Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

OpenAIRE

Miyaji, E.N.; Johnson, R.T.; Downes, C.S.; Eveno, E.; Mezzina, M.; Sarasin, A.; Menck, C.F.M.

2000-01-01

Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2) that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, po...

Cell cycle checkpoints: reversible when possible, irreversible when needed

NARCIS (Netherlands)

Krenning, L.

2015-01-01

Cell cycle checkpoints are reversible in nature, and can prevent progression into the next cell cycle phase if needed. In the case of DNA damage, cells can prevent progression from G1 into S phase, and from G2 into mitosis in the presence of DNA double strand breaks. Following DNA repair, these

DNA repair , cell repair and radiosensitivity

International Nuclear Information System (INIS)

Zhestyanikov, V.D.

1983-01-01

Data obtained in laboratory of radiation cytology and literature data testifying to a considerable role of DNA repair in cell sensitivity to radiation and chemical DNA-tropic agents have been considered. Data pointing to the probability of contribution of inducible repair of DNA into plant cells sensitivity to X-rays are obtained. Certain violations of DNA repair do not result in the increase of radiosensitivity. It is assumed that in the cases unknown mechanisms of DNA repair operate

The repair of damage to DNA in different cell types

International Nuclear Information System (INIS)

Karran, P.

1974-01-01

DNA single strand breaks induced by either X-ray irradiation or by methyl methanesulphonate (MMS) were studied in different lymphoid cell populations directly taken from the animal and maintained in tissue culture merely for the duration of the experiment. The results obtained from these cell populations were compared with those obtained with L5178Y cells maintained in tissue culture. All cell types studied were found to possess at least one class of enzymes required for repair of DNA damage, namely those enzymes involved in the rejoining of X-ray induced by MMS is different in each cell type. Repair replication was at much reduced levels and the endonucleolytic degradation was at much reduced levels and the endonucleolytic degradation was initiated at lower MMS concentration in the lymphoid cells as compared to L5178Y cells. It is suggested that the overall ''repair capacity'' of a population may be related to the number of cells in a cycle which, moreover, might be the only ones to have the ability to repair damage to DNA induced by MMS (G.G.)

Cell-cycle-dependent repair of heavy-ion damage

International Nuclear Information System (INIS)

Blakely, E.A.; Chang, P.Y.; Lommel, L.; Tobias, C.A.

1985-01-01

Synchronized human T-1 cells have been used to investigate the G1-phase age dependence of repair of potentially lethal damage (PLDR). The cells were irradiated with single doses of either 225 kVp X rays or Bragg-peak 425 MeV/μ neon ions at ages between 1.5 and 6.0 hrs after mitotic selection, and then either trypsinized and plated immediately, or held at 37 0 C for 6 hrs in PBS, or PBS containing 60μM of the DNA-polymerase-inhibitor 1-β-D-arabinofurano-syladenine (β-araA) before trypsinization and plating. Delayed plating showed significant PLDR at all ages irradiated with X rays, with the increase of survival varying between 2- to 8-fold. At equivalent survival levels, there was a reduced capacity for PLDT at each cell age irradiated with neon ions. In early G1 after neon-ion exposures, delayed plating actually enhanced cell killing; whereas, in late G1 the survival increased about 2-fold. β-araA almost completely eliminated the PLDR after X rays, reducing the survival to that measured with immediate plating. β-araA slightly enhanced neon-ion cell killing at all cell ages

Analysis of cell-cycle regulation following exposure of lung-derived cells to γ-rays

Science.gov (United States)

Trani, D.; Lucchetti, C.; Cassone, M.; D'Agostino, L.; Caputi, M.; Giordano, A.

Acute exposure of mammalian cells to ionizing radiation results in a delay of cell-cycle progression and/or augmentation of apoptosis. Following ionizing radiation-induced DNA damage, cell-cycle arrest in the G1- or G2-phase of the cell-cycle prevents or delays DNA replication or mitosis, providing time for the DNA repair machinery to exert its function. Deregulation or failing of cell-cycle checkpoints and/or DNA repair mechanisms may lead normal cells bearing chromosome mutations to acquire neoplastic autonomy, which in turn can trigger the onset of cancer. Existing studies have focused on the impact of p53 status on the radiation response of lung cancer (LC) cell lines in terms of both cell-cycle regulation and apoptosis, while no comparative studies have been performed on the radiation response of lung derived normal and cancerous epithelial cells. To investigate the radiation response in normal and cancerous phenotypes, along with the role and impact of p53 status, and possible correlations with pRb/p105 or other proteins involved in carcinogenesis and cell-cycle regulation, we selected two lung-derived epithelial cell lines, one normal (NL20, p53 wild-type) and one non-small cell lung cancer (NSCLC), H358 (known to be p53-deficient). We compared the levels of γ-induced cell proliferation ability, cell-cycle arrest, apoptotic index, and expression levels of cell-cycle regulating and regulated proteins. The different cell sensitivity, apoptotic response and protein expression profiles resulting from our study for NL20 and H358 cells suggest that still unknown mechanisms involving p53, pRb/p105 and their target molecules might play a pivotal role in determining cell sensitivity and resistance upon exposure to ionizing radiation.

Relationship between cell cycle stage in the fertilized egg of mice and repair capacity for X-ray-induced damage in the sperm

International Nuclear Information System (INIS)

Matsuda, Y.; Maemori, M.; Tobari, I.

1989-01-01

The potentiation effects of 3-aminobenzamide, caffeine, hydroxyurea and arabinofuranosyl cytosine on the yield of X-ray-induced chromosome aberrations of mouse sperm were examined at the first-cleavage metaphase, to clarify a correlation between chromosome aberrations and cell cycle dependency of repair capacity of the fertilized egg. The result provided evidence that there are two major types of DNA damage in X-irradiated sperm: (1) short-lived DNA lesions; the lesions are subject to repair inhibitions by agents added in G 1 and are converted into chromosome-type aberrations during G 1 , and (2) long-lived DNA lesions; the lesions persist until S phase and repair of the lesions is inhibited by caffeine, hydroxyurea and arabinofuranosyl cytosine in G 2 . (author)

Relationship between cell cycle stage in the fertilized egg of mice and repair capacity for X-ray-induced damage in the sperm

Energy Technology Data Exchange (ETDEWEB)

Matsuda, Y.; Maemori, M.; Tobari, I. (National Inst. of Radiological Sciences, Chiba (Japan))

1989-09-01

The potentiation effects of 3-aminobenzamide, caffeine, hydroxyurea and arabinofuranosyl cytosine on the yield of X-ray-induced chromosome aberrations of mouse sperm were examined at the first-cleavage metaphase, to clarify a correlation between chromosome aberrations and cell cycle dependency of repair capacity of the fertilized egg. The result provided evidence that there are two major types of DNA damage in X-irradiated sperm: (1) short-lived DNA lesions; the lesions are subject to repair inhibitions by agents added in G{sub 1} and are converted into chromosome-type aberrations during G{sub 1}, and (2) long-lived DNA lesions; the lesions persist until S phase and repair of the lesions is inhibited by caffeine, hydroxyurea and arabinofuranosyl cytosine in G {sub 2}. (author).

Repair and cell-cycle response in cells exposed to environmental biohazards. Comprehensive project report, June 1, 1979-May 31, 1982

International Nuclear Information System (INIS)

Billen, D.

1982-01-01

Agents which cause damage to DNA leading to inhibition of DNA synthesis or faulty DNA replication or repair may cause cell death or mutation. Many organisms possess the ability to circumvent some or all of this DNA damage. Many DNA mutants of E. coli and B. subtilis provide a genetic approach to measuring the role of individual components of the DNA repair and replicative system. The information obtained with prokaryotes provides leads to assess the details of DNA repair and replication in mammalian systems including man. Escherichia coli cells treated with a low concentration of toluene become permeable to a variety of compounds, including the precursors and cofactors necessary for DNA synthesis. By their manipulation various aspects of DNA replication and repair can be selectively emphasized. Observations made by use of this system include: (1) Repair synthesis induced by x irradiation or exposure to alkylating chemicals of toluene-treated cells is more extensive if polynucleotide ligase is inhibited. (2) DNA replication in E. coli is carried out by DNA polymerase III. The replication of DNA is strongly inhibited by methylmethansulfonate, N-methyl-N-nitrosourea, and N-methyl-N'-nitro-N-nitrosoguanidine. (3) Using a po1A1 po1B100 dnaB (po1I - , po1II - , po1III + ) mutant of E. coli, it was demonstrated that the dnaB gene product is not necessary for Po1III directed repair synthesis. (4) The physiological stage of cells and tissues affects their response to environmental hazards. (5) Procedures for permeabilizing mammalian cells have been developed or further refined; and (6) In earlier studies involving both alkylating agents and x rays, it was observed that the number of DNA single-strand breaks increased with dose along with repair synthesis. It appears that non-repaired sites do not serve as primer ends for Po1I-dependent repair synthesis in toluene-treated cells

Role of DNA lesions and repair in the transformation of human cells

International Nuclear Information System (INIS)

Maher, V.M.; McCormick, J.J.

1987-01-01

Results of studies on the transformation of diploid human fibroblasts in culture into tumor-forming cells by exposure to chemical carcinogens or radiation indicate that such transformation is multi-stepped process that at least one step, acquisition of anchorage independence, occurs as a mutagenic event. Studies comparing normal-repairing human cells with DNA repair-deficient cells, such as those derived from cancer-prone xeroderma pigmentosum patients, indicate that excision repair in human fibroblasts is essentially an error-free process that the ability to excise potentially cytotoxic, mutagenic, or transforming lesions induced DNA by carcinogens determines their ultimate biological consequences. Cells deficient in excision repair are abnormally sensitive to these agents. Studies with cells treated at various times in the cell cycle show that there is a certain limited amount of time available for DNA repair between the initial exposure and the onset of the cellular event responsible for mutation induction and transformation to anchorage independence. The data suggest that DNA replication on a template containing unexcised lesions (photoproducts, adducts) is the critical event

Repair and cell cycle response in cells exposed to environmental biohazards. Progress report, October 1, 1976--May 31, 1979

International Nuclear Information System (INIS)

Billen, D.

1979-01-01

A wide variety of environmental agents are known which induce damage in DNA leading to an inhibition of DNA synthesis or faulty replication. Both results may cause cell death or mutation. Both bacteria and mommalian cells are being used to assess the roles of the several known DNA polymerases and other DNA metabolic enzymes and factors in DNA repair, replication and recombination. The many DNA mutants of E. coli and B. subtilis provide a genetic approach to measuring the role of individual components of the DNA repair and replicative system. Because of the advantage of controlling pools and precursors of nucleic acid synthesis we have further developed the use of permeabilized cells for such studies. In addition a series of repair studies with Bacillus subtilis have been carried on because of the unique genetic manipulation of this system which includes the ability of cells to be easily transformed by exogenous DNA. The information obtained with prokaryotes provides leads to assess the details of DNA repair and replication in mammalian systems including man

Effect of cell cycle stage, dose rate and repair of sublethal damage of radiation-induced apoptosis in F9 teratocarcinoma cells

International Nuclear Information System (INIS)

Langley, R.E.; Quartuccio, S.G.; Kennealey, P.T.

1995-01-01

There are at least two different models of cell death after treatment with ionizing radiation. The first is a failure to undergo sustained cell division despite metabolic survival, and we refer to this end point as open-quotes classical reproductive cell death.close quotes The second is a process that results in loss of cell integrity. This second category includes cellular necrosis as well as apoptosis. Earlier studies in our laboratory showed that the predominant mechanism of cell death for irradiated F9 cell is apoptosis, and there is no indication that these cells die by necrosis. We have therefore used cells of this cell line to reassess basic radiobiological principles with respect to apoptosis. Classical reproductive cell death was determined by staining colonies derived from irradiated cells and scoring colonies of less than 50 cells as reproductively dead and colonies of more than 50 cells as survivors. Cells that failed to produce either type of colony (detached from the plate or disintegrated) were scored as having undergone apoptosis. Using these criteria we found that the fraction of the radiation-killed F9 cells that died by apoptosis did not vary when cells were irradiated at different stages of the cell cycle despite large variations in overall survival. This suggests that the factors that influence radiation sensitivity throughout the cell cycle have an equal impact on apoptosis and classical reproductive cell death. There was no difference in cell survival between split doses and single doses of X rays, suggesting that sublethal damage repair is not a factor in radiation-induced apoptosis of F9 cells. Apoptosis was not affected by changes in dose rate in the range of 0.038-4.96 Gy/min. 48 refs., 6 figs., 1 tab

Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation.

Science.gov (United States)

Weeden, Clare E; Asselin-Labat, Marie-Liesse

2018-01-01

Maintenance of genomic integrity in tissue-specific stem cells is critical for tissue homeostasis and the prevention of deleterious diseases such as cancer. Stem cells are subject to DNA damage induced by endogenous replication mishaps or exposure to exogenous agents. The type of DNA lesion and the cell cycle stage will invoke different DNA repair mechanisms depending on the intrinsic DNA repair machinery of a cell. Inappropriate DNA repair in stem cells can lead to cell death, or to the formation and accumulation of genetic alterations that can be transmitted to daughter cells and so is linked to cancer formation. DNA mutational signatures that are associated with DNA repair deficiencies or exposure to carcinogenic agents have been described in cancer. Here we review the most recent findings on DNA repair pathways activated in epithelial tissue stem and progenitor cells and their implications for cancer mutational signatures. We discuss how deep knowledge of early molecular events leading to carcinogenesis provides insights into DNA repair mechanisms operating in tumours and how these could be exploited therapeutically. Copyright © 2017 Elsevier B.V. All rights reserved.

Repair and cell cycle response in cells exposed to environmental biohazards. Progress report, February 1, 1976--May 31, 1977

International Nuclear Information System (INIS)

Billen, D.

1977-01-01

Progress is reported on the following research projects: DNA polymerase III dependent repair of x-ray damage in Escherichia coli; regulation of reinsertation of nucleotides by DNA ligase; DNA synthesis in permeabilized CHO cells; measurement of damage to DNA in Bacillus subtilis; repair defect in rec A cells; inactivation of transforming DNA; and mutagenesis of transforming DNA

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

Energy Technology Data Exchange (ETDEWEB)

Fan Jinshui; Robert, Carine [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States); Jang, Yoon-Young; Liu Hua; Sharkis, Saul; Baylin, Stephen Bruce [Johns Hopkins University School of Medicine, Department of Oncology, Baltimore, MD 21231-1000 (United States); Rassool, Feyruz Virgilia, E-mail: frassool@som.umaryland.edu [Department of Radiation Oncology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 7-023A, Baltimore, MD 21201 (United States)

2011-08-01

Highlights: {yields} iPSC and hESC demonstrate a similar cell cycle profile, with increased S phase cells and decreased G0/G1. {yields} iPSC and hESC increased ROS and decreased DSBs, compared with differentiated parental cells. {yields} iPSC and hESC demonstrate elevated DSB repair activity, including nonhomologous end-joining, compared with differentiated parental cells. {yields} iPSC however show a partial apoptotic response to DNA damage, compared to hESC. {yields} DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming. - Abstract: To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to 'dedifferentiate' into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived

Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining

International Nuclear Information System (INIS)

Fan Jinshui; Robert, Carine; Jang, Yoon-Young; Liu Hua; Sharkis, Saul; Baylin, Stephen Bruce; Rassool, Feyruz Virgilia

2011-01-01

Highlights: → iPSC and hESC demonstrate a similar cell cycle profile, with increased S phase cells and decreased G0/G1. → iPSC and hESC increased ROS and decreased DSBs, compared with differentiated parental cells. → iPSC and hESC demonstrate elevated DSB repair activity, including nonhomologous end-joining, compared with differentiated parental cells. → iPSC however show a partial apoptotic response to DNA damage, compared to hESC. → DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming. - Abstract: To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to 'dedifferentiate' into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

Energy Technology Data Exchange (ETDEWEB)

Daigaku, Yasukazu [Graduate School of Life Sciences, Tohoku University, Sendai 980-8577 (Japan); Mashiko, Satsuki [Graduate School of Life Sciences, Tohoku University, Sendai 980-8577 (Japan); Mishiba, Keiichiro [Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003 (Japan); Yamamura, Saburo [Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003 (Japan); Ui, Ayako [Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578 (Japan); Enomoto, Takemi [Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578 (Japan); Yamamoto, Kazuo [Graduate School of Life Sciences, Tohoku University, Sendai 980-8577 (Japan)]. E-mail: yamamot@mail.tains.tohoku.ac.jp

2006-08-30

A CAN1/can1{delta} heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle.

Science.gov (United States)

Daigaku, Yasukazu; Mashiko, Satsuki; Mishiba, Keiichiro; Yamamura, Saburo; Ui, Ayako; Enomoto, Takemi; Yamamoto, Kazuo

2006-08-30

A CAN1/can1Delta heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.

Repair and cell cycle response in cells exposed to environmental biohazards. Final report, January 1, 1973-December 31, 1984

International Nuclear Information System (INIS)

Hadden, C.T.; Billen, D.

1986-01-01

These studies have focussed on agents which cause damage to DNA leading to inhibition of DNA synthesis or faulty DNA replication or repair. The overall goal of this project has been to understand how environmental agents interact with the DNA of cells and how cells cope with any resulting damage. In particular we have been concerned with the nature of the repair systems involved in restoration of damaged DNA and the cellular responses to radiation or chemical damage

Origin of the effects of cell cycle age on radiation sensitivity

International Nuclear Information System (INIS)

Nelson, J.M.; Braby, L.A.; Metting, N.F.; Roesch, W.C.

1987-01-01

The observed differences in radiation sensitivity as a function of cell age could reflect either differences in cellular capacity to remove different types of damage, or differences in damage production. The authors used centrifugal elutriation methods to produce enriched populations of G/sub 1/-, S-, and G/sub 2/-phase cells from stationary phase CHO cultures. Recognizing the imperfect correlation between cell age and cross-sectional area (the criterion upon which the separation process is based) flow-cytometric methods were used to measure cellular DNA content and determine cell age distributions. Split-dose repair kinetics were then determined for cells in different phases of the cycle. The authors find no striking differences in total repair, indicating that damage is removed at generally the same rate and with essentially the same efficiency, throughout the cell cycle. This suggests that changes in the production of damage are likely responsible for the change in sensitivity

Studies of DNA repair in Saccharomyces cerevisiae. I. Characterization of a new allele of RAD6. II. Investigation of events in the first cell cycle after DNA damage

International Nuclear Information System (INIS)

Dolthwright-Fasse, J.A.

1980-01-01

Studies in two independent, but related, areas of DNA repair have been carried out in the eucaryotic yeast, Saccharomyces cerevisiae. The first is the characterization of a new allele in the RAD6 gene suggesting that the gene is multifunctional. The second is the utilization of photoreactivation as a probe of events occurring during the first cell cycle after DNA damage. Strains carrying the new allele, designated rad6-4, of the RAD6 locus are about as sensitive to uv and ionizing radiation as those carrying rad6-1 or rad6-3. Although rad6-4 may well be a missense mutation, the data suggest that the RAD6 gene is multifunctional. One function is necessary to recover from DNA damage in an error-free manner, and the other is concerned with mutagenic processes and sporulation. The loss of photoreversibility (LOP) of ultraviolet induced mutations to arginine independence in an excision defective strain carrying arg4-17 examines the events occurring in the first cell cycle. The post uv protein synthesis causes pyrimidine dimmers to become inaccessible to the photoreactivating enzyme in some unknown manner. There is no evidence indicating whether the normal function of the protein is involved in excision repair, or in one of the two repair processes believed to be inducible; induced mutagenesis or recombinational repair

Two pathways of DNA double-strand break repair in G1 cells of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Glazunov, A.V.

1988-01-01

The G1 cells of the diploid yeast Saccharomyces cerevislae are known to be capable of a slow repair of DNA double-strand breaks (DSB) during holding the cells in a non-nutrient medium. In the present paper, it has been shown that S. cerevislae cells γ-irradiated in the G1 phase of cell cycle are capable of fast repair of DNA DSB; this process is completed within 30-40 min of holding the cells in water at 28 deg C. For this reason, the kinetics of DNA DSB repair during holding the cells in a non-nutrient medium are biphasic, i.e., the first, ''fast'' phase is completed within 30-40 min; wheras the second, ''slow'' one, within 48 h. Mutations rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not practically influence this process. It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S, cerevislae are separate pathways of DNA DSB repair in yeast

Repair and cell cycle response in cells exposed to environmental biohazards. Progress report, 1 June 1982-31 May 1983

International Nuclear Information System (INIS)

Billen, D.; Hadden, C.T.

1983-01-01

The research program has attempted to determine low environmental agents, especially ionizing radiation, interact with DNA and how the cells respond to the resulting damage. The focus has been on the spectrum of damage generated in DNA, the kinds of damage that can be repaired, and the mechanisms of repair. The effects of radioprotective agents and certain sensitizing conditions on breakage of DNA in bacterial cells by ionizing radiation, and mechanisms of enzymatic excision of pyrimidine dimers and other DNA adducts have been studied

Evidence for multiple repair pathways of double-strand DNA breaks in Chinese hamster cells

International Nuclear Information System (INIS)

Giaccia, A.J.; Weistein, R.; Stamato, T.D.; Roosa, R.

1984-01-01

XR-1 is a mutant of the Chinese hamster cell (CHO-K1) which is abnormally sensitive to killing by gamma rays in G/sub 1/ (D37 = 27 rads vs. 318 for parent) and early S phases of the cell cycle but has near normal resistance in late S and early G/sub 2/ (Somatic Cell Genetics, 9:165-173, 1983). Complementation studies between XR-1 and its parent indicate that this sensitivity to gamma rays is a recessive phenotype. Both the XR-1 and its parent cell are able to repair single strand DNA breaks. However, in comparison to its parental cell, the XR-1 cell is markedly deficient in the repair of double strand DNA breaks introduced by gamma irradiation during the sensitive G/sub 1/-early S period, while in the late S-G/sub 2/ resistant period the repair is similar in both cells. This correlation suggests that an unrepaired double strand DNA break is the lethal lesion and that at least two pathways for the repair of these lesions exist in mammalian cells

Reprogramming Cells for Brain Repair

Directory of Open Access Journals (Sweden)

Randall D. McKinnon

2013-08-01

Full Text Available At present there are no clinical therapies that can repair traumatic brain injury, spinal cord injury or degenerative brain disease. While redundancy and rewiring of surviving circuits can recover some lost function, the brain and spinal column lack sufficient endogenous stem cells to replace lost neurons or their supporting glia. In contrast, pre-clinical studies have demonstrated that exogenous transplants can have remarkable efficacy for brain repair in animal models. Mesenchymal stromal cells (MSCs can provide paracrine factors that repair damage caused by ischemic injury, and oligodendrocyte progenitor cell (OPC grafts give dramatic functional recovery from spinal cord injury. These studies have progressed to clinical trials, including human embryonic stem cell (hESC-derived OPCs for spinal cord repair. However, ESC-derived allografts are less than optimal, and we need to identify a more appropriate donor graft population. The cell reprogramming field has developed the ability to trans-differentiate somatic cells into distinct cell types, a technology that has the potential to generate autologous neurons and glia which address the histocompatibility concerns of allografts and the tumorigenicity concerns of ESC-derived grafts. Further clarifying how cell reprogramming works may lead to more efficient direct reprogram approaches, and possibly in vivo reprogramming, in order to promote brain and spinal cord repair.

Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

Energy Technology Data Exchange (ETDEWEB)

Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

1988-10-25

DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

Phytochemicals radiosensitize cancer cells by inhibiting DNA repair

International Nuclear Information System (INIS)

Singh, Rana P.

2017-01-01

Solid tumors are mostly treated with radiotherapy. Radiotherapy is toxic to normal tissues and also promote the invasiveness and radioresistance in cancer cells. The resistance against radiotherapy and adverse effects to normal cells reduce the overall therapeutic effects of the treatment. Radiosensitizing agents usually show limited success during clinical trials. Therefore, the search and development of new radiosensitizers showing selective response to only cancer cells is desirable. We analyzed the radiosensitizing effects including cell death effect of silibinin, a phytochemical on prostate cancer cells. Silibinin enhanced gamma radiation (2.5-10 Gy) induced inhibition in colony formation selectively in prostate cancer cells. In cell cycle progression, G2/M phase is the most sensitive phase for radiation-induced damage which was delayed by the compound treatment in radiation exposed cells. The lower concentrations of silibinin substantially enhanced radiation-induced apoptosis. A prolonged reactive oxygen species production was also observed in these treatments EGFR signaling pathway can contribute to radiation-induced pro-survival mechanisms and to the therapeutic resistance. Agent treatment reduced the IR-induced EGFR phosphorylation and consequently reversed the resistance mediating mechanisms within the cancer cell. Thus, inhibiting DNA repair in cancer cells would enhance therapeutic response of radiation in cancer cells. Silibinin affected the localization of EGFR and DNA-dependent protein kinase, the DNA-PK is known to be an important mediator of DSB repair in human cells, and showed increased number of pH2AX (ser139) foci, and thus indicating lower DNA repair in these cancer cells. This was also confirmed in the tumor xenograft study. Our findings suggest that a combination of silibinin with radiation could be an effective treatment of radioresistant human prostate cancer and warrants further investigation. (author)

Assessment of single nucleotide polymorphisms in screening 52 DNA repair and cell cycle control genes in Fanconi anemia patients

Directory of Open Access Journals (Sweden)

Petrović Sandra

2015-01-01

Full Text Available Fanconi anemia (FA is a rare genetically heterogeneous disorder associated with bone marrow failure, birth defects and cancer susceptibility. Apart from the disease- causing mutations in FANC genes, the identification of specific DNA variations, such as single nucleotide polymorphisms (SNPs, in other candidate genes may lead to a better clinical description of this condition enabling individualized treatment with improvement of the prognosis. In this study, we have assessed 95 SNPs located in 52 key genes involved in base excision repair (BER, nucleotide excision repair (NER, mismatch repair (MMR, double strand break (DSB repair and cell cycle control using a DNA repair chip (Asper Biotech, Estonia which includes most of the common variants for the candidate genes. The SNP genotyping was performed in five FA-D2 patients and in one FA-A patient. The polymorphisms studied were synonymous (n=10, nonsynonymous (missense (n=52 and in non-coding regions of the genome (introns and 5 ‘and 3’ untranslated regions (UTR (n=33. Polymorphisms found at the homozygous state are selected for further analysis. Our results have shown a significant inter-individual variability among patients in the type and the frequency of SNPs and also elucidate the need for further studies of polymorphisms located in ATM, APEX APE 1, XRCC1, ERCC2, MSH3, PARP4, NBS1, BARD1, CDKN1B, TP53 and TP53BP1 which may be of great importance for better clinical description of FA. In addition, the present report recommends the use of SNPs as predictive and prognostic genetic markers to individualize therapy of FA patients. [Projekat Ministarstva nauke Republike Srbije, br. 173046

Combined effects of scaffold stiffening and mechanical preconditioning cycles on construct biomechanics, gene expression, and tendon repair biomechanics.

Science.gov (United States)

Nirmalanandhan, Victor Sanjit; Juncosa-Melvin, Natalia; Shearn, Jason T; Boivin, Gregory P; Galloway, Marc T; Gooch, Cynthia; Bradica, Gino; Butler, David L

2009-08-01

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construct and repair stiffness after surgery suggest that enhancing structural stiffness before surgery could not only accelerate repair stiffness but also prevent premature failures in culture due to poor mechanical integrity. In this study, we examined the combined effects of scaffold crosslinking and subsequent mechanical stimulation on construct mechanics and biology. Autologous tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 15 New Zealand white rabbits on type I collagen sponges that had undergone additional dehydrothermal crosslinking (termed ADHT in this manuscript). Both constructs from each rabbit were mechanically stimulated for 8h/day for 12 consecutive days with half receiving 100 cycles/day and the other half receiving 3000 cycles/day. These paired MSC-collagen autologous constructs were then implanted in bilateral full-thickness, full-length defects in the central third of rabbit patellar tendons. Increasing the number of in vitro cycles/day delivered to the ADHT constructs in culture produced no differences in stiffness or gene expression and no changes in biomechanical properties or histology 12 weeks after surgery. Compared to MSC-based repairs from a previous study that received no additional treatment in culture, ADHT crosslinking of the scaffolds actually lowered the 12-week repair stiffness. Thus, while ADHT crosslinking may initially stiffen a construct in culture, this specific treatment also appears to mask any benefits

Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

International Nuclear Information System (INIS)

Rutz, H. Peter; Little, John B.

1995-01-01

Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO 2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G 2 /M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

DNA hybrids suggesting a recombination process repairing radiation-induced DNA double-strand breaks in Ehrlich Ascites tumor cells

International Nuclear Information System (INIS)

Barthel, H.R.

