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

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

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.)

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

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

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.)

In vitro toxicity of polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons to cetacean cells and tissues

Energy Technology Data Exchange (ETDEWEB)

Carvan, M.J. III.

1993-01-01

Cetaceans bioaccumulate high aromatic hydrocarbon tissue residues, and elevated levels of PCB residues in tissues are proposed to have occurred concurrently with recent epizootic deaths of dolphins. The objectives of this study were: (1) to develop and characterize an epithelial cell line derived from dolphin tissues, (2) to investigate the effects of hydrocarbon pollutants on those cells, and (3) to analyze the toxicity of hydrocarbon pollutants on cetacean tissues in vitro. An epithelial cell line, Carvan dolphin kidney (CDK), isolated from a spontaneously aborted female bottlenose dolphin, Tursiops truncatus, grew rapidly. These cells were neither transformed nor immortal. Velocity sedimentation analysis showed CDK cells contained nuclear aryl hydrocarbon receptor, suggestive of cytochrome P450 inducibility. BaP inhibited mitosis in CDK cells in a dose-dependent manner. Data indicate that CDK cells metabolize BaP, that BaP metabolites bind to cellular DNA initiating unscheduled DNA synthesis, and that the inhibition of cytochrome P450 metabolism decrease the BaP-associated inhibition of mitosis in dolphin cells. The data also suggest that TCDD acts synergistically to increase the levels of DNA damage by the procarcinogen BaP. Cetacean liver microsomes was isolated and evaluated for the presence of cytochrome P450 proteins by SDS-PAGE, apparent minimum molecular weight determination, and immunoblot analysis. P450 activity was induced in cetacean tissue samples and CDK cells by exposure in vitro to one of several cytochrome P450-inducing chemicals. The data suggest that cetacean tissues and cells can be utilized to study the in vitro induction of cytochrome P450, resultant metabolism of xenobiotic contaminants, and the subsequent cellular and molecular responses. However, the identity of specific P450 isozymes involved in this process will remain undetermined until monoclonal antibodies that recognize cetacean P450s can be generated.

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

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.

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.

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

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.)

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

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.

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

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)

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.)

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...

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.)

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

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

Directory of Open Access Journals (Sweden)

Miyaji E.N.

2000-01-01

Full Text Available 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(6-4pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, possibly indicating a defect in preferential repair of actively transcribed genes, and a slower cell proliferation rate, including a longer S-phase. This phenotype reinforces the present notion that control of key mechanisms in cell metabolism, such as cell cycle control, repair, transcription and cell death, can be linked.

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)

Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor

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Baeza-Squiban Armelle

2010-07-01

Full Text Available Abstract Background Nowadays, effects of fine particulate matter (PM2.5 are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM2.5 exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM2.5 involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect. Results In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies. The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM2.5 suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis. Conclusions The PM2.5-antiapoptotic effect in addition

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

International Nuclear Information System (INIS)

Wei, Kuo-Liang; Chen, Fei-Yun; Lin, Chih-Yi; Gao, Guan-Lun; Kao, Wen-Ya; Yeh, Chi-Hui; Chen, Chang-Rong; Huang, Hao-Chun; Tsai, Wei-Ren; Jong, Koa-Jen; Li, Wan-Jung; Su, Jyan-Gwo Joseph

2016-01-01

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG 0 /G 1 population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy. - Highlights: • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element. • Carbendazim induced nuclear translocation of the aryl hydrocarbon

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

Energy Technology Data Exchange (ETDEWEB)

Wei, Kuo-Liang [Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan, ROC (China); College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan, ROC (China); Chen, Fei-Yun; Lin, Chih-Yi [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Gao, Guan-Lun [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Department of Biological Resources, National Chiayi University, Chiayi, 60004, Taiwan, ROC (China); Kao, Wen-Ya [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Yeh, Chi-Hui [Department of Environmental Engineering, College of Engineering, Da-Yeh University, Dacun, Changhua 51591, Taiwan, ROC (China); Chen, Chang-Rong [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Huang, Hao-Chun [Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan, ROC (China); Tsai, Wei-Ren [Division of Applied Toxicology, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture, Executive Yuan, Taichung 41358, Taiwan, ROC (China); Jong, Koa-Jen [Department of Biological Resources, National Chiayi University, Chiayi, 60004, Taiwan, ROC (China); Li, Wan-Jung [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Su, Jyan-Gwo Joseph, E-mail: jgjsu@mail.ncyu.edu.tw [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China)

2016-09-01

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG{sub 0}/G{sub 1} population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy. - Highlights: • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element. • Carbendazim induced nuclear translocation of the aryl

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.)

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.

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.

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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.

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

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

Energy Technology Data Exchange (ETDEWEB)

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.

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.

Science.gov (United States)

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.

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)

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

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.

Science.gov (United States)

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

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.

Science.gov (United States)

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)

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

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

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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.

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

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

Phytochemicals radiosensitize cancer cells by inhibiting DNA repair

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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)

DNA strand breakage repair in ataxia telangiectasia fibroblast-like cells

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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

Hydrocarbon pollution from marinas in estuarine sediments

Science.gov (United States)

Voudrias, Evangelos A.; Smith, Craig L.

1986-03-01

A measure of the impact of marinas on three Eastern Virginia estuarine creeks was obtained by a study of hydrocarbons in their sediments. Two of the creeks support considerable marine activity, including pleasure boat marinas, boat repair facilities, and commercial fishing operations. The third creek, which served as a control, is seldom used by boats, and is surrounded by marsh and woodland. Sediments from the creeks with marinas contained significantly higher levels of both aromatic and aliphatic hydrocarbons than did the control. Differences in the concentrations of certain oil-pollution indicators, such as the 17α,21β-hopane homologs and phytane, and low molecular weight aromatic hydrocarbons, are indicative of light petroleum fractions. Most of the aromatic hydrocarbons from all creeks, however, appear to have a pyrogenic origin. Although hydrocarbons from three probable origins (petroleum, pyrogenesis, and recent biosynthesis) were detected in all locations, the petroleum-derived and pyrogenic hydrocarbons were of only minor importance relative to the biogenic hydrocarbons in the control creek.

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

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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.

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.

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

Conversion of hydrocarbons in solid oxide fuel cells

DEFF Research Database (Denmark)

Mogensen, Mogens Bjerg; Kammer Hansen, K.

2003-01-01

Recently, a number of papers about direct oxidation of methane and hydrocarbon in solid oxide fuel cells (SOFC) at relatively low temperatures (about 700degreesC) have been published. Even though the conversion of almost dry CH4 at 1000degreesC on ceramic anodes was demonstrated more than 10 years...

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.

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...

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.

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

Science.gov (United States)

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.

Science.gov (United States)

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.

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

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...

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

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...

IMPLICATIONS OF MICROBIAL ADHESION TO HYDROCARBONS FOR EVALUATING CELL-SURFACE HYDROPHOBICITY .1. ZETA-POTENTIALS OF HYDROCARBON DROPLETS

NARCIS (Netherlands)

BUSSCHER, HJ; VANDEBELTGRITTER, B; VANDERMEI, HC

1995-01-01

Microbial adhesion to hydrocarbons (MATH) is generally considered to be a measure of the organisms cell surface hydrophobicity. As microbial adhesion is a complicated interplay of long-range van der Waals and electrostatic forces and various short-range interactions, the above statement only holds

Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

KAUST Repository

Cerezo, Maria I.; Linden, Matthew; Agusti, Susana

2017-01-01

Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

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

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

Science.gov (United States)

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

Energy Technology Data Exchange (ETDEWEB)

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

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

Science.gov (United States)

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.

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.

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

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.

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.

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)

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

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)

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.

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

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.

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

Mesenchymal Stem Cells in Tissue Repair

Directory of Open Access Journals (Sweden)

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.

Highly Efficient and Reproducible Nonfullerene Solar Cells from Hydrocarbon Solvents

KAUST Repository

Wadsworth, Andrew; Ashraf, Raja; Abdelsamie, Maged; Pont, Sebastian; Little, Mark; Moser, Maximilian; Hamid, Zeinab; Neophytou, Marios; Zhang, Weimin; Amassian, Aram; Durrant, James R.; Baran, Derya; McCulloch, Iain

2017-01-01

With chlorinated solvents unlikely to be permitted for use in solution-processed organic solar cells in industry, there must be a focus on developing nonchlorinated solvent systems. Here we report high-efficiency devices utilizing a low-bandgap donor polymer (PffBT4T-2DT) and a nonfullerene acceptor (EH-IDTBR) from hydrocarbon solvents and without using additives. When mesitylene was used as the solvent, rather than chlorobenzene, an improved power conversion efficiency (11.1%) was achieved without the need for pre- or post-treatments. Despite altering the processing conditions to environmentally friendly solvents and room-temperature coating, grazing incident X-ray measurements confirmed that active layers processed from hydrocarbon solvents retained the robust nanomorphology obtained with hot-processed chlorinated solvents. The main advantages of hydrocarbon solvent-processed devices, besides the improved efficiencies, were the reproducibility and storage lifetime of devices. Mesitylene devices showed better reproducibility and shelf life up to 4000 h with PCE dropping by only 8% of its initial value.

Highly Efficient and Reproducible Nonfullerene Solar Cells from Hydrocarbon Solvents

KAUST Repository

Wadsworth, Andrew

2017-06-01

With chlorinated solvents unlikely to be permitted for use in solution-processed organic solar cells in industry, there must be a focus on developing nonchlorinated solvent systems. Here we report high-efficiency devices utilizing a low-bandgap donor polymer (PffBT4T-2DT) and a nonfullerene acceptor (EH-IDTBR) from hydrocarbon solvents and without using additives. When mesitylene was used as the solvent, rather than chlorobenzene, an improved power conversion efficiency (11.1%) was achieved without the need for pre- or post-treatments. Despite altering the processing conditions to environmentally friendly solvents and room-temperature coating, grazing incident X-ray measurements confirmed that active layers processed from hydrocarbon solvents retained the robust nanomorphology obtained with hot-processed chlorinated solvents. The main advantages of hydrocarbon solvent-processed devices, besides the improved efficiencies, were the reproducibility and storage lifetime of devices. Mesitylene devices showed better reproducibility and shelf life up to 4000 h with PCE dropping by only 8% of its initial value.

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

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

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)

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.

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

Energy Technology Data Exchange (ETDEWEB)

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.

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)

Electrode Design for Low Temperature Direct-Hydrocarbon Solid Oxide Fuel Cells

Science.gov (United States)

Chen, Fanglin (Inventor); Zhao, Fei (Inventor); Liu, Qiang (Inventor)

2015-01-01

In certain embodiments of the present disclosure, a solid oxide fuel cell is described. The solid oxide fuel cell includes a hierarchically porous cathode support having an impregnated cobaltite cathode deposited thereon, an electrolyte, and an anode support. The anode support includes hydrocarbon oxidation catalyst deposited thereon, wherein the cathode support, electrolyte, and anode support are joined together and wherein the solid oxide fuel cell operates a temperature of 600.degree. C. or less.

Electrode design for low temperature direct-hydrocarbon solid oxide fuel cells

Science.gov (United States)

Chen, Fanglin; Zhao, Fei; Liu, Qiang

2015-10-06

In certain embodiments of the present disclosure, a solid oxide fuel cell is described. The solid oxide fuel cell includes a hierarchically porous cathode support having an impregnated cobaltite cathode deposited thereon, an electrolyte, and an anode support. The anode support includes hydrocarbon oxidation catalyst deposited thereon, wherein the cathode support, electrolyte, and anode support are joined together and wherein the solid oxide fuel cell operates a temperature of 600.degree. C. or less.

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)

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny

Directory of Open Access Journals (Sweden)

Leyla Vahidi Ferdousi

2014-11-01

Full Text Available The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

Science.gov (United States)

Vahidi Ferdousi, Leyla; Rocheteau, Pierre; Chayot, Romain; Montagne, Benjamin; Chaker, Zayna; Flamant, Patricia; Tajbakhsh, Shahragim; Ricchetti, Miria

2014-11-01

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs. Copyright © 2014. Published by Elsevier B.V.

