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PubMed2005

Bcl-2 protects against oxidative stress while inducing premature senescence.

Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.

Animals↗
PubMedDate not supplied

Caffeine, mental stress, and risk for hypertension: a cross-cultural replication.

Separate and combined effects of caffeine and mental arithmetic stress on systolic blood pressure (SBP) and heart rate (HR) were assessed in a double-blind, placebo-controlled study. Thirty-six Chinese nationals, half with and half without a family history of hypertension, received either 0, 125, or 250 mg of caffeine in three separate experimental sessions, each preceded by a 12-hour abstention from caffeine consumption. Caffeine and mental stress elevated SBP, but caffeine did not potentiate the SBP response to mental stress. Caffeine did not affect HR. A positive family history of hypertension was associated with larger SBP elevations to the higher dose of caffeine. Family history of hypertension was associated with larger blood pressure elevations during mental stress, but only under conditions of high-level stimulation provided by the initial exposure to the stress. These results support previous findings of greater cardiovascular reactivity to laboratory stress in individuals at high risk for hypertension.

Adult↗
PubMedDate not supplied

Interferon-gamma, tumor necrosis factor-alpha, and interleukin 1-beta suppress the replication of hepatitis B virus through oxidative stress.

The effect of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and H2O2, on hepatitis B virus (HBV) replication was analyzed in the HBV DNA transfected human hepatoblastoma-derived cell line HB 611. Secretion of HBV DNA from HB611 cells was inhibited by IFN-gamma, TNF-alpha, IL-1beta, and H2O2 in a dose-dependent manner. These cytokines and H2O2 also decreased HBV mRNA expression in HB611 cells, meaning that these reagents decreased the synthesis of all virally encoded components of the HBV virion. The level of manganese-SOD mRNA, indicative of occurrence of oxidative stress, increased immediately after treatment with IFN-gamma, TNF-alpha, IL-1beta, and H2O2. Moreover, the antioxidant N-acetyl-L-cysteine substantially inhibited the antiviral effect. Our findings suggest that oxidative stress may be a common factor in the antiviral effects of IFN-gamma, TNF-alpha, and IL-1beta on HBV.

Antiviral Agents↗
PubMed1985

Cytoskeletal features of rat aortic cells during development. An electron microscopic, immunohistochemical, and biochemical study.

Actin, vimentin, desmin, and tropomyosin distribution in rat aortic endothelial and smooth muscle cells has been studied during development using fetal (18 to 20 days of gestation), and 5- and 14-day-, and 5-, and 12-week-old rats. Endothelial cells of newborn animals actively replicate and contain many actin stress fibers, whereas, in adult animals, replication is minimal and actin stress fibers are rare. The actin, vimentin, desmin, and tropomyosin content of smooth muscle cells increases gradually from fetal to adult animals. The number of desmin-containing cells also increases from 13% in fetal rats to 51% in adult rats. The beta-actin isoform is predominant in fetal and newborn animals, but gradually the alpha-isoform becomes quantitatively the most important, as seen by bidimensional polyacrylamide gels. Several analogies exist between the features of developing smooth muscle and what is known for developing striated muscle cells. The evolution of cytoskeletal features from fetal to adult animals is remarkably the opposite of what takes place in: (1) rat aortic smooth muscle cells proliferating after an endothelial injury, (2) human arterial smooth muscle cells present in atheromas, and (3) actively growing rat aortic smooth muscle cells in vitro. Thus, the assumption that pathological or cultured smooth muscle cells are "dedifferentiated" is supported by our biochemical observations.

Actins↗
PubMed2005

CDK-dependent stabilization of Cdc6: linking growth and stress signals to activation of DNA replication.

Cyclin-dependent kinases (CDKs) play a crucial role in cell cycle progression by controlling the transition from G(1) phase into S phase where DNA is replicated. Key to this transition is the regulation of initiation of DNA replication at replication origins. CDKs are thought to regulate origins of replication both positively and negatively by phosphorylating replication proteins at origins. Several replication proteins that are potentially negatively regulated upon CDK phosphorylation have been identified. However, the mechanism by which CDKs activate replication is currently less well understood. New observations revealing that the initiation protein Cdc6 is stabilized by CDK2-dependent phosphorylation may give more insight in this process.

Cell Cycle Proteins↗
PubMed2005

Chromosomal telomere shortening of kidney cells in IgA nephropathy by the measurement of DNA in urinary sediment.