1984-01-01

The results presented suggest the possibility of repair of DNA double-strand breaks by recombination, at least in the S and G 2 -phases of the cell cycle, in mammalian cells. Further experiments with synchronized cell cultures will have to show whether this process may also occur in the G 1 -phase of the cell cycle. (orig./AJ) [de

Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair

Directory of Open Access Journals (Sweden)

Kmiec Eric B

2007-02-01

Full Text Available Abstract Background Single-stranded oligonucleotides (ssODN are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time. Results First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours. Conclusion Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes.

Regulation of the cell cycle by irradiation

International Nuclear Information System (INIS)

Akashi, Makoto

1995-01-01

The molecular mechanism of cell proliferation is extremely complex; deregulation results in neoplastic transformation. In eukaryotes, proliferation of cells is finely regulated through the cell cycle. Studies have shown that the cell cycle is regulated by s series of enzymes known as cyclin-dependent kinases (CDKs). The activities of CDKs are controlled by their association with regulatory subunits, cyclins; the expression of cyclins and the activation of the different cyclin-CDK complexes are required for the cell to cycle. Thus, the cell cycle is regulated by activating and inhibiting phosphorylation of the CDK subunits and this program has internal check points at different stages of the cell cycle. When cells are exposed to external insults such as DNA damaging agents, negative regulation of the cell cycle occurs; arrest in either G1 or G2 stage is induced to prevent the cells from prematurely entering into the next stage before DNA is repaired. Recently, a potent inhibitor of CDKs, which inhibits the phosphorylation of retinoblastoma susceptibility (Rb) gene product by cyclin A-CDK2, cyclin E-CDK2, cyclin D1-CDK4, and cyclin D2-CDK4 complexes has been identified. This protein named WAF1, Sdi1, Cip1, or p21 (a protein of Mr 21,000) contains a p53-binding site in its promoter and studies have reported that the expression of WAF1 was directly regulated by p53; cells with loss of p53 activity due to mutational alteration were unable to induce WAF1. This chapter will be focused on the mechanisms of the cell cycle including inhibitors of CDKs, and the induction of WAF1 by irradiation through a pathway independent of p53 will be also described. (author)

Molecular biological mechanism II. Molecular mechanisms of cell cycle regulation

International Nuclear Information System (INIS)

Jung, T.

2000-01-01

The cell cycle in eukaryotes is regulated by central cell cycle controlling protein kinase complexes. These protein kinase complexes consist of a catalytic subunit from the cyclin-dependent protein kinase family (CDK), and a regulatory subunit from the cyclin family. Cyclins are characterised by their periodic cell cycle related synthesis and destruction. Each cell cycle phase is characterised by a specific set of CDKs and cyclins. The activity of CDK/cyclin complexes is mainly regulated on four levels. It is controlled by specific phosphorylation steps, the synthesis and destruction of cyclins, the binding of specific inhibitor proteins, and by active control of their intracellular localisation. At several critical points within the cell cycle, named checkpoints, the integrity of the cellular genome is monitored. If damage to the genome or an unfinished prior cell cycle phase is detected, the cell cycle progression is stopped. These cell cycle blocks are of great importance to secure survival of cells. Their primary importance is to prevent the manifestation and heritable passage of a mutated genome to daughter cells. Damage sensing, DNA repair, cell cycle control and apoptosis are closely linked cellular defence mechanisms to secure genome integrity. Disregulation in one of these defence mechanisms are potentially correlated with an increased cancer risk and therefore in at least some cases with an increased radiation sensitivity. (orig.) [de

Nucleotide excision repair in differentiated cells

Energy Technology Data Exchange (ETDEWEB)

Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

2007-01-03

Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

NONO couples the circadian clock to the cell cycle.

Science.gov (United States)

Kowalska, Elzbieta; Ripperger, Juergen A; Hoegger, Dominik C; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A

2013-01-29

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

Cell cycle arrest and the evolution of chronic kidney disease from acute kidney injury.

Science.gov (United States)

Canaud, Guillaume; Bonventre, Joseph V

2015-04-01

For several decades, acute kidney injury (AKI) was generally considered a reversible process leading to complete kidney recovery if the individual survived the acute illness. Recent evidence from epidemiologic studies and animal models, however, have highlighted that AKI can lead to the development of fibrosis and facilitate the progression of chronic renal failure. When kidney injury is mild and baseline function is normal, the repair process can be adaptive with few long-term consequences. When the injury is more severe, repeated, or to a kidney with underlying disease, the repair can be maladaptive and epithelial cell cycle arrest may play an important role in the development of fibrosis. Indeed, during the maladaptive repair after a renal insult, many tubular cells that are undergoing cell division spend a prolonged period in the G2/M phase of the cell cycle. These tubular cells recruit intracellular pathways leading to the synthesis and the secretion of profibrotic factors, which then act in a paracrine fashion on interstitial pericytes/fibroblasts to accelerate proliferation of these cells and production of interstitial matrix. Thus, the tubule cells assume a senescent secretory phenotype. Characteristic features of these cells may represent new biomarkers of fibrosis progression and the G2/M-arrested cells may represent a new therapeutic target to prevent, delay or arrest progression of chronic kidney disease. Here, we summarize recent advances in our understanding of the biology of the cell cycle and how cell cycle arrest links AKI to chronic kidney disease. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

DNA repair in human cells

International Nuclear Information System (INIS)

Regan, J.D.; Carrier, W.L.; Kusano, I.; Furuno-Fukushi, I.; Dunn, W.C. Jr.; Francis, A.A.; Lee, W.H.

1982-01-01

Our primary objective is to elucidate the molecular events in human cells when cellular macromolecules such as DNA are damaged by radiation or chemical agents. We study and characterize (i) the sequence of DNA repair events, (ii) the various modalities of repair, (iii) the genetic inhibition of repair due to mutation, (iv) the physiological inhibition of repair due to mutation, (v) the physiological inhibition of repair due to biochemical inhibitors, and (vi) the genetic basis of repair. Our ultimate goals are to (i) isolate and analyze the repair component of the mutagenic and/or carcinogenic event in human cells, and (ii) elucidate the magnitude and significance of this repair component as it impinges on the practical problems of human irradiation or exposure to actual or potential chemical mutagens and carcinogens. The significance of these studies lies in (i) the ubiquitousness of repair (most organisms, including man, have several complex repair systems), (ii) the belief that mutagenic and carcinogenic events may arise only from residual (nonrepaired) lesions or that error-prone repair systems may be the major induction mechanisms of the mutagenic or carcinogenic event, and (iii) the clear association of repair defects and highly carcinogenic disease states in man [xeroderma pigmentosum (XP)

Arrest of irradiated G1, S, or G2 cells at mitosis using nocodazole promotes repair of potentially lethal damage

International Nuclear Information System (INIS)

Iliakis, G.; Nuesse, M.

1984-01-01

The ability of synchronized Ehrlich ascites tumor cells, irradiated in G1, S, and G2 phases, to repair potentially lethal damage when arrested at mitosis by using 0.4 μg/ml nocodazole, a specific inhibitor of microtubule polymerization, has been studied. Cells irradiated in these phases were found to repair potentially lethal damage at mitosis. The extent of this repair was similar to that observed for cells irradiated at the same stages in the cell cycle but allowed to repair potentially lethal damage by incubating in balanced salt solution for 6 hr after X irradiation

HIV-1 Tat depresses DNA-PKCS expression and DNA repair, and sensitizes cells to ionizing radiation

International Nuclear Information System (INIS)

Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun

2006-01-01

Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and γ-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and γH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy γ-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G 2 /M arrest as compared with parental cells. In addition, the G 2 /M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS

Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest

International Nuclear Information System (INIS)

Lo, Hsin-Lung; Nakajima, Satoshi; Ma, Lisa; Walter, Barbara; Yasui, Akira; Ethell, Douglas W; Owen, Laurie B

2005-01-01

UV-induced damage can induce apoptosis or trigger DNA repair mechanisms. Minor DNA damage is thought to halt the cell cycle to allow effective repair, while more severe damage can induce an apoptotic program. Of the two major types of UV-induced DNA lesions, it has been reported that repair of CPD, but not 6-4PP, abrogates mutation. To address whether the two major forms of UV-induced DNA damage, can induce differential biological effects, NER-deficient cells containing either CPD photolyase or 6-4 PP photolyase were exposed to UV and examined for alterations in cell cycle and apoptosis. In addition, pTpT, a molecular mimic of CPD was tested in vitro and in vivo for the ability to induce cell death and cell cycle alterations. NER-deficient XPA cells were stably transfected with CPD-photolyase or 6-4PP photolyase to specifically repair only CPD or only 6-4PP. After 300 J/m 2 UVB exposure photoreactivation light (PR, UVA 60 kJ/m 2 ) was provided for photolyase activation and DNA repair. Apoptosis was monitored 24 hours later by flow cytometric analysis of DNA content, using sub-G1 staining to indicate apoptotic cells. To confirm the effects observed with CPD lesions, the molecular mimic of CPD, pTpT, was also tested in vitro and in vivo for its effect on cell cycle and apoptosis. The specific repair of 6-4PP lesions after UVB exposure resulted in a dramatic reduction in apoptosis. These findings suggested that 6-4PP lesions may be the primary inducer of UVB-induced apoptosis. Repair of CPD lesions (despite their relative abundance in the UV-damaged cell) had little effect on the induction of apoptosis. Supporting these findings, the molecular mimic of CPD, (dinucleotide pTpT) could mimic the effects of UVB on cell cycle arrest, but were ineffective to induce apoptosis. The primary response of the cell to UV-induced 6-4PP lesions is to trigger an apoptotic program whereas the response of the cell to CPD lesions appears to principally involve cell cycle arrest. These

DNA replication and repair in Tilapia cells

International Nuclear Information System (INIS)

Yew, F.H.; Chang, L.M.

1984-01-01

The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-β-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor. (author)

Transfer of Chinese hamster DNA repair gene(s) into repair-deficient human cells (Xeroderma pigmentosum)

International Nuclear Information System (INIS)

Karentz, D.; Cleaver, J.E.

1985-01-01

Transfer of repair genes by DNA transfection into repair-deficient Xeroderma pigmentosum (XP) cells has thus far been unsuccessful, presenting an obstacle to cloning XP genes. The authors chose an indirect route to transfer repair genes in chromosome fragments. DNA repair-competent (UV resistant) hybrid cell lines were established by PEG-mediated fusions of DNA repair-deficient (UV sensitive) human fibroblasts (XP12RO) with wild type Chinese hamster (CHO) cells (AA8). CHO cells were exposed to 5 Krad X-rays prior to fusions, predisposing hybrid cells to lose CHO chromosome fragments preferentially. Repair-competent hybrids were selected by periodic exposures to UV light. Secondary and tertiary hybrid cell lines were developed by fusion of X-irradiated hybrids to XP12RO. The hybrid cell lines exhibit resistance to UV that is comparable to that of CHO cells and they are proficient at repair replication after UV exposure. Whole cell DNA-DNA hybridizations indicate that the hybrids have greater homology to CHO DNA than is evident between XP12RO and CHO. These observations indicate that CHO DNA sequences which can function in repair of UV-damaged DNA in human cells have been transferred into the genome of the repair-deficient XP12RO cells

Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

Energy Technology Data Exchange (ETDEWEB)

Ockey, C.H.

1982-04-01

DNA fibre autoradiography, after incorporation of high specific activity /sup 3/H-thymidine and /sup 3/H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) of G/sub 1/ (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G/sub 1/ cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.

Understanding cell cycle and cell death regulation provides novel weapons against human diseases.

Science.gov (United States)

Wiman, K G; Zhivotovsky, B

2017-05-01

Cell division, cell differentiation and cell death are the three principal physiological processes that regulate tissue homoeostasis in multicellular organisms. The growth and survival of cells as well as the integrity of the genome are regulated by a complex network of pathways, in which cell cycle checkpoints, DNA repair and programmed cell death have critical roles. Disruption of genomic integrity and impaired regulation of cell death may both lead to uncontrolled cell growth. Compromised cell death can also favour genomic instability. It is becoming increasingly clear that dysregulation of cell cycle and cell death processes plays an important role in the development of major disorders such as cancer, cardiovascular disease, infection, inflammation and neurodegenerative diseases. Research achievements in these fields have led to the development of novel approaches for treatment of various conditions associated with abnormalities in the regulation of cell cycle progression or cell death. A better understanding of how cellular life-and-death processes are regulated is essential for this development. To highlight these important advances, the Third Nobel Conference entitled 'The Cell Cycle and Cell Death in Disease' was organized at Karolinska Institutet in 2016. In this review we will summarize current understanding of cell cycle progression and cell death and discuss some of the recent advances in therapeutic applications in pathological conditions such as cancer, neurological disorders and inflammation. © 2017 The Association for the Publication of the Journal of Internal Medicine.

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells.

Science.gov (United States)

Bailis, Julie M; Weidmann, Alyson G; Mariano, Natalie F; Barton, Jacqueline K

2017-07-03

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)] 2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

DEFF Research Database (Denmark)

Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw

2005-01-01

repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment......-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions...

Cell-cycle phase specificity of chloroethylnitrosoureas

International Nuclear Information System (INIS)

Linfoot, P.A.

1986-01-01

Although the cancer chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is considered a non-cell cycle phase specific drug, it has been shown to produce differential cell killing in G 1 , S, and G 2 /M phase cells, with S phase cells appearing relatively resistant. Studies of cell cycle phase specific cell killing produced by nitrosoureas with different chemical reactivities, clearly indicated that the ability of compounds to cross-link DNA was important in determining their phase specificity. Cells that lacked guanine O 6 -alkytransferase activity showed similar patterns of BCNU phase specificity regardless of their intrinsic sensitivity to BCNU. DNA inter-strand cross-linking, as measured by alkaline elution, was similar in cells exposed to BCNU in G 1 or S phase. 3 H [1-chloroethyl-1nitrosourea] binding to DNA was the same in G 1 , S and G 2 /M phase cells indicating that phase-specific differences in drug uptake and intracellular drug dose were not responsible for phase specific cell kill. These studies suggest that cross-link lesions, other than DNA inter-strand cross-links, and/or effects on DNA repair, other than guanine O 6 -alkyltransferase, are additional important determinants of BCNU phase specific cell killing

Repair-misrepair model of cell survival

International Nuclear Information System (INIS)

Tobias, C.A.; Blakely, E.A.; Ngo, F.Q.H.

1980-01-01

During the last three years a new model, the repair-misrepair model (RMR) has been proposed, to interpret radiobiological experiments with heavy ions. In using the RMR model it became apparent that some of its features are suitable for handling the effects produced by a variety of environmental agents in addition to ionizing radiation. Two separate sequences of events are assumed to take place in an irradiated cell. The first sequence begins with an initial energy transfer consisting of ionizations and excitations, culminating via fast secondary physical and chemical processes in established macromolecular lesions in essential cell structures. The second sequence contains the responses of the cell to the lesions and consists of the processes of recognition and molecular repair. In normal cells there exists one repair process or at most a few enzymatic repair processes for each essential macromolecular lesion. The enzymatic repair processes may last for hours and minutes, and can be separated in time from the initial physicochemical and later genetic phases

Repair in Ehrlich ascites tumor cells

International Nuclear Information System (INIS)

Wanna-Nakamura, S.S.

1981-01-01

Unscheduled DNA synthesis (UDS), an indicator of excision repair, was induced in freshly drawn Ehrlich ascites tumor cells (EAT), using ionizing radiation, far ultraviolet light (254 nm) or near uv light (365 nm) in combination with 8-methoxypsoralen. UDS was scored by grain counts in autoradiographs following the incorporation of tritium-labelled thymidine. The amount of UDS after each of these agents was expressed in terms of two parameters, viz. numer of cells showing repair and the mean number of grains per nucleus. The influence of radiation dose and of the duration of radioactive thymidine incubation were also examined. To test for a possible relationship between low mitotic index and repair capability, EAT cells were incubated in buffered salt media to lower the mitotic index. Cells kept in a buffered salt solution for 7 h show a marked drop in mitotic index compared to those incubated in minimal medium containing 15% fetal calf serum (MEM + FCS). This drop in mitotic index was reversible for up to 5 h, if cells were returned to MEM + FCS. Cells incubated in MEM + FCS also showed a decrease in mitotic activity compared to freshly drawn cells. This reduced mitotic index is approximately constant for up to 24 h. With the drop in mitotic index, EAT cells also show a drop in repair compared to freshly drawn cells. The repair capability of cells incubated in buffer can be restored by returning cells to MEM + FCS

Repair of furocoumarin adducts in mammalian cells

International Nuclear Information System (INIS)

Zolan, M.E.; Smith, C.A.; Hanawalt, P.C.

1984-01-01

DNA repair was studied in cultured mammalian cells treated with the furocoumarins 8-methoxypsoralen (8-MOP), aminomethyl trioxsalen, or angelicin and irradiated with near UV light. The amount of DNA cross-linked by 8-MOP in normal human cells decreased by about one-half in 24 hours after treatment; no decrease was observed in xeroderma pigmentosum cells, group A. At present, it is not known to what extent this decrease represents complete repair events at the sites of cross-links. Furocoumarin adducts elicited excision repair in normal human and monkey cells but not in xeroderma pigmentosum group A cells. This excision repair resembled in several aspects that elicited by pyrimidine dimers, formed in DNA by irradiation with 254-nm UV light; however, it appeared that for at least 8-MOP and aminomethyl trioxsalen, removal of adducts was not as efficient as was the removal of pyrimidine dimers. A comparison was also made of repair in the 172-base-pair repetitive alpha-DNA component of monkey cells to repair in the bulk of the genome. Although repair elicited by pyrimidine dimers in alpha-DNA was the same as in the bulk DNA, that following treatment of cells with either aminomethyl trioxsalen or angelicin and near UV was markedly deficient in alpha-DNA. This deficiency reflected the removal of fewer adducts from alpha-DNA after the same initial adduct frequencies. These results could mean that each furocoumarin may produce several structurally distinct adducts to DNA in cells and that the capacity of cellular repair systems to remove these various adducts may vary greatly

Modeling of DNA damage-cluster, cell-cycle and repair pathway dependent radiosensitivity after low- and high-LET irradiation

International Nuclear Information System (INIS)

Guenther, Paul

2017-01-01

This work focuses on modeling of the effects of ionizing radiation on cells, primarily on, the influence of the DNA repair pathway availability and the radiation quality on the cell-survival probability. The availability of DNA repair pathways depends on the replication state and defects of the DNA repair pathways. The radiation quality manifests itself in the microscopic ionization pattern. The Giant LOop Binary LEsion (GLOBLE) model and the Local Effect Model (LEM) describe the cell-survival after photon and ion irradiation, respectively. Both models assume that cell survival can be modeled based on the spatial distribution of Double-Strand Breaks (DSB) of the DNA (damage pattern), within a higher order chromatin structure. Single DSB are referred to as isolated DSB (iDSB) and two or more DSB in close proximity (within 540 nm) are called complex DSB (cDSB). In order to predict the cell-survival, the GLOBLE-Model considers different iDSB repair-pathways and their availability. One central assumption of the LEM is that the same damage patterns imply same effects, regardless of the radiation quality. In order to predict the damage pattern the microscopic local dose distribution of ions, described by the amorphous track structure, is evaluated. The cell survival after ion irradiation is predicted from a comparison with corresponding damage patterns after photon irradiation. The cell-survival curves after high dose photon irradiation cannot be predicted from the Linear Quadratic (LQ) Model due to their transition towards a linear dose dependence. This work uses the GLOBLE-Model to introduce a novel mechanistic approach, which allows the threshold dose to be predicted for the transition from a linear quadratic dose dependence, of survival curves at low doses, to a linear dose dependence at high doses. Furthermore, a method is presented, which allows LEM to predict the survival of synchronous cells after ion irradiation based on the cell survival after photon

Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells.

Science.gov (United States)

Nojima, Kuniharu; Hochegger, Helfrid; Saberi, Alihossein; Fukushima, Toru; Kikuchi, Koji; Yoshimura, Michio; Orelli, Brian J; Bishop, Douglas K; Hirano, Seiki; Ohzeki, Mioko; Ishiai, Masamichi; Yamamoto, Kazuhiko; Takata, Minoru; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Yamazoe, Mitsuyoshi; Kawamoto, Takuo; Araki, Kasumi; Takahashi, Jun A; Hashimoto, Nobuo; Takeda, Shunichi; Sonoda, Eiichiro

2005-12-15

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polzeta, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy.

Enhancement of postreplication repair in ultraviolet-light-irradiated Chinese hamster cells by irradiation in G2 or s-phase

International Nuclear Information System (INIS)

D'Ambrosio, S.M.; Aebersold, P.M.; Setlow, R.B.

1978-01-01

Postreplication repair in synchronous Chinese hamster cells was determined after split doses of ultraviolet (uv) radiation. Repair was enhanced by irradiation of cells in G 2 or S-phase with a small dose of uv radiation at least 1.5 h before a three-fold larger dose of uv. There was significantly greater enhancement when the first dose was given in G 2 than when it was given in the S-phase 0.5 to 1.5 h before the test dose. These data indicate that enhancement of postreplication repair does not require active DNA replication and qualitatively is independent of when in the cell cycle the cells are irradiated

Cell cycle-related genes as modifiers of age of onset of colorectal cancer in Lynch syndrome: a large-scale study in non-Hispanic white patients.

Science.gov (United States)

Chen, Jinyun; Pande, Mala; Huang, Yu-Jing; Wei, Chongjuan; Amos, Christopher I; Talseth-Palmer, Bente A; Meldrum, Cliff J; Chen, Wei V; Gorlov, Ivan P; Lynch, Patrick M; Scott, Rodney J; Frazier, Marsha L

2013-02-01

Heterogeneity in age of onset of colorectal cancer in individuals with mutations in DNA mismatch repair genes (Lynch syndrome) suggests the influence of other lifestyle and genetic modifiers. We hypothesized that genes regulating the cell cycle influence the observed heterogeneity as cell cycle-related genes respond to DNA damage by arresting the cell cycle to provide time for repair and induce transcription of genes that facilitate repair. We examined the association of 1456 single nucleotide polymorphisms (SNPs) in 128 cell cycle-related genes and 31 DNA repair-related genes in 485 non-Hispanic white participants with Lynch syndrome to determine whether there are SNPs associated with age of onset of colorectal cancer. Genotyping was performed on an Illumina GoldenGate platform, and data were analyzed using Kaplan-Meier survival analysis, Cox regression analysis and classification and regression tree (CART) methods. Ten SNPs were independently significant in a multivariable Cox proportional hazards regression model after correcting for multiple comparisons (P Lynch syndrome.

Studies of DNA repair in saccharomyces cerevisiae. I. Characterization of a new allele of RAD6. II. Investigation of events in the first cell cycle after DNA damage

International Nuclear Information System (INIS)

Douthwright-Fasse, J.A.

1979-01-01

Studies in two independent, but related, areas of DNA repair have been carried out in Saccharomyces cerevisiae; characterization of a new allele in the RAD6 gene which suggests that the gene is multifunctional, and utilization of photoreactivation as a probe of events occurring during the first cell cycle after DNA damage. Strains carrying the new allele, designated rad6-4, are as sensitive to uv and ionizing radiation as those carrying rad6-1 or rad6-3 but, unlike them, are capable of induced mutagenesis and sporulation. Although rad6-4 may well be a missense mutation, the evidence shows that it is unlikely that this phenotype is due to leakiness. Instead, the data suggest that the RAD6 gene is multifunctional. One function is necessary to recover from DNA damage in an error-free manner, and the other is concerned with mutagenic processes and sporulation. Rad6-1 and rad6-3 strains are deficient in both of these functions, while rad6-4 strains are deficient only in the error-free function. The loss of photoreversibility (LOP) of ultraviolet induced mutations to arginine independence in an excision defective strain carrying arg4-17 examines the events occurring in the first cell cycle after DNA damage. LOP is dependent upon de novo protein synthesis. LOP begins immediately after UV irradiation, before semiconservative DNA synthesis takes place, and is complete after four hours in growth medium.There is no evidence indicating whether the normal function of the protein is involved in excision repair, or in one of the two repair processes believed to be inducible; induced mutagenesis or recombinational repair

Double-strand break repair-adox: Restoration of suppressed double-strand break repair during mitosis induces genomic instability.

Science.gov (United States)

Terasawa, Masahiro; Shinohara, Akira; Shinohara, Miki

2014-12-01

Double-strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G1 phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non-homologous end joining or homologous recombination, respectively, and also modulation of the non-homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

The epidermal cell kinetic response to ultraviolet B irradiation combines regenerative proliferation and carcinogen associated cell cycle delay

Energy Technology Data Exchange (ETDEWEB)

Olsen, W.M.; Kirkhus, B. (Oslo Univ. (Norway))

1989-09-01

The cell cycle traverse of epidermal basal cells 24 h after in vivo exposure of ultraviolet B (UVB) irradiation was studied by immunochemical staining of incorporated bromodeoxyuridine (BrdU) and bivariate BrdU/DNA flow cytometric analysis. The results were compared with the cell kinetic patterns following topical application of the skin carcinogen methylnitrosourea (MNU) as well as the skin irritant cantharidin. The cell cycle traverse in hairless mouse epidermis 24 h after in vivo exposure to UVB seemed to be a combination of the cell kinetic effects following chemical skin carcinogens and skin irritants. UVB irradiation induced both a delay in transit time through S phase, probably due to DNA damage and subsequent repair, as well as a reduction in the total cell cycle time consistent with rapid regenerative proliferation. (author).

Isolating human DNA repair genes using rodent-cell mutants

International Nuclear Information System (INIS)

Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

1987-01-01

The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab

DNA repair and radiation sensitivity in mammalian cells

International Nuclear Information System (INIS)

Chen, D.J.C.; Stackhouse, M.; Chen, D.S.

1993-01-01

Ionizing radiation induces various types of damage in mammalian cells including DNA single-strand breaks, DNA double-strand breaks (DSB), DNA-protein cross links, and altered DNA bases. Although human cells can repair many of these lesions there is little detailed knowledge of the nature of the genes and the encoded enzymes that control these repair processes. We report here on the cellular and genetic analyses of DNA double-strand break repair deficient mammalian cells. It has been well established that the DNA double-strand break is one of the major lesions induced by ionizing radiation. Utilizing rodent repair-deficient mutant, we have shown that the genes responsible for DNA double-strand break repair are also responsible for the cellular expression of radiation sensitivity. The molecular genetic analysis of DSB repair in rodent/human hybrid cells indicate that at least 6 different genes in mammalian cells are responsible for the repair of radiation-induced DNA double-strand breaks. Mapping and the prospect of cloning of human radiation repair genes are reviewed. Understanding the molecular and genetic basis of radiation sensitivity and DNA repair in man will provide a rational foundation to predict the individual risk associated with radiation exposure and to prevent radiation-induced genetic damage in the human population

Small Engine Repair. Two-Stroke and Four-Stroke Cycle.

Science.gov (United States)

Hires, Bill; And Others

This curriculum guide is intended to assist persons teaching a course in repairing two- and four-stroke cycle small engines. Addressed in the individual units of instruction are the following topics: safety, tools, fasteners, and measurement techniques; basic small engine theory (engine identification and inspection, basic engine principles and…

Change of mitotic cycle and DNA repair in embryonic cells of rat, immortalized by E1 A oncogene and transformated by E1 A and c-Ha-Ras oncogenes under ionizing radiation action

International Nuclear Information System (INIS)

Kirillova, T.V.

1997-01-01

Comparison investigation into the repair of mitotic cycle and the reunion of DN single- and double-strand breaks in gamma-ray irradiated initial E1 A oncogene immortalized and E1 A and c-Ha-Ras oncogene transformed (mutant form) lines of rat embryonic fibroblasts was carried out. Possible involvement of Ras gene product in DNA repair speed governing and absence of tumor suppression function of p 53 protein in the embryonic and E1 A oncogene immortalized cells of rat fibroblast, as well as, presence of the mentioned function of p 53 protein in E1 A and c-Ha-Ras oncogene transformed cells were studied [ru

Common genetic variations in cell cycle and DNA repair pathways associated with pediatric brain tumor susceptibility

DEFF Research Database (Denmark)

Fahmideh, Maral Adel; Lavebratt, Catharina; Schüz, Joachim

2016-01-01

Knowledge on the role of genetic polymorphisms in the etiology of pediatric brain tumors (PBTs) is limited. Therefore, we investigated the association between single nucleotide polymorphisms (SNPs), identified by candidate gene-association studies on adult brain tumors, and PBT risk. The study is...... cycle and DNA repair pathways variations associated with susceptibility to adult brain tumors also seem to be associated with PBT risk, suggesting pediatric and adult brain tumors might share similar etiological pathways....

Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

Science.gov (United States)

Reinhardt, H Christian; Yaffe, Michael B

2013-09-01

Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

International Nuclear Information System (INIS)

Yezzi, M.J.

1985-01-01

A temperature-sensitive mutant for protein synthesis, CHO-TSH1, was compared to the wild-type cell, CHO-SC1, in single- and split-radiation-dose schemes. When the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal X-ray damage. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Distinct perturbations in the cell-cycle progression were noted following heat alone or heat with radiation. A delay in the progression of synchronized G 1 -phase and S-phase cells was demonstrated autoradiographically after inhibition of protein synthesis. In addition, treated S-phase cells showed a transient increase in the percent labelled cells after the cells were returned to their normal growth temperature of 35 0 C. This observation was suggestive of an unusual pattern of DNA synthesis during the recovery period. Split-dose experiments were done using incubation with cycloheximide to chemically inhibit protein synthesis. Both the chemical and thermal inhibition of protein synthesis substantiate its necessity for the repair of sublethal damage

Increased radiosensitivity of HPV-positive head and neck cancer cell lines due to cell cycle dysregulation and induction of apoptosis

International Nuclear Information System (INIS)

Arenz, Andrea; Ziemann, Frank; Wittig, Andrea; Preising, Stefanie; Engenhart-Cabillic, Rita; Mayer, Christina; Wagner, Steffen; Klussmann, Jens-Peter; Wittekindt, Claus; Dreffke, Kirstin

2014-01-01

Human Papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) respond favourably to radiotherapy as compared to HPV-unrelated HNSCC. We investigated DNA damage response in HPV-positive and HPV-negative HNSCC cell lines aiming to identify mechanisms, which illustrate reasons for the increased sensitivity of HPV-positive cancers of the oropharynx. Radiation response including clonogenic survival, apoptosis, DNA double-strand break (DSB) repair, and cell cycle redistribution in four HPV-positive (UM-SCC-47, UM-SCC-104, 93-VU-147T, UPCI:SCC152) and four HPV-negative (UD-SCC-1, UM-SCC-6, UM-SCC-11b, UT-SCC-33) cell lines was evaluated. HPV-positive cells were more radiosensitive (mean SF2: 0.198 range: 0.22-0.18) than HPV-negative cells (mean SF2: 0.34, range: 0.45-0.27; p = 0.010). Irradiated HPV-positive cell lines progressed faster through S-phase showing a more distinct accumulation in G2/M. The abnormal cell cycle checkpoint activation was accompanied by a more pronounced increase of cell death after x-irradiation and a higher number of residual and unreleased DSBs. The enhanced responsiveness of HPV-related HNSCC to radiotherapy might be caused by a higher cellular radiosensitivity due to cell cycle dysregulation and impaired DNA DSB repair. (orig.) [de

Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

Science.gov (United States)

Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

Science.gov (United States)

Kannappan, Ramaswamy; Mattapally, Saidulu; Wagle, Pooja A; Zhang, Jianyi

2018-05-18

The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells. Exposure to low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) measured by RAD50 and MRE11 expression, Checkpoint kinase 2 activation and γH2A.X recruitment at DNA strand breaks in both the cell groups indicating silencing p53-TAD do not affect DDR mechanism upstream of p53. Following removal of Doxo p53-WT hiPS cells underwent DNA repair, corrected their damaged DNA and restored DNA integrity. Conversely, p53-TAD KO hiPS cells did not undergo complete DNA repair and failed to restore DNA integrity. More importantly continuous culture of p53-TAD KO hiPS cells underwent G2/M cell cycle arrest and expressed cellular senescent marker p16 INK4a . Our data clearly shows that silencing transactivation domain of p53 did not affect DDR but affected the DNA repair process implying the crucial role of p53 transactivation domain in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent senescence.

Caffeine-enhanced survival of radiation-sensitive, repair-deficient Chinese hamster cells

International Nuclear Information System (INIS)

Utsumi, H.; Elkind, M.M.

1983-01-01

A clone of V79 Chinese hamster cells (V79-AL162/S-10) with unique properties has been isolated after a challenge of parental cells (V79-AL162) with 1 mM ouabain. Compared with parental cells, or with other clones isolated after the ouabain challenge, these cells form smaller colonies, are more sensitive to both x rays and fission-spectrum neutrons, and respond atypically to a postirradiation treatment with caffeine. Their enhanced response to x rays results mainly from a large reduction in the shoulder of their survival curve, probably because in late S phase, the most resistant phase in the cell cycle, the survival curve of these cells has a reduced shoulder width. Caffeine, and to a lesser extent theophylline, added to the colony-forming medium immediately after exposure appreciably increases the width of the shoulder of these sensitive cells, whereas caffeine has the opposite effect on the response of normal V79 cells. Thus the unique response of the V79-AL162/S-10 cells to a radiation posttreatment with caffeine (increased survival) results from a net increase in their ability to repair damage that is otherwise lethal; caffeine treatment ordinarly prevents normal V79 cells from repairing damage that is only potentially lethal

Repair of traumatized mammalian hair cells via sea anemone repair proteins.

Science.gov (United States)

Tang, Pei-Ciao; Smith, Karen Müller; Watson, Glen M

2016-08-01

Mammalian hair cells possess only a limited ability to repair damage after trauma. In contrast, sea anemones show a marked capability to repair damaged hair bundles by means of secreted repair proteins (RPs). Previously, it was found that recovery of traumatized hair cells in blind cavefish was enhanced by anemone-derived RPs; therefore, the ability of anemone RPs to assist recovery of damaged hair cells in mammals was tested here. After a 1 h incubation in RP-enriched culture media, uptake of FM1-43 by experimentally traumatized murine cochlear hair cells was restored to levels comparable to those exhibited by healthy controls. In addition, RP-treated explants had significantly more normally structured hair bundles than time-matched traumatized control explants. Collectively, these results indicate that anemone-derived RPs assist in restoring normal function and structure of experimentally traumatized hair cells of the mouse cochlea. © 2016. Published by The Company of Biologists Ltd.

Effect of failures and repairs on multiple cell production lines

Energy Technology Data Exchange (ETDEWEB)

Legato, P.; Bobbio, A.; Roberti, L.

1989-01-01

This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

Repair of radiation damage in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Setlow, R.B.

1981-01-01

The responses, such as survival, mutation, and carcinogenesis, of mammalian cells and tissues to radiation are dependent not only on the magnitude of the damage to macromolecular structures - DNA, RNA, protein, and membranes - but on the rates of macromolecular syntheses of cells relative to the half-lives of the damages. Cells possess a number of mechanisms for repairing damage to DNA. If the repair systems are rapid and error free, cells can tolerate much larger doses than if repair is slow or error prone. It is important to understand the effects of radiation and the repair of radiation damage because there exist reasonable amounts of epidemiological data that permits the construction of dose-response curves for humans. The shapes of such curves or the magnitude of the response will depend on repair. Radiation damage is emphasized because: (a) radiation dosimetry, with all its uncertainties for populations, is excellent compared to chemical dosimetry; (b) a number of cancer-prone diseases are known in which there are defects in DNA repair and radiation results in more chromosomal damage in cells from such individuals than in cells from normal individuals; (c) in some cases, specific radiation products in DNA have been correlated with biological effects, and (d) many chemical effects seem to mimic radiation effects. A further reason for emphasizing damage to DNA is the wealth of experimental evidence indicating that damages to DNA can be initiating events in carcinogenesis.

Repair of radiation damage in mammalian cells

International Nuclear Information System (INIS)

Setlow, R.B.

1981-01-01

The responses, such as survival, mutation, and carcinogenesis, of mammalian cells and tissues to radiation are dependent not only on the magnitude of the damage to macromolecular structures - DNA, RNA, protein, and membranes - but on the rates of macromolecular syntheses of cells relative to the half-lives of the damages. Cells possess a number of mechanisms for repairing damage to DNA. If the repair systems are rapid and error free, cells can tolerate much larger doses than if repair is slow or error prone. It is important to understand the effects of radiation and the repair of radiation damage because there exist reasonable amounts of epidemiological data that permits the construction of dose-response curves for humans. The shapes of such curves or the magnitude of the response will depend on repair. Radiation damage is emphasized because: (a) radiation dosimetry, with all its uncertainties for populations, is excellent compared to chemical dosimetry; (b) a number of cancer-prone diseases are known in which there are defects in DNA repair and radiation results in more chromosomal damage in cells from such individuals than in cells from normal individuals; (c) in some cases, specific radiation products in DNA have been correlated with biological effects, and (d) many chemical effects seem to mimic radiation effects. A further reason for emphasizing damage to DNA is the wealth of experimental evidence indicating that damages to DNA can be initiating events in carcinogenesis

DNA repair in murine embryonic stem cells and differentiated cells

International Nuclear Information System (INIS)

Tichy, Elisia D.; Stambrook, Peter J.

2008-01-01

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells

Cytogenetic Response to Ionizing Radiation Exposure in Human Fibroblasts with Suppressed Expression of Non-DSB Repair Genes

Science.gov (United States)

Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Hammond, Dianne; Mehta, Satish K.; Jeevarajan, Antony S.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in radiation-induced chromosome aberrations and micronuclei formation. In the study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequencies of micronuclei (MN) formation and chromosome aberrations were measured to determine the efficiency of cytogenetic repair, and the fraction of bi-nucleated cells in the MN analysis was used as a marker for cell cycle progression. In response to gamma radiation, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR

Cell sensitivity to irradiation and DNA repair processes

International Nuclear Information System (INIS)

Kozubek, S.; Krasavin, E.A.

1984-01-01

A new model of oxygen effect realisation is proposed for E.coli cells. The model explains differencies in oxygen enhancement ratio (OER) between wild type cells and repair deficient mutants. These differencies are logically linked to corresponding defects in repair systems. A quantitative analysis has been performed. The dependence of OER and cell sensitivity on the properties of cultivation medium is considered, too. Decreasing OER and increasing sensitivity in poor conditions are explained as the consequence of the shift of repair capacity from slow to fast repair system

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Wyrobek, Andrew J.

2009-01-18

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

Roles of Rad51 protein in homologous recombination in mammalian cells: relation with repair, replication and cell cycle

International Nuclear Information System (INIS)

Lambert, S.

2001-01-01

Homologous recombination (HR) is a fundamental process, allowing a faithful repair. In mammalian, MmRAD51, which is the homologue of Saccharomyces cerevisiae ScRAD51 key protein for HR, is an essential gene. This work is based on the characterisation of viable hyper and hypo-recombinant cell lines specifically affected in the Rad51 pathway. By expressing wild type and dominant negative forms of MmRad51, we demonstrated that Rad51 pathway participates to the repair by HR to induced DNA damages. However, inhibition of the Rad 51 pathway does not affect cell viability, spontaneously or after irradiation, whereas, radiation induced HR is inhibited. In the presence of DNA damages during late S and G2/M phase, inhibition of Rad51 pathway induced chromosomal aberrations, leading to a transient arrest in mitosis. This arrest is associated with an increased of cell death. However, a fraction of cells can escape from this transient arrest by forming tetraploid cells, associated with an absence of chromalid separation. Thus, in response to impaired Rad51 pathway, mitotic checkpoints seems to play an essential role. In line with this, we showed that the essential function of Rad51 is p53-dependent, which is in agreement with the role of p53 in tetraploidy inhibition. Our results suggest that the Rad51 protein could participate to the control of mitotic checkpoints and thus to the maintenance of genetic stability. This function could involve other Rad51 partners such as the tumour suppressors BRCA1, BRCA2 and p53. (author) [fr

Capacity of ultraviolet-induced DNA repair in human glioma cells

Energy Technology Data Exchange (ETDEWEB)

Itoh, Hiroji

1987-04-01

A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment.

Induced repair and mutagenesis in animal cells

International Nuclear Information System (INIS)

Takimoto, Koichi

1981-01-01

Induced repair and mutagenesis of animal cells against UV were studied in contrast with SOS repair of E. coli primarily by the use of viruses. Since UV-enhanced reactivation is a phenomenon similar to UV-reactivation (mutagenesis) and the presence of lesion bypass synthsis has been suggested, UV-enhanced reactivation has several common aspects with SOS reactivation of E. coli. However, correlation is not necessarily noted between increase in the viral survival rate and mutagenesis, nor do protease blockers exert any effect. Therefore, SOS repair of E. coli may have different mechansms from induced repair and mutagenesis in animal cells. (Ueda, J.)

DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

Science.gov (United States)

Pinto-Fernandez, Adan; Kessler, Benedikt M

2016-01-01

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

DUBbing cancer: Deubiquitylating enzymes involved in epigenetics, DNA damage and the cell cycle as therapeutic targets

Directory of Open Access Journals (Sweden)

Benedikt M Kessler

2016-07-01

Full Text Available Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs, have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

MicroRNA-Related DNA Repair/Cell-Cycle Genes Independently Associated With Relapse After Radiation Therapy for Early Breast Cancer

Energy Technology Data Exchange (ETDEWEB)

Gee, Harriet E., E-mail: harriet.gee@sydney.edu.au [The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW (Australia); The Chris O' Brien Lifehouse, Missenden Road, Camperdown, NSW (Australia); Central Clinical School, Sydney Medical School, University of Sydney, NSW (Australia); Buffa, Francesca M.; Harris, Adrian L. [Department of Medical Oncology, The University of Oxford, Oxford (United Kingdom); Toohey, Joanne M.; Carroll, Susan L. [The Chris O' Brien Lifehouse, Missenden Road, Camperdown, NSW (Australia); Cooper, Caroline L. [Central Clinical School, Sydney Medical School, University of Sydney, NSW (Australia); Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW (Australia); Beith, Jane [The Chris O' Brien Lifehouse, Missenden Road, Camperdown, NSW (Australia); McNeil, Catriona [The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW (Australia); The Chris O' Brien Lifehouse, Missenden Road, Camperdown, NSW (Australia); Carmalt, Hugh; Mak, Cindy; Warrier, Sanjay [The Chris O' Brien Lifehouse, Missenden Road, Camperdown, NSW (Australia); Holliday, Anne [The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW (Australia); Selinger, Christina; Beckers, Rhiannon [Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW (Australia); Kennedy, Catherine [Central Clinical School, Sydney Medical School, University of Sydney, NSW (Australia); Graham, Peter [Department of Radiation Oncology, Cancer Care Centre, St. George Hospital, Kogarah, NSW (Australia); Swarbrick, Alexander [The Kinghorn Cancer Centre & Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW (Australia); St Vincent' s Clinical School, Faculty of Medicine, University of NSW, Kensington, NSW (Australia); and others

2015-12-01

Purpose: Local recurrence and distant failure after adjuvant radiation therapy for breast cancer remain significant clinical problems, incompletely predicted by conventional clinicopathologic markers. We had previously identified microRNA-139-5p and microRNA-1274a as key regulators of breast cancer radiation response in vitro. The purpose of this study was to investigate standard clinicopathologic markers of local recurrence in a contemporary series and to establish whether putative target genes of microRNAs involved in DNA repair and cell cycle control could better predict radiation therapy response in vivo. Methods and Materials: With institutional ethics board approval, local recurrence was measured in a contemporary, prospectively collected series of 458 patients treated with radiation therapy after breast-conserving surgery. Additionally, independent publicly available mRNA/microRNA microarray expression datasets totaling >1000 early-stage breast cancer patients, treated with adjuvant radiation therapy, with >10 years of follow-up, were analyzed. The expression of putative microRNA target biomarkers—TOP2A, POLQ, RAD54L, SKP2, PLK2, and RAG1—were correlated with standard clinicopathologic variables using 2-sided nonparametric tests, and to local/distant relapse and survival using Kaplan-Meier and Cox regression analysis. Results: We found a low rate of isolated local recurrence (1.95%) in our modern series, and that few clinicopathologic variables (such as lymphovascular invasion) were significantly predictive. In multiple independent datasets (n>1000), however, high expression of RAD54L, TOP2A, POLQ, and SKP2 significantly correlated with local recurrence, survival, or both in univariate and multivariate analyses (P<.001). Low RAG1 expression significantly correlated with local recurrence (multivariate, P=.008). Additionally, RAD54L, SKP2, and PLK2 may be predictive, being prognostic in radiation therapy–treated patients but not in untreated matched

Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs

International Nuclear Information System (INIS)

Savas, Sevtap; Ozcelik, Hilmi

2005-01-01

Phosphorylation is a reversible post-translational modification that affects the intrinsic properties of proteins, such as structure and function. Non-synonymous single nucleotide polymorphisms (nsSNPs) result in the substitution of the encoded amino acids and thus are likely to alter the phosphorylation motifs in the proteins. In this study, we used the web-based NetPhos tool to predict candidate nsSNPs that either introduce or remove putative phosphorylation sites in proteins that act in DNA repair and cell cycle pathways. Our results demonstrated that a total of 15 nsSNPs (16.9%) were likely to alter the putative phosphorylation patterns of 14 proteins. Three of these SNPs (CDKN1A-S31R, OGG1-S326C, and XRCC3-T241M) have already found to be associated with altered cancer risk. We believe that this set of nsSNPs constitutes an excellent resource for further molecular and genetic analyses. The novel systematic approach used in this study will accelerate the understanding of how naturally occurring human SNPs may alter protein function through the modification of phosphorylation mechanisms and contribute to disease susceptibility

Adaptive repair induced by small doses of γ radiation in repair-defective human cells

International Nuclear Information System (INIS)

Zasukhina, G.D.; L'vova, G.N.; Vasil'eva, I.M.; Sinel'shchikova, T.A.; Semyachkina, A.N.

1993-01-01

Adaptive repair induced by small doses of gamma radiation was studied in repair-defective xeroderma pigmentosum, gout, and homocystinuria cells. The adaptation of cells induced by small doses of radiation was estimated after subsequent exposure to gamma radiation, 4-nitroquinoline-1-oxide, and N-methyl-N-nitro-N-nitrosoguanidine by three methods: (1) by the reduction in DNA breaks; (2) by induction of resistant DNA synthesis; and (3) by increased reactivation of vaccinia virus. The three cell types in response to the three different mutagens revealed differences in the mechanism of cell defense in excision repair, in the adaptive response, and in Weigl reactivation

Toona Sinensis Extracts Induced Cell Cycle Arrest and Apoptosis in the Human Lung Large Cell Carcinoma

Directory of Open Access Journals (Sweden)

Cheng-Yuan Wang

2010-02-01

Full Text Available Toona sinensis extracts have been shown to exhibit anti-cancer effects in human ovarian cancer cell lines, human promyelocytic leukemia cells and human lung adenocarcinoma. Its safety has also been confirmed in animal studies. However, its anti-cancer properties in human lung large cell carcinoma have not been studied. Here, we used a powder obtained by freeze-drying the super-natant of centrifuged crude extract from Toona sinensis leaves (TSL-1 to treat the human lung carcinoma cell line H661. Cell viability was evaluated by the 3-(4-,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. Flow cytometry analysis revealed that TSL-1 blocked H661 cell cycle progression. Western blot analysis showed decreased expression of cell cycle proteins that promote cell cycle progression, including cyclin-dependent kinase 4 and cyclin D1, and increased the expression of proteins that inhibit cell cycle progression, including p27. Furthermore, flow cytometry analysis showed that TSL-1 induced H661 cell apoptosis. Western blot analysis showed that TSL-1 reduced the expression of the anti-apoptotic protein B-cell lymphoma 2, and degraded the DNA repair protein, poly(ADP-ribose polymerase. TSL-1 shows potential as a novel therapeutic agent or for use as an adjuvant for treating human lung large cell carcinoma.

DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells.

LENUS (Irish Health Repository)

Martin, Lynn M

2013-07-10

DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2 Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2 h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24 h after 0.2 Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR.

Cell-cycle variation in the induction of lethality and mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Davies, P.J.; Tippins, R.S.; Parry, J.M.

1978-01-01

Exponentially growing yeast cultures separated into discrete periods of the cell cycle by zonal rotor centrifugation show cyclic variation in both UV and nitrous acid induced cell lethality, mitotic gene conversion and mitotic crossing-over. Maximum cell survival after UV treatment was observed in the S and G2 phases of the cell cycle at a time when UV induction of both types of mitotic recombination was at a minumum. In contrast, cell inactivation by the chemical mutagen nitrous acid showed a single discrete period of sensitivity which occurred in S phase cells which are undergoing DNA synthesis. Mitotic gene conversion ahd mitotic crossing-over were induced by nitrous acid in cells at all stages of the cell cycle with a peak of induction of both events occurring at the time of maximum cell lethality. The lack of correlation observed between maximum cell survival and the maximum induction of mitotic intragenic recombination suggest that other DNA-repair mechanisms besides DNA-recombination repair are involved in the recovery of inactivated yeast cells during the cell cycle. (Auth.)

DNA repair in human bronchial epithelial cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

1982-01-01

The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

Directory of Open Access Journals (Sweden)

Katherine S Lawrence

2015-04-01

Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

Radioimmunoassay studies on repair of ultraviolet damaged DNA in cultured animal cells

International Nuclear Information System (INIS)

Yatani, Ryuichi; Tohgo, Yukihiro; Kunishima, Nobuyoshi.

1975-01-01

UV (ultraviolet) damaged DNA and its repair of various cultured animal cells were observed by radioimmunoassay using anti-serum against the UV irradiation induced heat-degenerated DNA. There is some difference among the cells of used animals according to their DNA repairabilities. The cells were divided into four groups according to the existence or strength of their repairabilities. 1) excision repair type: cells of men and chimpanzees. 2) photoreactivation type: cells derived from Tachydromus tachydromoides and chicks. 3) photoreactivation with excision repair: cells of rats, kangaroos and mosquitos. 4) non-excision repair type: cells of mice, Meriones and rats. Animal cells have plural types of repair. Main types of repair will differ according to the kind of animals. (Ichikawa, K.)

Regulation of DNA repair processes in mammalian cell

International Nuclear Information System (INIS)

Bil'din, V.N.; Sergina, T.B.; Zhestyanikov, V.D.

1992-01-01

A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase α and β (aphidicolin) and DNA polymerase β (2', 3'-dideoxythjymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gy) or by γ-ray irradiation (150 Gy) is more sensitive to aphidicolin and mitogen-simulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase α

DNA repair in mammalian cells exposed to combinations of carcinogenic agents

International Nuclear Information System (INIS)

Setlow, R.B.; Ahmed, F.E.

1979-01-01

Cells defective in one or more aspects of repair are killed and often mutagenized more readily than normal cells by DNA damaging agents, and humans whose cells are deficient in repair are at an increased carcinogenic risk compared to normal individuals. The excision repair of uv induced pyrimidine dimers is a well studied system, but the details of the steps in this repair system are far from being understood in human cells. We know that there are a number of chemicals that mimic uv in that normal human cells repair DNA damage from both these agents and from uv by a long patch excision repair system, and that xeroderma pigmentosum cells defective in repair of uv are also defective in the repair of damage from these chemicals. The chemicals we have investigated are AAAF, 4-NQO, DMBA-epoxide, and ICR-170. We describe experiments, using several techniques, in which DNA excision repair is measured after treatment of various human cell strains with combinations of uv and these agents. If two agents have a common rate limiting step then, at doses high enough to saturate the repair system, one would expect the observed repair after a treatment with a combination of agents to be equal to that from one agent alone. Such is not the case for normal human or excision-deficient XP cells. In the former repair is additive and in the latter repair is usually appreciably less than that observed with either agent alone. Models that attempt to explain these surprising results involve complexes of enzymes and cofactors

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

Science.gov (United States)

Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

2012-01-01

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

DNA-radiosensitivity and repair in mammolian cells

International Nuclear Information System (INIS)

Proskuryakov, S.Ya.; Ivannik, B.P.; Ryabchenko, N.I.

1979-01-01

Determination was made of the formation and repair of single-stranded DNA breaks (SB) in cells of rat thymus and liver and Ehrlich's ascites tumor (EAT) with the use of the method of low-gradient viscosimetry of alkaline cell lysates. The radiochemical yield of single-stranded breaks (Gsub(SB)) induced by irradiation of animals is 41.2 eV/break for hepatocytes, 96.8 eV/break, for thymocytes, and 129.7 eV/break, for EAT cells. The half-recovery time of single-stranded DNA breaks for cells of thymus and EAT exposed in vivo is 16.0 and 5.1 s -1 , correspondingly. In hepatocytes exposed in vivo and in vitro no repairs occurs for 3 h. Under conditions of inhibition of SB repair, when suspensions of thymocytes and hepatocytes were exposed in vitro at 4 deg C, Gsub(SB) is 35.5 and 38.7 eV/break, respectively. The analysis of the data obtained prompts the conclusion that under in vivo conditions, there is a correlation between DNA radiosensitivity and the rate of repair processes

Proliferating cell nuclear antigen (PCNA)-associated KIAA0101/PAF15 protein is a cell cycle-regulated anaphase-promoting complex/cyclosome substrate.

Science.gov (United States)

Emanuele, Michael J; Ciccia, Alberto; Elia, Andrew E H; Elledge, Stephen J

2011-06-14

The anaphase-promoting complex/cyclosome (APC/C) is a cell cycle-regulated E3 ubiquitin ligase that controls the degradation of substrate proteins at mitotic exit and throughout the G1 phase. We have identified an APC/C substrate and cell cycle-regulated protein, KIAA0101/PAF15. PAF15 protein levels peak in the G2/M phase of the cell cycle and drop rapidly at mitotic exit in an APC/C- and KEN-box-dependent fashion. PAF15 associates with proliferating cell nuclear antigen (PCNA), and depletion of PAF15 decreases the number of cells in S phase, suggesting a role for it in cell cycle regulation. Following irradiation, PAF15 colocalized with γH2AX foci at sites of DNA damage through its interaction with PCNA. Finally, PAF15 depletion led to an increase in homologous recombination-mediated DNA repair, and overexpression caused sensitivity to UV-induced DNA damage. We conclude that PAF15 is an APC/C-regulated protein involved in both cell cycle progression and the DNA damage response.

Repetitious nature of repaired DNA in mammalian cells

International Nuclear Information System (INIS)

1978-01-01

The report consists of three appendices, as follows: summary of preliminary studies of the comparative DNA repair in normal lymphoblastoid and Burkitt's lymphoma cell lines; nonuniform reassociation of human lymphoblastoid cell DNA repair replicated following methyl methane sulfonate treatment; and preliminary DNA single-strand breakage studies in the L5178Y cell line

Skin appendage-derived stem cells: cell biology and potential for wound repair.

Science.gov (United States)

Xie, Jiangfan; Yao, Bin; Han, Yutong; Huang, Sha; Fu, Xiaobing

2016-01-01

Stem cells residing in the epidermis and skin appendages are imperative for skin homeostasis and regeneration. These stem cells also participate in the repair of the epidermis after injuries, inducing restoration of tissue integrity and function of damaged tissue. Unlike epidermis-derived stem cells, comprehensive knowledge about skin appendage-derived stem cells remains limited. In this review, we summarize the current knowledge of skin appendage-derived stem cells, including their fundamental characteristics, their preferentially expressed biomarkers, and their potential contribution involved in wound repair. Finally, we will also discuss current strategies, future applications, and limitations of these stem cells, attempting to provide some perspectives on optimizing the available therapy in cutaneous repair and regeneration.

miR-24-mediated down-regulation of H2AX suppresses DNA repair in terminally differentiated blood cells

Science.gov (United States)

Lal, Ashish; Pan, Yunfeng; Navarro, Francisco; Dykxhoorn, Derek M.; Moreau, Lisa; Meire, Eti; Bentwich, Zvi; Lieberman, Judy; Chowdhury, Dipanjan

2010-01-01

Terminally differentiated cells have reduced capacity to repair double strand breaks (DSB), but the molecular mechanism behind this down-regulation is unclear. Here we find that miR-24 is consistently up-regulated during post-mitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a key DSB repair protein that activates cell cycle checkpoint proteins and retains DSB repair factors at DSB foci. The H2AX 3’UTR contains conserved miR-24 binding sites regulated by miR-24. Both H2AX mRNA and protein are substantially reduced during hematopoietic cell terminal differentiation by miR-24 up-regulation both in in vitro differentiated cells and primary human blood cells. miR-24 suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs. Antagonizing miR-24 in differentiating cells protects them from DNA damage-induced cell death, while transfecting miR-24 mimics in dividing cells increases chromosomal breaks and unrepaired DNA damage and reduces viability in response to DNA damage. This DNA repair phenotype can be fully rescued by over-expressing miR-24-insensitive H2AX. Therefore, miR-24 up-regulation in post-replicative cells reduces H2AX and thereby renders them highly vulnerable to DNA damage. PMID:19377482

Repair processes for photochemical damage in mammalian cells

International Nuclear Information System (INIS)

Cleaver, J.E.

1974-01-01

Repair processes for photochemical damage in cells following uv irradiation are reviewed. Cultured fibroblast cells from human patients with xeroderma pigmentosum were used as an example to illustrate aspects of repair of injuries to DNA and proteins. (250 references) (U.S.)

DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

Energy Technology Data Exchange (ETDEWEB)

Ford, M.D.; Houldsworth, J.; Lavin, M.F. (Queensland Univ., Brisbane (Australia). Dept. of Biochemistry)

1981-12-01

The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair ..gamma..-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to ..gamma..-radiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrated to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10-40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.

Exonuclease 1 is a critical mediator of survival during DNA double strand break repair in nonquiescent hematopoietic stem and progenitor cells.

Science.gov (United States)

Desai, Amar; Qing, Yulan; Gerson, Stanton L

2014-02-01

Hematopoietic stem cell (HSC) populations require DNA repair pathways to maintain their long-term survival and reconstitution capabilities, but mediators of these processes are still being elucidated. Exonuclease 1 (Exo1) participates in homologous recombination (HR) and Exo1 loss results in impaired 5' HR end resection. We use cultured Exo1(mut) fibroblasts and bone marrow to demonstrate that loss of Exo1 function results in defective HR in cycling cells. Conversely, in Exo1(mut) mice HR is not required for maintenance of quiescent HSCs at steady state, confirming the steady state HSC reliance on nonhomologous end joining (NHEJ). Exo1(mut) mice sustained serial repopulation, displayed no defect in competitive repopulation or niche occupancy, and exhibited no increased sensitivity to whole body ionizing radiation. However, when Exo1(mut) HSCs were pushed into cell cycle in vivo with 5-fluorouracil or poly IC, the hematopoietic population became hypersensitive to IR, resulting in HSC defects and animal death. We propose Exo1-mediated HR is dispensable for stem cell function in quiescent HSC, whereas it is essential to HSC response to DNA damage processing after cell cycle entry, and its loss is not compensated by intact NHEJ. In HSCs, the maintenance of stem cell function after DNA damage is dependent on the DNA repair capacity, segregated by active versus quiescent points in cell cycle. © AlphaMed Press.

Distinctive adaptive response to repeated exposure to hydrogen peroxide associated with upregulation of DNA repair genes and cell cycle arrest

Directory of Open Access Journals (Sweden)

Gloria A. Santa-Gonzalez

2016-10-01

Full Text Available Many environmental and physiological stresses are chronic. Thus, cells are constantly exposed to diverse types of genotoxic insults that challenge genome stability, including those that induce oxidative DNA damage. However, most in vitro studies that model cellular response to oxidative stressors employ short exposures and/or acute stress models. In this study, we tested the hypothesis that chronic and repeated exposure to a micromolar concentration of hydrogen peroxide (H2O2 could activate DNA damage responses, resulting in cellular adaptations. For this purpose, we developed an in vitro model in which we incubated mouse myoblast cells with a steady concentration of ~50 μM H2O2 for one hour daily for seven days, followed by a final challenge of a 10 or 20X higher dose of H2O2 (0.5 or 1 mM. We report that intermittent long-term exposure to this oxidative stimulus nearly eliminated cell toxicity and significantly decreased genotoxicity (in particular, a >5-fold decreased in double-strand breaks resulting from subsequent acute exposure to oxidative stress. This protection was associated with cell cycle arrest in G2/M and induction of expression of nine DNA repair genes. Together, this evidence supports an adaptive response to chronic, low-level oxidative stress that results in genomic protection and up-regulated maintenance of cellular homeostasis.

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

International Nuclear Information System (INIS)

Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

1991-01-01

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

Low concentrations of antimony impair DNA damage signaling and the repair of radiation-induced DSB in HeLa S3 cells.

Science.gov (United States)

Koch, Barbara; Maser, Elena; Hartwig, Andrea

2017-12-01

Antimony is utilized in a large variety of industrial applications, leading to significant environmental and occupational exposure. Mainly based on animal experiments, the IARC and MAK Commission have classified antimony and its inorganic compounds as Group 2B or 2 carcinogens, respectively. However, the underlying mode(s) of action are still largely unknown. In the present study, we investigated the impact of non-cytotoxic up to cytotoxic concentrations of SbCl 3 on DNA DSB repair and cell cycle control in HeLa S3 cells. We induced DSB by γ-irradiation and analyzed inhibitory actions of antimony on potential molecular targets of the DSB repair machinery. Antimony disturbed cell cycle control, affecting phosphorylation of Chk1. Furthermore, the repair of DSB was impaired in the presence of antimony, as monitored by pulsed-field gel electrophoresis and γH2AX foci formation of cells in G1 and G2 phase. Specifically, BRCA1 and RAD51 were identified as molecular targets. Our results point towards an interference with both non-homologous end-joining (NHEJ) and homologous recombination (HR), and inhibitory effects may be explained by interactions with critical cysteine groups; this needs to be further investigated. Altogether, the results provide further evidence for the impairment of DNA repair processes as one underlying mechanism in antimony-induced carcinogenicity.

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

Science.gov (United States)

Ogara, María F; Sirkin, Pablo F; Carcagno, Abel L; Marazita, Mariela C; Sonzogni, Silvina V; Ceruti, Julieta M; Cánepa, Eduardo T

2013-01-01

The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

Chromatin relaxation-mediated induction of p19INK4d increases the ability of cells to repair damaged DNA.

Directory of Open Access Journals (Sweden)

María F Ogara

Full Text Available The maintenance of genomic integrity is of main importance to the survival and health of organisms which are continuously exposed to genotoxic stress. Cells respond to DNA damage by activating survival pathways consisting of cell cycle checkpoints and repair mechanisms. However, the signal that triggers the DNA damage response is not necessarily a direct detection of the primary DNA lesion. In fact, chromatin defects may serve as initiating signals to activate those mechanisms. If the modulation of chromatin structure could initiate a checkpoint response in a direct manner, this supposes the existence of specific chromatin sensors. p19INK4d, a member of the INK4 cell cycle inhibitors, plays a crucial role in regulating genomic stability and cell viability by enhancing DNA repair. Its expression is induced in cells injured by one of several genotoxic treatments like cis-platin, UV light or neocarzinostatin. Nevertheless, when exogenous DNA damaged molecules are introduced into the cell, this induction is not observed. Here, we show that p19INK4d is enhanced after chromatin relaxation even in the absence of DNA damage. This induction was shown to depend upon ATM/ATR, Chk1/Chk2 and E2F activity, as is the case of p19INK4d induction by endogenous DNA damage. Interestingly, p19INK4d improves DNA repair when the genotoxic damage is caused in a relaxed-chromatin context. These results suggest that changes in chromatin structure, and not DNA damage itself, is the actual trigger of p19INK4d induction. We propose that, in addition to its role as a cell cycle inhibitor, p19INK4d could participate in a signaling network directed to detecting and eventually responding to chromatin anomalies.

125IdUrd-induced chromosome fragments, assayed by premature chromosome condensation, and DNA double-strand breaks have similar repair kinetics in G1-phase CHO-cells

International Nuclear Information System (INIS)

Iliakis, George; Pantelias, G.E.; Okayasu, Ryuichi; Seaner, Robert

1987-01-01

The effect of 125 I-decay on cell lethality, and induction of chromosome and DNA damage, was studied in synchronous non-cycling, G 1 -phase CHO-cells. Neutral filter elution was used to assay repair of DNA double-strand breaks (dsbs), and premature chromosome condensation was used to assay repair of chromosome fragments and induction of ring chromosomes. The results indicate very little repair at the cell survival level (repair of PLD). At the DNA level an efficient repair of DNA dsbs was observed, with kinetics similar to those observed after exposure to X-rays. At the chromosome level a fast repair of prematurely condensed chromosome fragments was observed, with a concomitant increase in the number of ring chromosomes induced. The repair kinetics of chromosome fragments and DNA dsbs were very similar, suggesting that DNA dsbs may underlie chromosome fragmentation. (author)

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

Science.gov (United States)

Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

2016-01-01

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

PLD-Repair after ''holding'' of rodent cell cultures: A critical annotation

International Nuclear Information System (INIS)

Stevenson, A.F.G.; Lange, C.S.

1987-01-01

The radiosensitivity of cells in vitro has been reported to be influenced by culture-conditions both before and after exposure to ionising radiations. The effect of ''Stationary-Holding'' and/or delayed plating on the repair of potentially lethal damage (PLDR) are such examples. The so-called Stationary or Plateau-Phase of commonly used cell lines like the V79 are often quiescent (non-mitotic). The authors analysis of the V79 ''Plateau-Phase'' (S-phase cytocide through VrUdR incorporation with FuDR block and light exposure) reveals that the cultures have merely reached a state of maximal permissible density and are, in fact, in kinetic equilibrium with at least half the population of cells, even in densely confluent cultures, going through cycle. Attempts at holding the cells from cycling (through nutrient-depletion and serum-privation) were unsuccessful, although the turn-over-rate was reduced. Their assays for X-irradiated clonogenic survivors after attempted holding combined with delayed plating (DP) showed differences in the survival curves for exponentially growing (Log) and confluent cultures, Although DP improved survival in general, it abolished the shoulder in Log cultures. These observations may explain why most PLDR is seen in the second and lower survival-decades. The significance of the persistent cycling fraction in the interpretation of PLDR after delayed plating must be considered

DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

International Nuclear Information System (INIS)

Inoue, Masao; Takebe, Hiraku.

1978-01-01

Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells

International Nuclear Information System (INIS)

Freed, J.J.; Hoess, R.H.; Angelosanto, F.A.; Massey, H.C. Jr.

1979-01-01

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (D 0 ) in the range 1.5-1.7 Jm -2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dsub(q)), the value for exponentially multiplying diploid cells (3.0 Jm -2 ) being higher than that found for haploids (1.2 Jm -2 ). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromoatographically during up to 48 h incubation in the dark and since autoradiographically detected 'unscheduled DNA synthesis' was limited to a 2-fold increase saturated at 10 Jm -2 . These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield. (Auth.)

Cell-free assay measuring repair DNA synthesis in human fibroblasts

International Nuclear Information System (INIS)

Ciarrocchi, G.; Linn, S.

1978-01-01

Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks

International Nuclear Information System (INIS)

Turney, Benjamin W.; Kerr, Martin; Chitnis, Meenali M.; Lodhia, Kunal; Wang, Yong; Riedemann, Johann; Rochester, Mark; Protheroe, Andrew S.; Brewster, Simon F.; Macaulay, Valentine M.

2012-01-01

Background and purpose: IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. Methods: We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. Results: We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30–40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. Conclusions: These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments.

Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks.

Science.gov (United States)

Turney, Benjamin W; Kerr, Martin; Chitnis, Meenali M; Lodhia, Kunal; Wang, Yong; Riedemann, Johann; Rochester, Mark; Protheroe, Andrew S; Brewster, Simon F; Macaulay, Valentine M

2012-06-01

IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30-40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Endogenous Cartilage Repair by Recruitment of Stem Cells.

Science.gov (United States)

Im, Gun-Il

2016-04-01

Articular cartilage has a very limited capacity for repair after injury. The adult body has a pool of stem cells that are mobilized during injury or disease. These cells exist inside niches in bone marrow, muscle, adipose tissue, synovium, and other connective tissues. A method that mobilizes this endogenous pool of stem cells will provide a less costly and less invasive alternative if these cells successfully regenerate defective cartilage. Traditional microfracture procedures employ the concept of bone marrow stimulation to regenerate cartilage. However, the regenerated tissue usually is fibrous cartilage, which has very poor mechanical properties compared to those of normal hyaline cartilage. A method that directs the migration of a large number of autologous mesenchymal stem cells toward injury sites, retains these cells around the defects, and induces chondrogenic differentiation that would enhance success of endogenous cartilage repair. This review briefly summarizes chemokines and growth factors that induce recruitment, proliferation, and differentiation of endogenous progenitor cells, endogenous cell sources for regenerating cartilage, scaffolds for delivery of bioactive factors, and bioadhesive materials that are necessary to bring about endogenous cartilage repair.

Inhibition of topoisomerase II activity in repair-proficient CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

International Nuclear Information System (INIS)

Grdina, D.J.; Constantinou, A.; Shigematsu, N.

1992-09-01

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector under in vitro conditions when it is administered 30 min prior to radiation exposure at a concentration of 4 mM to repair-proficient Chinese hamster ovary Kl cells (i.e., a dose modification factor of 1.4). In contrast, the DNA double-strand break, repair-deficient Chinese hamster ovary xrs-5 cell line is not protected under these conditions (i.e., a dose modification factor of 1.0). Topoisomerase (topo) I and II activities and protein contents were measured in both Kl and xrs-5 cell lines and were found to be similar in magnitude. Neither exposure to radiation, to WR-1065, or to both affected these variables in xrs-5 cells. WR 1065 was effective, however, in reducing topo 11 activity by a factor of 2 in the repair-proficient Kl cell line. Topo II protein content, however, was not affected by these exposure conditions. One of several mechanisms of radiation protection attributed to aminothiol compounds has been their ability to affect enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results demonstrate a modifying effect by 2-[(aminopropyl)amino]ethanethiol on a specific nuclear enzyme (i.e., type H topoisomerase), which is involved in DNA synthesis. These results also suggest that differences do exist between the topo 11 enzymes isolated from the parent repair-proficient Kl and the DNA double-strand break, repair-deficient xrs-5 mutant cell lines

Inhibition of topoisomerase II activity in repair-proficient CHO K1 cells by 2-[(aminopropyl)amino]ethanethiol (WR-1065)

Energy Technology Data Exchange (ETDEWEB)

Grdina, D.J.; Constantinou, A.; Shigematsu, N.

1992-09-01

The aminothiol 2-[(aminopropyl)amino]ethanethiol (WR-1065) is the active thiol of the clinically studied radioprotective agent S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721). WR-1065 is an effective radiation protector under in vitro conditions when it is administered 30 min prior to radiation exposure at a concentration of 4 mM to repair-proficient Chinese hamster ovary Kl cells (i.e., a dose modification factor of 1.4). In contrast, the DNA double-strand break, repair-deficient Chinese hamster ovary xrs-5 cell line is not protected under these conditions (i.e., a dose modification factor of 1.0). Topoisomerase (topo) I and II activities and protein contents were measured in both Kl and xrs-5 cell lines and were found to be similar in magnitude. Neither exposure to radiation, to WR-1065, or to both affected these variables in xrs-5 cells. WR 1065 was effective, however, in reducing topo 11 activity by a factor of 2 in the repair-proficient Kl cell line. Topo II protein content, however, was not affected by these exposure conditions. One of several mechanisms of radiation protection attributed to aminothiol compounds has been their ability to affect enzymatic reactions involved in DNA synthesis, repair, and cell cycle progression. These results demonstrate a modifying effect by 2-[(aminopropyl)amino]ethanethiol on a specific nuclear enzyme (i.e., type H topoisomerase), which is involved in DNA synthesis. These results also suggest that differences do exist between the topo 11 enzymes isolated from the parent repair-proficient Kl and the DNA double-strand break, repair-deficient xrs-5 mutant cell lines.

Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells

International Nuclear Information System (INIS)

Tsujimura, T.; Maher, V.M.; McCormick, J.J.; Godwin, A.R.; Liskay, R.M.

1990-01-01

To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10-to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination

Economic Analysis for Setting Appropriate Repair Cycles on the Fixed Materials and Facilities in the Public Rental Housing

Directory of Open Access Journals (Sweden)

Sung-Min Choi

2016-01-01

Full Text Available Currently, repair and maintenance cycles that follow the completion of construction facilities lead to the necessitation of subsequent data on the analysis of study and plan for maintenance. As such, an index of evaluation was drafted and a plan of maintenance cycle was computed using the investigation data derived from surveying target housing units in permanent rental environmental conditions, with a minimum age of 20 years, and their maintenance history. Optimal maintenance and replacement methods were proposed based on this data. Economic analysis was conducted through the Risk-Weighted Life Cycle Cost (RWLCC method in order to determine the cost analysis of maintenance life cycle methods used for repair. Current maintenance cycle methods that have been used for 20 years were also compared with alternative maintenance cycles.

Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

International Nuclear Information System (INIS)

Yezzi, M.J.

1985-04-01

A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40 0 C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40 0 C for 2 hrs before a first dose and maintained at 40 0 C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G 1 -phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35 0 C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs

Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

Energy Technology Data Exchange (ETDEWEB)

Yezzi, M.J.

1985-04-01

A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

International Nuclear Information System (INIS)

Marie, Melanie; Hafner, Sophie; Moratille, Sandra; Vaigot, Pierre; Rigaud, Odile; Martin, Michele T.; Mine, Solene

2012-01-01

Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2. (authors)

Protein phosphatase 5 is necessary for ATR-mediated DNA repair

International Nuclear Information System (INIS)

Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

2011-01-01

Research highlights: → Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. → However, it is not clear exactly how PP5 participates in this process. → Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

Relationship of DNA repair and chromosome aberrations to potentially lethal damage repair in X-irradiated mammalian cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Nagasawa, H.; Little, J.B.

1980-01-01

By the alkaline elution technique, the repair of x-ray-induced DNA single strand breaks and DNA-protein cross-links was investigated in stationary phase, contact-inhibited mouse cells. During the first hour of repair, approximately 90% of x-ray induced single strand breaks were rejoined whereas most of the remaining breaks were rejoined more slowly during the next 5 h. The number of residual non-rejoined single strand breaks was approximately proportional to the x-ray dose at early repair times. DNA-protein cross-links were removed at a slower rate - T 1/2 approximately 10 to 12 h. Cells were subcultured at low density at various times after irradiation and scored for colony survival, and chromosome aberrations in the first mitosis after sub-culture. Both cell lethality and the frequency of chromosome aberrations decreased during the first several hours of repair, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA strand breaks. The possible relationship of DNA repair to changes in survival and chromosome aberrations is discussed

X-ray repair replication in L1210 leukemia cells

International Nuclear Information System (INIS)

Lee, Y.C.; Byfield, J.E.; Bennett, L.R.; Chan, P.Y.M.

1974-01-01

Repair replication has been studied in detail in mouse L1210 leukemia cells. A method of identifying and quantitating repair replication using a pre- and postradiation block of normal replication with cytosine arabinoside is illustrated. The method derived does not require isolation of DNA per se and appears to be satisfactory for screening for inhibitors of repair replication. Repair replication can be demonstrated at doses in the 1000-rad range in bromouridine deoxyriboside-substituted cells and at slightly higher doses in nonsubstituted cells. Drugs that are known to bind to DNA inhibit this x-ray-induced repair replication. Drugs with these properties may be identified by the methods described and compared quantitatively in their ability to inhibit this type of x-ray damage. Since these phenomena can be demonstrated for low radiation doses and at drug concentrations attainable in vivo during human cancer chemotherapy this class of anticancer agent may be worthy of closer study. Application to the L1210 leukemia system should permit comparison of in vitro and in vivo drug effects in the context of the extensive in vivo pharmacological data already available for L1210 cells. (U.S.)

Cell cycle phases in the unequal mother/daughter cell cycles of Saccharomyces cerevisiae.

Science.gov (United States)

Brewer, B J; Chlebowicz-Sledziewska, E; Fangman, W L

1984-11-01

During cell division in the yeast Saccharomyces cerevisiae mother cells produce buds (daughter cells) which are smaller and have longer cell cycles. We performed experiments to compare the lengths of cell cycle phases in mothers and daughters. As anticipated from earlier indirect observations, the longer cell cycle time of daughter cells is accounted for by a longer G1 interval. The S-phase and the G2-phase are of the same duration in mother and daughter cells. An analysis of five isogenic strains shows that cell cycle phase lengths are independent of cell ploidy and mating type.

The Bright and the Dark Sides of DNA Repair in Stem Cells

OpenAIRE

Frosina, Guido

2010-01-01

DNA repair is a double-edged sword in stem cells. It protects normal stem cells in both embryonic and adult tissues from genetic damage, thus allowing perpetuation of intact genomes into new tissues. Fast and efficient DNA repair mechanisms have evolved in normal stem and progenitor cells. Upon differentiation, a certain degree of somatic mutations becomes more acceptable and, consequently, DNA repair dims. DNA repair turns into a problem when stem cells transform and become cancerous. Tran...

High levels of BRC4 induced by a Tet-On 3G system suppress DNA repair and impair cell proliferation in vertebrate cells.

Science.gov (United States)

Abe, Takuya; Branzei, Dana

2014-10-01

Transient induction or suppression of target genes is useful to study the function of toxic or essential genes in cells. Here we apply a Tet-On 3G system to DT40 lymphoma B cell lines, validating it for three different genes. Using this tool, we then show that overexpression of the chicken BRC4 repeat of the tumor suppressor BRCA2 impairs cell proliferation and induces chromosomal breaks. Mechanistically, high levels of BRC4 suppress double strand break-induced homologous recombination, inhibit the formation of RAD51 recombination repair foci, reduce cellular resistance to DNA damaging agents and induce a G2 damage checkpoint-mediated cell-cycle arrest. The above phenotypes are mediated by BRC4 capability to bind and inhibit RAD51. The toxicity associated with BRC4 overexpression is exacerbated by chemotherapeutic agents and reversed by RAD51 overexpression, but it is neither aggravated nor suppressed by a deficit in the non-homologous end-joining pathway of double strand break repair. We further find that the endogenous BRCA2 mediates the cytotoxicity associated with BRC4 induction, thus underscoring the possibility that BRC4 or other domains of BRCA2 cooperate with ectopic BRC4 in regulating repair activities or mitotic cell division. In all, the results demonstrate the utility of the Tet-On 3G system in DT40 research and underpin a model in which BRC4 role on cell proliferation and chromosome repair arises primarily from its suppressive role on RAD51 functions. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

Energy Technology Data Exchange (ETDEWEB)

Abbasi, Rashda [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Efferth, Thomas [Institute of Pharmacy und Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Kuhmann, Christine [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Opatz, Till [Institute of Organic Chemistry, Johannes Gutenberg University, Duesbergweg 10-14, 55128 Mainz (Germany); Hao, Xiaojiang [Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204 (China); Popanda, Odilia, E-mail: o.popanda@dkfz.de [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Schmezer, Peter [Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

2012-03-15

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC{sub 50} values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC{sub 50} values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

International Nuclear Information System (INIS)

Abbasi, Rashda; Efferth, Thomas; Kuhmann, Christine; Opatz, Till; Hao, Xiaojiang; Popanda, Odilia; Schmezer, Peter

2012-01-01

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC 50 values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC 50 values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes. -- Highlights: ► Thousand-fold higher Ascaridol activity in NER-deficient versus proficient cells. ► Impaired repair of Ascaridol-induced oxidative DNA damage in NER-deficient cells. ► Selective activity of Ascaridol opens new therapy options in

High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

Directory of Open Access Journals (Sweden)

Hee Jin Shim

Full Text Available Ionizing radiation (IR treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

Increased DNA-repair in spleen cells of M. Hodgkin

International Nuclear Information System (INIS)

Frischauf, H.; Neumann, E.; Howanietz, L.; Dolejs, I.; Tuschl, H.; Altmann, H.

1974-11-01

In spleen cells of control patients and cells of Morbus Hodgkin, DNA-repair after gamma- and UV-irradiation was determined measuring the incorporated 3H-thymidine activity in the DNA. Additionally, the ratio of labeled cells compared to non-labeled cells and the grains per cell were evaluated by autoradiographic investigations. DNA-content per cell was measured using pulsecytophotometry. A significant increase of DNA-repair capacity after gamma-irradiation was found by density gradient centrifugation in alkaline sucrose. The same trend could be shown by investigations of unscheduled DNA-synthesis using autoradiographic method. (author)

Deficient repair of chemical adducts in alpha DNA of monkey cells

International Nuclear Information System (INIS)

Zolan, M.E.; Cortopassi, G.A.; Smith, C.A.; Hanawalt, P.C.

1982-01-01

Researchers have examined excision repair of DNA damage in the highly repeated alpha DNA sequence of cultured African green monkey cells. Irradiation of cells with 254 nm ultraviolet light resulted in the same frequency of pyrimidine dimers in alpha DNA and the bulk of the DNA. The rate and extent of pyrimidine dimer removal, as judged by measurement of repair synthesis, was also similar for alpha DNA and bulk DNA. In cells treated with furocoumarins and long-wave-length ultraviolet light, however, repair synthesis in alpha DNA was only 30% of that in bulk DNA, although it followed the same time course. Researchers found that this reduced repair was not caused by different initial amounts of furocoumarin damage or by different sizes of repair patches, as researchers found these to be similar in the two DNA species. Direct quantification demonstrated that fewer furocoumarin adducts were removed from alpha DNA than from bulk DNA. In cells treated with another chemical DNA-damaging agent, N-acetoxy-2-acetylaminofluorene, repair synthesis in alpha DNA was 60% of that in bulk DNA. These results show that the repair of different kinds of DNA damage can be affected to different extents by some property of this tandemly repeated heterochromatic DNA. To our knowledge, this is the first demonstration in primate cells of differential repair of cellular DNA sequences

Repair of potentially lethal and sublethal radiation damage in x-irradiated ascites tumor cells

International Nuclear Information System (INIS)

Tsuboi, Atsushi; Okamoto, Mieko; Tsuchiya, Takehiko.

1985-01-01

The ability of cells to repair cellular radiation damage during the growth of TMT-3 ascites tumor and the effect of host reaction on the repair ability were examined by using an in vitro assay of cell clonogenicity after in situ irradiation of tumor cells. In single-dose experiments, the repair of potentially lethal radiation damage (PLD) was observed in stationary phase cells (12-day tumor) of the unirradiated host, but not in exponential phase cells (3-day tumor) of the unirradiated host animals. However, if previously irradiated host animals were used, even the exponentially growing tumor cells showed repair of PLD. In two-dose experiments, the ability to repair sublethal radiation damage (SLD) in exponential phase tumor cells was less than that of stationary phase cells in the unirradiated host. In the pre-irradiated host, the extent of the repair in exponential phase cells was somewhat enhanced. These results suggest that irradiation of host animals might suppress a factor that inhibits repair, resulting in enhancement of the repair capability of tumor cells. (author)

Repair and cell cycle response in cells exposed to environmental biohazards. Progress report, June 1, 1979-May 31, 1980

International Nuclear Information System (INIS)

Billen, D.; Hadden, C.T.

1980-01-01

Progress is reported in the following research areas: (1) DNA repair in Bacillus subtilis; (2) postreplication repair in Rec - mutants of B. subtilis; (3) photobiology of halogenated DNA; (4) effects of caffeine on pyrimidine dimer excision and postreplication repair in B. subtilis; and (5) DNA repair in toluene-treated Escherichia coli

Cell cycle regulation of DNA polymerase beta in rotenone-based Parkinson's disease models.

Directory of Open Access Journals (Sweden)

Hongcai Wang

Full Text Available In Parkinson's disease (PD, neuronal cells undergo mitotic catastrophe and endoreduplication prior to cell death; however, the regulatory mechanisms remain to be defined. In this study, we investigated cell cycle regulation of DNA polymerase β (poly β in rotenone-based dopaminergic cellular and animal models. Incubation with a low concentration (0.25 µM of rotenone for 1.5 to 7 days resulted in a flattened cell body and decreased DNA replication during S phase, whereas a high concentration (2 µM of rotenone exposure resulted in enlarged, multi-nucleated cells and converted the mitotic cycle into endoreduplication. Consistently, DNA poly β, which is mainly involved in DNA repair synthesis, was upregulated to a high level following exposure to 2 µM rotenone. The abrogation of DNA poly β by siRNA transfection or dideoxycytidine (DDC treatment attenuated the rotenone-induced endoreduplication. The cell cycle was reactivated in cyclin D-expressing dopaminergic neurons from the substantia nigra (SN of rats following stereotactic (ST infusion of rotenone. Increased DNA poly β expression was observed in the substantia nigra pars compacta (SNc and the substantia nigra pars reticulate (SNr of rotenone-treated rats. Collectively, in the in vitro model of rotenone-induced mitotic catastrophe, the overexpression of DNA poly β promotes endoreduplication; in the in vivo model, the upregulation of DNA poly β and cell cycle reentry were also observed in the adult rat substantia nigra. Therefore, the cell cycle regulation of DNA poly β may be involved in the pathological processes of PD, which results in the induction of endoreduplication.

Saturation of DNA repair in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Ahmed, F E; Setlow, R B

1979-01-01

Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20 J/m/sup 2/. We measured excision repair in normal human fibroblasts up to 80 J/m/sup 2/. The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus, and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24h following two 20 J/m/sup 2/ doses separated by 24h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the rate limiting steps in repair.

DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

International Nuclear Information System (INIS)

Yaki, T.