The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

International Nuclear Information System (INIS)

Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

2012-01-01

The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO 3 ). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate.

Science.gov (United States)

Bajinskis, Ainars; Olsson, Gunilla; Harms-Ringdahl, Mats

2012-03-01

The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO(3)). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure. Copyright © 2011 Elsevier B.V. All rights reserved.

The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

Energy Technology Data Exchange (ETDEWEB)

Bajinskis, Ainars, E-mail: ainars.bajinskis@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden); Olsson, Gunilla; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, S-10691 Stockholm (Sweden)

2012-03-01

The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO{sub 3}). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

Science.gov (United States)

Kienzler, Aude; Mahler, Barbara J.; Van Metre, Peter C.; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-01-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

Signaling factors in stem cell-mediated repair of infarcted myocardium

NARCIS (Netherlands)

Vandervelde, S; van Luyn, MJA; Tio, RA; Harmsen, MC

Myocardial infarction leads to scar formation and subsequent reduced cardiac performance. The ultimate therapy after myocardial infarction would pursue stem cell-based regeneration. The aim of stem cell-mediated cardiac repair embodies restoration of cardiac function by regeneration of healthy

Slow mitochondrial repair of 5'-AMP renders mtDNA susceptible to damage in APTX deficient cells

DEFF Research Database (Denmark)

Akbari, Mansour; Sykora, Peter; Bohr, Vilhelm A

2015-01-01

deficient cells. Moreover, the removal of 5'-AMP from DNA was significantly slower in the mitochondrial extracts from human cell lines and mouse tissues compared with their corresponding nuclear extracts. These results suggest that, contrary to nuclear DNA repair, mitochondrial DNA repair is not able...... elucidated. Here, we monitored the repair of 5'-AMP DNA damage in nuclear and mitochondrial extracts from human APTX(+/+) and APTX(-/-) cells. The efficiency of repair of 5'-AMP DNA was much lower in mitochondrial than in nuclear protein extracts, and resulted in persistent DNA repair intermediates in APTX......Aborted DNA ligation events in eukaryotic cells can generate 5'-adenylated (5'-AMP) DNA termini that can be removed from DNA by aprataxin (APTX). Mutations in APTX cause an inherited human disease syndrome characterized by early-onset progressive ataxia with ocular motor apraxia (AOA1). APTX...

Adipose, Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair

Science.gov (United States)

Fellows, Christopher R.; Matta, Csaba; Zakany, Roza; Khan, Ilyas M.; Mobasheri, Ali

2016-01-01

Current cell-based repair strategies have proven unsuccessful for treating cartilage defects and osteoarthritic lesions, consequently advances in innovative therapeutics are required and mesenchymal stem cell-based (MSC) therapies are an expanding area of investigation. MSCs are capable of differentiating into multiple cell lineages and exerting paracrine effects. Due to their easy isolation, expansion, and low immunogenicity, MSCs are an attractive option for regenerative medicine for joint repair. Recent studies have identified several MSC tissue reservoirs including in adipose tissue, bone marrow, cartilage, periosteum, and muscle. MSCs isolated from these discrete tissue niches exhibit distinct biological activities, and have enhanced regenerative potentials for different tissue types. Each MSC type has advantages and disadvantages for cartilage repair and their use in a clinical setting is a balance between expediency and effectiveness. In this review we explore the challenges associated with cartilage repair and regeneration using MSC-based cell therapies and provide an overview of phenotype, biological activities, and functional properties for each MSC population. This paper also specifically explores the therapeutic potential of each type of MSC, particularly focusing on which cells are capable of producing stratified hyaline-like articular cartilage regeneration. Finally we highlight areas for future investigation. Given that patients present with a variety of problems it is unlikely that cartilage regeneration will be a simple “one size fits all,” but more likely an array of solutions that need to be applied systematically to achieve regeneration of a biomechanically competent repair tissue. PMID:28066501

Adipose, Bone Marrow and Synovial Joint-derived Mesenchymal Stem Cells for Cartilage Repair

Directory of Open Access Journals (Sweden)

Christopher Fellows

2016-12-01

Full Text Available Current cell-based repair strategies have proven unsuccessful for treating cartilage defects and osteoarthritic lesions, consequently advances in innovative therapeutics are required and mesenchymal stem cell-based (MSC therapies are an expanding area of investigation. MSCs are capable of differentiating into multiple cell lineages and exerting paracrine effects. Due to their easy isolation, expansion and low immunogenicity, MSCs are an attractive option for regenerative medicine for joint repair. Recent studies have identified several MSC tissue reservoirs including in adipose tissue, bone marrow, cartilage, periosteum and muscle. MSCs isolated from these discrete tissue niches exhibit distinct biological activities, and have enhanced regenerative potentials for different tissue types. Each MSC type has advantages and disadvantages for cartilage repair and their use in a clinical setting is a balance between expediency and effectiveness. In this review we explore the challenges associated with cartilage repair and regeneration using MSC-based cell therapies and provide an overview of phenotype, biological activities and functional properties for each MSC population. This paper also specifically explores the therapeutic potential of each type of MSC, particularly focusing on which cells are capable of producing stratified hyaline-like articular cartilage regeneration. Finally we highlight areas for future investigation. Given that patients present with a variety of problems it is unlikely that cartilage regeneration will be a simple ‘one size fits all’, but more likely an array of solutions that need to applied systematically to achieve regeneration of a biomechanically competent repair tissue.

Viability in holder of irradiated cells: distinguish between repair and cell multiplication

International Nuclear Information System (INIS)

Araujo, A.C. de.

1980-01-01

In experiments in which liquid holding recovery (LHR) was measured, the majority of cellular population is formed by non-viable cells and cell multiplication may be important for LHR expression. In order to distinguish between recuperation of viability (true LHR) and cell multiplication, it was necessary to employ improved plating techniques and a fluctuation test based on Poisson distribution. Our results are an indication that this fluctuation test, used together with the traditional method, is a good tool to distinguish repair from cell multiplication. (author)

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.

Repair of potentially lethal damage in unfed plateau phase cultures of Ehrlich ascited tumour cells

International Nuclear Information System (INIS)

Illiakis, G.

1980-01-01

Plateau phase EAT-cells have been irradiated at different times in the plateau phase and their ability to repair PLD has been measured. A large capacity to repair PLD has been observed if the cultures were kept in the plateau phase for some hours after irradiation before diluting and plating to measure the survival. In combination with theoretical considerations it is concluded that almost all the PLD produced under these conditions can be repaired. The reaction rate of this repair was independent of the dose and the age of the culture. The results also indicate that PLD repair is independent of the intercellular contact of EAT-cells. (author)

Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome

International Nuclear Information System (INIS)

Ringborg, U.; Lambert, B.; Landergen, J.; Lewensohn, R.

1981-01-01

The uv-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed

Repair of γ-irradiation-induced DNA single-strand breaks in human bone marrow cells. Analysis of unfractionated and CD34+ cells using single-cell gel electrophoresis

International Nuclear Information System (INIS)

Lankinen, Maarit H.; Vilpo, Juhani A.

1997-01-01

Human bone marrow mononuclear cells (BMMNCs) were separated by density gradient centrifugation, and a subpopulation of progenitor cells was further isolated using anti-CD34-coated magnetic beads. The cells were irradiated with γ-rays (0.93-5.43 Gy) from a 137 Cs source. The extent of DNA damage, i.e., single-strand breaks (SSBs) and alkali-labile lesions of individual cells, was investigated using the alkaline single-cell gel electrophoresis technique. The irradiation resulted in a dose-dependent increase in DNA migration, reflecting the number of detectable DNA lesions. An approximately similar extent of SSB formation was observed in BMMNCs and CD34+ cells. Damage was repaired when the cells were incubated at 37C: a fast initial repair phase was followed by a slower rejoining of SSBs in both BMMNC and CD34+ cell populations. A significantly longer time was required to repair the lesions caused by 5.43 Gy than those caused by 0.93 Gy. In the present work we report, for the first time, the induction and repair of DNA SSBs at the level of single human bone marrow cells when exposed to ionizing radiation at clinically relevant doses. These data, together with our previous results with human blood granulocytes and lymphocytes, indicate an approximately similar extent of formation and repair of γ-irradiation-induced DNA SSBs in immature and mature human hematopoietic cells

Effect of polyamine depletion on DNA damage and repair following UV irradiation of HeLa cells

International Nuclear Information System (INIS)

Snyder, R.D.; Sunkara, P.S.

1990-01-01

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggest that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair. (author)

Effect of polyamine depletion on DNA damage and repair following UV irradiation of HeLa cells

Energy Technology Data Exchange (ETDEWEB)

Snyder, R.D.; Sunkara, P.S. (Merrell Dow Research Inst., Cincinnati, OH (USA))

1990-09-01

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggest that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair. (author).

Stem cells for brain repair in neonatal hypoxia-ischemia.

Science.gov (United States)

Chicha, L; Smith, T; Guzman, R

2014-01-01

Neonatal hypoxic-ischemic insults are a significant cause of pediatric encephalopathy, developmental delays, and spastic cerebral palsy. Although the developing brain's plasticity allows for remarkable self-repair, severe disruption of normal myelination and cortical development upon neonatal brain injury are likely to generate life-persisting sensory-motor and cognitive deficits in the growing child. Currently, no treatments are available that can address the long-term consequences. Thus, regenerative medicine appears as a promising avenue to help restore normal developmental processes in affected infants. Stem cell therapy has proven effective in promoting functional recovery in animal models of neonatal hypoxic-ischemic injury and therefore represents a hopeful therapy for this unmet medical condition. Neural stem cells derived from pluripotent stem cells or fetal tissues as well as umbilical cord blood and mesenchymal stem cells have all shown initial success in improving functional outcomes. However, much still remains to be understood about how those stem cells can safely be administered to infants and what their repair mechanisms in the brain are. In this review, we discuss updated research into pathophysiological mechanisms of neonatal brain injury, the types of stem cell therapies currently being tested in this context, and the potential mechanisms through which exogenous stem cells might interact with and influence the developing brain.

DNA replication and repair of Tilapia cells: Pt. 2

International Nuclear Information System (INIS)

Chen, J.D.; Yew, F.H.

1988-01-01

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34 0 C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 0 C to the sublethal high temperature of 37 0 C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37 0 C the rate of DNA synthesis overshot the 31 0 C control level by 180%. When moved to low temperature (18 0 C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25 0 C, but not at 18 0 C. Initiation of nascent DNA synthesis was blocked at 4Jm -2 in TO-2 cells compared with ≤ 1Jm -2 in mammalian cells. After 9Jm -2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair. (author)

Effects of cytotoxic chemotherapeutic agents on split-dose repair in intestinal crypt cells

International Nuclear Information System (INIS)

Phillips, Theodore L.; Ross, Glenda Y.

