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PubMed1987

Inhibition of simian virus 40 DNA replication in vitro by poly(ADP-ribosyl)ated diadenosine tetraphosphate.

Poly(ADP-ribosyl)ated diadenosine tetraphosphate was found to inhibit the in vitro replication of SV40 DNA. This inhibition was sensitive to preincubation of the polymer with either poly(ADP-ribose) glycohydrolase, diadenosine tetraphosphate (Ap4A):ADP phosphohydrolase, or an excess of free Ap4A. In contrast, the general catalytic activity of DNA polymerase was not inhibited by the poly(ADP-ribosyl)ated Ap4A when activated salmon sperm DNA was used as a template. These data suggest that inhibition of SV40 DNA replication by poly(ADP-ribosyl)ated Ap4A requires both the intact polymer and intact Ap4A moiety and is specific to events occurring during the initiation or elongation of a double-stranded template. Since both poly(ADP-ribose) and Ap4A accumulate in cultured mammalian cells following stresses which are accompanied by DNA strand breaks, these data are consistent with a model in which poly(ADP-ribosyl)ated Ap4A inhibits DNA replication following DNA damage.

Adenine Nucleotides↗
PubMed2006

Protein expression dynamics during replicative senescence of endothelial cells studied by 2-D difference in-gel electrophoresis.

Endothelial senescence contributes to endothelium dysfunctionality and is thereby linked to vascular aging. A dynamic proteomic study on human umbilical vein endothelial cells, isolated from three umbilical cords, was performed. The cells were cultured towards replicative senescence and whole cell lysates were subjected to 2-D difference gel electrophoresis (DIGE). Despite the biological variability of the three independent isolations, a set of proteins was found that showed senescence-dependent expression patterns in all isolations. We focused on those proteins that showed significant changes, with a paired analysis of variance (RM-ANOVA) p-value of < or =0.05. Thirty-five proteins were identified with LC-Fourier transform MS, and functional annotation revealed that endothelial replicative senescence is accompanied by increased cellular stress, protein biosynthesis and reduction in DNA repair and maintenance. Nuclear integrity becomes affected and cytoskeletal structure is also changed. Such important changes in the cell infrastructure might accelerate endothelium dysfunctionality. This study provides biological information that will initiate studies to further unravel endothelial senescence and gain more knowledge about the consequences of this process in the in vivo situation.

Cell Proliferation↗
PubMed1992

The effect of age on the response of the detrusor to intracellular mechanical stimulus: DNA replication and the cell actin matrix.

Benign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myocyte ultrastructure and consequent effects these might have in modifying the response of the detrusor to mechanical stimulus, we studied differences in dynamic nuclear-cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which specifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2-3 week) and old (8-12 mon) guinea pigs. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracellular stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti-alpha-smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor myocytes showed dose dependent disruption of the actin component of the cytoskeleton. These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the detrusor in response to outflow obstruction in infancy and adulthood. Furthermore, they suggest that 1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; 2) there appears to be a decrease in myocyte nuclear elasticity with ageing; 3) release of nuclear template restrictions increases activity of DNA polymerase alpha in young, but not old, detrusor myocytes; and 4) mechanico-chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that 1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and 2) integrity of nuclear actin may function to maintain the spatial template restriction on DNA polymerase alpha activity.

Actins↗
PubMed2001

Oxidative biochemical markers; clues to understanding aging in long-lived species.

Clues as to why long-lived species live so much longer than short-lived species may reside in the amount of reactive oxygen species (ROS) produced and their effect on damaging cell components (especially proteins) and alterations of crucial cellular processes. Rigorous evaluation of these concepts required critical comparisons of oxidative damage markers and/or parameters with assess difference in ROS flux and the critical age-modifying processes they influence. The limited experimental comparative results available implicate that ROS production per unit weight of total oxygen consumed is much less in the longer-lived species than in shorter-lived species. Mitochondria are the major site of ROS production. They are also the functional nexus for intracellular signaling thus modulating stress and growth factor mediated cellular survival, proliferation and apoptotic processes. Mitochondrial DNA mutations, perhaps caused by ROS, increase with age. Mutant mitochondria possess comparative replicative advantage, which leads to age-specific intracellular swarms. General inflammatory stress tends to increase with age. Disruption in coordinated cell-to-cell signaling triggered by alterations in intracellular signaling may be the basis of the age-related increases in tissue inflammation, which may explain some of the differences between long-lived species and short-lived species.