1982-01-01

DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

Science.gov (United States)

Burke, Russell T; Marcus, Joshua M; Orth, James D

2017-06-13

Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

Chapter 10 the primary cilium coordinates signaling pathways in cell cycle control and migration during development and tissue repair

DEFF Research Database (Denmark)

Christensen, Søren T; Pedersen, Stine F; Satir, Peter

2008-01-01

Cell cycle control and migration are critical processes during development and maintenance of tissue functions. Recently, primary cilia were shown to take part in coordination of the signaling pathways that control these cellular processes in human health and disease. In this review, we present...... an overview of the function of primary cilia and the centrosome in the signaling pathways that regulate cell cycle control and migration with focus on ciliary signaling via platelet-derived growth factor receptor alpha (PDGFRalpha). We also consider how the primary cilium and the centrosome interact...... with the extracellular matrix, coordinate Wnt signaling, and modulate cytoskeletal changes that impinge on both cell cycle control and cell migration....

DNA replication and repair in Tilapia cells. 1. The effect of ultraviolet radiation

Energy Technology Data Exchange (ETDEWEB)

Yew, F.H.; Chang, L.M. (National Taiwan Univ., Taipei (China))

1984-12-01

The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-..beta..-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor.

[Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

Science.gov (United States)

Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

2007-01-01

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology

SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

Energy Technology Data Exchange (ETDEWEB)

Sadetaporn, D [Rice University, Houston, TX (United States); The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Flint, D; McFadden, C; Sawakuchi, G [The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Asaithamby, A [UT Southwestern Medical Center, Dallas, TX (United States)

2016-06-15

Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

SU-F-T-665: Confocal Microscopy Imaging of Cell Cycle Distribution in Cells Treated with Pegylated Gold Nanoshells

International Nuclear Information System (INIS)

Sadetaporn, D; Flint, D; McFadden, C; Sawakuchi, G; Asaithamby, A

2016-01-01

Purpose: To use confocal microscopy to distinguish cells in different phases of the cell cycle before and after treatment with pegylated gold nanoshells (PEG-AuNSs). Methods: Transfected fibrosarcoma cells (HT1080-EYFP-53BP1-FUCCI) were cultured in T-25 flasks and seeded in glass bottom dishes. These cells express the fluorescent probe AmCyan during the G2/S phases of the cell cycle, mCherry during the G1 phase, and EYFP tagged to the DNA repair protein 53BP1. After allowing cells 4 h to adhere to dishes, PEG-AuNS (Nanospectra Biosciences, Houston, TX) at a concentration of 0.15 OD were administered. At time points of 8, 16 and 24 h following treatment, the PEG-AuNS-treated and control samples were washed with phosphate buffered saline (PBS) and fixed using 4% paraformaldehyde in PBS. Samples were imaged with an Olympus FV1200 confocal microscope using 473, 543, and 641 nm excitation lasers. We used band-pass filters to select AmCyan and mCherry fluorescence. Reflection from the 641 nm laser was used to detect PEG-AuNSs. Z-stack images were analyzed to assess cell cycle distribution through fluorescent probe expression. Live cells were imaged after PEG-AuNS treatment using a confocal microscope with a stage top CO2 incubator. Results: We were able to obtain high-resolution images of cells with internalized AuNSs. We were also able to distinguish cells in different phases of the cell cycle. Conclusion: This work demonstrates a new assay to investigate the effect of AuNSs on the cell cycle phase in live cells. Future work will employ confocal microscopy and flow cytometry to focus on effects of AuNS treatment on cell cycle distribution. This research was supported by the Sister Institution Network Fund and the Center for Radiation Oncology Research at The University of Texas MD Anderson Cancer Center and Cancer Prevention and Research Institute of Texas. Gabriel Sawakuchi has research support from Elekta Inc.

Chromatin dynamics during cell cycle mediate conversion of DNA damage into chromatid breaks and affect formation of chromosomal aberrations: Biological and clinical significance

International Nuclear Information System (INIS)

Terzoudi, Georgia I.; Hatzi, Vasiliki I.; Donta-Bakoyianni, Catherine; Pantelias, Gabriel E.

2011-01-01

The formation of diverse chromosomal aberrations following irradiation and the variability in radiosensitivity at different cell-cycle stages remain a long standing controversy, probably because most of the studies have focused on elucidating the enzymatic mechanisms involved using simple DNA substrates. Yet, recognition, processing and repair of DNA damage occur within the nucleoprotein complex of chromatin which is dynamic in nature, capable of rapid unfolding, disassembling, assembling and refolding. The present work reviews experimental work designed to investigate the impact of chromatin dynamics and chromosome conformation changes during cell-cycle in the formation of chromosomal aberrations. Using conventional cytogenetics and premature chromosome condensation to visualize interphase chromatin, the data presented support the hypothesis that chromatin dynamic changes during cell-cycle are important determinants in the conversion of sub-microscopic DNA lesions into chromatid breaks. Consequently, the type and yield of radiation-induced chromosomal aberrations at a given cell-cycle-stage depends on the combined effect of DNA repair processes and chromatin dynamics, which is cell-cycle-regulated and subject to up- or down-regulation following radiation exposure or genetic alterations. This new hypothesis is used to explain the variability in radiosensitivity observed at various cell-cycle-stages, among mutant cells and cells of different origin, or among different individuals, and to revisit unresolved issues and unanswered questions. In addition, it is used to better understand hypersensitivity of AT cells and to provide an improved predictive G2-assay for evaluating radiosensitivity at individual level. Finally, experimental data at single cell level obtained using hybrid cells suggest that the proposed hypothesis applies only to the irradiated component of the hybrid.

Repair and regeneration: opportunities for carcinogenesis from tissue stem cells

OpenAIRE

Perryman, Scott V; Sylvester, Karl G

2007-01-01

This review will discuss the mechanisms of repair and regeneration in various tissue types and how dysregulation of these mechaisms may lead to cancer. Normal homeostasis involves a careful balance between cell loss and cell renewal. Stem and progenitor cells perform these biologic processes as the functional units of regeneration during both tissue homeostasis and repair. The concept of tissue stem cells capable of giving rise to all differentiated cells within a given tissue led to the conc...

Defective bone repair in mast cell-deficient Cpa3Cre/+ mice.

Directory of Open Access Journals (Sweden)

Jose Luis Ramirez-GarciaLuna

Full Text Available In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient KitW-sh/W-sh mice (KitW-sh implicated mast cells in bone repair but KitW-sh mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (WT and Cpa3Cre/+ mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1 mast cells appear in healing bone of WT mice but not Cpa3Cre/+ mice, beginning 14 days after surgery; 2 re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in Cpa3Cre/+ mice compared with WT mice; 3 the defects in Cpa3Cre/+ mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice and fibrous mal-union in most Cpa3Cre/+ mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.

Defective bone repair in mast cell-deficient Cpa3Cre/+ mice.

Science.gov (United States)

Ramirez-GarciaLuna, Jose Luis; Chan, Daniel; Samberg, Robert; Abou-Rjeili, Mira; Wong, Timothy H; Li, Ailian; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Henderson, Janet E; Martineau, Paul A

2017-01-01

In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient KitW-sh/W-sh mice (KitW-sh) implicated mast cells in bone repair but KitW-sh mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (WT) and Cpa3Cre/+ mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1) mast cells appear in healing bone of WT mice but not Cpa3Cre/+ mice, beginning 14 days after surgery; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in Cpa3Cre/+ mice compared with WT mice; 3) the defects in Cpa3Cre/+ mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most WT mice and fibrous mal-union in most Cpa3Cre/+ mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.

Expression profile of cell cycle genes in the fish CATLA CATLA (Ham.) exposed to gamma radiation

International Nuclear Information System (INIS)

Anbumani, S.; Mohankumar Mary, N.

2012-01-01

The International Commission on Radiological Protection (ICRP) emphasized the need to protect non-human biota from the potential effects of ionizing radiation and proposed to include molecular effects such as DNA damage as endpoints. Molecular effects of ionizing radiation exposure in representative non-humans are largely unexplored and sufficient data is not available in fishes. Gene expression is a fast and sensitive end point in detecting the molecular cues as a result of ionizing radiation exposure in a wide variety of aquatic organisms under suspected environmental contamination. Exposure to ionizing radiation transiently alters gene expression profiles as cells regulate certain genes to protect cellular structures and repair damage. The present study focused on genes like Gadd45á, Cdk1 and Bcl-2 in DNA damage repair and cell cycle machinery and its implication as molecular markers of radiation exposure. This study is first of its kind showing the in vivo expression profile of cell cycle genes in fish exposed to gamma radiation. Although this preliminary investigation points to certain molecular markers of ionizing radiation, elaborate studies with various doses and dose-rates are required before these markers find application as prospective molecular markers in aquatic radiation biodosimetry

A New Real-Time Cycle Slip Detection and Repair Method under High Ionospheric Activity for a Triple-Frequency GPS/BDS Receiver.

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Liu, Wanke; Jin, Xueyuan; Wu, Mingkui; Hu, Jie; Wu, Yun

2018-02-01

Cycle slip detection and repair is a prerequisite for high-precision global navigation satellite system (GNSS)-based positioning. With the modernization and development of GNSS systems, more satellites are available to transmit triple-frequency signals, which allows the introduction of additional linear combinations and provides new opportunities for cycle slip detection and repair. In this paper, we present a new real-time cycle slip detection and repair method under high ionospheric activity for undifferenced Global Positioning System (GPS)/BeiDou Navigation Satellite System (BDS) triple-frequency observations collected with a single receiver. First, three optimal linearly independent geometry-free pseudorange minus phase combinations are selected to correctly and uniquely determine the cycle slips on the original triple-frequency carrier phase observations. Then, a second-order time-difference algorithm is employed for the pseudorange minus phase combinations to mitigate the impact of between-epoch ionospheric residuals on cycle slip detection, which is especially beneficial under high ionospheric activity. The performance of the approach is verified with static GPS/BDS triple-frequency observations that are collected with a 30 s sampling interval under active ionospheric conditions, and observations are manually inserted with simulated cycle slips. The results show that the method can correctly detect and repair cycle slips at a resolution as small as 1 cycle. Moreover, kinematic data collected from car-driven and airborne experiments are also processed to verify the performance of the method. The experimental results also demonstrate that the method is effective in processing kinematic data.

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

International Nuclear Information System (INIS)

Desai, Amar; Webb, Bryan; Gerson, Stanton L.

2014-01-01

Background: Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells. Materials and methods: A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ϒ-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes. Results: A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells. Conclusions: CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs

Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

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Komaki, Yukako; Mariñas, Benito J; Plewa, Michael J

2014-10-07

Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 μM IAN, 12 μM BAN and 900 μM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of

Cell Cycle Control by PTEN.

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Brandmaier, Andrew; Hou, Sheng-Qi; Shen, Wen H

2017-07-21

Continuous and error-free chromosome inheritance through the cell cycle is essential for genomic stability and tumor suppression. However, accumulation of aberrant genetic materials often causes the cell cycle to go awry, leading to malignant transformation. In response to genotoxic stress, cells employ diverse adaptive mechanisms to halt or exit the cell cycle temporarily or permanently. The intrinsic machinery of cycling, resting, and exiting shapes the cellular response to extrinsic stimuli, whereas prevalent disruption of the cell cycle machinery in tumor cells often confers resistance to anticancer therapy. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a guardian of the genome that is frequently mutated or deleted in human cancer. Moreover, it is increasingly evident that PTEN deficiency disrupts the fundamental processes of genetic transmission. Cells lacking PTEN exhibit cell cycle deregulation and cell fate reprogramming. Here, we review the role of PTEN in regulating the key processes in and out of cell cycle to optimize genomic integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Summary report on development of bilateral servo manipulator (BSM) for nuclear fuel cycle facilities in the Japan Nuclear Cycle Development Institute

International Nuclear Information System (INIS)

Miki, Yasuo; Koizumi, Tsutomu; Aoshima, Atsushi; Kawanobe, Kazunori; Kobayashi, Yuichi

2000-03-01

In order to improve availability of nuclear fuel cycle facilities such as fuel reprocessing plants, reduce occupational radiation exposure, the Japan Nuclear Cycle Development Institute (JNC) has been developing an advanced remote manipulative system for fully remote maintenance and repair tasks in large volume repair cells. Fully remote maintenance and repair task is performed primarily by the utilization of overhead bridge cranes, mechanical master-slave manipulators and electro-mechanical power manipulators. This system requires also that plant process and remote processing equipment should be designed to provide modular or unit replacement based on the potential mode of system failures. Repair of equipment is performed following removal of the failed component from process line and transfer to the repair cell. Equipment repair in the cell is commonly carried out by the use of remote manipulators. However, the realization of fully remote maintenance facility requires so remote manipulative systems as to provide excellent controllability, durability and remote maintenance capability, development of a bilateral servo-manipulator was initiated in 1982. Two of BSM were installed in the Tokai Vitrification Facility (TVF) cell and their remote maintenance feasibility was evaluated. Following installation in the TVF, developing efforts toward achieving advanced remote maintenance capability for the Recycle Equipment Test Facility (RETF) have been made. This report summarizes mainly mechanical and control system design for improvement, particularly upgrading controllability. (Itami, H.)

DNA repair and its coupling to DNA replication in eukaryotic cells. [UV, x ray

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Cleaver, J.E.

1978-01-01

This review article with 184 references presents the view that mammalian cells have one major repair system, excision repair, with many branches (nucleotide excision repair, base excision repair, crosslink repair, etc.) and a multiplicity of enzymes. Any particular carcinogen makes a spectrum of damaged sites and each kind of damage may be repaired by one or more branches of excision repair. Excision repair is rarely complete, except at very low doses, and eukaryotic cells survive and replicate DNA despite the presence of unrepaired damage. An alteration in a specific biochemical pathway seen in damaged or mutant cells will not always be the primary consequence of damage or of the biochemical defect of the cells. Detailed kinetic data are required to understand comprehensively the various facets of excision repair and replication. Correlation between molecular events of repair and cytological and cellular changes such as chromosomal damage, mutagenesis, transformation, and carcinogenesis are also rudimentary.

A robust network of double-strand break repair pathways governs genome integrity during C. elegans development.

NARCIS (Netherlands)

Pontier, D.B.; Tijsterman, M.

2009-01-01

To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs). Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ)

Cloning human DNA repair genes

International Nuclear Information System (INIS)

Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

1994-01-01

Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

Differing levels of excision repair in human fetal dermis and brain cells

International Nuclear Information System (INIS)

Gibson, R.E.; D'Ambrosio, S.M.; Ohio State Univ., Columbus

1982-01-01

The levels of DNA excision repair, as measured by unscheduled DNA synthesis (UDS) and the UV-endonuclease sensitive site assay, were compared in cells derived from human fetal brain and dermal tissues. The level of UDS induced following ultraviolet (UV) irradiation was found to be lower (approx. 60%) in the fetal brain cells than in fetal dermal cells. It was determined, using the UV-endonuclease sensitive site assay to confirm the UDS observation, that 50% of the dimers induced by UV in fetal dermal cells were repaired in 8 h. while only 15% were removed in the fetal brain cells during the same period of time. Even after 24 h. only 44% of the dimers induced by UV in the fetal brain cells were repaired, while 65% were removed in the dermal cells. These data suggest that cultured human fetal brain cells exhibit lower levels of excision repair compared to cultured human fetal dermal cells. (author)

Roles of neural stem cells in the repair of peripheral nerve injury.

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Wang, Chong; Lu, Chang-Feng; Peng, Jiang; Hu, Cheng-Dong; Wang, Yu

2017-12-01

Currently, researchers are using neural stem cell transplantation to promote regeneration after peripheral nerve injury, as neural stem cells play an important role in peripheral nerve injury repair. This article reviews recent research progress of the role of neural stem cells in the repair of peripheral nerve injury. Neural stem cells can not only differentiate into neurons, astrocytes and oligodendrocytes, but can also differentiate into Schwann-like cells, which promote neurite outgrowth around the injury. Transplanted neural stem cells can differentiate into motor neurons that innervate muscles and promote the recovery of neurological function. To promote the repair of peripheral nerve injury, neural stem cells secrete various neurotrophic factors, including brain-derived neurotrophic factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor and hepatocyte growth factor. In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways.

Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

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Giddings Ian

2011-06-01

Full Text Available Abstract Background Benzo[a]pyrene (BaP is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP 1A1 and CYP1B1, which are involved in BaP metabolism. Results Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE, the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. Conclusions This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell

Quantitation of DNA repair in brain cell cultures: implications for autoradiographic analysis of mixed cell populations

International Nuclear Information System (INIS)

Dambergs, R.; Kidson, C.

1979-01-01

Quantitation of DNA repair in the mixed cell population of mouse embryo brain cultures has been assessed by autoradiographic analysis of unscheduled DNA synthesis following UV-irradiation. The proportion of labelled neurons and the grain density over neuronal nuclei were both less than the corresponding values for glial cells. The nuclear geometries of these two classes of cell are very different. Partial correction for the different geometries by relating grain density to nuclear area brought estimates of neuronal and glial DNA repair synthesis more closely in line. These findings have general implications for autoradiographic measurement of DNA repair in mixed cell populations and in differentiated versus dividing cells. (author)

Multiparameter Cell Cycle Analysis.

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Jacobberger, James W; Sramkoski, R Michael; Stefan, Tammy; Woost, Philip G

2018-01-01

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

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Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

2011-01-01

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

Directory of Open Access Journals (Sweden)

Monika Hlavová

Full Text Available DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.

Cell Based Meniscal Repair Using an Aligned Bioactive Nanofibrous Sheath

Science.gov (United States)

2017-07-01

to subsequently guide tissue regeneration , for example, by seeded tissue progenitor cells . To achieve this objective, the first step is to develop...AWARD NUMBER: W81XWH-15-1-0104 TITLE: Cell -Based Meniscal Repair Using an Aligned Bioactive Nanofibrous Sheath PRINCIPAL INVESTIGATOR...SUBTITLE 5a. CONTRACT NUMBER Cell -Based Meniscal Repair Using an Aligned Bioactive Nanofibrous Sheath 5b. GRANT NUMBER W81XWH-15-1-0104 5c. PROGRAM

The stem cell secretome and its role in brain repair.

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Drago, Denise; Cossetti, Chiara; Iraci, Nunzio; Gaude, Edoardo; Musco, Giovanna; Bachi, Angela; Pluchino, Stefano

2013-12-01

Compelling evidence exists that non-haematopoietic stem cells, including mesenchymal (MSCs) and neural/progenitor stem cells (NPCs), exert a substantial beneficial and therapeutic effect after transplantation in experimental central nervous system (CNS) disease models through the secretion of immune modulatory or neurotrophic paracrine factors. This paracrine hypothesis has inspired an alternative outlook on the use of stem cells in regenerative neurology. In this paradigm, significant repair of the injured brain may be achieved by injecting the biologics secreted by stem cells (secretome), rather than implanting stem cells themselves for direct cell replacement. The stem cell secretome (SCS) includes cytokines, chemokines and growth factors, and has gained increasing attention in recent years because of its multiple implications for the repair, restoration or regeneration of injured tissues. Thanks to recent improvements in SCS profiling and manipulation, investigators are now inspired to harness the SCS as a novel alternative therapeutic option that might ensure more efficient outcomes than current stem cell-based therapies for CNS repair. This review discusses the most recent identification of MSC- and NPC-secreted factors, including those that are trafficked within extracellular membrane vesicles (EVs), and reflects on their potential effects on brain repair. It also examines some of the most convincing advances in molecular profiling that have enabled mapping of the SCS. Copyright © 2013 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Repair of human DNA: radiation and chemical damage in normal and xeroderma pigmentosum cells

International Nuclear Information System (INIS)

Regan, J.D.; Setlow, R.B.

1976-01-01

We present the experimental evidence we have gathered, using a particular assay for DNA repair in human cells, the photolysis of bromodeoxyuridine (BrdUrd) incorporated during repair. This assay characterizes the sequence of repair events that occur in human cells after radiation, both ultraviolet and ionizing, and permits an estimation of the size of the average repaired region after these physical insults to DNA. We will discuss chemical insults to DNA and attempt to liken the repair processes after chemical damages of various kinds to those repair processes that occur in human DNA after damage from physical agents. We will also show results indicating that, under certain conditions, repair events resembling those seen after uv-irradiation can be observed in normal human cells after ionizing radiation. Furthermore the XP cells, defective in the repair of uv-induced DNA damage, show defective repair of these uv-like DNA lesions induced by ionizing radiation

Repair and cell cycle response in cells exposed to environmental biohazards. Progress report, June 1, 1980-May 31, 1981

International Nuclear Information System (INIS)

Billen, D.; Hadden, C.T.

1981-01-01

Research progress for the period June 1, 1980 through May 31, 1981 is reported. Topics include: (1) repair of damage by psoralen plus near-uv light; (2) in vitro repair of transforming DNA; (3) photobiology of halogenated DNA; and (4) DNA repair in toluene-treated Escherichia coli containing BU-DNA

DNA repair in human cells exposed to combinations of carcinogenic agents

International Nuclear Information System (INIS)

Setlow, R.B.; Ahmed, F.E.

1980-01-01

Normal human and XP 2 fibroblasts were treated with uv plus uv-mimetic chemicals. The uv dose used was sufficient to saturate the uv excision repair system. Excision repair after combined treatments was estimated by unscheduled DNA synthesis, BrdUrd photolysis, and the loss of sites sensitive to a uv specific endonuclease. Since the repair of damage from uv and its mimetics is coordinately controlled we expected that there would be similar rate-limiting steps in the repair of uv and chemical damage and that after a combined treatment the total amount of repair would be the same as from uv or the chemicals separately. The expectation was not fulfilled. In normal cells repair after a combined treatment was additive whereas in XP cells repair after a combined treatment was usually less than after either agent separately. The chemicals tested were AAAF, DMBA-epoxide, 4NQO, and ICR-170

STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

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Benito, Cristina; Davis, Catherine M; Gomez-Sanchez, Jose A; Turmaine, Mark; Meijer, Dies; Poli, Valeria; Mirsky, Rhona; Jessen, Kristjan R

2017-04-19

After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population. SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal

DNA replication and post-replication repair in U.V.-sensitive mouse neuroblastoma cells

International Nuclear Information System (INIS)

Lavin, M.F.; McCombe, P.; Kidson, C.

1976-01-01

Mouse neuroblastoma cells differentiated when grown in the absence of serum; differentiation was reversed on the addition of serum. Differentiated cells were more sensitive to U.V.-radiation than proliferating cells. Whereas addition of serum to differentiated neuroblastoma cells normally resulted in immediate, synchronous entry into S phase, irradiation just before the addition of serum resulted in a long delay in the onset of DNA replication. During this lag period, incorporated 3 H-thymidine appeared in the light density region of CsCl gradients, reflecting either repair synthesis or abortive replication. Post-replication repair (gap-filling) was found to be present in proliferating cells and at certain times in differentiated cells. It is suggested that the sensitivity of differentiated neuroblastoma cells to U.V.-radiation may have been due to ineffective post-replication repair or to deficiencies in more than one repair mechanism, with reduction in repair capacity beyond a critical threshold. (author)

Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle.

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Pesavento, James J; Yang, Hongbo; Kelleher, Neil L; Mizzen, Craig A

2008-01-01

Methylation of histone H4 at lysine 20 (K20) has been implicated in transcriptional activation, gene silencing, heterochromatin formation, mitosis, and DNA repair. However, little is known about how this modification is regulated or how it contributes to these diverse processes. Metabolic labeling and top-down mass spectrometry reveal that newly synthesized H4 is progressively methylated at K20 during the G(2), M, and G(1) phases of the cell cycle in a process that is largely inescapable and irreversible. Approximately 98% of new H4 becomes dimethylated within two to three cell cycles, and K20 methylation turnover in vivo is undetectable. New H4 is methylated regardless of prior acetylation, and acetylation occurs predominantly on K20-dimethylated H4, refuting the hypothesis that K20 methylation antagonizes H4 acetylation and represses transcription epigenetically. Despite suggestions that it is required for normal mitosis and cell cycle progression, K20 methylation proceeds normally during colchicine treatment. Moreover, delays in PR-Set7 synthesis and K20 methylation which accompany altered cell cycle progression during sodium butyrate treatment appear to be secondary to histone hyperacetylation or other effects of butyrate since depletion of PR-Set7 did not affect cell cycle progression. Together, our data provide an unbiased perspective of the regulation and function of K20 methylation.

Radiation studies on sensitivity and repair of human mammary epithelial cells

International Nuclear Information System (INIS)

Tracy Chuihsu Yang; Stampfer, M.R.; Tobias, C.A.

1989-01-01

The authors present results indicating that normal breast epithelial cells and fibroblasts respond to X-rays similarly, lacking significant repair of sublethal damage when 2 Gy was used as the conditioning dose. Epithelial cells from tumor and from parenchymal tissue peripheral to the tumor, however, did show an efficient repair of sublethal damage. The reasons for this difference is unknown. Heavy-ion studies suggest energetic carbon and neon particles can be more effective in killing normal and tumour cells. The RBE for normal cells, however, appeared to be slightly less than for tumor cells. The repair of sublethal damage in tumor cells was less for neon particles than for X-rays. These findings suggest that heavy ions might be more advantageous than X-rays in treating breast tumors. (author)

Some important advances in DNA repair study on the mammalian cells

International Nuclear Information System (INIS)

Xia Shouxuan.

1991-01-01

In the recent years the study of DNA damage and repair in the mammalian cells has gone deeply at gene level and got the following advances: (1) For a long time DNA has been considered to be an uniform unit in case of damage and repair. Now this concept should be replaced by the non-random distribution of damage and heterogenous repair in the genome. These would allow us to study cellular mutagenesis, carcinogenesis, aging and dying processes in great detail, and would be beneficial to the elucidation of mechanisms of radiation sickness and chemical toxicology. (2) The advent of new techniques in molecular biology has made it possible to isolate and clone the human DNA repair genes. Up to now more than ten human DNA repair genes have been cloned and these works would have an important impact on the theoretical and practical study in this field. Because DNA repair system is very complicate, voluminous work should be done in the future. (3) The technique of gene transfer has been efficiently used in the study of DNA repair in mammalian cells and has made great contribution in the cellular engineering. It could modify the genetic behavior of the gene-accepting cells, and enhance the DNA repair ability to physical and chemical damages. Human gene therapy for DNA deficient diseases is now on the day

Mitochondrial DNA repair and aging

Energy Technology Data Exchange (ETDEWEB)

Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

2002-11-30

The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis.

Mitochondrial DNA repair and aging

International Nuclear Information System (INIS)

Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

2002-01-01

The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis

Modern problems of DNA repair in mammalian cells and some unsettled questions

International Nuclear Information System (INIS)

Gaziev, A.I.

1978-01-01

A comparison of DNA repair process in the cells of mammals and E. coli revealed no principal differences in the enzymic mechanisms of DNA repair in the cells of higher and lower organisms. It has been found that when given is the same number of impairments in the section of DNA chain in the cells of mammals and bacteria the regeneration in the former occurs more slowly than in the latter. Low rate elimination of impairments of DNA in the cells of mammals is due to a more complex intracellular and permolecular organization. It is stressed that the investigation into the mechanisms of fixing impairments in case of postreplication DNA repair is a very important and unresolved problem, especially in terms of radiation mutagenesis and cancerogenesis. Much thought is given to the problem of repairing double stranded ruptures of DNA. It is proposed that DNA repair should be considered not only in terms of functioning of enzymes in DNA metabolism, but also permolecular organization of genome in the cell

Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death.

Science.gov (United States)

Ur Rahman, Muhammad Saif; Zhang, Ling; Wu, Lingyan; Xie, Yuqiong; Li, Chunchun; Cao, Jiang

2017-01-01

Severe side effects are major problems with chemotherapy of gastric cancer (GC). These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB) was used with other DNA linker agents mitomycin C (MMC), cisplatin (DDP), or cyclophosphamide (CTX) to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC 50 ) by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G 0 /G 1 and G 1 /S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC cells to the alkylating agents via arresting the cell cycle and enhancing cell death. This is of significant therapeutic importance in the reduction of side effects associated with these drugs.

Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

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Elise Darmon

Full Text Available DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

Repair of tracheal epithelium by basal cells after chlorine-induced injury

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Musah Sadiatu

2012-11-01

Full Text Available Abstract Background Chlorine is a widely used toxic compound that is considered a chemical threat agent. Chlorine inhalation injures airway epithelial cells, leading to pulmonary abnormalities. Efficient repair of injured epithelium is necessary to restore normal lung structure and function. The objective of the current study was to characterize repair of the tracheal epithelium after acute chlorine injury. Methods C57BL/6 mice were exposed to chlorine and injected with 5-ethynyl-2′-deoxyuridine (EdU to label proliferating cells prior to sacrifice and collection of tracheas on days 2, 4, 7, and 10 after exposure. Airway repair and restoration of a differentiated epithelium were examined by co-localization of EdU labeling with markers for the three major tracheal epithelial cell types [keratin 5 (K5 and keratin 14 (K14 for basal cells, Clara cell secretory protein (CCSP for Clara cells, and acetylated tubulin (AcTub for ciliated cells]. Morphometric analysis was used to measure proliferation and restoration of a pseudostratified epithelium. Results Epithelial repair was fastest and most extensive in proximal trachea compared with middle and distal trachea. In unexposed mice, cell proliferation was minimal, all basal cells expressed K5, and K14-expressing basal cells were absent from most sections. Chlorine exposure resulted in the sloughing of Clara and ciliated cells from the tracheal epithelium. Two to four days after chlorine exposure, cell proliferation occurred in K5- and K14-expressing basal cells, and the number of K14 cells was dramatically increased. In the period of peak cell proliferation, few if any ciliated or Clara cells were detected in repairing trachea. Expression of ciliated and Clara cell markers was detected at later times (days 7–10, but cell proliferation was not detected in areas in which these differentiated markers were re-expressed. Fibrotic lesions were observed at days 7–10 primarily in distal trachea. Conclusion

Regulation of cell cycle progression by cell-cell and cell-matrix forces

NARCIS (Netherlands)

Uroz, Marina; Wistorf, Sabrina; Serra-Picamal, Xavier; Conte, Vito; Sales-Pardo, Marta; Roca-Cusachs, Pere; Guimerà, Roger; Trepat, Xavier

2018-01-01

It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces 1-12 . However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression

DNA strand breakage repair in ataxia telangiectasia fibroblast-like cells

Energy Technology Data Exchange (ETDEWEB)

Vincent, Jr, R A; Sheridan, III, R B; Huang, P C [Johns Hopkins Univ., Baltimore, Md. (USA). Dept. of Environmental and Biophysical Sciences

1975-12-01

Human diploid fibroblast-like cells derived from four patients with the genetic disease ataxia telangiectasia and from two non-mutant donors were examined for the repair of x-ray induced strand breaks in DNA. The ataxia telangiectasia cultures showed no significant differences from the non-mutant cultures in the kinetics and extent of strand repair. This suggests that the increased spontaneous and x-ray induced chromatid aberrations observed in ataxia telangiectasia cells are not caused by a defect in the repair of single strand breaks as might be suspected from a general model of aberration production.

Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate

International Nuclear Information System (INIS)

Cleaver, J.E.

1982-01-01

Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex

Comparison between major repair and replacement options for a bridge deck life cycle assessment: A case study

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Abu Dabous Saleh

2017-01-01

Full Text Available Material production, manufacturing, transportation, usage, and end of lifeprocessing are usually the main contributors defining the life cycle assessment (LCA. Bridge infrastructure is important to the economy and the society. Over their life cycle, highway bridges experience several stressors that can significantly affect their structural performance and therefore require rehabilitation. This paper discusses the life cycle analysis of bridge rehabilitation decisions and demonstrates the analysis with a case study of a bridge located in Ontario, Canada. The LCA of the bridge deck is analyzed for two rehabilitation strategies: major repair and replacement. The study focuses on evaluating the different life cycle phases of the bridge deck by assessing their carbon dioxide emission, energy consumption and cost. Also, the paper presents the impact of the different elements within each phase to identify the most contributing elements. The LCA of the bridge deck is analyzed and estimated with the aid of CES EduPack 2016 software that includes a database of more than 4000 different materials and more than 200 manufacturing processes. Analysis of the case study shows that material phase causes significant life cycle impact. The study concluded that the deck replacement yields higher environmental impact and life cycle cost compared to repairing and strengthening the deck.

Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

International Nuclear Information System (INIS)

Marchese, M.J.; Minarik, L.; Hall, E.J.; Zaider, M.

1985-01-01

Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant-two melanomas and one osteosarcoma-as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells. (author)

A cell-free scaffold-based cartilage repair provides improved function hyaline-like repair at one year.

Science.gov (United States)

Siclari, Alberto; Mascaro, Gennaro; Gentili, Chiara; Cancedda, Ranieri; Boux, Eugenio

2012-03-01

Bone marrow stimulation techniques in cartilage repair such as drilling are limited by the formation of fibrous to hyaline-like repair tissue. It has been suggested such techniques can be enhanced by covering the defect with scaffolds. We present an innovative approach using a polyglycolic acid (PGA)-hyaluronan scaffold with platelet-rich-plasma (PRP) in drilling. We asked whether (1) PRP immersed in a cell-free PGA-hyaluronan scaffold improves patient-reported 1-year outcomes for the Knee injury and Osteoarthritis Score (KOOS), and (2) implantation of the scaffold in combination with bone marrow stimulation leads to the formation of hyaline-like cartilage repair tissue. We reviewed 52 patients who had arthroscopic implantation of the PGA-hyaluronan scaffold immersed with PRP in articular cartilage defects of the knee pretreated with Pridie drilling. Patients were assessed by KOOS. At 9 months followup, histologic staining was performed in specimens obtained from five patients to assess the repair tissue quality. The KOOS subscores improved for pain (55 to 91), symptoms (57 to 88), activities of daily living (69 to 86), sports and recreation (36 to 70), and quality of life (38 to 73). The histologic evaluation showed a homogeneous hyaline-like cartilage repair tissue. The cell-free PGA-hyaluronan scaffold combined with PRP leads to cartilage repair and improved patient-reported outcomes (KOOS) during 12 months of followup. Histologic sections showed morphologic features of hyaline-like repair tissue. Long-term followup is needed to determine if the cartilage repair tissue is durable. Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

An Investigation of the Repair Cycle for H-53 and H-60 Helicopter Main Gearboxes - Physical Movement and Information Flows

Science.gov (United States)

1989-09-01

15 The Pipeline . . 17 The Repair Cycle 20 The Order Cycle ard Order Processing 24 Summary . . . . . . . . . . . 27 III. Methodology...cycles, and order processing . The literature is found in business logistics books, business logistics periodicals, and military periodicals. 14 II...differences and problems previously outlined (1:19). The Order Cycle and Order Processing The Nature of the Order Cycle and Order Processing . Order

Sulforaphane enhances irradiation effects in terms of perturbed cell cycle progression and increased DNA damage in pancreatic cancer cells.

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Patrick Naumann

Full Text Available Sulforaphane (SFN, an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT.In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT.Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4.Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.

Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography.

Science.gov (United States)

Thompson, Larry H

2012-01-01

The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships. Copyright © 2012 Elsevier B.V. All rights reserved.

Ku80-deleted cells are defective at base excision repair

International Nuclear Information System (INIS)

Li, Han; Marple, Teresa; Hasty, Paul

2013-01-01

Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H 2 O 2 and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs

Ku80-deleted cells are defective at base excision repair

Energy Technology Data Exchange (ETDEWEB)

Li, Han [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain); Marple, Teresa [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [The University of Texas Health Science Center at San Antonio, The Institute of Biotechnology, The Department of Molecular Medicine, 15355 Lambda Drive, San Antonio, TX 78245-3207 (United States); Tumor Suppression Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029 (Spain)

2013-05-15

Graphical abstract: - Highlights: • Ku80-deleted cells are hypersensitive to ROS and alkylating agents. • Cells deleted for Ku80, but not Ku70 or Lig4, have reduced BER capacity. • OGG1 rescues hypersensitivity to H{sub 2}O{sub 2} and paraquat in Ku80-mutant cells. • Cells deleted for Ku80, but not Lig4, are defective at repairing AP sites. • Cells deleted for Ku80, but not Lig4 or Brca2 exon 27, exhibit increased PAR. - Abstract: Ku80 forms a heterodimer with Ku70, called Ku, that repairs DNA double-strand breaks (DSBs) via the nonhomologous end joining (NHEJ) pathway. As a consequence of deleting NHEJ, Ku80-mutant cells are hypersensitive to agents that cause DNA DSBs like ionizing radiation. Here we show that Ku80 deletion also decreased resistance to ROS and alkylating agents that typically cause base lesions and single-strand breaks (SSBs). This is unusual since base excision repair (BER), not NHEJ, typically repairs these types of lesions. However, we show that deletion of another NHEJ protein, DNA ligase IV (Lig4), did not cause hypersensitivity to these agents. In addition, the ROS and alkylating agents did not induce γ-H2AX foci that are diagnostic of DSBs. Furthermore, deletion of Ku80, but not Lig4 or Ku70, reduced BER capacity. Ku80 deletion also impaired BER at the initial lesion recognition/strand scission step; thus, involvement of a DSB is unlikely. Therefore, our data suggests that Ku80 deletion impairs BER via a mechanism that does not repair DSBs.

The influence of inhibitors on dimer removal and repair of single-strand breaks in normal and bromodeoxyuridine substituted DNA of HeLa cells

International Nuclear Information System (INIS)

Cornelis, J.J.

1978-01-01

The elimination of cyclobutane pyrimidine dimers from the nuclear DNA of ultraviolet irradiated HeLa cells has been examined by means of chromatography and immunoautoradiography. The extent and duration of the process was similar when dimers were assayed by both methods, proving that the antisera recognized pyrimidine dimers. The rate of dimer excision did not differ through the cell cycle with the exception of mitosis during which no dimers were removed. Dimer excision is a relatively fast process which is terminated within a few hours, but it leaves many dimers in the DNA. Excision is depressed by inhibitors of semiconservative DNA synthesis that affect the DNA precursor pool or DNA polymerases. Cells whose DNA is partly substituted with bromodeoxyuridine instead of thymidine, repair single-strand breaks and remove dimers at the same rate but to different extents. On the other hand, inhibitors limit repair of breaks and removal of dimers to the same degree suggesting that the repair of the two types of lesion is coordinated. (Auth.)

Indirect immobilized Jagged1 suppresses cell cycle progression and induces odonto/osteogenic differentiation in human dental pulp cells.

Science.gov (United States)

Manokawinchoke, Jeeranan; Nattasit, Praphawi; Thongngam, Tanutchaporn; Pavasant, Prasit; Tompkins, Kevin A; Egusa, Hiroshi; Osathanon, Thanaphum

2017-08-31

Notch signaling regulates diverse biological processes in dental pulp tissue. The present study investigated the response of human dental pulp cells (hDPs) to the indirect immobilized Notch ligand Jagged1 in vitro. The indirect immobilized Jagged1 effectively activated Notch signaling in hDPs as confirmed by the upregulation of HES1 and HEY1 expression. Differential gene expression profiling using an RNA sequencing technique revealed that the indirect immobilized Jagged1 upregulated genes were mainly involved in extracellular matrix organization, disease, and signal transduction. Downregulated genes predominantly participated in the cell cycle, DNA replication, and DNA repair. Indirect immobilized Jagged1 significantly reduced cell proliferation, colony forming unit ability, and the number of cells in S phase. Jagged1 treated hDPs exhibited significantly higher ALP enzymatic activity, osteogenic marker gene expression, and mineralization compared with control. Pretreatment with a γ-secretase inhibitor attenuated the Jagged1-induced ALP activity and mineral deposition. NOTCH2 shRNA reduced the Jagged1-induced osteogenic marker gene expression, ALP enzymatic activity, and mineral deposition. In conclusion, indirect immobilized Jagged1 suppresses cell cycle progression and induces the odonto/osteogenic differentiation of hDPs via the canonical Notch signaling pathway.

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

International Nuclear Information System (INIS)

Bigdeli, Bahareh; Goliaei, Bahram; Masoudi-Khoram, Nastaran; Jooyan, Najmeh; Nikoofar, Alireza; Rouhani, Maryam; Haghparast, Abbas; Mamashli, Fatemeh

2016-01-01

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel radiosensitizer for

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

Energy Technology Data Exchange (ETDEWEB)

Bigdeli, Bahareh, E-mail: bhr.bigdeli@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Goliaei, Bahram, E-mail: goliaei@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Masoudi-Khoram, Nastaran, E-mail: n.masoudi@alumni.ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Jooyan, Najmeh, E-mail: n.jooyan@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Nikoofar, Alireza, E-mail: nikoofar@iums.ac.ir [Department of Radiotherapy, Iran University of Medical Sciences (IUMS), Shahid Hemmat Highway, Tehran (Iran, Islamic Republic of); Rouhani, Maryam, E-mail: rouhani@iasbs.ac.ir [Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Prof. Yousef Sobouti Blvd., Gava Zang, Zanjan (Iran, Islamic Republic of); Haghparast, Abbas, E-mail: Haghparast@sbmu.ac.ir [Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Daneshjo St., Evin, Tehran (Iran, Islamic Republic of); Mamashli, Fatemeh, E-mail: mamashli@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of)

2016-12-15

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel radiosensitizer for

Differential recruitment of DNA Ligase I and III to DNA repair sites

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Mortusewicz, Oliver; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

2006-01-01

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair. PMID:16855289

Impact of cycling cells and cell cycle regulation on Hydra regeneration.

Science.gov (United States)

Buzgariu, Wanda; Wenger, Yvan; Tcaciuc, Nina; Catunda-Lemos, Ana-Paula; Galliot, Brigitte

2018-01-15

Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

International Nuclear Information System (INIS)

Karentz, D.; Cleaver, J.E.

1986-01-01

Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia

Aging impairs double-strand break repair by homologous recombination in Drosophila germ cells.

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Delabaere, Laetitia; Ertl, Henry A; Massey, Dashiell J; Hofley, Carolyn M; Sohail, Faraz; Bienenstock, Elisa J; Sebastian, Hans; Chiolo, Irene; LaRocque, Jeannine R

2017-04-01

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Repair models of cell survival and corresponding computer program for survival curve fitting

International Nuclear Information System (INIS)

Shen Xun; Hu Yiwei

1992-01-01

Some basic concepts and formulations of two repair models of survival, the incomplete repair (IR) model and the lethal-potentially lethal (LPL) model, are introduced. An IBM-PC computer program for survival curve fitting with these models was developed and applied to fit the survivals of human melanoma cells HX118 irradiated at different dose rates. Comparison was made between the repair models and two non-repair models, the multitar get-single hit model and the linear-quadratic model, in the fitting and analysis of the survival-dose curves. It was shown that either IR model or LPL model can fit a set of survival curves of different dose rates with same parameters and provide information on the repair capacity of cells. These two mathematical models could be very useful in quantitative study on the radiosensitivity and repair capacity of cells

Repair of radiation damage in mammalian cells: its relevance to environmental effects

International Nuclear Information System (INIS)

Han, A.; Elkind, M.M.

1979-01-01

Assessment of the potential biological hazards associated with energy production technologies involves the quantitation of risk on the basis of dose-effect dependencies, from which, it is hoped, some safety guidelines can be developed. Our current knowledge of the biological importance of damage/repair processes stems by and large from radiation studies which clearly demonstrate that cellular response to radiation depends upon the ability of cells to repair the damage. Apparently, the same is true for cellular response to different chemical agents. Drawing upon our experiences from radiation studies, we demonstrate the relevance of ongoing repair processes, as evident in the studies of radiation induced cell killing and neoplastic transformation, to the type of risk estimates that might be associated with the hazards from energy production technologies. The effect of repair on cell survival is considered. It is evident from our studies that in the region of small doses, repair of damage relative to cell lethality is of importance in estimating the magnitude of effect. Aside from the cytotoxic effects in terms of cell killing, one of the greatest concerns associated with energy production is the potential of a given technology, or its effluents, to produce cancer. It is therefore of importance to quantify the risk in this context of damage registration and possible effect of repair on damage expression. It has been generally established that exposure of normal cells in culture to a variety of known carcinogens results in neoplastic transformation. Our observations with C3H/10T1/2 cells in culture lend direct evidence for the hypothesis that reduced tumor incidences at low dose rates of radiation could be due to the repair of induced damage

Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

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Quy Le

2015-09-01

Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

'Regular' and 'emergency' repair

International Nuclear Information System (INIS)

Luchnik, N.V.

1975-01-01

Experiments on the combined action of radiation and a DNA inhibitor using Crepis roots and on split-dose irradiation of human lymphocytes lead to the conclusion that there are two types of repair. The 'regular' repair takes place twice in each mitotic cycle and ensures the maintenance of genetic stability. The 'emergency' repair is induced at all stages of the mitotic cycle by high levels of injury. (author)

Selective induction of DNA repair pathways in human B cells activated by CD4+ T cells.

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Xiaosheng Wu

Full Text Available Greater than 75% of all hematologic malignancies derive from germinal center (GC or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID, GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR. Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID-/-mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells.

DNA repair in lens cells during chick embryo development

International Nuclear Information System (INIS)

Counis, M.F.; Chaudun, E.; Simonneau, L.; Courtois, Y.

1979-01-01

When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenon of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [ 3 H)thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in the experimental system the synthesis of delta- and αcrystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case. (Auth.)

Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

Science.gov (United States)

Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

2009-05-01

Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

Radiosensitivity of Nicotiana protoplasts. Action on cell; cycle effects of low dose and fractionated irradiations; biological repair

International Nuclear Information System (INIS)

Magnien, E.

1981-10-01

Leaf protoplasts of Nicotiana plumbaginifolia and Nicotiana sylvestris demonstrate five main qualities: they can be maintained as haploid lines; they constitute starting populations with a remarkable cytological homogeneity; they show a transient initial lag-phase; they yield very high plating efficiencies and retain permanently a complete differentiation capacity; being derived of a cell wall, they appear well adapted for fusion experiments or enzymatic dosages. The resumption of mitotic activity was followed by cytophotometric measurements, labelling experiments, nuclear sizing and enzymatic assays. The action of 5 Gy gamma-ray irradiations delayed entrance in the S-phase, provoked an otherwise not verified dependency between transcription, translation and protein synthesis, increased nuclear volumes in the G2-phase, and slightly stimulated the activity of a repair enzyme. The plating efficiency was a sensitive end-point which allowed the evaluation of the biological effectiveness of low to medium radiation-doses after gamma-ray and fast neutron irradiations. The neutron dose-RBE relationship increased from 3 to 25 when the dose decreased from 5 Gy to 5 mGy. When fractionated into low single doses only, a neutron dose of 300 mGy markedly increased its biological effectiveness: this phenomenon could not be explained by cell progression, and necessitated additional hypotheses involving other mechanisms in the specific action of low radiation doses. Radiation-induced UDS was measured in presence of aphidicolin. A beta-like DNA-polymerase was shown to be definitely involved in nuclear repair synthesis [fr

Defective double-strand DNA break repair and chromosomal translocations by MYC overexpression.

Science.gov (United States)

Karlsson, Asa; Deb-Basu, Debabrita; Cherry, Athena; Turner, Stephanie; Ford, James; Felsher, Dean W

2003-08-19

DNA repair mechanisms are essential for the maintenance of genomic integrity. Disruption of gene products responsible for DNA repair can result in chromosomal damage. Improperly repaired chromosomal damage can result in the loss of chromosomes or the generation of chromosomal deletions or translocations, which can lead to tumorigenesis. The MYC protooncogene is a transcription factor whose overexpression is frequently associated with human neoplasia. MYC has not been previously implicated in a role in DNA repair. Here we report that the overexpression of MYC disrupts the repair of double-strand DNA breaks, resulting in a several-magnitude increase in chromosomal breaks and translocations. We found that MYC inhibited the repair of gamma irradiation DNA breaks in normal human cells and blocked the repair of a single double-strand break engineered to occur in an immortal cell line. By spectral karyotypic analysis, we found that MYC even within one cell division cycle resulted in a several-magnitude increase in the frequency of chromosomal breaks and translocations in normal human cells. Hence, MYC overexpression may be a previously undescribed example of a dominant mutator that may fuel tumorigenesis by inducing chromosomal damage.

Cell Cycle Regulation of Stem Cells by MicroRNAs.

Science.gov (United States)

Mens, Michelle M J; Ghanbari, Mohsen

2018-06-01

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

Radioadaptive response. Efficient repair of radiation-induced DNA damage in adapted cells

International Nuclear Information System (INIS)

Ikushima, Takaji; Aritomi, Hisako; Morisita, Jun

1996-01-01

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage

A UV-sensitive human clonal cell line, RSa, which has low repair activity

International Nuclear Information System (INIS)

Suzuki, N.; Fuse, A.

1981-01-01

The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 mm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl- 3 H]thymidine ([ 3 H]dThd) or 5-[6- 3 H]bromodeoxyuridine ([ 3 H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed. (orig.)

Enhancement of postreplication repair in Chinese hamster cells

International Nuclear Information System (INIS)

D'Ambrosio, S.M.; Setlow, R.B.

1976-01-01

Alkaline sedimentation profiles of pulse-labeled DNA from Chinese hamster cells showed that DNA from cells treated with N-acetoxy-acetylaminofluorene or ultraviolet radiation was made in segments smaller than those from untreated cells. Cells treated with a small dose (2.5 μM) of N-acetoxy-acetylaminofluorene or(2.5 J . m -2 ) 254-nm radiation, several hours before a larger dose (7 to 10 μM) of N-acetoxy-acetylaminofluorene or 5.0 J . m -2 of 254-nm radiation, also synthesized small DNA after the second dose. However, the rate at which this small DNA was joined together into parental size was appreciably greater than in absence of the small dose. This enhancement of postreplication repair (as a result of the initial small dose) was not observed when cells were incubated with cycloheximide between the two treatments. The results suggest that N-acetoxy-acetylaminofluorene and ultraviolet-damaged DNA from Chinese hamster cells are repaired by similar postreplicative mechanisms that require de novo protein synthesis for enhancement

Helicobacter pylori Disrupts Host Cell Membranes, Initiating a Repair Response and Cell Proliferation

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Hsueh-Fen Juan

2012-08-01

Full Text Available Helicobacter pylori (H. pylori, the human stomach pathogen, lives on the inner surface of the stomach and causes chronic gastritis, peptic ulcer, and gastric cancer. Plasma membrane repair response is a matter of life and death for human cells against physical and biological damage. We here test the hypothesis that H. pylori also causes plasma membrane disruption injury, and that not only a membrane repair response but also a cell proliferation response are thereby activated. Vacuolating cytotoxin A (VacA and cytotoxin-associated gene A (CagA have been considered to be major H. pylori virulence factors. Gastric cancer cells were infected with H. pylori wild type (vacA+/cagA+, single mutant (ΔvacA or ΔcagA or double mutant (ΔvacA/ΔcagA strains and plasma membrane disruption events and consequent activation of membrane repair components monitored. H. pylori disrupts the host cell plasma membrane, allowing localized dye and extracellular Ca²⁺ influx. Ca²⁺-triggered members of the annexin family, A1 and A4, translocate, in response to injury, to the plasma membrane, and cell surface expression of an exocytotic maker of repair, LAMP-2, increases. Additional forms of plasma membrane disruption, unrelated to H. pylori exposure, also promote host cell proliferation. We propose that H. pylori activation of a plasma membrane repair is pro-proliferative. This study might therefore provide new insight into potential mechanisms of H. pylori-induced gastric carcinogenesis.

The Potential for Synovium-derived Stem Cells in Cartilage Repair

DEFF Research Database (Denmark)

Kubosch, Eva Johanna; Lang, Gernot Michael; Fürst, David

2018-01-01

for the treatment of large, isolated, full thickness cartilage defects. Several disadvantages such as the need for two surgical procedures or hypertrophic regenerative cartilage, underline the need for alternative cell sources. OBJECTIVE: Mesenchymal stem cells, particularly synovium-derived mesenchymal stem cells......, represent a promising cell source. Synovium-derived mesenchymal stem cells have attracted considerable attention since they display great chondrogenic potential and less hypertrophic differentiation than mesenchymal stem cells derived from bone marrow. The aim of this review was to summarize the current...... knowledge on the chondrogenic potential for synovial stem cells in regard to cartilage repair purposes. RESULTS: A literature search was carried out identifying 260 articles in the databases up to January 2017. Several in vitro and initial animal in vivo studies of cartilage repair using synovia stem cell...

Cyclin A2 promotes DNA repair in the brain during both development and aging.

Science.gov (United States)

Gygli, Patrick E; Chang, Joshua C; Gokozan, Hamza N; Catacutan, Fay P; Schmidt, Theresa A; Kaya, Behiye; Goksel, Mustafa; Baig, Faisal S; Chen, Shannon; Griveau, Amelie; Michowski, Wojciech; Wong, Michael; Palanichamy, Kamalakannan; Sicinski, Piotr; Nelson, Randy J; Czeisler, Catherine; Otero, José J

2016-07-01

Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation inCCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice.

Calvarial Suture-Derived Stem Cells and Their Contribution to Cranial Bone Repair

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Daniel H. Doro

2017-11-01

Full Text Available In addition to the natural turnover during life, the bones in the skeleton possess the ability to self-repair in response to injury or disease-related bone loss. Based on studies of bone defect models, both processes are largely supported by resident stem cells. In the long bones, the source of skeletal stem cells has been widely investigated over the years, where the major stem cell population is thought to reside in the perivascular niche of the bone marrow. In contrast, we have very limited knowledge about the stem cells contributing to the repair of calvarial bones. In fact, until recently, the presence of specific stem cells in adult craniofacial bones was uncertain. These flat bones are mainly formed via intramembranous rather than endochondral ossification and thus contain minimal bone marrow space. It has been previously proposed that the overlying periosteum and underlying dura mater provide osteoprogenitors for calvarial bone repair. Nonetheless, recent studies have identified a major stem cell population within the suture mesenchyme with multiple differentiation abilities and intrinsic reparative potential. Here we provide an updated review of calvarial stem cells and potential mechanisms of regulation in the context of skull injury repair.

Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor

International Nuclear Information System (INIS)

Waarde, Maria A.W.H. van; Assen, Annette J. van; Konings, Antonius W.T.; Kampinga, Harm H.

1996-01-01

Purpose: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radio responsiveness of tumors. Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared with DSB induction and repair in exponentially growing RIF-1 cells in culture (irradiated in vitro). Methods and Materials: A murine RIF-1 tumor grown in vivo was digested, and cells were exposed to x-rays (ex vivo) at doses of 1 to 75 Gy. DNA damage was measured using CHEF (clamped homogeneous electric fields) electrophoresis. Repair kinetics were studied at 37 deg. C for 4 h after irradiation. Radiosensitivity was determined by clonogenic assay, and cell cycle distributions by flow cytometry. For comparison, a trypsinized suspension of exponentially growing RIF-1 cells in vitro was run parallel with each ex vivo experiment. Results: Induction of DSBs, expressed as % DNA extracted from the plug, was similar in the in vitro and ex vivo irradiated cells. Compared to repair rates in in vitro cultured RIF-1 cells, repair kinetics in a freshly prepared cell suspension from the tumor were decreased, unrelated to differences in radiosensitivity. Differences in repair could not be explained by endogenous DNA degradation, nor by influences of enzymes used for digestion of the tumor. A lower plating efficiency and differences in ploidy (as revealed by flow cytometry) were the only reproducible differences between in vivo and in vitro grown cells that may explain the differences in repair kinetics. Conclusions: The current results do not support the idea that PFGE is a technique robust enough to be a predictive assay for the radiosensitivity of tumor cells

Enhanced capacity of DNA repair in human cytomegalovirus-infected cells

International Nuclear Information System (INIS)

Nishiyama, Y.; Rapp, F.

1981-01-01

Plaque formation in Vero cells by UV-irradiated herpes simplex virus was enhanced by infection with human cytomegalovirus (HCMV), UV irradiation, or treatment with methylmethanesulfonate. Preinfection of Vero cells with HCMV enhanced reactivation of UV-irradiated herpes simplex virus more significantly than did treatment with UV or methylmethanesulfonate alone. A similar enhancement by HCMV was observed in human embryonic fibroblasts, but not in xeroderma pigmentosum (XP12BE) cells. It was also found that HCMV infection enhanced hydroxyurea-resistant DNA synthesis induced by UV light or methylmethanesulfonate. Alkaline sucrose gradient sedimentation analysis revealed an enhanced rate of synthesis of all size classes of DNA in UV-irradiated HCMV-infected Vero cells. However, HCMV infection did not induce repairable lesions in cellular DNA and did not significantly inhibit host cell DNA synthesis, unlike UV or methylmethanesulfonate. These results indicate that HCMV enhanced DNA repair capacity in the host cells without producing detectable lesions in cellular DNA and without inhibiting DNA synthesis. This repair appeared to be error proof for UV-damaged herpes simplex virus DNA when tested with herpes simplex virus thymidine kinase-negative mutants

Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K., E-mail: sskumar@barc.gov.in

2015-09-15

Highlights: • Nrf2 inhibition in A549 cells led to attenuated DNA repair and radiosensitization. • Influence of Nrf2 on DNA repair is not linked to its antioxidant function. • Nrf2 influences DNA repair through homologous recombination (HR) repair pathway. • Many genes involved in HR pathway show ARE sequences in their upstream region. - Abstract: Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2

Electrical stimulation enhances cell migration and integrative repair in the meniscus

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Yuan, Xiaoning; Arkonac, Derya E.; Chao, Pen-hsiu Grace; Vunjak-Novakovic, Gordana

2014-01-01

Electrical signals have been applied towards the repair of articular tissues in the laboratory and clinical settings for over seventy years. We focus on healing of the meniscus, a tissue essential to knee function with limited innate repair potential, which has been largely unexplored in the context of electrical stimulation. Here we demonstrate for the first time that electrical stimulation enhances meniscus cell migration and integrative tissue repair. We optimize pulsatile direct current electrical stimulation parameters on cells at the micro-scale, and apply these to healing of full-thickness defects in explants at the macro-scale. We report increased expression of the adenosine A2b receptor in meniscus cells after stimulation at the micro- and macro-scale, and propose a role for A2bR in meniscus electrotransduction. Taken together, these findings advance our understanding of the effects of electrical signals and their mechanisms of action, and contribute to developing electrotherapeutic strategies for meniscus repair. PMID:24419206

Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

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Tichy, Elisia D; Pillai, Resmi; Deng, Li; Liang, Li; Tischfield, Jay; Schwemberger, Sandy J; Babcock, George F; Stambrook, Peter J

2010-11-01

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

Relevance of the nuclear division cycle to radiosensitivity in yeast

International Nuclear Information System (INIS)

Brunborg, G.; Williamson, D.H.