BACKGROUND: The histology and function of the kidney deteriorates with age and progressive renal failure, but the mechanisms involved in renal ageing are not known. In vitro studies suggest that telomere shortening is important in replicative senescence, and is accelerated by stress factors that increase replication. We investigated whether IgA nephropathy, a prototype chronic kidney disease, is associated with localized intrarenal cellular ageing. METHODS: We studied the mean length of terminal restriction fragments (TRF), a measure of average telomere size, in the DNA of peripheral blood mononuclear cells and urinary sediment of 15 patients with IgA nephropathy. RESULTS: The mean TRF lengths in peripheral blood is 7043.8 +/- 1 182.8 base pairs, and in urinary sediment is 6 749.7 +/- 636.5 base pairs. The mean TRF lengths of urinary DNA significantly correlate with the serum creatinine (r = -0.525, p = 0.044) and estimated glomerular filtration rate (GFR) (r = 0.651, p = 0.009). The mean TRF lengths of urinary DNA had an insignificant inverse correlation with patient age (r = -0.364, p = 0.2), and do not correlate with the degree of glomerulosclerosis (r = 0.004, p = 0.9) or tubulointerstitial scarring in renal biopsy (r =-0.032, p = 0.9). After 30 months of follow-up, the rate of decline of estimated GFR has an inverse correlation with the mean TRF lengths of urinary DNA (r = -0.699, p = 0.004). The TRF lengths of peripheral blood DNA do not correlate with any clinical or histological parameter or the rate of renal function decline. CONCLUSIONS: Although this is a pilot study, our observation indicates that the TRF lengths of genomic DNA extracted from urinary sediment is related to the degree of renal impairment. However, a long telomere length of genomic DNA in urinary sediment is associated with a more rapid decline of renal function. Our findings might be relevant to the pathogenesis of progressive renal failure.

Adult↗
PubMed2006

Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase.

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 microM(-1) s(-1), a value approximately 10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 microM(-1) s(-1), a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA.

Human diploid fibroblast cells can divide for only a limited number of times in vitro, a phenomenon known as replicative senescence or the Hayflick limit. Variability in doubling potential is observed within a clone of cells, and between two sister cells arising from a single mitotic division. This strongly suggests that the process by which cells become senescent is intrinsically stochastic. Among the various biochemical mechanisms that have been proposed to explain replicative senescence, particular interest has been focussed on the role of telomere reduction. In the absence of telomerase--an enzyme switched off in normal diploid fibro-blasts-cells lose telomeric DNA at each cell division. According to the telomere hypothesis of cell senescence, cells eventually reach a critically short telomere length and cell cycle arrest follows. In support of this concept, forced expression of telomerase in normal fibroblasts appears to prevent cell senescence. Nevertheless, the telomere hypothesis in its basic form has some difficulty in explaining the marked stochastic variations seen in the replicative lifespans of individual cells within a culture, and there is strong empirical and theoretical support for the concept that other kinds of damage may contribute to cellular ageing. We describe a stochastic network model of cell senescence in which a primary role is played by telomere reduction but in which other mechanisms (oxidative stress linked particularly to mitochondrial damage, and nuclear somatic mutations) also contribute. The model gives simulation results that are in good agreement with published data on intra-clonal variability in cell doubling potential and permits an analysis of how the various elements of the stochastic network interact. Such integrative models may aid in developing new experimental approaches aimed at unravelling the intrinsic complexity of the mechanisms contributing to human cell ageing.

Cell Culture Techniques↗
PubMedDate not supplied

Activation of p53/p21waf1 pathway is associated with senescence during v-Ha-ras transformation of immortal C2C12 myoblasts.

It has recently been shown that tumor cells can retain the ability to undergo senescence, while the capacity of bypassing senescence has been associated with tumor progression. In this report, we showed that v-Ha-ras-mediated transformation of already immortal C2C12 myoblasts can be associated with senesence, in a low amount during in vitro passages and, to a higher extent, affer cellular stress (cell culture alkalinkation), or DNA damage (doxorubicin treatment). The capacity to undergo replicative senescence is associated with a strong increase of wt-p53 transcriptional activity and p21WAF1 up-regulation. These biochemical activities are down-modulated in the cells that evade the massive replicative senescence after stressing stimuli. Altogether, these findings show that active ras can cause senescence during the transformation of already immortal cells in associaton with p53/p21WAF1 pathway activation and support the hypothesis that p53/p21WAF1 functional activity is important in maintaining the integrity of the senescence pathway during cellular transformation.

Animals↗
PubMed2002

Endothelial cells maintain a reduced redox environment even as mitochondrial function declines.

Human umbilical vein endothelial cells (HUVECs) are an endothelial model of replicative senescence. Oxidative stress, possibly due to dysfunctional mitochondria, is believed to play a key role in replicative senescence and atherosclerosis, an age-related vascular disease. In this study, we determined the effect of cell division on genomic instability, mitochondrial function, and redox status in HUVECs that were able to replicate for approximately 60 cumulative population doublings (CPD). After 20 CPD, the nuclear genome deteriorated and the protein content of the cell population increased. This indicated an increase in cell size, which was accompanied by an increase in oxygen consumption, ATP production, and mitochondrial genome copy number and approximately 10% increase in mitochondrial mass. The antioxidant capacity increased, as seen by an increase in reduced glutathione, glutathione peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase. However, by CPD 52, the latter two enzymes decreased, as well as the ratio of mitochondrial-to-nuclear genome copies, the mitochondrial mass, and the oxygen consumption per milligram of protein. Our results signify that HUVECs maintain a highly reducing (GSH) environment as they replicate despite genomic instability and loss of mitochondrial function.

