of the retinoblastoma protein (pRb) and induces a significant reduction in the pRb half-life which can be blocked by epoxomicin. Moreover, HPV-45 E7 induces anchorage-independent cell cycle progression of NIH3T3 cells and extends the lifespan of primary human keratinocytes. HPV-45 E7C28G did not bind pRb and could neither induce pRb-proteolysis nor promote cell cycle progression. HPV-45 E7Δ87LQQLF91 had intermediate pRb-binding affinity and retained a residual activity to induce the degradation of pRb but lost the capability to promote cell cycle progression in suspension. Another carboxyl-terminal mutant, HPV-45 E7Δ81AEDL84, showed a trend to reduced transforming activity, had reduced pRb-binding activity and lost the capability to induce pRb-degradation; however, this mutant could induce anchorage-independent cell cycle progression with the same efficiency as HPV-45 E7 wild type. In summary, these data suggest that HPV-45 E7 is a transforming protein and that abrogation of cell cycle control contributes to its oncogenic potential. [Copyright &y& Elsevier]

Abstract: The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a “public” mechanism of aging. [Copyright &y& Elsevier]

Abstract: According to the ‘free radical theory of ageing’, the generation and accumulation of reactive oxygen species are key events during ageing of biological systems. Mitochondria are a major source of ROS and prominent targets for ROS-induced damage. Whereas mitochondrial DNA and membranes were shown to be oxidatively modified with ageing, mitochondrial protein oxidation is not well understood. The purpose of this study was an unbiased investigation of age-related changes in mitochondrial proteins and the molecular pathways by which ROS-induced protein oxidation may disturb cellular homeostasis. In a differential comparison of mitochondrial proteins from young and senescent strains of the fungal ageing model Podospora anserina, from brains of young (5 months) vs. older rats (17 and 31 months), and human cells, with normal and chemically accelerated in vitro ageing, we found certain redundant posttranslationally modified isoforms of subunits of ATP synthase affected across all three species. These appear to represent general susceptible hot spot targets for oxidative chemical changes of proteins accumulating during ageing, and potentially initiating various age-related pathologies and processes. This type of modification is discussed using the example of SAM-dependent O-methyltransferase from P. anserina (PaMTH1), which surprisingly was found to be enriched in mitochondrial preparations of senescent cultures. [Copyright &y& Elsevier]

According to the free radical theory of aging, reactive oxygen species (ROS) act as a driving force of the aging process, and it is generally believed that mitochondrial dysfunction is a major source of increased oxidative stress in tissues with high content of mitochondria, such as muscle or brain. However, recent experiments in mouse models of premature aging have questioned the role of mitochondrial ROS production in premature aging. To address the role of mitochondrial impairment and ROS production for aging in human muscles, we have analyzed mitochondrial properties in muscle fibres isolated from the vastus lateralis of young and elderly donors. Mitochondrial respiratory functions were addressed by high-resolution respirometry, and ROS production was analyzed by in situ staining with the redox-sensitive dye dihydroethidium. We found that aged human skeletal muscles contain fully functional mitochondria and that the level of ROS production is higher in young compared to aged muscle. Accordingly, we could not find any increase in oxidative modification of proteins in muscle from elderly donors. However, the accumulation of lipofuscin was identified as a robust marker of human muscle aging. The data support a model, where ROS-induced molecular damage is continuously removed, preventing the accumulation of dysfunctional mitochondria despite ongoing ROS production. [ABSTRACT FROM AUTHOR]

The cell-cycle regulating gene, p16INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence. Although there is an age-dependent increase of p16INK4A expression in human fibroblast senescence in vitro, no data are available regarding the age dependency of p16INK4A in vivo. To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0–20, 21–70, and 71–95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67. In immunofluorescence co-expression studies, Ki67-positive cells were negative for p16INK4A and BMI1-expressing cells also stained negatively for Ki67. In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin. [ABSTRACT FROM AUTHOR]

Abstract: The mitochondrial theory of aging predicts that functional alterations in mitochondria contribute to the aging process. Whereas this hypothesis implicates increased production of reactive oxygen species (ROS) as a driving force of the aging process, little is known about molecular mechanisms by which mitochondrial impairment might contribute to aging. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between mitochondrial impairment and the appearance of a senescence-like phenotype in young cells. We found that treatment by antimycin A delays proliferation and induces premature senescence in a subset of the cells, associated with increased reactive oxygen species (ROS) production. Quenching of ROS by antioxidants did however not restore proliferation capacity nor prevent premature senescence. Premature senescence is also induced upon chronic exposure to oligomycin, irrespective of ROS production, and oligomycin treatment induced the up-regulation of the cdk inhibitors p16, p21 and p27, which are also up-regulated in replicative senescence. Thus, besides the well-established influence of ROS on proliferation and senescence, a reduction in the level of oxidative phosphorylation is causally related to reduced cell proliferation and the induction of premature senescence. [Copyright &y& Elsevier]