1978-01-01

To investigate whether the nuclear division cycle could be related to cycle-specific changes in repair of ionizing radiation damage, we have determined the survival curves after γ-irradiation of samples taken frequently from synchronously dividing cultures of Saccharomyces cerevisiae cells. Survival was low in G1 and increased during S, reaching a maximum around the end of the S phase, which was maintained in G2. The shape of the survival curves for samples taken from later stages revealed a rapid cycle-specific drop in the radioresistance of individual cells. A simple model was formulated on the assumption that survival is greatly enhanced by the action of an enzymatic repair mechanism which requires duplicated but unsegregated DNA as a substrate. (orig.) [de

Cell growth and division cycle

International Nuclear Information System (INIS)

Darzynkiewicz, Z.

1986-01-01

The concept of the cell cycle in its present form was introduced more than three decades ago. Studying incorporation of DNA precursors by autoradiography, these authors observed that DNA synthesis in individual cells was discontinuous and occupied a discrete portion of the cell life (S phase). Mitotic division was seen to occur after a certain period of time following DNA replication. A distinct time interval between mitosis and DNA replication was also apparent. Thus, the cell cycle was subdivided into four consecutive phases, G/sub 1/, S, G/sub 2/, and M. The G/sub 1/ and G/sub 2/ phases represented the ''gaps'' between mitosis and the start of DNA replication, and between the end of DNA replication and the onset of mitosis, respectively. The cell cycle was defined as the interval between the midpoint of mitosis and the midpoint of the subsequent mitosis of the daughter cell(s). The authors' present knowledge on the cell cycle benefited mostly from the development of four different techniques: autoradiography, time-lapse cinematography, cell synchronization and flow cytometry. Of these, autoradiography has been the most extensively used, especially during the past two decades. By providing a means to analyse incorporation of precursors of DNA, RNA or proteins by individual cells and, in combination with various techniques of cell synchronization, autoradiography yielded most of the data fundamental to the current understanding of the cell cycle-related phenomena. Kinetics of cell progression through the cell cycle could be analysed in great detail after development of such sophisticated autoradiographic approaches as measurements of the fraction of labeled mitoses (''FLM curves'') or multiple sequential cell labelling with /sup 3/H- and /sup 14/C-TdR

Repair of UV-induced DNA damage and its inhibition by etoposide in Sf9 insect cells: comparison with human cells

International Nuclear Information System (INIS)

Chandna, Sudhir; Dwarakanath, B.S.; Moorthy, Ganesh; Jain, Charu

2004-01-01

In the present investigation, the kinetics of DNA repair in a lepidopteran cell line Sf9 (derived from the ovaries of Spodoptera frugiperda) following UV-irradiation was compared with the responses in a human embryonic kidney cell. DNA repair was studied by analyzing the kinetics of induction and removal of repair related strand breaks using the alkaline single cell gel electrophoresis and Halo assays. Since topoisomerases play important roles in the cellular responses to UV-induced damage, the effects of etoposideon DNA repair kinetics was also studied

Cell cycle gene expression under clinorotation

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Artemenko, Olga

2016-07-01

Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

Repair capability and the cellular age response for killing and mutation induction after UV

International Nuclear Information System (INIS)

Wood, R.D.; Burki, H.J.; California Univ., Berkeley

1982-01-01

The cell-cycle response for killing and mutation induction by ultraviolet irradiation was measured in synchronous Chinese hamster ovary cells (CHO wild-type) and in a UV-hypersensitive mutant (43-3B) derived from this line. The CHO 43-3B line shows a greatly enhanced sensitivity to killing (D 0 of 0.3 as compared to 3.2 J/m 2 for the wild-type), is hypermutable, and deficient in DNA repair. For the wild-type, a characteristic age response is seen for killing by UV, with maximum sensitivity in early-S and resistance increasing through the S-phase. There is also a life-cycle specificity for induction of diphtheria-toxin resistance in late-G 1 and early-S. Relatively little variation is seen through the cell cycle for induced 6-thioguanine and ouabain resistance. In contrast, the 43-3B cell line shows a relatively 'flat' response to UV throughout the cell cycle, for both killing and mutation induction. Therefore it appears that the characteristic age responses seen in the wild-type CHO are associated with the function of an essentially error-free repair process. (orig./AJ)

Skin appendage-derived stem cells: cell biology and potential for wound repair

OpenAIRE

Xie, Jiangfan; Yao, Bin; Han, Yutong; Huang, Sha; Fu, Xiaobing

2016-01-01

Stem cells residing in the epidermis and skin appendages are imperative for skin homeostasis and regeneration. These stem cells also participate in the repair of the epidermis after injuries, inducing restoration of tissue integrity and function of damaged tissue. Unlike epidermis-derived stem cells, comprehensive knowledge about skin appendage-derived stem cells remains limited. In this review, we summarize the current knowledge of skin appendage-derived stem cells, including their fundament...

DNA repair in a Fanconi's anemia fibroblast cell strain

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Little, J.B.; Weichselbaum, R.R.

1979-01-01

DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution. (Auth.)

DNA repair in a Fanconi's anemia fibroblast cell strain

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Fornace, Jr, A J; Little, J B [Harvard School of Public Health, Boston, MA (USA); Weichselbaum, R R [Harvard Medical School, Boston, MA (USA)

1979-01-26

DNA repair and colony survival were measured in fibroblasts from a patient with Fanconi's anemia, HG 261, and from normal human donors after exposure to these cells to the cross-linking agent mitomycin C, X-rays or ultraviolet light. Survival was similar in HG 261 and normal cells after X-ray or ultraviolet radiation, but was reduced in the Fanconi's anemia cells after treatment with mitomycin C. The level of DNA cross-linking, as measured by the method of alkaline elution, was the same in both cell strains after exposure to various doses of mitomycin C. With incubation after drug treatment, a gradual decrease in the amount of cross-linking was observed, the rate of this apparent repair of cross-link damage was the same in both normal and HG 261 cells. The rejoining of DNA single strand breaks after X-irradiation and the production of excision breaks after ultraviolet radiation were also normal in HG 261 cells as determined by alkaline elution.

Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells.

Science.gov (United States)

Chang, Hsin-Yi; Shih, Meng-Her; Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

2013-01-01

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3-5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G(2)/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G(2)/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.

Cell cycle control by components of cell anchorage

OpenAIRE

Gad, Annica

2005-01-01

Extracellular factors, such as growth factors and cell anchorage to the extracellular matrix, control when and where cells may proliferate. This control is abolished when a normal cell transforms into a tumour cell. The control of cell proliferation by cell anchorage was elusive and less well studied than the control by growth factors. Therefore, we aimed to clarify at what points in the cell cycle and through which molecular mechanisms cell anchorage controls cell cycle pro...

Measurement of DNA double-strand breaks in CHO cells at various stages of the cell cycle using pulsed field gel electrophoresis: calibration by means of 125I decay

International Nuclear Information System (INIS)

Iliakis, G.E.; Cicilioni, O.; Metzger, L.

1991-01-01

Experiments were performed to calibrate a recently developed pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (AFIGE), for the measurement of double-strand breaks (dsb) in the DNA of mammalian cells. Calibration was carried out by means of 125 I decay accumulation, under conditions preventing repair, based on the observation that each 125 I decay in the DNA produces approximately one dsb. Results suggest that that observed fluctuations in the fraction of DNA activity released (FAR) per Gy throughout the cycle reflect cell-cycle-associated differences in the physicochemical properties of the DNA molecules that alter their electrophoretic mobility, rather than variations in the induction of dsb per Gy, i.e. the sensitivity of the assay fluctuates throughout the cycle. (author)

Quantitative aspects of repair of potentially lethal damage in mammalian cells

International Nuclear Information System (INIS)

Iliakis, G.; Pohlit, W.

1979-01-01

Stationary cultures of Ehrlich ascites tumour cells were irradiated with X-rays and then immediately or after a time interval tsub(rep) plated to measure the survival. The increase in survival observed after delayed plating was interpreted as repair of potentially lethal damage. A cybernetic model was used to analyse these data. Three states of damage were assumed for the cells. In state A the cells could grow to macrocolonies, in state B the cells suffered potentially lethal damage and could grow to macrocolonies only if they were allowed to repair the damage and in state C the cells were lethally damaged. A method of deriving the values of the parameters of the model from the experimental data was given. The dependence of the reaction rate constant of the repair potentially lethal damage on the dose D was used to derive a possible mechanism for the production of the shoulder in the dose effect curve. Finally this model was compared with other models of radiation action in living cells. (author)

Inhibition of DNA replication and repair by anthralin or danthron in cultured human cells

International Nuclear Information System (INIS)

Clark, J.M.; Hanawalt, P.C.

1982-01-01

The comparative effects of the tumor promoter anthralin and its analog, danthron, on semiconservative DNA replication and DNA repair synthesis were studied in cultured human cells. Bromodeoxyuridine was used as density label together with 3 H-thymidine to distinguish replication from repair synthesis in isopycnic CsCl gradients. Anthralin at 1.1 microgram inhibited replication in T98G cells by 50%. In cells treated with 0.4 or 1.3 microM anthralin and additive effect was observed on the inhibition of replication by ultraviolet light (254 nm). In cells irradiated with 20 J/m2, 2.3 microM anthralin was required to inhibit repair synthesis by 50%. Thus there was no selective inhibitory effect of anthralin on repair synthesis. Danthron exhibited no detectable effect on either semiconservative replication or repair synthesis at concentrations below about 5.0 microM. Neither compound stimulated repair synthesis in the absence of ultraviolet irradiation. Thus, anthralin and danthron do not appear to react with DNA to form adducts that are subject to excision repair. Although both compounds appear to intercalate into supercoiled DNA in vitro to a limited extent, the degree of unwinding introduced by the respective drugs does not correlate with their relative effects on DNA synthesis in vivo. Therefore the inhibitory effect of anthralin on DNA replication and repair synthesis in T98G cells does not appear to result from the direct interaction of the drug with DNA

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

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King, Harry O; Brend, Tim; Payne, Helen L; Wright, Alexander; Ward, Thomas A; Patel, Karan; Egnuni, Teklu; Stead, Lucy F; Patel, Anjana; Wurdak, Heiko; Short, Susan C

2017-01-10

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines

International Nuclear Information System (INIS)

Bedford, P.; Fichtinger-Schepman, A.M.; Shellard, S.A.; Walker, M.C.; Masters, J.R.; Hill, B.T.

1988-01-01

The formation and removal of four platinum-DNA adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (RT112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 microM (5 micrograms/ml) cisplatin. RT112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinated DNA may account for their hypersensitivity to cisplatin

Repair of soft X-ray damage to mammalian cell DNA

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Meldrum, R.A.; Wharton, C.W. (Birmingham Univ. (UK). Dept. of Biochemistry)

1990-10-01

Inhibitors of polymerase {alpha} (hydroxyurea and cytosine arabinoside) and an inhibitor of polymerase {beta} and ''delta (di-deoxythymidine) had equal inhibitory effects on repair synthesis in the first 15 min after irradiation of Chinese hamster ovary cells with soft x-rays produced from a laser plasma. Polymerase {alpha} inhibitors had considerably more effect after 15 min following irradiation. This implies that polymerase {alpha}, {beta}, and/or {delta} are all equally active in the initial stages of repair synthesis after soft X-radiation, but {alpha}-activity is more prominent in later stages of repair synthesis. Polymerase {alpha} is thought to catalyse long-patch repair synthesis, while polymerase {beta} is thought to catalyse short-patch repair. Polymerase {delta} has been shown to be active in DNA repair synthesis, but its precise function is as yet uncertain. (author).

DNA repair

International Nuclear Information System (INIS)

Van Zeeland, A.A.

1984-01-01

In this chapter a series of DNA repair pathways are discussed which are available to the cell to cope with the problem of DNA damaged by chemical or physical agents. In the case of microorganisms our knowledge about the precise mechanism of each DNA repair pathway and the regulation of it has been improved considerably when mutants deficient in these repair mechanisms became available. In the case of mammalian cells in culture, until recently there were very little repair deficient mutants available, because in almost all mammalian cells in culture at least the diploid number of chromosomes is present. Therefore the frequency of repair deficient mutants in such populations is very low. Nevertheless because replica plating techniques are improving some mutants from Chinese hamsters ovary cells and L5178Y mouse lymphoma cells are now available. In the case of human cells, cultures obtained from patients with certain genetic diseases are available. A number of cells appear to be sensitive to some chemical or physical mutagens. These include cells from patients suffering from xeroderma pigmentosum, Ataxia telangiectasia, Fanconi's anemia, Cockayne's syndrome. However, only in the case of xeroderma pigmentosum cells, has the sensitivity to ultraviolet light been clearly correlated with a deficiency in excision repair of pyrimidine dimers. Furthermore the work with strains obtained from biopsies from man is difficult because these cells generally have low cloning efficiencies and also have a limited lifespan in vitro. It is therefore very important that more repair deficient mutants will become available from established cell lines from human or animal origin

Breast cancer stem cell-like cells are more sensitive to ionizing radiation than non-stem cells: role of ATM.

Directory of Open Access Journals (Sweden)

Seog-Young Kim

Full Text Available There are contradictory observations about the different radiosensitivities of cancer stem cells and cancer non-stem cells. To resolve these contradictory observations, we studied radiosensitivities by employing breast cancer stem cell (CSC-like MDA-MB231 and MDA-MB453 cells as well as their corresponding non-stem cells. CSC-like cells proliferate without differentiating and have characteristics of tumor-initiating cells [1]. These cells were exposed to γ-rays (1.25-8.75 Gy and survival curves were determined by colony formation. A final slope, D(0, of the survival curve for each cell line was determined to measure radiosensitivity. The D(0 of CSC-like and non-stem MDA-MB-453 cells were 1.16 Gy and 1.55 Gy, respectively. Similar results were observed in MDA-MB-231 cells (0.94 Gy vs. 1.56 Gy. After determination of radiosensitivity, we investigated intrinsic cellular determinants which influence radiosensitivity including cell cycle distribution, free-radical scavengers and DNA repair. We observed that even though cell cycle status and antioxidant content may contribute to differential radiosensitivity, differential DNA repair capacity may be a greater determinant of radiosensitivity. Unlike non-stem cells, CSC-like cells have little/no sublethal damage repair, a low intracellular level of ataxia telangiectasia mutated (ATM and delay of γ-H2AX foci removal (DNA strand break repair. These results suggest that low DNA repair capacity is responsible for the high radiosensitivity of these CSC-like cells.

The role of undifferentiated adipose-derived stem cells in peripheral nerve repair.

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Zhang, Rui; Rosen, Joseph M

2018-05-01

Peripheral nerve injuries impose significant health and economic consequences, yet no surgical repair can deliver a complete recovery of sensory or motor function. Traditional methods of repair are less than ideal: direct coaptation can only be performed when tension-free repair is possible, and transplantation of nerve autograft can cause donor-site morbidity and neuroma formation. Cell-based therapy delivered via nerve conduits has thus been explored as an alternative method of nerve repair in recent years. Stem cells are promising sources of the regenerative core material in a nerve conduit because stem cells are multipotent in function, abundant in supply, and more accessible than the myelinating Schwann cells. Among different types of stem cells, undifferentiated adipose-derived stem cell (uASC), which can be processed from adipose tissue in less than two hours, is a promising yet underexplored cell type. Studies of uASC have emerged in the past decade and have shown that autologous uASCs are non-immunogenic, easy to access, abundant in supply, and efficacious at promoting nerve regeneration. Two theories have been proposed as the primary regenerative mechanisms of uASC: in situ trans-differentiation towards Schwann cells, and secretion of trophic and anti-inflammatory factors. Future studies need to fully elucidate the mechanisms, side effects, and efficacy of uASC-based nerve regeneration so that uASCs can be utilized in clinical settings.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

Science.gov (United States)

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Immediate and repair induced DNA double strand breaks in mammalian cells

International Nuclear Information System (INIS)

Bryant, P.E.

1986-01-01

It seems logical to postulate that double strand breaks (dsb) arising both at the time of irradiation and via repair processes are potentially equally damaging for a cell in terms of the potential to induce chromosomal aberrations. However, in some cell systems the repair of double es or es-ssb sites may run concurrently with the incision so that these lesions do not remain open for long: hence the lack of accumulation of dsb during repair. The rate of incision will thus determine both the accumulation and the probability of exchanges leading to chromosomal aberrations between these and other frank dsb. Rapid incision leading to a large additional pool of dsb appears to be the case in Chinese hamster V79 cells. Some evidence also exists for the conversion of base damage, via dsb, into deletion type chromatid aberrations which accumulate in irradiated G2 human cells treated with ara C. A small fraction of dsb, probably arising both at the time of irradiation as well as enzymatically during repair of base or sugar damage, appears to be either left unrepaired, yielding deletion type chromosomal aberrations, or is misrepaired, yielding exchange aberrations. The induction of these aberrations appears to be of central importance in the biological effects of ionizing radiation such as mutations, oncogenic transformation, and cell death. 52 refs., 5 figs

Annexins are instrumental for efficient plasma membrane repair in cancer cells.

Science.gov (United States)

Lauritzen, Stine Prehn; Boye, Theresa Louise; Nylandsted, Jesper

2015-09-01

Plasma membrane stress can cause damage to the plasma membrane, both when imposed by the extracellular environment and by enhanced oxidative stress. Cells cope with these injuries by rapidly activating their plasma membrane repair system, which is triggered by Ca(2+) influx at the wound site. The repair system is highly dynamic, depends on both lipid and protein components, and include cytoskeletal reorganization, membrane replacements, and membrane fusion events. Cancer cells experience enhanced membrane stress when navigating through dense extracellular matrix, which increases the frequency of membrane injuries. In addition, increased motility and oxidative stress further increase the risk of plasma membrane lesions. Cancer cells compensate by overexpressing Annexin proteins including Annexin A2 (ANXA2). Annexin family members can facilitate membrane fusion events and wound healing by binding to negatively charged phospholipids in the plasma membrane. Plasma membrane repair in cancer cells depends on ANXA2 protein, which is recruited to the wound site and forms a complex with the Ca(2+)-binding EF-hand protein S100A11. Here they regulate actin accumulation around the wound perimeter, which is required for wound closure. In this review, we will discuss the requirement for Annexins, S100 proteins and actin cytoskeleton in the plasma membrane repair response of cancer cells, which reveals a novel avenue for targeting metastatic cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

Science.gov (United States)

El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

Mesenchymal Stem Cells in Tissue Growth and Repair

OpenAIRE

Kalinina, N.I.; Sysoeva, V.Yu.; Rubina, K.A.; Parfenova, Ye.V.; Tkachuk, V.A.

2011-01-01

It has been established in the recent several decades that stem cells play a crucial role in tissue renewal and regeneration. Mesenchymal stem cells (MSCs) are part of the most important population of adult stem cells. These cells have hereby been identified for the very first time and subsequently isolated from bone marrow stroma. Bone marrow-derived MSCs have been believed to play the role of a source of cells for the renewal and repair of connective tissues, including bone, cartilage and a...

Molecular dosimetry of chemical mutagens: measurement of molecular dose and DNA repair germ cells

International Nuclear Information System (INIS)

Sega, G.A.

1975-01-01

Molecular dosimetry in the germ cells of male mice is reviewed with regard to in vivo alkylation of sperm heads, in vivo alkylation of sperm DNA, and possible alkylation of sperm protamine. DNA repair in male germ cells is reviewed with regard to basic design of experiments, DNA repair in various stages of spermatogenesis, effect of protamine on DNA repair following treatment with EMS or x radiation, and induction of DNA repair by methyl methanesulfonate, propyl methanesulfonate, and isopropyl methanesulfonate

Lack of effect of inhibitors of DNA synthesis/repair on the ionizing radiation-induced chromosomal damage in G[sub 2] stage of ataxia telangiectasia cells

Energy Technology Data Exchange (ETDEWEB)

Antoccia, A. (Univ. ' La Sapienza' , Rome (Italy). Dipt. di Genetica e Biologia Molecolare); Palitti, F.; Raggi, T. (Univ. del Tuscia, Viterbo (Italy). Dipt. di Agrobiologia ed Agrochimica); Catena, C. (ENEA, Casaccia (Italy). Centro Ricerche Energia); Tanzarella, C. (Rome Univ. 3 (Italy). Dipt. di Biologia)

1994-09-01

The relationship between the repair processes occurring at the G[sub 2] phase of the cell cycle and cytogenetic damage in ataxia telangiectasia (AT) cells was studied. Lymphoblastoid cells derived from normal, heterozygote AT (HzAT) and three AT patients were exposed to X-rays or fission neutrons and post-treated with inhibitors of DNA synthesis/repair, such as inhibitors of DNA polymerases [alpha], [sigma] and [epsilon] (cytosine arabinoside, ara-C; aphidicolin, APC; buthylphenyl-guanine, BuPdG) or ribonucleotide reductase (hydroxyurea HU). A strong increase of radiation-induced chromosomal aberrations was observed in normal and HzAT cells post-treated with ara-C, APC and HU, but not in the presence of BuPdG. No enhancing effect was observed in cells derived from AT patients, except for HU post-irradiation treatment. These results suggest that the enzymes that can be inhibited by these agents are not directly involved in the repair of radiation damage induced in G[sub 2] cells from AT patients, indicating that probably the AT cells that we used lack the capability to transform the primary DNA lesions into reparable products, or that AT cells might contain a mutated form of DNA polymerase resistant to the inhibitors. (author).

Visualization of DNA double-strand break repair: From molecules to cells

NARCIS (Netherlands)

Krawczyk, Przemek M.; Stap, Jan; Aten, Jacob A.

2008-01-01

DNA double-strand break (DSB) signaling and repair processes are positioned at the crossroad of nuclear pathways that regulate DNA replication, cell division, senescence and apoptosis. Importantly, errors in DSB repair may lead to lethal or potentially tumorigenic chromosome rearrangements.

Cell Seeding Densities in Autologous Chondrocyte Implantation Techniques for Cartilage Repair.

Science.gov (United States)

Foldager, Casper Bindzus; Gomoll, Andreas H; Lind, Martin; Spector, Myron

2012-04-01

Cartilage repair techniques have been among the most intensively investigated treatments in orthopedics for the past decade, and several different treatment modalities are currently available. Despite the extensive research effort within this field, the generation of hyaline cartilage remains a considerable challenge. There are many parameters attendant to each of the cartilage repair techniques that can affect the amount and types of reparative tissue generated in the cartilage defect, and some of the most fundamental of these parameters have yet to be fully investigated. For procedures in which in vitro-cultured autologous chondrocytes are implanted under a periosteal or synthetic membrane cover, or seeded onto a porous membrane or scaffold, little is known about how the number of cells affects the clinical outcome. Few published clinical studies address the cell seeding density that was employed. The principal objective of this review is to provide an overview of the cell seeding densities used in cell-based treatments currently available in the clinic for cartilage repair. Select preclinical studies that have informed the use of specific cell seeding densities in the clinic are also discussed.

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

DEFF Research Database (Denmark)

Larsen, Dorthe Helena; Poinsignon, Catherine; Gudjonsson, Thorkell

2010-01-01

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate...

A hybrid mammalian cell cycle model

Directory of Open Access Journals (Sweden)

Vincent Noël

2013-08-01

Full Text Available Hybrid modeling provides an effective solution to cope with multiple time scales dynamics in systems biology. Among the applications of this method, one of the most important is the cell cycle regulation. The machinery of the cell cycle, leading to cell division and proliferation, combines slow growth, spatio-temporal re-organisation of the cell, and rapid changes of regulatory proteins concentrations induced by post-translational modifications. The advancement through the cell cycle comprises a well defined sequence of stages, separated by checkpoint transitions. The combination of continuous and discrete changes justifies hybrid modelling approaches to cell cycle dynamics. We present a piecewise-smooth version of a mammalian cell cycle model, obtained by hybridization from a smooth biochemical model. The approximate hybridization scheme, leading to simplified reaction rates and binary event location functions, is based on learning from a training set of trajectories of the smooth model. We discuss several learning strategies for the parameters of the hybrid model.

Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Wang, Z.; Wu, X.; Friedberg, E.C.

1993-01-01

A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg 2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

Repair of DNA double-strand breaks and cell killing by charged particles

Science.gov (United States)

Eguchi-Kasai, K.; Murakami, M.; Itsukaichi, H.; Fukutsu, K.; Yatagai, F.; Kanai, T.; Ohara, H.; Sato, K.

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing. We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/mum and were even smaller than unity for the LET region greater than 300 keV/mum. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/mum, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main cause of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

Energy Technology Data Exchange (ETDEWEB)

Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

2007-12-31

Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells

International Nuclear Information System (INIS)

Gedik, C.M.; Collins, A.R.; Ewen, S.W.B.

1992-01-01

The authors have adapted procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m -2 and incubation for as little as 5 min. The authors also studied UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, key incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examined consequent breaks both by SCGE and by alkaline unwinding to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10 9 daltons are detected. (Author)

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

Science.gov (United States)

JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

2016-06-17

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

The inhibition of repair in UV irradiated human cells

International Nuclear Information System (INIS)

Collins, A.R.S.; Schor, S.L.; Johnson, R.T.

1977-01-01

Three different assay procedures are used to determine the effects of hydroxyurea on excision repair in UV-irradiated HeLa cells. At the cytological level, incubation of UV-irradiated metaphase cells with hydroxyurea caused chromosome decondensation. Using a modified alkaline sucrose gradient sedimentation technique involving minimal lysis before centrifugation, a marked retardation was found in the sedimentation of DNA from UV-irradiated cells incubated for a short period with hydroxyurea. The effect of hydroxyurea on the incorporation of [ 3 H]thymidine by UV-irradiated G1 cells was found to depend on the concentration of thymidine present in the medium. The results point to an inhibition of repair DNA synthesis by hydroxyurea (or deoxyadenosine), at the level of the supply of DNA precursors, i.e. in the same way that these agents inhibit semiconservative DNA synthesis. In the presence of these inhibitors, single-strand gaps accumulate in the DNA

Intrinsic radiosensitivity and PLD repair in osteosarcoma cell lines

International Nuclear Information System (INIS)

Sugimoto, M.; Toguchida, J.; Kotoura, Y.; Yamamuro, T.; Utsumi, H.

1992-01-01

The response to radiation of seven osteosarcoma cell lines was analysed by in vitro colony-forming assay and compared with that of eight human fibroblast strains. The values of D 0 , the surviving fraction after 2 Gy (S2Gy), and the mean inactivation dose (D-bar) of osteosarcoma cells in log-phase culture were significantly higher than those of fibroblast strains (p<0.01). PLD (potentially lethal damage) repair of osteosarcoma cells evaluated in the plateau phase of growth showed great variation for enhancement of survival, although all of the values were maximised within 12 h after irradiation. In the osteosarcoma, intrinsic radiosensitivity in vitro reflected the clinical response to radiation. However, the capacity for PLD repair might not be a good indicator for predicting the results of radiation therapy. (author)

DNA repair and induction of plasminogen activator in human fetal cells treated with ultraviolet light

International Nuclear Information System (INIS)

Ben-Ishai, R.; Sharon, R.; Rothman, M.; Miskin, R.