1997-01-01

Purpose: Many cancer chemotherapeutic agents interact with radiation to enhance the amount of radiation damage observed in both tumor and normal tissues. It is important to predict this interaction and to determine the effect of drug on sublethal damage repair. To evaluate for effects in rapid renewing normal tissues, the intestinal crypt cell in vivo assay is an excellent one to employ. These studies investigate the effect of eleven cancer chemotherapeutic drugs on split-dose repair in the intestinal crypt cell of the mouse. Methods and Materials: LAF1 male mice, age 10-12 weeks, were exposed to whole-body irradiation with orthovoltage x-rays delivered as a single dose or as equally divided doses delivered with intervals between the two exposures of 2 to 24 h. In the experimental group, the cancer chemotherapeutic agent was administered intraperitoneally 2 h before the first radiation dose. At 3.6 days after the second irradiation, the mice were sacrificed; the jejunum was removed, fixed, and sectioned for light microscopy. The number of regenerating crypts were counted and corrected to represent the number of surviving cells per circumference. Results: Of the eleven drugs tested, only carmustine eliminated split-dose repair. Cisplatin delayed repair, and methotrexate caused marked synchronization obliterating the observation of split-dose repair. Conclusions: Most cytotoxic chemotherapeutic agents do not inhibit sublethal damage repair in intestinal crypt cells when given 2 h before the first radiation exposure. Absence of the initial increase in survival seen with split-dose radiation is noted with carmustine and high-dose methotrexate

Bone marrow stromal cell : mediated neuroprotection for spinal cord repair

NARCIS (Netherlands)

Ritfeld, Gaby Jane

2014-01-01

Currently, there is no treatment available that restores anatomy and function after spinal cord injury. This thesis explores transplantation of bone marrow-derived mesenchymal stem cells (bone marrow stromal cells; BMSCs) as a therapeutic approach for spinal cord repair. BMSCs secrete neurotrophic

Un-repairable DNA damage in cell due to irradiation

International Nuclear Information System (INIS)

Yoshii, Giichi

1992-01-01

Radiation-induced cell reproductive deactivation is caused by damage to DNA. In a cell, cellular DNA radical reacts with diffusion controlled rate and generates DNA peroxide radical. The chemical repair of DNA radical with hydrogen donation by thiol competes with the reaction of oxygen with same radicals in the DNA molecules. From the point reaction rates, the prolongation of radical life time is not as great as expected from the reduction in the glutathione content of the cell. This indicates that further reducting compounds (protein bound thiol) are present in the cell. The residual radicals are altered to strand breaks, base damages and so on. The effective lesions for a number of endpoints is un-repaired double strand break, which has been discovered in a cluster. This event gives risk to high LET radiation or to a track end of X-rays. For X- or electron irradiations the strand breaks are frequently induced by the interactions between sublesions on two strands in DNA. A single strand break followed by radical action may be unstable excited state, because of remaining sugar radical action and of having negative charged phosphates, in which strands breaks will be rejoined in a short time to stable state. On the same time, a break in the double helix will be immediately produced if two breaks are on either or approximately opposite locations. The formation of a double strand break in the helix depends on the ion strength of the cell. The potassium ions are largely released from polyanionic strand during irradiation, which results in the induction of denatured region. Double strand break with the denatured region seems to be un-repairable DNA damage. (author)

Three-Dimensional Printing and Cell Therapy for Wound Repair.

Science.gov (United States)

van Kogelenberg, Sylvia; Yue, Zhilian; Dinoro, Jeremy N; Baker, Christopher S; Wallace, Gordon G

2018-05-01

Significance: Skin tissue damage is a major challenge and a burden on healthcare systems, from burns and other trauma to diabetes and vascular disease. Although the biological complexities are relatively well understood, appropriate repair mechanisms are scarce. Three-dimensional bioprinting is a layer-based approach to regenerative medicine, whereby cells and cell-based materials can be dispensed in fine spatial arrangements to mimic native tissue. Recent Advances: Various bioprinting techniques have been employed in wound repair-based skin tissue engineering, from laser-induced forward transfer to extrusion-based methods, and with the investigation of the benefits and shortcomings of each, with emphasis on biological compatibility and cell proliferation, migration, and vitality. Critical issues: Development of appropriate biological inks and the vascularization of newly developed tissues remain a challenge within the field of skin tissue engineering. Future Directions: Progress within bioprinting requires close interactions between material scientists, tissue engineers, and clinicians. Microvascularization, integration of multiple cell types, and skin appendages will be essential for creation of complex skin tissue constructs.

Altered Gene Expressions and Cytogenetic Repair Efficiency in Cells with Suppressed Expression of XPA after Proton Exposure

Science.gov (United States)

Zhang, Ye; Rohde, Larry H.; Gridley, Daila S.; Mehta, Satish K.; Pierson, Duane L.; Wu, Honglu

2009-01-01

Cellular responses to damages from ionizing radiation (IR) exposure are influenced not only by the genes involved in DNA double strand break (DSB) repair, but also by non- DSB repair genes. We demonstrated previously that suppressed expression of several non-DSB repair genes, such as XPA, elevated IR-induced cytogenetic damages. In the present study, we exposed human fibroblasts that were treated with control or XPA targeting siRNA to 250 MeV protons (0 to 4 Gy), and analyzed chromosome aberrations and expressions of genes involved in DNA repair. As expected, after proton irradiation, cells with suppressed expression of XPA showed a significantly elevated frequency of chromosome aberrations compared with control siRNA treated (CS) cells. Protons caused more severe DNA damages in XPA knock-down cells, as 36% cells contained multiple aberrations compared to 25% in CS cells after 4Gy proton irradiation. Comparison of gene expressions using the real-time PCR array technique revealed that expressions of p53 and its regulated genes in irradiated XPA suppressed cells were altered similarly as in CS cells, suggesting that the impairment of IR induced DNA repair in XPA suppressed cells is p53-independent. Except for XPA, which was more than 2 fold down regulated in XPA suppressed cells, several other DNA damage sensing and repair genes (GTSE1, RBBP8, RAD51, UNG and XRCC2) were shown a more than 1.5 fold difference between XPA knock-down cells and CS cells after proton exposure. The possible involvement of these genes in the impairment of DNA repair in XPA suppressed cells will be further investigated.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures

Directory of Open Access Journals (Sweden)

Melissa Lo Monaco

2018-01-01

Full Text Available Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs or induced pluripotent stem cells (iPSCs have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures.

Science.gov (United States)

Lo Monaco, Melissa; Merckx, Greet; Ratajczak, Jessica; Gervois, Pascal; Hilkens, Petra; Clegg, Peter; Bronckaers, Annelies; Vandeweerd, Jean-Michel; Lambrichts, Ivo

2018-01-01

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain uncertain. Stem cells can contribute to cartilage repair via chondrogenic differentiation, via immunomodulation, or by the production of paracrine factors and extracellular vesicles. But before novel cell-based therapies for cartilage repair can be introduced into the clinic, rigorous testing in preclinical animal models is required. Preclinical models used in regenerative cartilage studies include murine, lapine, caprine, ovine, porcine, canine, and equine models, each associated with its specific advantages and limitations. This review presents a summary of recent in vitro data and from in vivo preclinical studies justifying the use of MSCs and iPSCs in cartilage tissue engineering. Moreover, the advantages and disadvantages of utilizing small and large animals will be discussed, while also describing suitable outcome measures for evaluating cartilage repair.

A preliminary investigation into the extent of increased radioresistance or hyper-radiosensitivity in cells of hamster cell lines known to be deficient in DNA repair

International Nuclear Information System (INIS)

Skov, K.; Marples, B.; Matthews, J.B.; Zhou, H.; Joiner, M.C.

1994-01-01

The response to low doses of X rays was assessed in cells of three hamster cell lines which are defective in DNA repair and was compared with their parental lines. Cells of the V79-derived double-strand break repair-deficient line XR-V15B showed no radioresistance in the 0.5-Gy range compared with the V79B wild type, but instead showed an exponential response. Cells of the single-strand break repair-deficient line EM9 showed hyper-radiosensitivity and exhibited increased radioresistance. Most interestingly, cells of the UV-20 cell line appeared to respond exponentially, as a continuation of the hyper-radiosensitive portion of the curve, with no evidence of increased radioresistance. This line is defective in an incision step of excision repair and is sensitive to crosslinking agents. Further studies are warranted to address the possible role of single- and double-strand break repair and excision repair in hyper-radiosensitivity and increased radioresistance. 24 refs., 4 figs

DNA replication and repair of Tilapia cells: Pt. 2. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells

Energy Technology Data Exchange (ETDEWEB)

Chen, J.D.; Yew, F.H.

1988-02-01

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15-34/sup 0/C). We report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31/sup 0/C to the sublethal high temperature of 37/sup 0/C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37/sup 0/C the rate of DNA synthesis overshot the 31/sup 0/C control level by 180%. When moved to low temperature (18/sup 0/C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. U.V. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3h at 37, 31 and 25/sup 0/C, but not at 18/sup 0/C. Initiation of nascent DNA synthesis was blocked at 4Jm/sup -2/ in TO-2 cells compared with less than or equal to 1Jm/sup -2/ in mammalian cells. After 9Jm/sup -2/ u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate. TO-2 cells showed low levels of u.v.-induced excision repair.

Aryl hydrocarbon receptor ligand effects in RBL2H3 cells

DEFF Research Database (Denmark)

Maaetoft-Udsen, Kristina; Shimoda, Lori M. N.; Frøkiær, Hanne

2012-01-01

The aryl hydrocarbon receptor (AHR) mediates toxic effects of dioxin and xenobiotic metabolism. AHR has an emerging role in the immune system, but its physiological ligands and functional role in immunocytes remain poorly understood. Mast cells are immunocytes that are central to inflammatory...

Stem cells and repair of lung injuries

Directory of Open Access Journals (Sweden)

Randell Scott H

2004-07-01

Full Text Available Abstract Fueled by the promise of regenerative medicine, currently there is unprecedented interest in stem cells. Furthermore, there have been revolutionary, but somewhat controversial, advances in our understanding of stem cell biology. Stem cells likely play key roles in the repair of diverse lung injuries. However, due to very low rates of cellular proliferation in vivo in the normal steady state, cellular and architectural complexity of the respiratory tract, and the lack of an intensive research effort, lung stem cells remain poorly understood compared to those in other major organ systems. In the present review, we concisely explore the conceptual framework of stem cell biology and recent advances pertinent to the lungs. We illustrate lung diseases in which manipulation of stem cells may be physiologically significant and highlight the challenges facing stem cell-related therapy in the lung.

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

Translational Application of Microfluidics and Bioprinting for Stem Cell-Based Cartilage Repair

Directory of Open Access Journals (Sweden)

Silvia Lopa

2018-01-01

Full Text Available Cartilage defects can impair the most elementary daily activities and, if not properly treated, can lead to the complete loss of articular function. The limitations of standard treatments for cartilage repair have triggered the development of stem cell-based therapies. In this scenario, the development of efficient cell differentiation protocols and the design of proper biomaterial-based supports to deliver cells to the injury site need to be addressed through basic and applied research to fully exploit the potential of stem cells. Here, we discuss the use of microfluidics and bioprinting approaches for the translation of stem cell-based therapy for cartilage repair in clinics. In particular, we will focus on the optimization of hydrogel-based materials to mimic the articular cartilage triggered by their use as bioinks in 3D bioprinting applications, on the screening of biochemical and biophysical factors through microfluidic devices to enhance stem cell chondrogenesis, and on the use of microfluidic technology to generate implantable constructs with a complex geometry. Finally, we will describe some new bioprinting applications that pave the way to the clinical use of stem cell-based therapies, such as scaffold-free bioprinting and the development of a 3D handheld device for the in situ repair of cartilage defects.

Translational Application of Microfluidics and Bioprinting for Stem Cell-Based Cartilage Repair

Science.gov (United States)

Mondadori, Carlotta; Mainardi, Valerio Luca; Talò, Giuseppe; Candrian, Christian; Święszkowski, Wojciech

2018-01-01

Cartilage defects can impair the most elementary daily activities and, if not properly treated, can lead to the complete loss of articular function. The limitations of standard treatments for cartilage repair have triggered the development of stem cell-based therapies. In this scenario, the development of efficient cell differentiation protocols and the design of proper biomaterial-based supports to deliver cells to the injury site need to be addressed through basic and applied research to fully exploit the potential of stem cells. Here, we discuss the use of microfluidics and bioprinting approaches for the translation of stem cell-based therapy for cartilage repair in clinics. In particular, we will focus on the optimization of hydrogel-based materials to mimic the articular cartilage triggered by their use as bioinks in 3D bioprinting applications, on the screening of biochemical and biophysical factors through microfluidic devices to enhance stem cell chondrogenesis, and on the use of microfluidic technology to generate implantable constructs with a complex geometry. Finally, we will describe some new bioprinting applications that pave the way to the clinical use of stem cell-based therapies, such as scaffold-free bioprinting and the development of a 3D handheld device for the in situ repair of cartilage defects. PMID:29535776

APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

Science.gov (United States)

Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

2012-07-12

APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

Clustered DNA lesion repair in eukaryotes: Relevance to mutagenesis and cell survival

Energy Technology Data Exchange (ETDEWEB)

Sage, Evelyne [Institut Curie, Bat. 110, Centre Universitaire, 91405 Orsay (France); CNRS UMR3348, Bat. 110, Centre Universitaire, 91405 Orsay (France); Harrison, Lynn, E-mail: lclary@lsuhsc.edu [Department of Molecular and Cellular Physiology, LSUHSC-S, 1501 Kings Highway, Shreveport, LA 71130 (United States)

2011-06-03

A clustered DNA lesion, also known as a multiply damaged site, is defined as {>=}2 damages in the DNA within 1-2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed.