Cells, Cultured↗
PubMed2006

Papillomavirus E1 protein binds to and stimulates human topoisomerase I.

The papillomavirus (PV) E1 helicase plays a direct role in recruiting cellular DNA replication factors, such as replication protein A or polymerase alpha-primase, to replicate PV genomes. Here, E1 is shown to bind to human topoisomerase I and stimulate its relaxation activity up to sevenfold. The interaction between E1 and topoisomerase I was mapped to the E1 DNA binding domain and C terminus. These findings imply a mechanism for the recruitment of topoisomerase I to PV DNA replication forks and for stimulating topoisomerase I to allow for efficient relaxation of the torsional stress induced by replication fork progression.

Animals↗
PubMedDate not supplied

Hardiness and support at work as predictors of work stress and job satisfaction.

PURPOSE: To test a theoretically and empirically based model linking potential protective resources (hardiness, coworker and supervisor support) to the outcomes of work stress and job satisfaction and replicating the relationship of work stress to job satisfaction while accounting for the potential influence of negative affectivity. DESIGN: A cross-sectional research design using survey data collected from two convenience samples. SETTING: Two worksites: (1) a high-tech company and (2) a government agency. SUBJECTS: High-tech employees (N = 310; response rate, 73.8%) and government agency employees (N = 745; response rate, 49.7%). MEASURES: The Dispositional Resilience Scale measured hardiness and the Positive and Negative Affect Schedule measured negative affectivity. Coworker and supervisor support were measured using the Coworker Support Scale and the Supervisor Support Scale, respectively. The Perceived Work Stress Scale measured work stress, and a single item from the Job Satisfaction Scale assessed overall job satisfaction. RESULTS: A multiple-group path analysis examined the proposed model. Similar patterns of association were found for both samples and suggested a more parsimonious model without the path from negative affectivity to job satisfaction. The model supports the protective nature of hardiness and support at work with regard to work stress and job satisfaction. CONCLUSION: Explanations of relationships depicted in the model, practical implications for reducing work stress and enhancing job satisfaction, limitations and future directions are discussed.

Adult↗
PubMed1995

Mutual adaptation of bacteriophage fd, pfd plasmids and their host strains.

The synthetic plasmid pfdC1 with the replication origin of phage fd and fd gene 2 grows autonomously in E. coli cells. DNA sequencing revealed several mutations compared to the fd genome causing reduced expression of viral gene 2 protein, which can be toxic for the host cell. Another adaptation was noticed for E. coli strains with a copy of fd gene 2 on the F-episome and a pfdA-plasmid with a minimal fd replication origin, when maintained at 42 degrees C. The carrier cells adjusted their cellular metabolism to these stress conditions, whereas replication functions of the plasmid or expression of fd gene 2 on the F-episome were not changed. The filamentous bacteriophages tend to reduce their genome size into miniphages, which was also observed for phages with an antibiotic resistance gene. Bacteriophages with a transposon insertion in the viral gene 2 had a tendency to restore the mutated gene by exchange with the functional gene 2 carried in recA-host cells. Mobilization of pfd-plasmids with RP4 transfer functions was reduced due to interference of replication and transfer in the rolling circle mode. The vectors used in these studies can also be applied as cloning vectors, which are compatible with many other plasmid vectors.

Adaptation, Physiological↗
PubMed1996

Differential response to UV stress and DNA damage during the yeast replicative life span.

The yeast Saccharomyces cerevisiae is mortal. Before they die, individual yeasts bud repeatedly producing a finite number of progeny, which have the capacity for a full life span. A feature of aging in many species is the waning of resistance to stress. To determine whether this is the case in yeast, we have examined the survival (viability) of age-synchronized populations of yeasts of various ages, spanning youth, midlife, and old age, after irradiation with ultraviolet light (UV). Resistance to UV was biphasic. There was an increase through midlife, followed by a precipitous decline. For comparison, another mutagenic agent, ethyl methanesulfonate (EMS), was tested in the same way. The response was very different. A uniphase decrease in resistance to this DNA-alkylating agent was found with a plateau later in life. The results argue that the increase in resistance to UV with age is an active process and not simply a monotonic age change. RAS2 is among the genes that determine yeast longevity. This gene is preferentially expressed in young cells and has a life span-extending effect on yeasts. One known function of RAS2 is to mount a protective response to irradiation by UV, which occurs independently of DNA damage. The distinction between UV and EMS found here is consistent with the notion that resistance to UV plays a role in yeast longevity in a manner not related to DNA damage. Furthermore, it suggests that RAS2 may participate in this response. We have found that RAS2 expression and UV resistance coincide in middle-aged yeasts bolstering this possibility. These data and the eclipse in activity of several longevity determining genes at midlife in yeasts also raise the possibility that active life maintenance processes function through this period, after which the organism operates on any remaining reserves until death.