Abstract: E7 proteins are major oncoproteins of human papillomaviruses (HPVs) which play a key role in virus-associated cervical carcinogenesis. The E7 oncoprotein of HPV-16 has been shown to interact with a variety of cellular target proteins and these interactions are considered essential for the transforming properties of this oncoprotein. Several additional HPV types associated etiologically to cervical cancer have been described, the second most common being HPV-18. Less is known about the biochemical functions and interactions of HPV-18 E7. As a first step to determine biochemical properties common to the E7 proteins of the high-risk HPV types 16 and 18 these E7 proteins were expressed in bacteria and purified to homogeneity. Purified E7 proteins were used to investigate the in vitro interaction with the pocket protein p107 and insulin-like growth factor-binding protein-3 (IGFBP-3) that are known to interact with the amino-terminal and the carboxyl-terminal part of IGFBP-3, respectively. Both purified E7 proteins interacted strongly with p107 and, as demonstrated here for the first time, HPV-18 E7 was capable of binding to IGFBP-3, albeit to a lesser extent than HPV-16 E7. These findings suggest that the purified recombinant E7 proteins retain, at least in part, their biochemical activities. [Copyright &y& Elsevier]

Signaling through the insulin/IGF axis plays a major role in determining the rate of aging in many species. IGF-binding proteins (IGFBPs) modulate the IGF pathway in higher organisms. IGFBP-3 accumulates in conditioned medium of senescent human fibroblasts, suggesting that it may contribute to the senescent phenotype. IGFBP-3 can enhance apoptotic cell death in tumor cells due to its ability to target intracellular regulators of apoptosis, including nuclear transcription factors. Senescent fibroblasts are highly resistant to apoptosis, suggesting that IGFBP-3 fails to induce apoptosis in this cell type; however, mechanisms of apoptosis resistance in senescent cells are poorly understood. To address this question, we studied the production and intracellular localization of IGFBP-3 in senescent fibroblasts. Whereas IGFBP-3 is highly overexpressed by senescent fibroblasts, IGFBP-3 was not detectable in the nucleus of senescent fibroblasts. In tumor cells, IGFBP-3 can be internalized by endocytosis, which is considered as a prerequisite for the intracellular functions of IGFBP-3 and probably also for its transport to the nucleus; we show here that endocytotic uptake of IGFBP-3 does not occur in senescent human fibroblasts. This is correlated with a generally decreased endocytotic activity of these cells, as shown with the model substrate transferrin. The data are consistent with a model where IGFBP-3 accumulation in conditioned medium of senescent fibroblasts contributes to growth arrest of these cells, whereas the failure to endocytose IGFBP-3 and the absence of nuclear IGFBP-3 may contribute to the well-established apoptosis resistance of senescent human fibroblasts. [ABSTRACT FROM AUTHOR]

Abstract: Aging of human cells can be reproduced in monolayer cultures, revealing the phenotype of replicative senescence. It was shown that diploid human fibroblasts enter a stable growth arrest phenotype at the end of their lifespan and, in particular, these cells are resistant to various apoptotic stimuli. In contrast, human endothelial cells from the umbilical vein (HUVEC) acquire a proapoptotic phenotype when reaching senescence and this probably results from reactive oxygen species (ROS) induced damage and associated signaling. Ceramides were shown to accumulate in senescent fibroblasts and are also known as potent regulators of apoptotic cell death. To further study age-associated changes in proneness to apoptosis between fibroblasts and endothelial cells, both cell types were challenged by administration of exogenous ceramide and apoptotic cell death was determined. While ceramide can efficiently induce apoptosis in both young and senescent cells of either histotype, quantitative evaluation of the data show that senescent fibroblasts are more resistant to apoptosis induction when compared to their young counterparts, whereas in the case of endothelial cells proneness for apoptosis is increased in senescent cells. Together, these data suggest significant differences in the regulation of apoptosis associated with senescence in fibroblasts and endothelial cells. [Copyright &y& Elsevier]