Aging↗
PubMed1996

The Clp ATPases define a novel class of molecular chaperones.

The Clp ATPases were originally identified as a regulatory component of the bacterial ATP-dependent Clp serine proteases. Proteins homologous to the Escherichia coli Clp ATPases (ClpA, B, X or Y) have been identified in every organism examined so far. Recent data suggest that the Clp ATPases are not only specificity factors which help to 'present' various protein substrates to the ClpP or other catalytic proteases, but are also molecular chaperones which can function independently of ClpP. This review discusses the recent evidence that the Clp ATPases are indeed molecular chaperones capable of either repairing proteins damaged during stress conditions or activating the initiation proteins for Mu, lambda or P1 DNA replication. A mechanism is suggested to explain how the Clp ATPases 'decide' whether to repair or destroy their protein substrates.

Adenosine Triphosphatases↗
PubMed2005

Frag1, a homolog of alternative replication factor C subunits, links replication stress surveillance with apoptosis.

We report the identification and characterization of a potent regulator of genomic integrity, mouse and human FRAG1 gene, a conserved homolog of replication factor C large subunit that is homologous to the alternative replication factor C subunits Elg1, Ctf18/Chl12, and Rad24 of budding yeast. FRAG1 was identified in a search for key caretaker genes involved in the regulation of genomic stability under conditions of replicative stress. In response to stress, Atr participates in the down-regulation of FRAG1 expression, leading to the induction of apoptosis through the release of Rad9 from damaged chromatin during the S phase of the cell cycle, allowing Rad9-Bcl2 association and induction of proapoptotic Bax protein. We propose that the Frag1 signal pathway, by linking replication stress surveillance with apoptosis induction, plays a central role in determining whether DNA damage is compatible with cell survival or whether it requires cell elimination by apoptosis.

Amino Acid Sequence↗
PubMed2005

Recent progress in spinocerebellar ataxia type-10 (SCA10).

Spinocerebellar ataxia type 10 (SCA10) is a dominantly inherited ataxia caused by expansion of ATTCT pentanucleotide repeat in intron 9 of a novel gene, E46L, on chromosome 22q13.3. SCA10 is a complex neurodegenerative condition. Initial studies characterized SCA10 as pure cerebellar ataxia associated with seizures. Recent identification of new SCA10 families revealed more diverse phenotypes, including polyneuropathy, pyramidal signs, cognitive and neuropsychiatric impairment. Moreover, several families manifest with ataxia without seizures. Thus a complete clinical spectrum is emerging. Progress has also been made in understanding the molecular and genetic mechanisms of pathogenesis. The length of expanded ATTCT repeats is variable in different tissues and highly unstable during paternal transmission, revealing complex genetic and pathogenetic processes. Under torsional stress, ATTCT repeats form unpaired DNA structure and may serve as an erroneous DNA replication origin, potentially contributing to repeat instability and aberrant cell cycle entry. E46L is a cytoplasmic protein with unknown function. Reduced expression of E46L in primary neuronal cultures from cerebellum and cortex by small interfering RNAs (siRNAs) caused increased apoptosis, raising the possibility that reduced expression of E46L might also play an important role in SCA10 pathogenesis.

Animals↗
PubMed1999

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling.

Animals↗
PubMed1995

MDM2 transformation in the absence of p53 and abrogation of the p107 G1 cell-cycle arrest.

