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At least 307 records · Page 17Linked to original sources
PubMed2005

Germline genomic instability in PCNA mutants of Drosophila: DNA fingerprinting and microsatellite analysis.

PCNA participates in multiple processes of DNA metabolism with an essential role in DNA replication and intervening in DNA repair. Temperature-sensitive PCNA mutants of Drosophila (mus209) are sensitive to mutagens, impair developmental processes and suppress positional-effect variegation. To investigate the role of proliferating cell nuclear antigen (PCNA) in germline genomic stability, independent mus209-defective and mus209-normal lines were established and maintained over six generations. A time course study was carried out and general genomic alterations were analyzed in the progeny by using arbitrarily primed PCR (AP-PCR) and microsatellite analysis. The AP-PCR analysis has shown that a dysfunctional PCNA leads to germline genomic instability, being the amount of genomic alterations transmitted to the progeny directly related to the number of mus209B1 mutant alleles. In addition, we have found that the frequency of genomic alterations tends to increase over successive generations. Surprisingly, the highest microsatellite instability was found in the heterozygous mus209-defective lines, suggesting a greater mutation rate in these individuals, in comparison with the homozygous mus209-defective lines. In conclusion, our results clearly indicate that PCNA is an important factor to maintain genomic stability in germinal cells, both in the overall genome and in simple repeated sequences. The implication of PCNA mutations in transgenerational genomic instability and related to cancer susceptibility is also discussed.

Animals↗
PubMed1994

Stability of genomic DNA fragment patterns in methicillin resistant Staphylococcus aureus (MRSA) during the course of intra- and interhospital spread.

The analysis of genomic DNA fragment patterns has revealed as a powerful tool for strain discrimination in Staphylococcus aureus; for use as an epidemiological marker, stability during the course of an outbreak is an essential prerequisite. Genomic DNA fragment patterns (SmaI restriction, pulsed-field electrophoresis) of four different epidemic MRSA strains were compared along with intra- and interhospital and country-wide spread over more than 12 months in Germany. Strain I was isolated from infections in 8 hospitals. In one hospital a subclone arised which differed from the original strain by 4 fragments. Strain II was spread among 4 hospitals, isolates from three of these hospitals exhibited a variability of one to three fragments in the 150-200 kb range. Two hospitals in the Hannover-area were affected by strain III; in 17 isolates of this strain a variability up to three fragments was found in the 170-200 kb range. Strain IV was isolated from 19 cases of infections in 3 hospitals in Berlin. The fragment patterns were completely stable. When S. aureus strains are typed by genomic DNA fragment patterns, a variability in a definite range of molecular masses during the course of an epidemic should be taken into consideration.

Cross Infection↗
PubMed1999

The role of mismatch repair in DNA damage-induced apoptosis.

DNA mismatch repair plays a critical role in maintaining genomic integrity. Defects in human mismatch repair are the primary cause of certain types of cancer, including hereditary nonpolyposis colorectal cancer. In the past, the ability of mismatch repair proteins to correct DNA mismatches that occur during DNA replication, repair, and recombination was considered the primary mechanism by which it contributes to genomic stability. However, increasing evidence supports the idea that the mismatch repair system also contributes to genome stability by stimulating DNA damage-induced apoptosis as part of the cytotoxic response to physical and chemical agents. MutS/MutL homologues mediate the process of apoptosis by binding to DNA adducts and either provoking futile repair events or blocking steps in DNA metabolism (i.e., DNA replication and/or repair). This damage recognition step by mismatch repair (MMR) proteins stimulates a signaling cascade for apoptosis, resulting in activation of protein kinase(s) that phosphorylate p53 and/or the related protein p73. Activated p53 and p73 in turn transmit a signal to the apoptotic machinery to execute cell death. The goal of this commentary is to discuss the molecular mechanism(s) by which mismatch repair proteins stimulate apoptosis.

Adenosine Triphosphatases↗
PubMed2004

Sex- and tissue-specific expression of maintenance and de novo DNA methyltransferases upon low dose X-irradiation in mice.