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

Osteocalcin expressing cells from tendon sheaths in mice contribute to tendon repair by activating Hedgehog signaling

OpenAIRE

Wang, Yi; Zhang, Xu; Huang, Huihui; Xia, Yin; Yao, YiFei; Mak, Arthur Fuk-Tat; Yung, Patrick Shu-Hang; Chan, Kai-Ming; Wang, Li; Zhang, Chenglin; Huang, Yu; Mak, Kingston King-Lun

2017-01-01

Both extrinsic and intrinsic tissues contribute to tendon repair, but the origin and molecular functions of extrinsic tissues in tendon repair are not fully understood. Here we show that tendon sheath cells harbor stem/progenitor cell properties and contribute to tendon repair by activating Hedgehog signaling. We found that Osteocalcin (Bglap) can be used as an adult tendon-sheath-specific marker in mice. Lineage tracing experiments show that Bglap-expressing cells in adult sheath tissues pos...

Altering Cell Survival by Modulating Levels of Mitochondrial DNA Repair Enzymes

National Research Council Canada - National Science Library

Shokolenko, Inna

2002-01-01

.... Our previous results demonstrated that stable expression of E.coli Exonuclease III in mitochondria of breast cancer cells diminishes mtDNA repair capacity following oxidative stress, which leads to a decrease in long-term cell survival...

Prospect of Induced Pluripotent Stem Cell Genetic Repair to Cure Genetic Diseases

Directory of Open Access Journals (Sweden)

Jeanne Adiwinata Pawitan

2012-01-01

Full Text Available In genetic diseases, where the cells are already damaged, the damaged cells can be replaced by new normal cells, which can be differentiated from iPSC. To avoid immune rejection, iPSC from the patient’s own cell can be developed. However, iPSC from the patients’s cell harbors the same genetic aberration. Therefore, before differentiating the iPSCs into required cells, genetic repair should be done. This review discusses the various technologies to repair the genetic aberration in patient-derived iPSC, or to prevent the genetic aberration to cause further damage in the iPSC-derived cells, such as Zn finger and TALE nuclease genetic editing, RNA interference technology, exon skipping, and gene transfer method. In addition, the challenges in using the iPSC and the strategies to manage the hurdles are addressed.

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.

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

International Nuclear Information System (INIS)

Stark, M.; Naiman, T.; Canaani, D.

1989-01-01

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [ 3 H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line

A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

Directory of Open Access Journals (Sweden)

Gebhard Daniel

2010-06-01

Full Text Available Abstract Background The Host Cell Reactivation Assay (HCRA is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances. A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing. Results In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis. The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. Conclusions The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity

DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz(a)anthracene 5,6-oxide

Energy Technology Data Exchange (ETDEWEB)

Ahmed, F.E.; Gentil, A.; Renstein, B.S.; Setlow, R.B.

1980-01-01

Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz(a)anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification and bromodeoxyuridine photolysis and endonuclease-sensitive sites assay. Radiautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of the epoxide to be 0.1 to 0.2, that after a saturating ultraviolet dose, though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive. The data indicate that there are different rate-limiting steps in the removal of the ultraviolet and the epoxide damages, and that the residual repair activity in xeroderma pigmentosum cells is accomplished by different, not just fewer, enzymes than in normal cells.

DNA repair

International Nuclear Information System (INIS)

Setlow, R.

1978-01-01

Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

Mammalian cell biology

International Nuclear Information System (INIS)

Anon.

1976-01-01

Progress is reported on studies of the molecular biology and functional changes in cultured mammalian cells following exposure to x radiation, uv radiation, fission neutrons, or various chemical environmental pollutants alone or in combinations. Emphasis was placed on the separate and combined effects of polycyclic aromatic hydrocarbons released during combustion of fossil fuels and ionizing and nonionizing radiations. Sun lamps, which emit a continuous spectrum of near ultraviolet light of 290 nm to 315 nm were used for studies of predictive cell killing due to sunlight. Results showed that exposure to uv light (254 nm) may not be adequate to predict effects produced by sunlight. Data are included from studies on single-strand breaks and repair in DNA of cultured hamster cells exposed to uv or nearultraviolet light. The possible interactions of the polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)-anthracene (DmBA) alone or combined with exposure to x radiation, uv radiation (254 nm) or near ultraviolet simulating sunlight were compared for effects on cell survival

Stem cell-derived angiogenic/vasculogenic cells: Possible therapies for tissue repair and tissue engineering

NARCIS (Netherlands)

Zwaginga, J. J.; Doevendans, P.

2003-01-01

1. The recent ability to isolate stem cells and study their specific capacity of self-renewal with the formation of different cell types has opened up exciting vistas to help the repair of damaged tissue and even the formation of new tissue. In the present review, we deal with the characteristics

Repair and replication of DNA in hereditary (bilateral) retinoblastoma cells after X-irradiation

International Nuclear Information System (INIS)

Cleaver, J.E.; Char, D.; Charles, W.C.; Rand, N.

1982-01-01

Fibroblasts from patients with hereditary retinoblastoma reportedly exhibit increased sensitivity to killing by X-rays. Although some human syndromes with similar or greater hypersensitivity to DNA-damaging agents (e.g., X-rays, ultraviolet light, and chemical carcinogens), such as xeroderma pigmentosum, are deficient in DNA repair, most do not have such clearly demonstrable defects in repair. Retinoblastoma cells appear to be normal in repairing single-strand breaks and performing repair replication after X-irradiation and also in synthesizing poly(adenosine diphosphoribose). Semiconservative DNA replication in these cells, however, is slightly more resistant than normal after X-irradiation, suggesting that continued replication of damaged parental DNA could contribute to the pathogenesis of the disease. This effect is small, however, and may be a consequence rather than a cause of the fundamental enzymatic abnormality in retinoblastoma that causes the tumorigenesis

Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair

DEFF Research Database (Denmark)

Boye, Theresa Louise; Maeda, Kenji; Pezeshkian, Weria

2017-01-01

Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca2+- and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate...... that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane...... that induction of curvature force around wound edges is an early key event in cell membrane repair....

Uninduced adipose-derived stem cells repair the defect of full-thickness hyaline cartilage.

Science.gov (United States)

Zhang, Hai-Ning; Li, Lei; Leng, Ping; Wang, Ying-Zhen; Lv, Cheng-Yu

2009-04-01

To testify the effect of the stem cells derived from the widely distributed fat tissue on repairing full-thickness hyaline cartilage defects. Adipose-derived stem cells (ADSCs) were derived from adipose tissue and cultured in vitro. Twenty-seven New Zealand white rabbits were divided into three groups randomly. The cultured ADSCs mixed with calcium alginate gel were used to fill the full-thickness hyaline cartilage defects created at the patellafemoral joint, and the defects repaired with gel or without treatment served as control groups. After 4, 8 and 12 weeks, the reconstructed tissue was evaluated macroscopically and microscopically. Histological analysis and qualitative scoring were also performed to detect the outcome. Full thickness hyaline cartilage defects were repaired completely with ADSCs-derived tissue. The result was better in ADSCs group than the control ones. The microstructure of reconstructed tissue with ADSCs was similar to that of hyaline cartilage and contained more cells and regular matrix fibers, being better than other groups. Plenty of collagen fibers around cells could be seen under transmission electron microscopy. Statistical analysis revealed a significant difference in comparison with other groups at each time point (t equal to 4.360, P less than 0.01). These results indicate that stem cells derived from mature adipose without induction possess the ability to repair cartilage defects.

Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

Directory of Open Access Journals (Sweden)

Kalpana Mujoo

2017-11-01

Full Text Available The nitric oxide (NO-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1. Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18, which causes stem cell differentiation has no effect on double-strand break (DSB repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line.

Science.gov (United States)

Kienzler, Aude; Mahler, Barbara J; Van Metre, Peter C; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-07-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity. Copyright © 2015 Elsevier B.V. All rights reserved.

After Nerve Injury, Lineage Tracing Shows That Myelin and Remak Schwann Cells Elongate Extensively and Branch to Form Repair Schwann Cells, Which Shorten Radically on Remyelination.

Science.gov (United States)

Gomez-Sanchez, Jose A; Pilch, Kjara S; van der Lans, Milou; Fazal, Shaline V; Benito, Cristina; Wagstaff, Laura J; Mirsky, Rhona; Jessen, Kristjan R

2017-09-13

There is consensus that, distal to peripheral nerve injury, myelin and Remak cells reorganize to form cellular columns, Bungner's bands, which are indispensable for regeneration. However, knowledge of the structure of these regeneration tracks has not advanced for decades and the structure of the cells that form them, denervated or repair Schwann cells, remains obscure. Furthermore, the origin of these cells from myelin and Remak cells and their ability to give rise to myelin cells after regeneration has not been demonstrated directly, although these conversions are believed to be central to nerve repair. Using genetic lineage-tracing and scanning-block face electron microscopy, we show that injury of sciatic nerves from mice of either sex triggers extensive and unexpected Schwann cell elongation and branching to form long, parallel processes. Repair cells are 2- to 3-fold longer than myelin and Remak cells and 7- to 10-fold longer than immature Schwann cells. Remarkably, when repair cells transit back to myelinating cells, they shorten ∼7-fold to generate the typically short internodes of regenerated nerves. The present experiments define novel morphological transitions in injured nerves and show that repair Schwann cells have a cell-type-specific structure that differentiates them from other cells in the Schwann cell lineage. They also provide the first direct evidence using genetic lineage tracing for two basic assumptions in Schwann cell biology: that myelin and Remak cells generate the elongated cells that build Bungner bands in injured nerves and that such cells can transform to myelin cells after regeneration. SIGNIFICANCE STATEMENT After injury to peripheral nerves, the myelin and Remak Schwann cells distal to the injury site reorganize and modify their properties to form cells that support the survival of injured neurons, promote axon growth, remove myelin-associated growth inhibitors, and guide regenerating axons to their targets. We show that the

Repair of Avascular Meniscus Tears with Electrospun Collagen Scaffolds Seeded with Human Cells.

Science.gov (United States)

Baek, Jihye; Sovani, Sujata; Glembotski, Nicholas E; Du, Jiang; Jin, Sungho; Grogan, Shawn P; D'Lima, Darryl D

2016-03-01

The self-healing capacity of an injured meniscus is limited to the vascularized regions and is especially challenging in the inner avascular regions. As such, we investigated the use of human meniscus cell-seeded electrospun (ES) collagen type I scaffolds to produce meniscal tissue and explored whether these cell-seeded scaffolds can be implanted to repair defects created in meniscal avascular tissue explants. Human meniscal cells (derived from vascular and avascular meniscal tissue) were seeded on ES scaffolds and cultured. Constructs were evaluated for cell viability, gene expression, and mechanical properties. To determine potential for repair of meniscal defects, human meniscus avascular cells were seeded and cultured on aligned ES collagen scaffolds for 4 weeks before implantation. Surgical defects resembling "longitudinal tears" were created in the avascular zone of bovine meniscus and implanted with cell-seeded collagen scaffolds and cultured for 3 weeks. Tissue regeneration and integration were evaluated by histology, immunohistochemistry, mechanical testing, and magentic resonance imaging. Ex vivo implantation with cell-seeded collagen scaffolds resulted in neotissue that was significantly better integrated with the native tissue than acellular collagen scaffolds or untreated defects. Human meniscal cell-seeded ES collagen scaffolds may therefore be useful in facilitating meniscal repair of avascular meniscus tears.