The p53 tumour-suppressor guards the genome in response to genotoxic stress by transcriptional regulation of genes involved in cell-cycle control, DNA replication, repair and apoptosis such as p21, GADD45, bax and mdm2 (Cox and Lane, 1995). Mdm2 is classically considered to be an inhibitor of p53, that forms an auto-regulatory loop (Momand et al., 1992; Oliner et al., 1993; Wu et al., 1993; Chen et al., 1994; Chen and Levine, 1995). It immortalises cells containing wild type p53 and transforms them together with Ras (Finlay, 1993). We show that, in the absence of p53, mdm2 confers a growth advantage to cells (i.e. "transforms" them) and can overcome a G1 cell-cycl arrest induced by p107, a member of the pRb tumour-suppressor family (Adams and Kaelin, 1995). The minimum "transforming" and p107 inhibiting region of Mdm2 corresponds to its p53 binding domain. p53 inhibits transformation by Mdm2, apparently without requiring transcription. p53 can be considered to be a suppressor of Mdm2, a positive effector of the cell cycle. Mdm2 over-expression in tumours is reminiscent of p53 mutations with gain of function, in that Mdm2 both transforms cells and inhibits p53 activity.

Cell Adhesion↗
PubMed1999

[Replicative senescence as a model of aging: the role of oxidative stress and telomere shortening--an overview].

Replicative senescence is characterized by the irreversible loss of division potential of cultivated human and animal cells. Correlations between the replicative potential in vitro and the age of the donor or the maximal lifespan of the species suggest replicative senescence to be an appropriate model for aging. Telomeres of human somatic cells shorten with each cell division but are stabilized at constant length in tumors and immortal cells by the enzyme telomerase. The assumption of a causal role of telomere shortening for the limited lifespan of cells in vitro was borne out recently. We could demonstrate oxidative stress as a main reason for telomere shortening. Telomeres are sensors for oxidative damage in the genome. Telomeres shorten during in vivo aging as well; however, there are significant differences between individuals. Telomere erosion might play a major role for the aging of the immune system. Our data suggest that telomere shortening in vivo could reflect the cumulative amount of oxidative damage to the organism. It might be useful as a biomarker of aging.

Animals↗
PubMed2006

Exposure to 5-bromo-2'-deoxyuridine induces oxidative stress and activator protein-1 DNA binding activity in the embryo.

BACKGROUND: During organogenesis the embryo is highly sensitive to oxidative stress. We hypothesize that oxidative stress and activation of a redox-sensitive transcription factor, activator protein-1 (AP-1), are early indicators of embryonic stress in response to a teratogenic insult. 5-Bromo-2'-deoxyuridine (BrdU) was chosen as a model teratogen to test this hypothesis; BrdU is a thymidine analog that is incorporated into replicating DNA. METHODS: Timed pregnant CD1 mice were given vehicle or BrdU (400, 600, 800, or 1000 mg of BrdU/kg of body weight) on gestation day 9 (GD 9). Oxidative stress, assessed as the ratio of glutathione disulfide (GSSG) to reduced glutathione (GSH), and AP-1 DNA binding activity (c-Fos- and c-Jun-dependent DNA binding) were measured in the maternal livers and embryos 0.5, 3, and 6 hr after treatment. External and skeletal malformations were assessed on GD 18. N-acetylcysteine, a glutathione precursor, was coadministered with BrdU to further explore the relationship between teratogenicity and redox homeostasis. RESULTS: BrdU exposure produced a dose-dependent increase in skeletal malformations, which included polydactyly, and delayed ossification of the sternebrae and vertebrae. Exposure to teratogenic doses of BrdU depleted GSH concentrations and increased oxidative stress, as assessed by the GSSG:GSH ratio, in both maternal livers and embryos. While c-Jun DNA binding activity in embryos was not affected, c-Fos DNA binding activity was elevated significantly 3 hr after BrdU exposure. Coadministration of N-acetylcysteine decreased the skeletal malformations and AP-1 DNA binding activity induced by BrdU. CONCLUSIONS: BrdU exposure induced an embryonic stress response manifested as an increase in oxidative stress and AP-1 DNA binding activity; these data support the hypothesis that disturbances in redox homeostasis mediate the response of the conceptus to a teratogenic insult.

Abnormalities, Drug-Induced↗
PubMed2001

Integrating stress-response and cell-cycle checkpoint pathways.