DNA methylation is crucial for normal development, proliferation, and proper maintenance of genome stability for a given organism. A variety of DNA damaging agents that are known to affect genome stability were also shown to alter DNA methylation patterns. We have recently pioneered the studies in the area of the radiation effects on DNA methylation, and found that radiation exposure led to substantial dose-dependent and tissue-specific DNA hypomethylation, which was much more pronounced in spleen and liver of female animals. The exact mechanisms of radiation-induced DNA hypomethylation are still to be uncovered. We have previously shown that one of those mechanisms may potentially be DNA repair related. Another possible mechanism may be linked to changes in the expression of DNA methyltransferases (DNMTs). In the current study, we examined the radiation-induced changes in expression of maintenance DNMT1, and de novo methyltransferases DNMT3a and DNMT3b in spleen and liver of irradiated animals. This was paralleled by the studies of acute and chronic IR-induced methylation changes in spleen and liver of intact animals, as well as in animals with altered sex hormone status. Here we report that radiation-induced DNA methylation changes correlated with radiation-induced alterations in expression of DNA methyltransferases. We present the data on tissue-specificity in radiation-induced expression of DNA methyltransferases, and prove that changes in the expression of de novo methyltransferases DNMT3a and DNMT3b are the most important in radiation-induced DNA methylation alterations. We also discuss the role of sex hormones, especially estrogen, in the generation of the sex-specific radiation-induced methylation changes.

Animals↗
PubMed2003

ATM and related protein kinases: safeguarding genome integrity.

Maintenance of genome stability is essential for avoiding the passage to neoplasia. The DNA-damage response--a cornerstone of genome stability--occurs by a swift transduction of the DNA-damage signal to many cellular pathways. A prime example is the cellular response to DNA double-strand breaks, which activate the ATM protein kinase that, in turn, modulates numerous signalling pathways. ATM mutations lead to the cancer-predisposing genetic disorder ataxia-telangiectasia (A-T). Understanding ATM's mode of action provides new insights into the association between defective responses to DNA damage and cancer, and brings us closer to resolving the issue of cancer predisposition in some A-T carriers.

Animals↗
PubMed2026

Multivalent cations stabilize DNA duplexes beyond charge neutralization.

Multivalent cations are abundant in cells and play essential roles in DNA duplex stability, genome packaging, and DNA-protein interactions. They can also condense DNA, making it challenging to determine their influence on DNA duplex stability. To overcome this challenge, we studied DNA unpeeling at equilibrium under high tension using magnetic tweezers, thereby preventing condensation. Experiments show that DNA duplex stability first increases and then decreases as cation concentration increases and the maximum DNA duplex stability increases with cation valence. The maximum free energy change of DNA was 3.33 k B T/bp for Na+ and increased to 3.98 k B T/bp for protamine, which is a small arginine-rich protein with a highly positive charge (≈21 for salmon sperm), corresponding to a relative increase of 19.5%. Consistently, all-atom molecular dynamics simulations show that higher-valent cations preferentially embed in the minor groove of DNA and clamp the minor groove, in contrast to the major-groove clamping reported for RNA, thereby stabilizing the helix more efficiently. These findings establish a single-molecule framework for quantifying DNA thermodynamics in complex ionic environments, which contributes to understanding ionic control of genome stability and to designing ion-tunable DNA-based nanostructures and delivery systems.

Journal Article↗
PubMed2003

siRNA depletion of BRCA1, but not BRCA2, causes increased genome instability in Fanconi anemia cells.

BRCA1 and BRCA2 proteins act in repair of interstrand crosslinks (ICLs) and maintenance of genome stability and are known to be part of the Fanconi anemia (FA) pathway. We have investigated the role of the BRCA1 and BRCA2 genes in genome stability following ICL damage in normal and FA cells. To circumvent cell lethality of complete disruptions in BRCA1 or BRCA2, small inhibitory RNA (siRNA) was used to transiently deplete the expression of the proteins. Using chromosomal stability after ICL damage as the end point, we find that BRCA1 functions in more than just the FA pathway for genome maintenance, whereas BRCA2 appears to act predominantly in the FA pathway. Depletion of BRCA1 causes a marked decrease, although not a complete absence of, ubiquitination of FANCD2. In contrast to BRCA1, BRCA2 is not needed for normal ubiquitination of FANCD2 after DNA damage, a requirement for the FA pathway to function. Thus, BRCA2 is epistatic to FA genes for ICL repair, but not for damage-induced modification of FANCD2 and may act downstream form FANCD2.

BRCA1 Protein↗
PubMed2006

Association between the MLH1 gene and longevity.