Inhibition of X-ray-induced potentially lethal damage (PLD) repair in aerobic plateau-phase Chinese hamster cells by misonidazole

International Nuclear Information System (INIS)

Brown, D.M.

1984-01-01

The effect of the 2-nitroimidazole radiosensitizer misonidazole (MISO) and the hydrophilic analog SR-2508 on the repair of X-ray-induced potentially lethal damage (PLD) was studied in plateau-phase Chinese Hamster ovary (HA-1) cells. It was found that although MISO does not radiosensitize aerobic cells, it inhibits the repair of PLD. However, under hypoxic conditions, MISO has no effect on PLD repair. The major portion of the inhibition of PLD repair in aerobic cells requires the presence of MISO only during irradiation; little or no additional inhibition occurs when MISO is present during the postirradiation repair period. Also, treatment of aerobic cells with 5 mM MISO for either 5 or 30 min prior to irradiation is equally inhibitory. This suggests that the presence of MISO in some way modifies the initial lesion under aerobic conditions since it does not increase cell killing as determined by immediate plating but inhibits subsequent repair. The inhibition is concentration dependent; 0.5 mM MISO inhibits PLD repair by one-half while 5-10 mM totally inhibits the repair measured 6 hr postirradiation. This phenomenon suggests that radiosensitization of tissue in vivo by MISO and other 2-nitroimidazoles may not be unequivocal proof of the presence of hypoxic cells

Mesenchymal stem cells overexpressing Ihh promote bone repair.

Science.gov (United States)

Zou, Shasha; Chen, Tingting; Wang, Yanan; Tian, Ruhui; Zhang, Lingling; Song, Pingping; Yang, Shi; Zhu, Yong; Guo, Xizhi; Huang, Yiran; Li, Zheng; Kan, Lixin; Hu, Hongliang

2014-10-28

Indian hedgehog (Ihh) signaling pathway is known to play key roles in various aspects of normal endochondral bone development. This study tested the potential roles of high Ihh signaling in the context of injury-induced bone regeneration. A rabbit tibia defect model was established to test the effects of the implant of Ihh/mesenchymal stem cells (MSCs)/scaffold complex. Computed tomography (CT), gross observation, and standard histological and immunohistological techniques were used to evaluate the effectiveness of the treatment. In vitro studies with MSCs and C3H10T1/2 cells were also employed to further understand the cellular and molecular mechanisms. We found that the implanted Ihh/MSCs/scaffold complex promoted bone repair. Consistently, in vitro study found that Ihh induced the upregulation of chondrocytic, osteogenic, and vascular cell markers, both in C3H10T1/2 cells and MSCs. Our study has demonstrated that high Ihh signaling in a complex with MSCs enhanced bone regeneration effectively in a clinically relevant acute injury model. Even though the exact underlying mechanisms are still far from clear, our primary data suggested that enhanced chondrogenesis, osteogenesis, and angiogenesis of MSCs at least partially contribute to the process. This study not only has implications for basic research of MSCs and Ihh signaling pathway but also points to the possibility of direct application of this specific paradigm to clinical bone repair.

Propofol promotes spinal cord injury repair by bone marrow mesenchymal stem cell transplantation

OpenAIRE

Zhou, Ya-jing; Liu, Jian-min; Wei, Shu-ming; Zhang, Yun-hao; Qu, Zhen-hua; Chen, Shu-bo

2015-01-01

Propofol is a neuroprotective anesthetic. Whether propofol can promote spinal cord injury repair by bone marrow mesenchymal stem cells remains poorly understood. We used rats to investigate spinal cord injury repair using bone marrow mesenchymal stem cell transplantation combined with propofol administration via the tail vein. Rat spinal cord injury was clearly alleviated; a large number of newborn non-myelinated and myelinated nerve fibers appeared in the spinal cord, the numbers of CM-Dil-l...

The role of mismatch repair in small-cell lung cancer cells

DEFF Research Database (Denmark)

Hansen, L T; Thykjaer, T; Ørntoft, T F

2003-01-01

The role of mismatch repair (MMR) in small-cell lung cancer (SCLC) is controversial, as the phenotype of a MMR-deficiency, microsatellite instability (MSI), has been reported to range from 0 to 76%. We studied the MMR pathway in a panel of 21 SCLC cell lines and observed a highly heterogeneous...... pattern of MMR gene expression. A significant correlation between the mRNA and protein levels was found. We demonstrate that low hMLH1 gene expression was not linked to promoter CpG methylation. One cell line (86MI) was found to be deficient in MMR and exhibited resistance to the alkylating agent MNNG...

Aging and photo-aging DNA repair phenotype of skin cells-Evidence toward an effect of chronic sun-exposure

Energy Technology Data Exchange (ETDEWEB)

Prunier, Chloe; Masson-Genteuil, Gwenaeelle [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); Ugolin, Nicolas [Laboratoire de Cancerologie Experimentale, CEA, DSV, IRCM, SREIT, BP6, Fontenay-aux-Roses Cedex F-92265 (France); Sarrazy, Fanny [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France); Sauvaigo, Sylvie, E-mail: sylvie.sauvaigo@cea.fr [Laboratoire Lesions des Acides Nucleiques, CEA, DSM, INAC, SCIB, UMR-E CEA/UJF-Grenoble 1, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9 (France)

2012-08-01

Several studies have demonstrated the deleterious effect of aging on the capacity of cells to repair their DNA. However, current existing assays aimed at measuring DNA repair address only a specific repair step dedicated to the correction of a specific DNA lesion type. Consequently they provide no information regarding the repair pathways that handle other types of lesions. In addition to aging, consequences of photo-exposure on these repair processes remain elusive. In this study we evaluated the consequence of aging and of chronic and/or acute photo-exposure on DNA repair in human skin fibroblasts using a multiplexed approach, which provided detailed information on several repair pathways at the same time. The resulting data were analyzed with adapted statistics/bioinformatics tools. We showed that, irrespective of the repair pathway considered, excision/synthesis was less efficient in non-exposed cells from elderly compared to cells from young adults and that photo-exposure disrupted this very clear pattern. Moreover, it was evidenced that chronic sun-exposure induced changes in DNA repair properties. Finally, the identification of a specific signature at the level of the NER pathway in cells repeatedly exposed to sun revealed a cumulative effect of UVB exposure and chronic sun irradiation. The uses of bioinformatics tools in this study was essential to fully take advantage of the large sum of data obtained with our multiplexed DNA repair assay and unravel the effects of environmental exposure on DNA repair pathways.

Stem Cells for Cartilage Repair: Preclinical Studies and Insights in Translational Animal Models and Outcome Measures

OpenAIRE

Lo Monaco, Melissa; Merckx, Greet; Ratajczak, Jessica; Gervois, Pascal; Hilkens, Petra; Clegg, Peter; Bronckaers, Annelies; Vandeweerd, Jean-Michel; Lambrichts, Ivo

2018-01-01

Due to the restricted intrinsic capacity of resident chondrocytes to regenerate the lost cartilage postinjury, stem cell-based therapies have been proposed as a novel therapeutic approach for cartilage repair. Moreover, stem cell-based therapies using mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) have been used successfully in preclinical and clinical settings. Despite these promising reports, the exact mechanisms underlying stem cell-mediated cartilage repair remain...

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

Science.gov (United States)

Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-04-03

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. Copyright © 2017 Zapotoczny and Sekelsky.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

Directory of Open Access Journals (Sweden)

Grzegorz Zapotoczny

2017-04-01

Full Text Available DNA double-strand breaks (DSBs are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells.

In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype.

Science.gov (United States)

Walker, J L; Bleaken, B M; Romisher, A R; Alnwibit, A A; Menko, A S

2018-05-02

Following injury, mesenchymal repair cells are activated to function as leader cells that modulate wound healing. These cells have the potential to differentiate to myofibroblasts, resulting in fibrosis and scarring. The signals underlying these differing pathways are complex and incompletely understood. The ex vivo mock cataract surgery cultures are an attractive model with which to address this question. With this model we study, concurrently, the mechanisms that control mesenchymal leader cell function in injury repair within their native microenvironment, and the signals that induce this same cell population to acquire a myofibroblast phenotype when these cells encounter the environment of the adjacent tissue culture platform. Here, we show that upon injury, the cytoskeletal protein vimentin is released into the extracellular space, binds to the cell surface of the mesenchymal leader cells located at the wound edge in the native matrix environment, and supports wound closure. In pro-fibrotic environments, the extracellular vimentin pool also links specifically to the mesenchymal leader cells, and has an essential role in signaling their fate change to a myofibroblast. These findings suggest a novel role for extracellular, cell-surface-associated vimentin in mediating repair-cell function in wound repair and in transitioning these cells to a myofibroblast phenotype. Movie S1 Movie S1 Collective movement of mesenchymal leader and epithelial follower cells across the tissue culture substrate (ECZ) in response to injury was followed by time-lapse imaging from D0-D3. The mesenchymal cells at the leading edge were easily distinguished morphologically from the lens epithelial follower cells.

Investigations on the mechanism of DNA excision repair in tissue culture cells

International Nuclear Information System (INIS)

Wawra, E.; Dolejs, I.; Ott, E.

1976-12-01

Semiconservative DNA- synthesis and repair- synthesis was measured in HeLa cells and spleen cells under different conditions (i.e. different temperatures, addition of p-chloromercuribenzoate or cytosine-arabinoside). In order to obtain more information about the enzymatic background of these steps of DNA metabolism, parallel in vitro experiments were done with two different types of DNA polymerase, which had been isolated from pig spleen. At least the experiments at different temperatures are showing some correlations of α-polymerase with semiconservative synthesis and of β-polymerase with repair synthesis. (author)

[SOS response of DNA repair and genetic cell instability under hypoxic conditions].

Science.gov (United States)

Vasil'eva, S V; Strel'tsova, D A

2011-01-01

The SOS DNA repair pathway is induced in E. coli as a multifunctional cell response to a wide variety of signals: UV, X or gamma-irradiation, mitomycin C or nalidixic acid treatment, thymine starvation, etc. Triggering of the system can be used as a general and early sign of DNA damage. Additionally, the SOS-response is known to be an "error-prone" DNA repair pathway and one of the sources of genetic instability. Hypoxic conditions are established to be the major factor of genetic instability as well. In this paper we for the first time studied the SOS DNA repair response under hypoxic conditions induced by the well known aerobic SOS-inducers. The SOS DNA repair response was examined as a reaction of E. coli PQ37 [sfiA::lacZ] cells to UVC, NO-donating agents and 4NQO. Here we provide evidence that those agents were able to induce the SOS DNA repair response in E. coli at anaerobic growth conditions. The process does not depend on the transcriptional activity of the universal protein of E. col anaerobic growth Fnr [4Fe-4S]2+ or can not be referred to as an indicator of genetic instability in hypoxic conditions.