The DNA integrity checkpoint and stress kinase (SAPK/JNK and p38) pathways function to modulate cell-cycle, apoptotic and transcriptional responses to stress. Although initially considered to function independently, recent advances indicate a number of links between the stress-response and checkpoint pathways. Here, we consider the relationship between the stress-response and checkpoint pathways and how they interact to modulate cell-cycle control.

Animals↗
PubMed2003

Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner.

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress-activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in the nucleus during re-entry and progression through S phase. These results are consistent with a role of kin17 protein in DNA replication. About 50% of the total amount of kin17 protein was detected on nuclear structures and could not be released by detergents. Furthermore, the amount of kin17 protein greatly increased in both G(1)/S and S phase-arrested cells in fractions containing proteins anchored to nuclear structures. The detection of kin17 protein showed for the first time its preferential assembly within non-chromatin nuclear structures in G(1)/S and S phase-arrested cells, while the association with these structures was found to be less stable in the G(2)/M phase, as judged by fractionation of human cells and immunostaining. In asynchronous growing cells, kin17 protein interacted with both chromatin DNA and non-chromatin nuclear structures, while in S phase-arrested cells it interacted mostly with non-chromatin nuclear structures, as judged by DNase I treatment and in vivo UV cross-linking. In the presence of DNA damage in S phase cells, the distribution of kin17 protein became mainly associated with chromosomal DNA, as judged by limited formaldehyde cross-linking of living cells. The physical interaction of kin17 protein with components of the nuclear matrix was confirmed and visualized by indirect immunofluorescence and immunoelectron microscopy. Our results indicate that, during S phase, a fraction of the human kin17 protein preferentially associates with the nuclear matrix, a fundamentally non-chromatin higher order nuclear structure, and to chromatin DNA in the presence of DNA damage.

Cell Cycle↗
PubMed1999

Bulky endogenous DNA modifications (I-compounds) -possible structural origins and functional implications.

I-compounds are bulky covalent DNA modifications which increase with age in tissues of unexposed laboratory animals and are derived from endogenous DNA-reactive intermediates of nutrient and oxygen metabolism. They have been classified into 2 major groups, i.e., type I and type II. Profiles and levels of type I I-compounds show considerable variation depending on species, strain, tissue, and gender, but are also affected by diet and chemical and hormonal exposures, indicating their formation to be determined by genetic and environmental factors. For example, sex hormones, dietary oat lipids, and isoprenoids affect their profiles and/or levels in tissue DNA. A gradual depletion of many type I I-compounds occurs during carcinogenesis, as many carcinogens/tumor promoters significantly reduce their levels, and neoplasms display very low levels, apparently independent of growth rate, indicating a loss of the ability to form these modified nucleotides. Conversely, dietary restriction, the most effective method to retard carcinogenesis and aging, significantly elevates type I I-compound levels, as compared to age-matched ad libitum-fed animals. Levels of many liver and kidney I-compounds exhibit genotype- and diet-dependent positive linear correlations with median life span. Formation of high levels of oat-related type I I-compounds has been associated with reduced formation of carcinogen-induced preneoplastic hepatic foci. These results suggest that such DNA modifications may not represent DNA lesions but may rather be functionally important. This view is supported by circadian rhythms displayed by some I-compounds. Thus, certain type I I-compounds may play a protective role against carcinogenesis and age-associated degenerative processes. Type II I-compounds, on the other hand, represent DNA damage and include several bulky lesions, which are enhanced by pro-oxidant carcinogens such as ferric nitrilotri- acetate (Fe-NTA) in target organ (kidney) DNA of rodents and are identical to products generated by oxidizing DNA or oligonucleotides under Fenton reaction conditions in vitro. Some of these products appear to be base-base or base-sugar intrastrand crosslinks. Notably, Fe-NTA reduces the levels of type I I-compounds in renal DNA. Type II I-compound levels are increased in tissue DNA of normal newborn rats. The formation of oxidative DNA lesions in neonates is most likely caused by oxidative stress associated with the sudden increase of partial oxygen pressure in arterial blood and tissues at birth. In view of the rapid cell replication at this developmental stage, endogenous oxidative DNA lesions sustained early in life may contribute to the development of cancer and degenerative diseases later in life.