Perturbations in genomic stability result in cancer, a reduced life span, and premature aging. MLH1 is a mismatch repair enzyme that acts to maintain genomic stability, and a loss of MLH1 increases cancer incidence and apoptosis resistance, which suggests a link between MLH1 and longevity. We found here that MLH1 is associated with longevity by comparing a centenarian group with a control group. Our data indicate a critical role for MLH1 in longevity.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Pharmacological manipulation of ataxia-telangiectasia kinase activity as a treatment for Parkinson's disease.

Parkinson's disease (PD) is a major cause of morbidity and mortality among older individuals. Although the causes of Parkinson's disease are multifactorial, considerable evidence indicates that elevated labile iron in the substantia nigra pars compacta plays an important role in producing oxyradicals which subsequently damage nigro-striatal neurons. Based on this several researchers have suggested that blood-brain barrier crossing iron chelators might have clinical efficacy in treating PD. Work demonstrating that iron chelators protect nigro-striatal neurons in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine-induced rodent PD models supports this hypothesis. Recently, we found that the ATM gene product (mutated in ataxia-telangiectasia, A-T), is required for cell survival and genomic stability maintenance following exposure to low labile iron concentrations. Iron chelators (desferal, quercetin, and apoferritin) also increase A-T cell genomic stability and viability, and activate ATM-dependent cellular events in normal cells. Additionally Atm-deficient mice exhibit a selective loss of dopaminergic nigro-striatal neurons. Based on this, we propose that iron chelators protect the substantia nigra pars compacta not only by chelating labile iron and reducing oxyradical formation, but also by inducing ATM activity, leading to increased oxidative stress resistance and DNA repair. Support for this hypothesis comes from the recent observation that the iron chelating flavonoid quercetin both directly activates ATM and protects neuronal cells from the toxic effects of the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Therefore since; (1) ATM is required for iron toxicity resistance, (2) iron chelators such as quercetin, desferal, and apoferritin induce ATM activity and/or ATM-dependent events, and (3), Atm-deficient mice preferentially lose dopaminergic nigro-striatal neurons, we propose that ATM activity has an important function in PD. Furthermore, pharmacological manipulation of ATM activity via iron chelation might have clinical efficacy in PD treatment.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2005

The fission yeast Crb2/Chk1 pathway coordinates the DNA damage and spindle checkpoint in response to replication stress induced by topoisomerase I inhibitor.

Living organisms experience constant threats that challenge their genome stability. The DNA damage checkpoint pathway coordinates cell cycle progression with DNA repair when DNA is damaged, thus ensuring faithful transmission of the genome. The spindle assembly checkpoint inhibits chromosome segregation until all chromosomes are properly attached to the spindle, ensuring accurate partition of the genetic material. Both the DNA damage and spindle checkpoint pathways participate in genome integrity. However, no clear connection between these two pathways has been described. Here, we analyze mutants in the BRCT domains of fission yeast Crb2, which mediates Chk1 activation, and provide evidence for a novel function of the Chk1 pathway. When the Crb2 mutants experience damaged replication forks upon inhibition of the religation activity of topoisomerase I, the Chk1 DNA damage pathway induces sustained activation of the spindle checkpoint, which in turn delays metaphase-to-anaphase transition in a Mad2-dependent fashion. This new pathway enhances cell survival and genome stability when cells undergo replicative stress in the absence of a proficient G(2)/M DNA damage checkpoint.

Alleles↗
PubMed1998

Investigating the biological functions of DNA topoisomerases in eukaryotic cells.

DNA topoisomerases participate in nearly all events relating to DNA metabolism including replication, transcription, and chromosome segregation. Recent studies in eukaryotic cells have led to the discovery of several novel topoisomerases, and to new questions concerning the roles of these enzymes in cellular processes. Gene knockout studies are helping to delineate the roles of topoisomerases in mammalian cells, just as similar studies in yeast established paradigms concerning the functions of topoisomerases in lower eukaryotes. The application of new technologies for identifying interacting proteins has connected the studies on topoisomerases to other areas of human biology including genome stability and aging. These studies highlight the importance of understanding how topoisomerases participate in the normal processes of transcription, DNA replication, and genome stability.

Animals↗
PubMed2004

p53's double life: transactivation-independent repression of homologous recombination.