γ-ray dose rate effect in DNA double-strand break repair deficient murine cells

International Nuclear Information System (INIS)

Li Liya; Li Peiwen

2002-01-01

Objective: To analyze the dose rate effect and potentially lethal damage repair in DNA double-strand break repair deficient murine cells (SCID) irradiated by γ-ray. Methods: The wild type (CB.17+/+) and SCID cells were exposed to γ-ray at high and low dose rates. The high dose rate exposure was fractionated into two equal doses at 24 h intervals. The survival rates of irradiated cells were calculated by clone-forming analysis. Results: When γ-ray was given to wild type (CB.17+/+) cells in two fractions at 24 h intervals, the survival rate was significantly higher than that when the same total dose was given singly. In contrast, there was no difference in the survival rates between the single and fractionated exposure in SCID cells. SCID cells were more sensitive than CB.17+/+ cells to both low and high dose rates γ-ray exposure for cell killing. The survival rate by low dose rate exposure was significantly higher than that by high dose rate exposure, not only in CB.17+/+ cells but also in SCID cells. Conclusions: SCID cells are deficient in repairing γ-ray induced double-strand breaks. There is dose rate effect in both SCID and CB.17+/+ cells

The studies of effects of 60Co γ-rays on DNA damage and repair in tumor cells

International Nuclear Information System (INIS)

Su Liaoyuan; Huang Hanxian; Cen Jiannong

1997-06-01

The effects of 60 Co γ-rays and its combination with hyperthermia on DNA strand breaks and their repair in L 5178y cells were studied using alkaline elution technique. When the cells were heated at 43 degree C for 30 min, there was obvious inhibition of repair of DNA damage caused by γ-irradiation. The hyperthermia before irradiation produced better inhibiting effect than that after irradiation. The effects of radiation on DNA strand breaks and its repair in HL-60 cells and HL-60 (VCR) cells were analyzed using technique of hydroxyapatite chromatography and the results sowed that the extent of DNA strand breaks in HL-60 cells and HL-60 (VCR) cells induced by irradiation was not markedly different, but the power of repair of strand breaks and the radioresistance of function of DNA synthesis in HL-60 (VCR) cells were higher than those in HL-60 cells. The difference was obvious. The results suggest that the heterogeneity of radiosensitivity of leukemia cells is correlated with its drug resistance. (10 refs., 3 tabs.)

Repair methods for damaged pipeline beyond diving depth

OpenAIRE

Mohammadi, Keramat

2011-01-01

Master's thesis in Offshore Technology Mechanical damage of a subsea pipeline is found as one of the most severe concern in management of pipeline integrity. The need to reach and bring the hydrocarbons from the fields located in deep and ultra-deep waters, imposes the need to improve the technologies and techniques in order to repair any unacceptable damage in pipeline. The main objective of this work is to investigate various methods for repairing a subsea pipeline that has been damaged ...

Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

Directory of Open Access Journals (Sweden)

De-Tao Yin

Full Text Available DNA damage response (DDR is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili(-/- MEFs were defective in cyclobutane pyrimidine dimers (CPD repair after UV treatment. As a result, the UV-treated mili(-/- MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose polymerase (PARP and Bik. The impaired DNA repair in the mili(-/- MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine-guanine (Pt-[GG] and double strand break (DSB repair were also defective in the mili(-/- MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR, respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

Prolonged particulate chromate exposure does not inhibit homologous recombination repair in North Atlantic right whale (Eubalaena glacialis) lung cells.

Science.gov (United States)

Browning, Cynthia L; Wise, Catherine F; Wise, John Pierce

2017-09-15

Chromosome instability is a common feature of cancers that forms due to the misrepair of DNA double strand breaks. Homologous recombination (HR) repair is a high fidelity DNA repair pathway that utilizes a homologous DNA sequence to accurately repair such damage and protect the genome. Prolonged exposure (>72h) to the human lung carcinogen, particulate hexavalent chromium (Cr(VI)), inhibits HR repair, resulting in increased chromosome instability in human cells. Comparative studies have shown acute Cr(VI) exposure induces less chromosome damage in whale cells than human cells, suggesting investigating the effect of this carcinogen in other species may inform efforts to prevent Cr(VI)-induced chromosome instability. Thus, the goal of this study was to determine the effect of prolonged Cr(VI) exposure on HR repair and clastogenesis in North Atlantic right whale (Eubalaena glacialis) lung cells. We show particulate Cr(VI) induces HR repair activity after both acute (24h) and prolonged (120h) exposure in North Atlantic right whale cells. Although the RAD51 response was lower following prolonged Cr(VI) exposure compared to acute exposure, the response was sufficient for HR repair to occur. In accordance with active HR repair, no increase in Cr(VI)-induced clastogenesis was observed with increased exposure time. These results suggest prolonged Cr(VI) exposure affects HR repair and genomic stability differently in whale and human lung cells. Future investigation of the differences in how human and whale cells respond to chemical carcinogens may provide valuable insight into mechanisms of preventing chemical carcinogenesis. Copyright © 2017. Published by Elsevier Inc.

Radiation Induced G2 Chromatic Break and Repairs Kinetics in Human Lymphoblastoid Cells

International Nuclear Information System (INIS)

Seong, Jin Sil

1993-01-01

In understanding radiosensitivity a new concept of inherent radiosensitivity based on individuality and heterogeneity within a population has recently beer explored. There has been some discussion of possible mechanism underlying differences in radiosensitivity between cells. Ataxia telangiectasia(AT), a rare autosomal recessive genetic disorder, is characterized by hypersensitivity to lonizing radiation and other DNA damaging agents at the cellular level. There have been a lot of efforts to describe the cause of this hypersensitivity to radiation. At the cellular level, chromosome repair kinetics study would be an appropriate approach. The purpose of this study was to better understand radiosensitivity in an approach to investigate kinetics of induction and repair of G2 chromatic breaks using normal, AT heterozygous(ATH), and AT homozygous lymphoblastoid cell lines. In an attempt to estimate initial damage, 9-β-D-arabinosyl-2-fluoroadenine, an inhibitor of DNA synthesis and repair, was used in this study. It was found from this study that radiation induces higher chromatid breaks in AT than in normal and ATH cells. There was no significant differences of initial chromatid breaks between normal and ATH cells. Repair kinetics was the same for all. So the higher level of breaks in AT G2 cells is thought to be a reflection of the increased initial damage. The amount of initial damage correlated well with survival fraction at 2 Gy of cell survival curve following radiation. Therefore, the difference of radiosensitivity in terms of G2 chromosomal sensitivity is thought to result from the difference of initial damage

Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells

International Nuclear Information System (INIS)

Lee-Chen, S.F.; Yu, C.T.; Jan, K.Y.

1992-01-01

Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase α in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine β-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts. (author)

Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells

Energy Technology Data Exchange (ETDEWEB)

Lee-Chen, S.F.; Yu, C.T.; Jan, K.Y. (Academia Sinica, Taipei, (Taiwan). Institute of Zoology)

1992-01-01

Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase [alpha] in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine [beta]-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts. (author).

Relationship between DNA repair and cell recovery: Importance of competing biochemical and metabolic processes

International Nuclear Information System (INIS)

Van Ankeren, S.C.; Wheeler, K.T.; Kansas Univ., Lawrence

1985-01-01

The relationship between the inhibition of repair of radiation-induced DNA damage and the inhibition of recovery from radiation-induced potentially lethal damage (PLD) by hypertonic treatment was compared in 9L/Ro rat brain tumor cells. Fed plateau phase cultures were γ-irradiated with 1500 rad and then immediately treated for 20 min with a 37 0 C isotonic (0.15 M) or hypertonic (0.50 M) salt solution. The kinetics of repair of radiation-induced DNA damage as assayed using alkaline filter elution were compared to those of recovery from radiation-induced PLD as assayed by colony formation. hypertonic treatment of unirradiated cells produced neither DNA damage nor cell kill. Post-irradiation hypertonic treatment inhibited both DNA repair and PLD recovery, while post-irradiation istonic treatment inhibited neither phenomenon. However, by 2 h after irradiation, the amount of DNA damage remaining after a 20 min hypertonic treatment was equivalent to that remaining after a 20 min isotonic treatment. In contrast, cell survival after hypertonic treatment remained 2 logs lower than after isotonic treatment even at times up to 24 h. These results suggest that the repair of radiation-induced DNA damage per per se is not causally related to recovery from radiation-induced PLD. However, the data are consistent with the time of DNA repair as an important parameter in determining cell survival and, therefore, tend to support the hypothesis that imbalances in sets of competing biochemical or metabolic processes determine survival rather than the presence of a single class of unrepaired DNA lesions. (orig.)

Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

KAUST Repository

Cerezo, Maria I.

2017-02-17

Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

DEFF Research Database (Denmark)

Akbari, Mansour; Otterlei, Marit; Pena Diaz, Javier

2004-01-01

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using......, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG...

Insulin-like growth factor-1 sustains stem cell mediated renal repair.

NARCIS (Netherlands)

Imberti, B.; Morigi, M.; Tomasoni, S.; Rota, C.; Corna, D.; Longaretti, L.; Rottoli, D.; Valsecchi, F.; Benigni, A.; Wang, J.; Abbate, M.; Zoja, C.; Remuzzi, G.

2007-01-01

In mice with cisplatin-induced acute kidney injury, administration of bone marrow-derived mesenchymal stem cells (MSC) restores renal tubular structure and improves renal function, but the underlying mechanism is unclear. Here, we examined the process of kidney cell repair in co-culture experiments

Repetitious nature of repaired DNA in mammalian cells. Progress report, June 1, 1976--February 28, 1977

International Nuclear Information System (INIS)

Meltz, M.L.

1977-02-01

Progress is reported on studies of DNA repair in cultured mouse L fibroblasts, human diploid fibroblasts, and cultured human lymphoblastoid cell lines. Data are included on the effects of methyl methanesulfonate treatment, uv light, and age of cell donors on repair replication of DNA

Stem cell death and survival in heart regeneration and repair.

Science.gov (United States)

Abdelwahid, Eltyeb; Kalvelyte, Audrone; Stulpinas, Aurimas; de Carvalho, Katherine Athayde Teixeira; Guarita-Souza, Luiz Cesar; Foldes, Gabor

2016-03-01

Cardiovascular diseases are major causes of mortality and morbidity. Cardiomyocyte apoptosis disrupts cardiac function and leads to cardiac decompensation and terminal heart failure. Delineating the regulatory signaling pathways that orchestrate cell survival in the heart has significant therapeutic implications. Cardiac tissue has limited capacity to regenerate and repair. Stem cell therapy is a successful approach for repairing and regenerating ischemic cardiac tissue; however, transplanted cells display very high death percentage, a problem that affects success of tissue regeneration. Stem cells display multipotency or pluripotency and undergo self-renewal, however these events are negatively influenced by upregulation of cell death machinery that induces the significant decrease in survival and differentiation signals upon cardiovascular injury. While efforts to identify cell types and molecular pathways that promote cardiac tissue regeneration have been productive, studies that focus on blocking the extensive cell death after transplantation are limited. The control of cell death includes multiple networks rather than one crucial pathway, which underlies the challenge of identifying the interaction between various cellular and biochemical components. This review is aimed at exploiting the molecular mechanisms by which stem cells resist death signals to develop into mature and healthy cardiac cells. Specifically, we focus on a number of factors that control death and survival of stem cells upon transplantation and ultimately affect cardiac regeneration. We also discuss potential survival enhancing strategies and how they could be meaningful in the design of targeted therapies that improve cardiac function.

Effects of aphidicolin on repair replication and induced chromosomal aberrations in mammalian cells

International Nuclear Information System (INIS)

Zeeland, A.A. van; Filon, A.R.; Natarajan, A.T.; Bussmann, C.J.M.; Degrassi, F.; Kesteren-van Leeuwen, A.C. van; Palitti, F.; Rome Univ.

1982-01-01

The influence of aphidicolin, an inhibitor of polymerase α, on UV-induced repair replication in human skin fibroblasts, as well as in HeLa cells, was determined. In growing fibroblasts and in HeLa cells, aphidicolin had a potentiating effect on UV-induced repair replication, whereas in fibroblasts grown to confluency, aphidicolin had an inhibitory effect. This inhibitory effect was stronger when measured in the presence of hydroxyurea. In HeLa cells the presence of both aphidicolin and hydroxyurea also had an inhibitory effect, but in the presence of hydroxyurea alone, UV-induced repair replication was enhanced. The results of these studies can be explained on the basis of differences in deoxyribonucleotide triphosphate pool sizes in growing and confluent cells. Post-treatment of X-irradiated human lymphocytes in the G 0 and G 1 stages with aphidicolin increased the frequencies of X-ray-induced chromosomal aberrations. Such an increase was not observed in G 1 cells of CHO after similar treatment with X-rays and aphidicolin. However, treatment with aphidicolin, in the G 2 stage, increased the frequencies of induced chromatid breaks. The significance of these results is discussed. (orig.)