Animals↗
PubMed1996

[Evolution modes of eukaryote retrotransposons].

A number of general problems of molecular macroevolution of retroposons were examined, including the question of the ratio of the contributions of genomic replication and retrotransposition to mutational variability of retrotransposons, as well as that of the influence of stress-induced transpositional variability on the rate of evolution and the phylogenetic trees of retrotransposons. It is thought that the substitution fixation rate in genes of retrotransposons is determined by the transposition rate and the probability of mutation upon replication of copies of retransposons in the genome and upon retrotransposition, as well as by selection stabilizing the function of proteins of the retroreplicative mechanism. By means of molecular-evolutionary parameters, estimated for Drosophila retrotransposons and animal retroviruses, it was shown that spontaneous retrotransposition makes the dominant contribution to the frequency of mutation, and the expected fixation rate of nucleotide substations is on the order of 2 x 10(-8) per position per year. This version of evolution of retrotransposons agrees with the results of phylogenetic analysis of trees of macroevolution. Another version, associated with stress-induced transpositions, gives a very high rate of evolution, on the order 3 x 10(-6) fixations of nucleotide substitutions per position per year. This version seems improbable, as it leads to a significant hidden genetic load.

Animals↗
PubMedDate not supplied

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells.

Thymidylate synthase-negative mutant mouse cells starved of thymidine or their parental FM3A cells treated with 5-fluoro-2'-deoxyuridine produced DNA fragments ranging from 50 to 200 kilobase pairs with a peak at 100 kb in length as determined by pulsed-field agarose gel electrophoresis. Accumulation of the DNA fragments following such thymidylate stress was time-dependent but their size distribution did not change in either case. Regions of the chromosomal DNA breaks seemed to be restricted to those where DNA replication was in progress as shown by pulse-labeling of the DNA synthesis. Emetine, an inhibitor of protein synthesis, blocked the accumulation of the DNA fragments when present during thymidylate stress. Cell-free extracts prepared from the thymidylate-stressed cells derived by either of the above means were capable of degrading DNA in chromatins prepared from normally growing cells in vitro. The resulting DNA fragments were similar but with a somewhat broader size distribution compared to those produced in vivo. The broader distribution of the fragments produced in the in vitro reaction became closer to the pattern obtained in vivo when ATP and 4 deoxyribonucleotides were added to the reaction.

Adenosine Triphosphate↗
PubMed2006

Cotransport of the heterodimeric small subunit of the Saccharomyces cerevisiae ribonucleotide reductase between the nucleus and the cytoplasm.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis and is essential in DNA replication and repair. Cells have evolved complex mechanisms to modulate RNR activity during normal cell cycle progression and in response to genotoxic stress. A recently characterized mode of RNR regulation is DNA damage-induced RNR subunit redistribution. The RNR holoenzyme consists of a large subunit, R1, and a small subunit, R2. The Saccharomyces cerevisiae R2 is an Rnr2:Rnr4 heterodimer. Rnr2 generates a diferric-tyrosyl radical cofactor required for catalysis; Rnr4 facilitates cofactor assembly and stabilizes the resulting holo-heterodimer. Upon DNA damage, Rnr2 and Rnr4 undergo checkpoint-dependent, nucleus-to-cytoplasm redistribution, resulting in colocalization of R1 and R2. Here we present evidence that Rnr2 and Rnr4 are transported between the nucleus and the cytoplasm as one protein complex. Tagging either Rnr2 or Rnr4 with a nuclear export sequence causes cytoplasmic localization of both proteins. Moreover, mutations at the Rnr2:Rnr4 heterodimer interface can affect the localization of both proteins without disrupting the heterodimeric complex. Finally, the relocalization of Rnr4 appears to involve both active export and blockage of nuclear import. Our findings provide new insights into the mechanism of DNA damage-induced RNR subunit redistribution.

Biological Transport, Active↗