DNA Damage↗
PubMed2006

Turning the replication checkpoint on and off.

The replication checkpoint monitors the progress of DNA replication forks during S phase, and delays the firing of later replication origins when active replication forks are stalled due to collisions with damaged or abnormally structured DNA. Key components of the replication checkpoint pathway are the apical protein kinase, ATR, and its downstream target kinase, Chk1. Defects in either ATR or Chk1 function result in loss of DNA replication fidelity and cell viability, even in the absence of extrinsic genotoxic stress. Moreover, several clinically important antitumor agents, such as the camptothecins (CPTs), exert their antitumor effects by interfering with DNA replication, and hence the therapeutic response to these drugs is intimately related to signaling through the replication checkpoint. A recent report from this laboratory adds a new facet to the regulatory mechanisms that control the function and duration of checkpoint signaling through the ATR-Chk1 pathway. The results indicate that replication stress induced by a variety of agents, including CPT and deep hypoxia, triggers the ubiquitin-dependent degradation of the checkpoint kinase Chk1 in both normal and transformed human cells. This review provides an overview of the study's major findings, together with their implications for both replication checkpoint function and tumor responsiveness to CPT and related anticancer drugs.

Checkpoint Kinase 1↗
PubMed2004

Diminished S-phase cyclin-dependent kinase function elicits vital Rad53-dependent checkpoint responses in Saccharomyces cerevisiae.

Cyclin-dependent kinase (CDK) is required for the initiation of chromosomal DNA replication in eukaryotes. In Saccharomyces cerevisiae, the Clb5 and Clb6 cyclins activate Cdk1 and drive replication origin firing. Deletion of CLB5 reduces initiation of DNA synthesis from late-firing origins. We have examined whether checkpoints are activated by loss of Clb5 function and whether checkpoints are responsible for the DNA replication defects associated with loss of Clb5 function. We present evidence for activation of Rad53 and Ddc2 functions with characteristics suggesting the presence of DNA damage. Deficient late origin firing in clb5Delta cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5Delta cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5Delta cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5Delta cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage.

CDC2 Protein Kinase↗
PubMed2000

Regulation of DNA replication during the cell cycle: roles of Cdc7 kinase and coupling of replication, recombination, and repair in response to replication fork arrest.

DNA replication is central to cell growth, development, and generation of tissues and organs. Recent advances in understanding replication machinery have revealed striking conservation of components involved in the processes of DNA replication, from yeasts to human. The conservation extends even to bacteria for some basic components of replication apparatus. Eukaryotic DNA replication is regulated at various stages to ensure strict regulation during cell cycle. We have identified a novel mammalian kinase, Cdc7-ASK (Activator of S phase Kinase), that plays a key role at the entry into S phase as a molecular switch for DNA replication. This kinase is specifically activated during S phase and triggers the firing of DNA replication by phosphorylating an essential DNA helicase component of the replication complex. Environmental stresses such as DNA damages or depletion of essential nutrients for DNA synthesis lead to unscheduled arrest of DNA replication forks. In bacteria, this leads to induction of altered modes of DNA replication, which may repair DNA damages, facilitate reassembly of replication machinery at the stalled replication fork, or do both. In eukaryotes, blocking replication forks usually induces both checkpoint responses, which prevent premature progression of cell cycle events before precise completion of the preceding cell cycle stage, and the recombinational repair system for the lesions. Possible common bases in recognition of stalled replication forks in bacteria and eukaryotes will be discussed. Furthermore, we will discuss the potential of replication and checkpoint proteins as targets of anticancer agents as well as possible novel technology for stem cell amplification through manipulation of DNA replication.

Animals↗
PubMed1984

Enzymology of DNA in replication in prokaryotes.

This review stresses recent developments in the in vitro study of DNA replication in prokaryotes. New insights into the enzymological mechanisms of initiation and elongation of leading and lagging strand DNA synthesis in ongoing studies are emphasized. Data from newly developed systems, such as those replicating oriC containing DNA or which are dependent on the lambda, O, and P proteins, are presented and the information compared to existing mechanisms. Evidence bearing on the coupling of DNA synthesis on both parental strands through protein-protein interactions and on the turnover of the elongation systems are analyzed. The structure of replication origins, and how their tertiary structure affects recognition and interaction with the various replication proteins is discussed.

Bacterial Proteins↗