The effect of caffeine on x-ray repair of radioresistant HeLa cells

International Nuclear Information System (INIS)

Kubo, Kihei; Koiwai, Soichiro; Morita, Kazuo

1985-01-01

The contribution of caffeine-modifiable repair process to the radiosensitivity of a radioresistant HeLa strain (RC-355) has been investigated in comparison with control HeLa strain (CC-24). Both the final slope and the shoulder of X-ray survival curve for log-phase cells were affected by caffeine posttreatment. When the treatment with 10 mM caffeine delayed, an increase in survival was observed with increasing interval between irradiation and the treatment. During first several hours of the repair interval, the steepness of the final slope of survival curve decreased rapidly, and rate of the decrease was found to be higher in RC-355 than in CC-24 cells. Longer time (24 hours or more) before the initiation of caffeine treatment was required for the complete recovery of the shoulder. When the cells were incubated in plateau-phase after irradiation, an appreciable increase in survival was observed in comparison with when plated immediately following X-ray. The increase was found to be greater for RC-355 than for CC-24. The results suggest that the radioresistant RC-355 cells repaired more X-ray-induced PLD than CC-24 cells did. (author)

Radiobiological study on DNA strand breaks and repair using single cell gel electrophoresis

International Nuclear Information System (INIS)

Ikushima, Takaji

1994-01-01

Single cell gel electrophoresis (SCGE) provides a novel method to measure DNA damage in individual cells and more importantly, to assess heterogeneity in response within a mixed population of cells. Cells embedded in agarose are lysed, subjected to electrophoresis, stained with a fluorescent DNA-specific dye, and viewed under a fluorescence microscope. Damaged cells display 'comets', broken DNA migrating farther to the anode in the electric field. We have previously used this technique to quantify DNA damage induced by moderate doses of low and high LET radiations in cultured Chinese hamster cells. The assay has been optimized in terms of lysing and electrophoresis conditions, and applied to analyse the DNA strand breaks, their repair kinetics and heterogeneity in response in individual Chinese hamster cells exposed to gamma-rays, and to KUR thermal neutrons with and without 10 B or to KEK PF monochromatic soft X-rays as well as to a radio-mimetic agent, neocarzinostatin. The DNA double-strand breaks induced by boron-neutron captured reactions were repaired at a slower rate, but a heterogeneity in response might not contribute to the difference. The neocarzinostatin-induced DNA damage were efficiently repaired in a dose-dependent fashion. The initial amount of gamma-ray induced DNA double-strand breaks was not significantly altered in cells pre-exposed to very low adapting dose. (author)

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

Ultra-fast repair of single-strand breaks in DNA of. gamma. -irradiated Chinese hamster cells

Energy Technology Data Exchange (ETDEWEB)

Leontjeva, G A; Mantzighin, Yu A; Gaziev, A I [AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki

1976-12-01

Studies of the effect of thermal treatment of Chinese hamster cells on sedimentation of DNA in the alkaline sucrose gradient showed that heating the cells to 68/sup 0/C for 15 min caused the same degradation as ..gamma..-irradiation with 5 to 7 krad at 37/sup 0/C. The inhibition of cellular repair enzymes by heating was therefore unacceptable. The process of ultra-fast repair is essentially determined by the DNA-ligase reaction, which is activated in the presence of Mg ions, and inhibited in mammalian cells in the presence of EDTA and pyrophosphate. Sedimentation profiles were therefore measured for the DNA of Chinese hamster cells ..gamma..-irradiated (5 krad) at 0/sup 0/C or 22/sup 0/C in the presence of Mg/sup + +/, or EDTA and pyrophosphate, and the results demonstrated ultra-fast repair only at 20 to 37/sup 0/C, in contrast to bacteria. A study was made of the temperature dependence of the activity of the DNA ligases isolated from E.coli and rabbit bone marrow. The NAD-dependent bacterial DNA ligase was active at temperatures from 0 to 40/sup 0/C, whereas ATP-dependent DNA ligase of mammals only showed activity in the range 15 to 40/sup 0/C. The differing temperature dependences of ultra-fast repair in bacterial and mammalian cells are in agreement with the temperature dependences of the activities of isolated enzymes, and the results suggest that the process of ultra-fast repair of single-strand breaks of DNA takes place in both bacterial and mammalian cells.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

International Nuclear Information System (INIS)

Kienzler, Aude; Mahler, Barbara J.; Van Metre, Peter C.; Schweigert, Nathalie; Devaux, Alain; Bony, Sylvie

2015-01-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity. - Highlights: • Co-exposure to runoff from coal-tar-sealcoated pavement and UVA caused DNA damage. • Significant genotoxicity occurred with a 1:100 dilution of runoff. • Runoff collected up to 36 d following coal-tar-sealcoat application was genotoxic. • Exposure to runoff from sealed pavement impaired an important DNA repair pathway. • Repair capacity was impaired with a 1:10 dilution of runoff (1:100 not

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line

Energy Technology Data Exchange (ETDEWEB)

Kienzler, Aude, E-mail: aude.kienzler@entpe.fr [Université de Lyon, UMR LEHNA 5023, USC INRA, ENTPE, rue Maurice Audin, Vaulx-en-Velin F-69518 (France); Mahler, Barbara J., E-mail: bjmahler@usgs.gov [U.S. Geological Survey, 1505 Ferguson Lane, Austin, TX 78754 (United States); Van Metre, Peter C., E-mail: pcvanmet@usgs.gov [U.S. Geological Survey, 1505 Ferguson Lane, Austin, TX 78754 (United States); Schweigert, Nathalie [Université de Lyon, UMR LEHNA 5023, USC INRA, ENTPE, rue Maurice Audin, Vaulx-en-Velin F-69518 (France); Devaux, Alain, E-mail: alain.devaux@entpe.fr [Université de Lyon, UMR LEHNA 5023, USC INRA, ENTPE, rue Maurice Audin, Vaulx-en-Velin F-69518 (France); Bony, Sylvie, E-mail: bony@entpe.fr [Université de Lyon, UMR LEHNA 5023, USC INRA, ENTPE, rue Maurice Audin, Vaulx-en-Velin F-69518 (France)

2015-07-01

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity. - Highlights: • Co-exposure to runoff from coal-tar-sealcoated pavement and UVA caused DNA damage. • Significant genotoxicity occurred with a 1:100 dilution of runoff. • Runoff collected up to 36 d following coal-tar-sealcoat application was genotoxic. • Exposure to runoff from sealed pavement impaired an important DNA repair pathway. • Repair capacity was impaired with a 1:10 dilution of runoff (1:100 not

Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

International Nuclear Information System (INIS)

Raffin, A.L.

2009-06-01

DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

Transplantation of bone marrow derived cells promotes pancreatic islet repair in diabetic mice

International Nuclear Information System (INIS)

Gao Xiaodong; Song Lujun; Shen Kuntang; Wang Hongshan; Niu Weixin; Qin Xinyu

2008-01-01

The transplantation of bone marrow (BM) derived cells to initiate pancreatic regeneration is an attractive but as-yet unrealized strategy. Presently, BM derived cells from green fluorescent protein transgenic mice were transplanted into diabetic mice. Repair of diabetic islets was evidenced by reduction of hyperglycemia, increase in number of islets, and altered pancreatic histology. Cells in the pancreata of recipient mice co-expressed BrdU and insulin. Double staining revealed β cells were in the process of proliferation. BrdU + insulin - PDX-1 + cells, Ngn3 + cells and insulin + glucagon + cells, which showed stem cells, were also found during β-cell regeneration. The majority of transplanted cells were mobilized to the islet and ductal regions. In recipient pancreas, transplanted cells simultaneously expressed CD34 but did not express insulin, PDX-1, Ngn3, Nkx2.2, Nkx6.1, Pax4, Pax6, and CD45. It is concluded that BM derived cells especially CD34 + cells can promote repair of pancreatic islets. Moreover, both proliferation of β cells and differentiation of pancreatic stem cells contribute to the regeneration of β cells

A comparative study of radiation induced DNA damage and repair in buccal cells and lymphocytes assessed by single cell gel electrophoresis (comet) assay

International Nuclear Information System (INIS)

Dhillon, V.S.; Fenech, M.

2003-01-01

Full text: During the past few years, there has been increasing interest in epithelial cells from buccal mucosa for genotoxicity evaluation of different chemical and/or physical agents. In the present study we used the buccal and sublingual epithelial cells to detect both inter- and intra-individual variation in radiation induced DNA damage and repair. For this purpose we used the single cell gel electrophoresis assay which over the years has gained wide spread acceptance as a simple, sensitive and reliable assay to measure genotoxicity related effects as well as kinetics of DNA repair. Buccal and sublingual epithelial cells from six individuals (3 male and 3 females; 35-45 years old) were collected. Cells were then irradiated for 0, 2 and 4 Gy doses using 137 Cs-source (5.58 Gy min-1). After irradiation the cells were either placed immediately on ice or incubated at 37 deg C for 2 1/2 hour to allow cellular repair. We also studied G0 and G1 lymphocytes from the same individuals to compare the radiation-induced DNA damage and repair potential with the two types of buccal cells. Baseline DNA damage rate was significantly greater (p < 0.001) in buccal (28.18%) and sublingual epithelial cells (30.66) as compared to G0 (22.02%) and G1 (21.46%) lymphocytes. Radiation-induced DNA damage in buccal (19.34%, 2Gy; 21.41%, 4 Gy) and sublingual epithelial cells (18.11% and 20.60%) was very similar and significantly lower than that observed in lymphocytes (29.76%, 56.77% for G0 and 32.66%, 59.32% for G1). The extent of DNA repair in buccal and sublingual epithelial cells was significantly lower than that observed in lymphocytes. The results for buccal and sublingual epithelial cells were highly correlated with each other (r 0.9541) as were those of G0 and G1 lymphocytes (r 0.9868). The results suggest a much reduced capacity for cellular repair in buccal and sublingual epithelial cells

Differential repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks

Energy Technology Data Exchange (ETDEWEB)

Smeets, M.F.M.A.; Mooren, E.H.M.; Abdel-Wahab, A.H.A.; Begg, A.C. [Netherlands Cancer Institute, Amsterdam (Netherlands)

1994-11-01

The goal of these experiments was to investigate further the relationship between DNA double-strand breaks and cell killing in human tumor cells, first by comparing different cell lines, and second by radiomodification studies. Field-inversion gel electrophoresis was used to quantify double-strand breaks. Two subclones of the radioresistant human squamous cell carcinoma line SQ20B (SQD9 and SQG6) were compared. These subclones differed in DNA index by a factor of 1.7 but showed the same resistance to radiation as cells of the parental cell line. It was found that, although induction of DSBs was not significantly different in the two cell lines, the t{sub 1/2} of the fast component of repair was significantly shorter for SQD9 cells, leading to greater overall repair which was not reflected in increased survival. Caffeine and cysteamine were tested as modifiers of radiosensitivity, using the radioresistant SQ20B line and the radiosensitive SCC61 cell line. No effect of caffeine was seen when the drug was present only during irradiation. Postirradiation incubations with caffeine, however, resulted in a dose reduction factor greater than 2.0 in cell survival for both cell lines. In contrast, induction of DSBs was reduced by caffeine, and no effect on DSB repair was observed. Cysteamine led to a dose protection factor greater than 1.8 in cell survival in both cell lines. A reduction in induced DSBs was found at high doses corresponding approximately with the increase in cell survival. Over the same (low) dose range, however, the correlation between DSB induction and cell killing was poor. These data indicate that DSB induction does not correlate well with cell killing either for different cell lines, for radiochemical modification (cysteamine) or for some other types of modification (caffeine). 31 refs., 8 figs.