1984-01-01

We have tested human fetal fibroblasts for development associated changes in DNA repair by utilizing nucleoid sedimentation as an assay for excision repair. Among skin fibroblasts the rate of excision repair was significantly higher in non-fetal cells than in fibroblasts derived from an 8 week fetus; this was evident by a delay in both the relaxation and the restoration of DNA supercoiling in nucleoids after irradiation. Skin fibroblasts derived at 12 week gestation were more repair proficient than those derived at 8 week gestation. However, they exhibited a somewhat lower rate of repair than non-fetal cells. The same fetal and non-fetal cells were also tested for induction of the protease plasminogen activator (PA) after u.v. irradiation. Enhancement of PA was higher in skin fibroblasts derived at 8 week than in those derived at 12 week gestation and was absent in non-fetal skin fibroblasts. These results are consistent with our previous findings that in human cells u.v. light-induced PA synthesis is correlated with reduced DNA repair capacity. Excision repair and PA inducibility were found to depend on tissue of origin in addition to gestational stage, as shown for skin and lung fibroblasts from the same 12 week fetus. Lung compared to skin fibroblasts exhibited lower repair rates and produced higher levels of PA after irradiation. The sedimentation velocity of nucleoids, prepared from unirradiated fibroblasts, in neutral sucrose gradients with or without ethidium bromide, indicated the presence of DNA strand breaks in fetal cells. It is proposed that reduced DNA repair in fetal cells may result from alterations in DNA supercoiling, and that persistent DNA strand breaks enhance transcription of PA gene(s)

Modelling cell cycle synchronisation in networks of coupled radial glial cells.

Science.gov (United States)

Barrack, Duncan S; Thul, Rüdiger; Owen, Markus R

2015-07-21

Radial glial cells play a crucial role in the embryonic mammalian brain. Their proliferation is thought to be controlled, in part, by ATP mediated calcium signals. It has been hypothesised that these signals act to locally synchronise cell cycles, so that clusters of cells proliferate together, shedding daughter cells in uniform sheets. In this paper we investigate this cell cycle synchronisation by taking an ordinary differential equation model that couples the dynamics of intracellular calcium and the cell cycle and extend it to populations of cells coupled via extracellular ATP signals. Through bifurcation analysis we show that although ATP mediated calcium release can lead to cell cycle synchronisation, a number of other asynchronous oscillatory solutions including torus solutions dominate the parameter space and cell cycle synchronisation is far from guaranteed. Despite this, numerical results indicate that the transient and not the asymptotic behaviour of the system is important in accounting for cell cycle synchronisation. In particular, quiescent cells can be entrained on to the cell cycle via ATP mediated calcium signals initiated by a driving cell and crucially will cycle in near synchrony with the driving cell for the duration of neurogenesis. This behaviour is highly sensitive to the timing of ATP release, with release at the G1/S phase transition of the cell cycle far more likely to lead to near synchrony than release during mid G1 phase. This result, which suggests that ATP release timing is critical to radial glia cell cycle synchronisation, may help us to understand normal and pathological brain development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

Science.gov (United States)

Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

2011-05-01

Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. Copyright © 2011 Elsevier Ltd. All rights reserved.

Stem Cells in Tissue Repair and Regeneration

OpenAIRE

Falanga, Vincent

2012-01-01

The field of tissue repair and wound healing has blossomed in the last 30 years. We have gone from recombinant growth factors, to living tissue engineering constructs, to stem cells. The task now is to pursue true regeneration, thus achieving full restoration of structures and their function.

Repair-dependent cell radiation survival and transformation: an integrated theory

International Nuclear Information System (INIS)

Sutherland, John C

2014-01-01

The repair-dependent model of cell radiation survival is extended to include radiation-induced transformations. The probability of transformation is presumed to scale with the number of potentially lethal damages that are repaired in a surviving cell or the interactions of such damages. The theory predicts that at doses corresponding to high survival, the transformation frequency is the sum of simple polynomial functions of dose; linear, quadratic, etc, essentially as described in widely used linear-quadratic expressions. At high doses, corresponding to low survival, the ratio of transformed to surviving cells asymptotically approaches an upper limit. The low dose fundamental- and high dose plateau domains are separated by a downwardly concave transition region. Published transformation data for mammalian cells show the high-dose plateaus predicted by the repair-dependent model for both ultraviolet and ionizing radiation. For the neoplastic transformation experiments that were analyzed, the data can be fit with only the repair-dependent quadratic function. At low doses, the transformation frequency is strictly quadratic, but becomes sigmodial over a wider range of doses. Inclusion of data from the transition region in a traditional linear-quadratic analysis of neoplastic transformation frequency data can exaggerate the magnitude of, or create the appearance of, a linear component. Quantitative analysis of survival and transformation data shows good agreement for ultraviolet radiation; the shapes of the transformation components can be predicted from survival data. For ionizing radiations, both neutrons and x-rays, survival data overestimate the transforming ability for low to moderate doses. The presumed cause of this difference is that, unlike UV photons, a single x-ray or neutron may generate more than one lethal damage in a cell, so the distribution of such damages in the population is not accurately described by Poisson statistics. However, the complete

Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.

Science.gov (United States)

Ahn, Eunyong; Kumar, Praveen; Mukha, Dzmitry; Tzur, Amit; Shlomi, Tomer

2017-11-06

Cellular metabolic demands change throughout the cell cycle. Nevertheless, a characterization of how metabolic fluxes adapt to the changing demands throughout the cell cycle is lacking. Here, we developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle. This is achieved by combining pulse-chase LC-MS-based isotope tracing in synchronized cell populations with computational deconvolution and metabolic flux modeling. We find that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes: Oxidation of glucose-derived flux peaks in late G1 phase, while oxidative and reductive glutamine metabolism dominates S phase. These complementary flux oscillations maintain a constant production rate of reducing equivalents and oxidative phosphorylation flux throughout the cell cycle. The shift from glucose to glutamine oxidation in S phase plays an important role in cell cycle progression and cell proliferation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Cell cycle regulation of hematopoietic stem or progenitor cells.

Science.gov (United States)

Hao, Sha; Chen, Chen; Cheng, Tao

2016-05-01

The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

Effects of cell-to-collagen ratio in stem cell-seeded constructs for Achilles tendon repair.

Science.gov (United States)

Juncosa-Melvin, Natalia; Boivin, Gregory P; Galloway, Marc T; Gooch, Cindi; West, John R; Butler, David L

2006-04-01

The objective of the present study was to test the hypotheses that implantation of cell-seeded constructs in a rabbit Achilles tendon defect model would 1) improve repair biomechanics and matrix organization and 2) result in higher failure forces than measured in vivo forces in normal rabbit Achilles tendon (AT) during an inclined hopping activity. Autogenous tissue-engineered constructs were fabricated in culture between posts in the wells of silicone dishes at four cell-to-collagen ratios by seeding mesenchymal stem cells (MSC) from 18 adult rabbits at each of two seeding densities (0.1 x 10(6) and 1 x 10(6) cell/mL) in each of two collagen concentrations (1.3 and 2.6 mg/mL). After 5 days of contraction, constructs having the two highest ratios (0.4 and 0.8 M/mg) were damaged by excessive cell traction forces and could not be used in subsequent in vivo studies. Constructs at the lower ratios (0.04 and 0.08 M/mg) were implanted in bilateral, 2 cm long gap defects in the rabbit's lateral Achilles tendon. At 12 weeks after surgery, both repair tissues were isolated and either failed in tension (n = 13) to determine their biomechanical properties or submitted for histological analysis (n = 5). No significant differences were observed in any structural or mechanical properties or in histological appearance between the two repair conditions. However, the average maximum force and maximum stress of these repairs achieved 50 and 85% of corresponding values for the normal AT and exceeded the largest peak in vivo forces (19% of failure) previously recorded in the rabbit AT. Average stiffness and modulus were 60 and 85% of normal values, respectively. New constructs with lower cell densities and higher scaffold stiffness that do not excessively contract and tear in culture and that further improve the repair stiffness needed to withstand various levels of expected in vivo loading are currently being investigated.

Repair of sublethal damage in mammalian cells irradiated at ultrahigh dose rates

International Nuclear Information System (INIS)

Gerweck, L.E.; Epp, E.R.; Michaels, H.B.; Ling, C.C.; Peterson, E.C.

1979-01-01

The lethal response of asynchronous Chinese hamster ovary (CHO) cells exposed to single and split doses of radiation at conventional or ultrahigh dose rates has been examined to determine whether repair of sublethal damage occurs in cells irradiated at ultrahigh dose rates. The high-intensity irradiations were performed with electrons delivered in single 3-nsec pulses from a 600-kV field emission source under medium-removed, thin-layer conditions. Conventional dose-rate experiments were done under identical thin-layer conditions with 50-kVp x rays, or under full-medium conditions with 280-kVp x rays. Oxygenated cells were irradiated and maintained at 22 to 24 0 C between exposures. Survival did not increase as the time between two doses of pulsed electrons increased from 0 to 4 min, indicating no evidence of fast repair. However, increased survival was observed when 30 to 90 min was allowed to elapse between the split doses. The half-time for maximum repair was approx. = 30 min irrespective of the exposure conditions and radiation modality used. Observed repair ratios increased from approx. = 2 to 4 as the single-dose surviving fraction decreased from 10 -2 to 5 x 10 -4 . Over this survival range the repair ratios, measured at the same value of surviving fraction, were independent of dose rate. The observed repair ratios imply that the shoulder regions of the nonfractionated x-ray and pulsed-electron survival curves were not completely restored between the split doses. However, the fraction of the shoulder restored between split doses of radiation was dose-rate-independent. It is concluded that sublethal damage can be repaired in oxygenated CHO cells irradiated at dose rates of the order of 10 11 rad/sec

Localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.

1987-01-01

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. We have investigated whether the repair of DNA damage occurs in the nuclear matrix compartment. Normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m/sup -2/ and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix. 5 J m/sup -2/ repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m/sup -2/) normal human fibroblast post-u.v. incubated in the presence of hydroxyurea and arabinsosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C) in contrast to XP-D cells and Syrian hamster embryonic cells.

Epigenetic dynamics across the cell cycle

DEFF Research Database (Denmark)

Kheir, Tony Bou; Lund, Anders H.

2010-01-01

Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

Analysis of X-ray induced cell-cycle perturbations in mouse osteosarcoma cells: a two-signal cell-cycle model

International Nuclear Information System (INIS)

Meeteren, A. van; Wijk, R. van; Stap, J.; Deys, B.F.

1984-01-01

The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. The first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. Of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. The X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cell-cycle-dependent. A qualitative model to account for the observations is presented. (author)

Analysis of X-ray induced cell-cycle perturbations in mouse osteosarcoma cells: a two-signal cell-cycle model

Energy Technology Data Exchange (ETDEWEB)

Meeteren, A van; Wijk, R van [Rijksuniversiteit Utrecht (Netherlands); Stap, J; Deys, B F [Amsterdam Univ. (Netherlands)

1984-03-01

The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. The first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. Of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. The X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cell-cycle-dependent. A qualitative model to account for the observations is presented.

Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

International Nuclear Information System (INIS)

Gustafsson, Ann-Sofie; Abramenkovs, Andris; Stenerlöw, Bo

2014-01-01

Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

Energy Technology Data Exchange (ETDEWEB)

Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

2014-11-15

Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

Repair capability of mammalian cell fractions demonstrated using infectivity of bacteriophage DNA

International Nuclear Information System (INIS)

Lai, S.P.; Lytle, C.D.; Benane, S.G.

1976-01-01

Extracts of Potoroo kidney cells (PtK2) were examined for ability to provide a repair function in vitro. The biological activity (infectivity) of uv-irradiated replicative form (RF) DNA of bacteriophage phiX174 was restored during incubation of the DNA with a nuclear extract but not with a cytoplasmic extract. The infectivity of the RF-DNA was determined in spheroplasts of E. coli C/sub s/, which is HCR - . This system for biological assay of uv-irradiated DNA repaired in vitro may be used to complement biochemical and biophysical investigations of molecular repair mechanisms in mammalian cells

Recent progress with the DNA repair mutants of Chinese hamster ovary cells

International Nuclear Information System (INIS)

Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

1986-01-01

Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs

Reserve stem cells: Reprogramming of differentiated cells fuels repair, metaplasia, and neoplasia in the adult gastrointestinal tract

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Mills, Jason C.; Sansom, Owen J.

2016-01-01

It has long been known that differentiated cells can switch fates, especially in vitro, but only recently has there been a critical mass of publications describing the mechanisms adult, post-mitotic cells use in vivo to reverse their differentiation state. We propose that this sort of cellular reprogramming is a fundamental cellular process akin to apoptosis or mitosis. Because reprogramming can invoke regenerative cells from mature cells, it is critical to the longterm maintenance of tissues like the pancreas, which encounter large insults during adulthood but lack constitutively active adult stem cells to repair the damage. However, even in tissues with adult stem cells, like stomach and intestine, reprogramming may allow mature cells to serve as reserve (“quiescent”) stem cells when normal stem cells are compromised. We propose that the potential downside to reprogramming is that it increases risk for cancers that occur late in adulthood. Mature, long-lived cells may have years of exposure to mutagens. Mutations that affect the physiological function of differentiated, post-mitotic cells may lead to apoptosis, but mutations in genes that govern proliferation might not be selected against. Hence, reprogramming with reentry into the cell cycle might unmask those mutations, causing an irreversible progenitor-like, proliferative state. We review recent evidence showing that reprogramming fuels irreversible metaplastic and precancerous proliferations in stomach and pancreas. Finally, we illustrate how we think reprogrammed differentiated cells are likely candidates as cells of origin for cancers of the intestine. PMID:26175494

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

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Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents.

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Rasmussen, L J; Rasmussen, M; Lützen, A; Bisgaard, H C; Singh, K K

2000-05-25

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.

Mesenchymal Stem Cells in Tissue Repair

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Amy M DiMarino

2013-09-01

Full Text Available The advent of mesenchymal stem cell (MSC based therapies for clinical therapeutics has been an exciting and new innovation for the treatment of a variety of diseases associated with inflammation, tissue damage and subsequent regeneration and repair. Application-based ability to measure MSC potency and fate of the cells post-MSC therapy are the variables that confound the use of MSCs therapeutics in human diseases. An evaluation of MSC function and applications with attention to detail in the preparation as well as quality control (QC and quality assurance (QA are only as good as the assays that are developed. In vivo measures of efficacy and potency require an appreciation of the overall pathophysiology of the model and standardization of outcome measures. The new concepts of how MSC’s participate in the tissue regeneration and wound repair process and further, how this is impacted by estimates of efficacy and potency Are important new topics. In this regard,,, this chapter will review some of the in vitro and in vivo assays for MSC function and activity and their application to the clinical arena.

Combined Effects of Scaffold Stiffening and Mechanical Preconditioning Cycles on Construct Biomechanics, Gene Expression, and Tendon Repair Biomechanics

OpenAIRE

Nirmalanandhan, Victor Sanjit; Juncosa-Melvin, Natalia; Shearn, Jason T.; Boivin, Gregory P.; Galloway, Marc T.; Gooch, Cynthia; Bradica, Gino; Butler, David L.

2009-01-01

Our group has previously reported that in vitro mechanical stimulation of tissue-engineered tendon constructs significantly increases both construct stiffness and the biomechanical properties of the repair tissue after surgery. When optimized using response surface methodology, our results indicate that a mechanical stimulus with three components (2.4% strain, 3000 cycles/day, and one cycle repetition) produced the highest in vitro linear stiffness. Such positive correlations between construc...

DNA repair in human cells: Methods for the determination of calmodulin involvement

International Nuclear Information System (INIS)

Charp, P.A.

1987-01-01

Exposure of DNA to either physical or chemical agents can result in the formation of a number of different lesions which must be repaired enzymatically in order for DNA to carry on normal replication and transcription. In most cases, the enzymes involved in this repair of damaged DNA include endonucleases, exonucleases, glycosylases, polymerases, and ligases. Each group of enzymes is involved in precise steps in DNA repair. Exposure to physical agents such as ultraviolet light (UV) at a wavelength of 254 nm is repaired by two distinct and different mechanisms. One mode of enzymatic repair of pyrimidine dimers is accomplished in situ by photoreactivation of UV-induced pyrimidine dimers by photoreactivating light. The second mode of enzymatic repair is the excision repair of pyrimidine dimers involving several different enzymes including endonuclease, exonuclease, and DNA ligase. A summary of the sequence of enzymatic steps involved is shown. It has been observed that specific drugs which bind to and alter the action of calmodulin in cells block DNA synthesis. This suggests that calmodulin may play a role both in normal DNA replication and repair. Others using an indirect method measuring the degree of DNA nucleoid sedimentation, showed that the specific anti-calmodulin agent W-13 slowed the rate of DNA repair. Others showed that DNA synthesis in T51B rat liver cells could be blocked with the addition of either chlorpromazine or trifluoperazine

Genetic polymorphisms in 85 DNA repair genes and bladder cancer risk.

Science.gov (United States)

Michiels, Stefan; Laplanche, Agnès; Boulet, Thomas; Dessen, Philippe; Guillonneau, Bertrand; Méjean, Arnaud; Desgrandchamps, François; Lathrop, Mark; Sarasin, Alain; Benhamou, Simone

2009-05-01

Several defense mechanisms have been developed and maintained during the evolution to protect human cells against damage produced from exogenous or endogenous sources. We examined the associations between bladder cancer and a panel of 652 polymorphisms from 85 genes involved in maintenance of genetic stability [base excision repair, nucleotide excision repair, double-strand break repair (DSBR) and mismatch repair, as well as DNA synthesis and cell cycle regulation pathways] in 201 incident bladder cancer cases and 326 hospital controls. Score statistics were used to test differences in haplotype frequencies between cases and controls in an unconditional logistic regression model. To account for multiple testing, we associated to each P-value the expected proportion of false discoveries (q-value). Haplotype analysis revealed significant associations (P genes (POLB and FANCA) with an associated q-value of 24%. A permutation test was also used to determine whether, in each pathway analyzed, there are more variants whose allelic frequencies are different between cases and controls as compared with what would be expected by chance. Differences were found for cell cycle regulation (P = 0.02) and to a lesser extent for DSBR (P = 0.05) pathways. These results hint to a few potential candidate genes; however, our study was limited by the small sample size and therefore low statistical power to detect associations. It is anticipated that genome-wide association studies will open new perspectives for interpretation of the results of extensive candidate gene studies such as ours.

Inhibition of X-ray induced DNA strand break repair in polyamine-depleted HeLa cells

Energy Technology Data Exchange (ETDEWEB)

Snyder, R.D.

1989-05-01

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, alpha-difluoromethylornithine (DFMO) or methylglyoxal bis(guanylhydrazone) (MGBG), results in, depending on the conditions, partial or complete depletion of the cellular polyamines: putrescine, spermidine and spermine. In this compromised state cells exhibited a distinct deficiency in repair of X-ray-induced DNA strand breaks. The half-time for return of normal DNA sedimentation following 1.6 Gy was 9.5 min for untreated control cells and 22, 32 and 50 min for cells treated with MGBG, DFMO+MGBG and DFMO, respectively. Normal repair kinetics were restored to these cells upon a short incubation in media containing all three polyamines. The rapid early phase of repair following higher X-ray doses (16 Gy) was also delayed in polyamine-depleted cells but later repair occurring 1-4 h post-irradiation, representing chromatin reconstitution, was apparently normal. (author).

Inhibition of X-ray induced DNA strand break repair in polyamine-depleted HeLa cells

International Nuclear Information System (INIS)

Snyder, R.D.

1989-01-01

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, alpha-difluoromethylornithine (DFMO) or methylglyoxal bis(guanylhydrazone) (MGBG), results in, depending on the conditions, partial or complete depletion of the cellular polyamines: putrescine, spermidine and spermine. In this compromised state cells exhibited a distinct deficiency in repair of X-ray-induced DNA strand breaks. The half-time for return of normal DNA sedimentation following 1.6 Gy was 9.5 min for untreated control cells and 22, 32 and 50 min for cells treated with MGBG, DFMO+MGBG and DFMO, respectively. Normal repair kinetics were restored to these cells upon a short incubation in media containing all three polyamines. The rapid early phase of repair following higher X-ray doses (16 Gy) was also delayed in polyamine-depleted cells but later repair occurring 1-4 h post-irradiation, representing chromatin reconstitution, was apparently normal. (author)

[The effect of 3-aminobenzamide on the mitotic cycle of Chinese hamster cells cultured on a medium with 5-bromodeoxyuridine following ionizing radiation action].

Science.gov (United States)

Kirillova, T V; Rozanov, Iu M; Spivak, I M

1992-01-01

A specific inhibitor of poly(ADP-ribose)polymerase-3-aminobenzamide (6 mM) has been shown to: 1) reduce survival of non-irradiated CHO-K1 cells, cultivated in medium containing 5-bromodeoxyuridine (10 mkM, BDU cells), and increase their radiosensitivity; 2) induce G2 delay in BDU cells while progressing through the cell cycle as analysed by the DNA flow cytometry; 3) increase to a great degree G2 delay in X-irradiated BDU cells. 3-Aminobenzamide is primarily effective when it is present during the first or two first cell cycles after the initial addition of BDU. The above data confirm the involvement, presumably an indirect one, of ADP-ribosylation in the DNA repair through affecting the chromatin structure.

Sensitization of gastric cancer cells to alkylating agents by glaucocalyxin B via cell cycle arrest and enhanced cell death

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Ur Rahman MS

2017-08-01

Full Text Available Muhammad Saif Ur Rahman,1 Ling Zhang,2 Lingyan Wu,1 Yuqiong Xie,1 Chunchun Li,1 Jiang Cao1 1Clinical Research Center, 2Cardiovascular Key Laboratory of Zhejiang Province, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, People’s Republic of China Abstract: Severe side effects are major problems with chemotherapy of gastric cancer (GC. These side effects can be reduced by using sensitizing agents in combination with therapeutic drugs. In this study, the low/nontoxic dosage of glaucocalyxin B (GLB was used with other DNA linker agents mitomycin C (MMC, cisplatin (DDP, or cyclophosphamide (CTX to treat GC cells. Combined effectiveness of GLB with drugs was determined by proliferation assay. The molecular mechanisms associated with cell proliferation, migration, invasion, cell cycle, DNA repair/replication, apoptosis, and autophagy were investigated by immunoblotting for key proteins involved. Cell cycle and apoptosis analysis were performed by flow cytometry. Reactive oxygen species level was also examined for identification of its role in apoptosis. Proliferation assay revealed that the addition of 5 µM GLB significantly sensitizes gastric cancer SGC-7901 cells to MMC, DDP, and CTX by decreasing half-maximal inhibitory concentration (IC50 by up to 75.40%±5%, 45.10%±5%, and 52.10%±5%, respectively. GLB + drugs decreased the expression level of proteins involved in proliferation and migration, suggesting the anticancer potential of GLB + drugs. GLB + MMC, GLB + CTX, and GLB + DDP arrest the cells in G0/G1 and G1/S phase, respectively, which may be the consequence of significant decrease in the level of enzymes responsible for DNA replication and telomerase shortening. Combined use of GLB with these drugs also induces DNA damage and apoptosis by activating caspase/PARP pathways and increased production of reactive oxygen species and increased autophagy in GC cells. GLB dosage sensitizes GC

Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

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Vielfort, Katarina; Weyler, Linda; Söderholm, Niklas; Engelbrecht, Mattias; Löfmark, Sonja; Aro, Helena

2013-01-01

We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells. PMID:23675492

Lactobacillus decelerates cervical epithelial cell cycle progression.

Directory of Open Access Journals (Sweden)

Katarina Vielfort

Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

Protein tyrosine nitration in the cell cycle

International Nuclear Information System (INIS)

Jia, Min; Mateoiu, Claudia; Souchelnytskyi, Serhiy

2011-01-01

Highlights: → Enrichment of 3-nitrotyrosine containing proteins from cells synchronized in different phases of the cell cycle. → Identification of 76 tyrosine nitrated proteins that change expression during the cell cycle. → Nineteen identified proteins were previously described as regulators of cell proliferation. -- Abstract: Nitration of tyrosine residues in proteins is associated with cell response to oxidative/nitrosative stress. Tyrosine nitration is relatively low abundant post-translational modification that may affect protein functions. Little is known about the extent of protein tyrosine nitration in cells during progression through the cell cycle. Here we report identification of proteins enriched for tyrosine nitration in cells synchronized in G0/G1, S or G2/M phases of the cell cycle. We identified 27 proteins in cells synchronized in G0/G1 phase, 37 proteins in S phase synchronized cells, and 12 proteins related to G2/M phase. Nineteen of the identified proteins were previously described as regulators of cell proliferation. Thus, our data indicate which tyrosine nitrated proteins may affect regulation of the cell cycle.

Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

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Kashino, Genro [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom); Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Suzuki, Keiji [Division of Radiation Biology, Department of Radiology and Radiation Biology, Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Matsuda, Naoki [Division of Radiation Biology and Protection, Center for Frontier Life Sciences, Nagasaki University, Nagasaki 852-8102 (Japan); Kodama, Seiji [Radiation Biology Laboratory, Radiation Research Center, Frontier Science Innovation Center, Organization for University-Industry-Government Cooperation, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570 (Japan); Ono, Koji [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Watanabe, Masami [Laboratory of Radiation Biology, Division of Radiation Life Science, Department of Radiation Life Science and Radiation Medical Science, Kyoto University Research Reactor Institute, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Prise, Kevin M [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom) and Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Lisburn Road, Belfast BT9 7AB (United Kingdom)]. E-mail: prise@gci.ac.uk

2007-06-01

Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.

2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells.

OpenAIRE

Andreassen, P R; Margolis, R L

1992-01-01

BHK cells blocked at any of several points in the cell cycle override their drug-induced arrest and proceed in the cycle when exposed concurrently to the protein kinase inhibitor 2-aminopurine (2-AP). For cells arrested at various points in interphase, 2-AP-induced cell cycle progression is made evident by arrival of the drug-treated cell population in mitosis. Cells that have escaped from mimosine G1 arrest, from hydroxyurea or aphidicolin S-phase arrest, or from VM-26-induced G2 arrest subs...

DNA damage and repair in rabbit lens epithelial cells following UVA radiation

International Nuclear Information System (INIS)

Sidjanin, Duska; Zigman, Seymour; Reddan, John

1993-01-01

Since ultraviolet light may be a contributing factor to cataractogenesis, we investigated the response of the lens epithelium, a potential target for UV insult, to UVA radiation. Cell survival and the induction and repair of DNA single-strand breaks (SSBs) were measured in cultured rabbit lens epithelial cells following UVA exposure. A 30 min exposure to UVA (180 KJ/m 2 ) induced measurable SSBs. An increase in UVA fluenced measurable SSBs. An increase in UVA fluence brought about an increase in UVA fluence brought about an increase in the number of DNA SSBs. Rejoining of SSBs were measured after the cells were irradiated in Tyrode's for 2 hrs and allowed to repair in the dark for 4 hrs at 36 o C in MEM containing 10% serum. Eighty percent of the DNA SSBs were repaired within 4 hrs as determined by analysis of the alkaline elution profile. The repair kinetics were biphasic with an initial fast and subsequently slower component. The results indicate that UVA can induce SSBs in lens-induced SSBs, and that UVA treatment can be toxic to the epithelium. (Author)

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

Science.gov (United States)

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

Repairable-conditionally repairable damage model based on dual Poisson processes.

Science.gov (United States)

Lind, B K; Persson, L M; Edgren, M R; Hedlöf, I; Brahme, A

2003-09-01

The advent of intensity-modulated radiation therapy makes it increasingly important to model the response accurately when large volumes of normal tissues are irradiated by controlled graded dose distributions aimed at maximizing tumor cure and minimizing normal tissue toxicity. The cell survival model proposed here is very useful and flexible for accurate description of the response of healthy tissues as well as tumors in classical and truly radiobiologically optimized radiation therapy. The repairable-conditionally repairable (RCR) model distinguishes between two different types of damage, namely the potentially repairable, which may also be lethal, i.e. if unrepaired or misrepaired, and the conditionally repairable, which may be repaired or may lead to apoptosis if it has not been repaired correctly. When potentially repairable damage is being repaired, for example by nonhomologous end joining, conditionally repairable damage may require in addition a high-fidelity correction by homologous repair. The induction of both types of damage is assumed to be described by Poisson statistics. The resultant cell survival expression has the unique ability to fit most experimental data well at low doses (the initial hypersensitive range), intermediate doses (on the shoulder of the survival curve), and high doses (on the quasi-exponential region of the survival curve). The complete Poisson expression can be approximated well by a simple bi-exponential cell survival expression, S(D) = e(-aD) + bDe(-cD), where the first term describes the survival of undamaged cells and the last term represents survival after complete repair of sublethal damage. The bi-exponential expression makes it easy to derive D(0), D(q), n and alpha, beta values to facilitate comparison with classical cell survival models.

The localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.

1987-01-01

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. We have investigated whether the repair of DNA damage occurs in the nuclear matrix compartment. Normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m -2 and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix. 5 J m -2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m -2 ) normal human fibroblast post-u.v. incubated in the presence of hydroxyurea and arabinsosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C) in contrast to XP-D cells and Syrian hamster embryonic cells. (author)