The tumor suppressor protein p53 controls cell cycle checkpoints and apoptosis via the transactivation of several genes. However, data from various laboratories suggest an additional role for p53: transcription-independent suppression of homologous recombination (HR). Genetic and physical interactions among p53, HR proteins (e.g. RAD51 and RAD54) and HR-DNA intermediates show that p53 acts directly on HR during the early and late steps of recombination. Complementary to the MSH2 mismatch-repair system, p53 appears to impair excess HR by controlling the minimal efficiency processing segment and by reversing recombination intermediates. By controlling the balance between the BLM and the RAD51 pathways, this direct role of p53 could maintain genome stability when replication forks are stalled at regions of DNA damage. In this article, we discuss the direct role of p53 on HR and the consequences for genome stability, tumor protection and speciation.

Animals↗
PubMed2005

Chromokinesins: multitalented players in mitosis.

Molecular motors generate cellular forces and act in a multitude of intracellular transport processes. The chromokinesins are a subgroup of kinesin motors. Chromokinesins act in various steps of mitosis, including chromosome condensation, metaphase alignment, chromosome segregation, cytokinesis and they help maintain genome stability. The emerging multifunctional nature of the chromokinesins provides insights into the coordination of distinct mitotic steps, and their role in maintenance of genome stability makes them attractive potential targets for therapeutic intervention.

Animals↗
PubMedDate not supplied

The tumor suppressor p53 can reduce stable transfection in the presence of irradiation.

The tumor suppressor p53 is believed to play an essential role in maintaining genome stability. Although it is currently unknown how p53 is involved in this important biological safeguard, several previous publications indicate that p53 can help to maintain genome integrity through the recombination-mediated DNA repair process. The integration of linearized plasmid DNA into the host chromosome utilizes the same repair process, and the frequency can be measured by clonogenic assays in which cells that were stably transfected by plasmid integration can be scored by their colony-forming abilities. To gain insight into whether p53 has a direct role in plasmid integration into the host chromosome, we determined the frequency of stable transfection with CHO cells expressing either wild-type or mutant p53 in the presence and absence of irradiation. We found that low-dose irradiation ( approximately 50 to 100 cGy) increased stable transfection frequencies in CHO cells regardless of their p53 status. However, the increase of transfection frequency was significantly lower in CHO cells expressing wild-type p53. Our data thus suggest that wild-type p53 can suppress plasmid DNA integration into the host genome. This p53 function may play a direct and significant role in maintaining genome stability.

Animals↗
PubMed2004

DNA stability and serum selenium levels in a high-risk group for prostate cancer.

The essential micronutrient, selenium, is at low levels in the New Zealand diet. Selenium is a component of a number of proteins involved in the maintenance of genomic stability, and recommended daily allowances (RDA) are set on saturation levels for glutathione peroxidase (GPx), a key enzyme in surveillance against oxidative stress. It has been assumed but not proven that this level will be adequate for other key selenoenzymes. The "Negative Biopsy Trial" identifies a group of New Zealand individuals at high risk of prostate cancer, whose serum selenium levels will be monitored and who will be supplemented with a yeast-based tablet, with or without selenium, over an extended time. Access to patients on this trial provides the opportunity to ask the more generic question as to whether selenium levels in this population are adequate to maintain genomic stability. The single cell gel electrophoresis (comet) assay was used to study DNA damage in blood leukocytes harvested from these volunteers. Average serum selenium levels before randomization was 97.8 +/- 16.6 ng/ml, low by international standards. For the half of the population below this mean value, lower serum selenium levels showed a statistically significant inverse relationship (P = 0.02) with overall accumulated DNA damage. Although other interpretations cannot be excluded, the data suggest that the selenium intake in half of this population is marginal for adequate repair of DNA damage, increasing susceptibility to cancer and other degenerative diseases. It also raises the question as to whether glutathione peroxidase saturation levels are appropriate indicators of the optimal selenium levels for a given population.

Aged↗
PubMed2003

Paradoxical role of apoptosis in tumor progression.

Tumors frequently acquire resistance to apoptosis that is expected to contribute to malignant phenotype and reduce sensitivity to treatment. In fact, inactivation of p53 tumor suppressor gene resulting in suppression of apoptosis serves as a negative prognostic marker. Surprisingly, expression of a strong anti-apoptotic protein Bcl-2, another mechanism to avoid apoptosis, was found to be associated with a favorable prognosis. This paradoxical anti-progressor function of Bcl-2 has been explained in literature based on the negative effect of Bcl-2 on cell proliferation. Here, by analyzing accumulated experimental and clinical data, we provide evidence supporting another hypothesis that defines apoptosis as an accelerator of tumor progression. The mechanism of anti-progressor function of Bcl-2 is based on creation of tumors that maintain control of genomic stability by eliminating selective advantages for the cells that acquire resistance to apoptosis through loss of p53. Thus, inhibition of apoptosis does not lead to loss of genomic stability and creates tumor environment that no longer supports further tumor progression and inhibitors of apoptosis can be considered as factors suppressing tumor progression.

Animals↗
PubMed1995

p53 mediated tumor cell response to chemotherapeutic DNA damage: a preliminary study in matched pairs of breast cancer biopsies.

Wild type p53 plays a crucial role in maintaining genomic stability in both normal and tumor cells in vitro. When DNA damage occurs, p53 acts as a cell cycle checkpoint and induces a cellular response that aims at restoring genomic integrity. p53 may either allow the repair of damaged DNA by inducing a transient G1 arrest or may eliminate the damaged cells by triggering apoptosis. Mutant p53 fails to mediate any of these effects. From this, a p53 status-dependent response to therapy might be expected when tumors are treated with DNA-damaging genotoxic agents: Although wild type p53-harboring tumors have an intact checkpoint that might allow them to restore genomic integrity back to a pre-exposure level, mutant p53 tumors have a corrupted checkpoint that could lead to an accelerated loss of genomic stability. Until now, no studies have been described that examine such a p53-mediated effect in vivo. The authors tested this response model in vivo comparing 32 matched biopsy pairs from patients with breast cancer before and after rigorously standardized polychemotherapy. Four of the five drugs specifically induce a wild type p53-mediated checkpoint response. Tumor tissue from matched pairs of untreated and treated biopsies of the same patient were analyzed for treatment-associated changes of p53 protein expression by immunocytochemistry and, in a few available specimens, of p53 genotype changes by polymerase chain reaction-based DNA analysis. Treatment-associated changes of the p53 immunophenotype, which the authors speculate to reflect clonal selection, occurred in 39% (12 of 31) of the specimens. One specimen was not informative. Most tumors undergoing clonal selection originally harbored mutant p53 (nine of 12), and only three of 12 tumors were wild type. This study shows that exposure to genotoxic agents is commonly associated with a change in p53 immunophenotype. Although the limited material in this cohort prevented direct analysis of genetic instability, these results suggest that tumors with altered p53 may be genomically less stable and, therefore, may be more likely to undergo treatment-induced clonal changes than wild type tumors. This study also shows that the rigorous matched sample approach, although difficult to obtain, is an important tool that allows the in vivo assessment of the tumor response to genotoxic therapy in a controlled fashion.

Adult↗
PubMed2000

Laryngeal mucosa of head and neck cancer patients shows increased DNA damage as detected by single cell microgel electrophoresis.

Major risk factors for cancer of the oral cavity, pharynx and larynx are smoking and excess alcohol consumption. Since long-term survival rates of head and neck cancer patients have not substantially been improved, new preventive strategies including the use of cancer chemopreventive agents have to be developed. With the aim of developing biomarkers which can verify the efficacy of chemopreventive interventions, a standardised alkaline microgel electrophoresis (MGE) assay was applied as a sensitive and rapid tool to detect DNA damage on a single cell level. Macroscopically normal laryngeal mucosa biopsies obtained by surgery from head and neck cancer patients (n=29) and from hospital controls (n=22) were analysed by MGE in a pilot study. As compared to controls, cells from head and neck cancer patients showed a significantly elevated DNA damage without any further genotoxic treatment (P<0.01). We conclude that this increased background DNA damage in laryngeal epithelia could result from genetic alterations caused by smoking and alcohol leading, in accord with the field cancerisation hypothesis, to a gradual decrease of genomic stability and malignancy. MGE should now be explored as a rapid screening method in larger clinical studies: (i) to identify high-risk subjects carrying cells with decreased genomic stability and (ii) to verify the efficacy of chemopreventive regimens to prevent or slow down the development of head and neck cancer in high-risk persons.

Adult↗