Electrochemical cell with integrated hydrocarbon gas sensor for automobile exhaust gas; Elektrochemische Zelle mit integriertem Kohlenwasserstoff-Gassensor fuer das Automobilabgas

Energy Technology Data Exchange (ETDEWEB)

Biskupski, D.; Moos, R. [Univ. Bayreuth (Germany). Bayreuth Engine Research Center, Lehrstuhl fuer Funktionsmaterialien; Wiesner, K.; Fleischer, M. [Siemens AG, Corporate Technology, CT PS 6, Muenchen (Germany)

2007-07-01

In the future sensors will be necessary to control the compliance with hydrocarbon limiting values, allowing a direct detection of the hydrocarbons. Appropriate sensor-active functional materials are metal oxides, which have a hydrocarbon sensitivity but are also dependent on the oxygen partial pressure. It is proposed that the gas-sensing layer should be integrated into an electrochemical cell. The authors show that the integration of a resistive oxygen sensor into a pump cell allows a defined oxygen concentration level at the sensor layer in any exhaust gas.

Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

Science.gov (United States)

Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

2012-09-01

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

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

Quantitative Methods for Measuring Repair Rates and Innate-Immune Cell Responses in Wounded Mouse Skin.

Science.gov (United States)

Li, Zhi; Gothard, Elizabeth; Coles, Mark C; Ambler, Carrie A

2018-01-01

In skin wounds, innate-immune cells clear up tissue debris and microbial contamination, and also secrete cytokines and other growth factors that impact repair process such as re-epithelialization and wound closure. After injury, there is a rapid influx and efflux of immune cells at wound sites, yet the function of each innate cell population in skin repair is still under investigation. Flow cytometry is a valuable research tool for detecting and quantifying immune cells; however, in mouse back skin, the difficulty in extracting immune cells from small area of skin due to tissue complexity has made cytometric analysis an underutilized tool. In this paper, we provide detailed methods on the digestion of lesion-specific skin without disrupting antigen expression followed by multiplex cell staining that allows for identification of seven innate-immune populations, including rare subsets such as group-3 innate lymphoid cells (ILC3s), by flow-cytometry analysis. Furthermore, when studying the functions of immune cells to tissue repair an important metric to monitor is size of the wound opening. Normal wounds close steadily albeit at non-linear rates, while slow or stalled wound closure can indicate an underlying problem with the repair process. Calliper measurements are difficult and time-consuming to obtain and can require repeated sedation of experimental animals. We provide advanced methods for measuring of wound openness; digital 3D image capture and semi-automated image processing that allows for unbiased, reliable measurements that can be taken repeatedly over time.

Double strand break repair: two mechanisms in competition but tightly linked to cell cycle

International Nuclear Information System (INIS)

Delacote, F.

2002-11-01

DNA double strand breaks (DSB) are highly toxic damage although they can be induced to create genetic diversity. Two distinct pathways can repair DSB: Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). If un- or mis-repaired, this damage can lead to cancer. Thus, it is essential to investigate how these two pathways are regulated for DSB repair. NHEJ inhibition leads to HR DSB repair stimulation. However, this channeling to HR is tightly linked to cell cycle since NHEJ and HR are active in G1/early S and late S/G2, respectively. Our results suggest that G1-unrepaired DSB go through S phase to be repaired by HR in G2. Those results allow a better understanding of DSB repair mechanisms regulation. (author)

MICROBIAL CELL-SURFACE HYDROPHOBICITY - THE INVOLVEMENT OF ELECTROSTATIC INTERACTIONS IN MICROBIAL ADHESION TO HYDROCARBONS (MATH)

NARCIS (Netherlands)

GEERTSEMADOORNBUSCH, GI; VANDERMEI, HC; BUSSCHER, HJ

Microbial adhesion to hydrocarbons (MATH) is the most commonly used method to determine microbial cell surface hydrophobicity. Since, however, the assay is based on adhesion, it is questionable whether the results reflect only the cell surface hydrophobicity or an interplay of hydrophobicity and

Genetic characterization of cells of homocystinuria patients with disrupted DNA repair system

International Nuclear Information System (INIS)

Sinel'shchikova, T.A.; L'vova, G.N.; Shoniya, N.N.; Zasukhina, G.D.

1986-01-01

Fibroblasts obtained from biopsy material and lymphocytes of patients with homocystinuria were investigated for repair activity according to the following criteria: rejoined DNA breaks, induced by 4-nitroquinoline-1-oxide and γ-radiation; indices of reactivation and induced mutagenesis of smallpox vaccine virus treated with these mutagens. In lymphocytes a defect of DNA repair was observed according to all criteria investigated. During passage of fibroblast cultures, inhibition of repair activity of cells was preserved according to γ-type. Increase in the number of spontaneous and γ-induced mutations of virus was noted according to degree of passage of fibroblasts

DNA-repair, cell killing and normal tissue damage

International Nuclear Information System (INIS)

Dahm-Daphi, J.; Dikomey, E.; Brammer, I.

1998-01-01

Background: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. Material and methods: Review. Results: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. Conclusion: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly. (orig.) [de

Polycyclic aromatic hydrocarbons reciprocally regulate IL-22 and IL-17 cytokines in peripheral blood mononuclear cells from both healthy and asthmatic subjects.

Directory of Open Access Journals (Sweden)

Coline Plé

Full Text Available Pollution, including polycyclic aromatic hydrocarbons (PAH, may contribute to increased prevalence of asthma. PAH can bind to the Aryl hydrocarbon Receptor (AhR, a transcription factor involved in Th17/Th22 type polarization. These cells produce IL17A and IL-22, which allow neutrophil recruitment, airway smooth muscle proliferation and tissue repair and remodeling. Increased IL-17 and IL-22 productions have been associated with asthma. We hypothesized that PAH might affect, through their effects on AhR, IL-17 and IL-22 production in allergic asthmatics. Activated peripheral blood mononuclear cells (PBMCs from 16 nonallergic nonasthmatic (NA and 16 intermittent allergic asthmatic (AA subjects were incubated with PAH, and IL-17 and IL-22 productions were assessed. At baseline, activated PBMCs from AA exhibited an increased IL-17/IL-22 profile compared with NA subjects. Diesel exhaust particle (DEP-PAH and Benzo[a]Pyrene (B[a]P stimulation further increased IL-22 but decreased IL-17A production in both groups. The PAH-induced IL-22 levels in asthmatic patients were significantly higher than in healthy subjects. Among PBMCs, PAH-induced IL-22 expression originated principally from single IL-22- but not from IL-17- expressing CD4 T cells. The Th17 transcription factors RORA and RORC were down regulated, whereas AhR target gene CYP1A1 was upregulated. IL-22 induction by DEP-PAH was mainly dependent upon AhR whereas IL-22 induction by B[a]P was dependent upon activation of PI3K and JNK. Altogether, these data suggest that DEP-PAH and B[a]P may contribute to increased IL22 production in both healthy and asthmatic subjects through mechanisms involving both AhR -dependent and -independent pathways.

Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants.

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Adams, Bret R; Golding, Sarah E; Rao, Raj R; Valerie, Kristoffer

2010-04-02

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

Aryl hydrocarbon receptor protects lung adenocarcinoma cells against cigarette sidestream smoke particulates-induced oxidative stress

International Nuclear Information System (INIS)

Cheng, Ya-Hsin; Huang, Su-Chin; Lin, Chun-Ju; Cheng, Li-Chuan; Li, Lih-Ann

2012-01-01

Environmental cigarette smoke has been suggested to promote lung adenocarcinoma progression through aryl hydrocarbon receptor (AhR)-signaled metabolism. However, whether AhR facilitates metabolic activation or detoxification in exposed adenocarcinoma cells remains ambiguous. To address this question, we have modified the expression level of AhR in two human lung adenocarcinoma cell lines and examined their response to an extract of cigarette sidestream smoke particulates (CSSP). We found that overexpression of AhR in the CL1-5 cell line reduced CSSP-induced ROS production and oxidative DNA damage, whereas knockdown of AhR expression increased ROS level in CSSP-exposed H1355 cells. Oxidative stress sensor Nrf2 and its target gene NQO1 were insensitive to AhR expression level and CSSP treatment in human lung adenocarcinoma cells. In contrast, induction of AhR expression concurrently increased mRNA expression of xenobiotic-metabolizing genes CYP1B1, UGT1A8, and UGT1A10 in a ligand-independent manner. It appeared that AhR accelerated xenobiotic clearing and diminished associated oxidative stress by coordinate regulation of a set of phase I and II metabolizing genes. However, the AhR-signaled protection could not shield cells from constant oxidative stress. Prolonged exposure to high concentrations of CSSP induced G0/G1 cell cycle arrest via the p53–p21–Rb1 signaling pathway. Despite no effect on DNA repair rate, AhR facilitated the recovery of cells from growth arrest when CSSP exposure ended. AhR-overexpressing lung adenocarcinoma cells exhibited an increased anchorage-dependent and independent proliferation when recovery from exposure. In summary, our data demonstrated that AhR protected lung adenocarcinoma cells against CSSP-induced oxidative stress and promoted post-exposure clonogenicity. -- Highlights: ► AhR expression level influences cigarette sidestream smoke-induced ROS production. ► AhR reduces oxidative stress by coordinate regulation of

Aryl hydrocarbon receptor protects lung adenocarcinoma cells against cigarette sidestream smoke particulates-induced oxidative stress

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Cheng, Ya-Hsin [Graduate Institute of Basic Medical Science, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China); Huang, Su-Chin; Lin, Chun-Ju; Cheng, Li-Chuan [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China); Li, Lih-Ann, E-mail: lihann@nhri.org.tw [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China)

2012-03-15

Environmental cigarette smoke has been suggested to promote lung adenocarcinoma progression through aryl hydrocarbon receptor (AhR)-signaled metabolism. However, whether AhR facilitates metabolic activation or detoxification in exposed adenocarcinoma cells remains ambiguous. To address this question, we have modified the expression level of AhR in two human lung adenocarcinoma cell lines and examined their response to an extract of cigarette sidestream smoke particulates (CSSP). We found that overexpression of AhR in the CL1-5 cell line reduced CSSP-induced ROS production and oxidative DNA damage, whereas knockdown of AhR expression increased ROS level in CSSP-exposed H1355 cells. Oxidative stress sensor Nrf2 and its target gene NQO1 were insensitive to AhR expression level and CSSP treatment in human lung adenocarcinoma cells. In contrast, induction of AhR expression concurrently increased mRNA expression of xenobiotic-metabolizing genes CYP1B1, UGT1A8, and UGT1A10 in a ligand-independent manner. It appeared that AhR accelerated xenobiotic clearing and diminished associated oxidative stress by coordinate regulation of a set of phase I and II metabolizing genes. However, the AhR-signaled protection could not shield cells from constant oxidative stress. Prolonged exposure to high concentrations of CSSP induced G0/G1 cell cycle arrest via the p53–p21–Rb1 signaling pathway. Despite no effect on DNA repair rate, AhR facilitated the recovery of cells from growth arrest when CSSP exposure ended. AhR-overexpressing lung adenocarcinoma cells exhibited an increased anchorage-dependent and independent proliferation when recovery from exposure. In summary, our data demonstrated that AhR protected lung adenocarcinoma cells against CSSP-induced oxidative stress and promoted post-exposure clonogenicity. -- Highlights: ► AhR expression level influences cigarette sidestream smoke-induced ROS production. ► AhR reduces oxidative stress by coordinate regulation of

Conversion of hydrocarbons and alcohols for fuel cells

Science.gov (United States)

Joensen, Finn; Rostrup-Nielsen, Jens R.

The growing demand for clean and efficient energy systems is the driving force in the development of fuel processing technology for providing hydrogen or hydrogen-containing gaseous fuels for power generation in fuel cells. Successful development of low cost, efficient fuel processing systems will be critical to the commercialisation of this technology. This article reviews various reforming technologies available for the generation of such fuels from hydrocarbons and alcohols. It also briefly addresses the issue of carbon monoxide clean-up and the question of selecting the appropriate fuel(s) for small/medium scale fuel processors for stationary and automotive applications.

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

Science.gov (United States)

Muiños-López, Emma; Hermida-Gómez, Tamara; Fuentes-Boquete, Isaac; de Toro-Santos, Javier; Blanco, Francisco Javier; Díaz-Prado, Silvia María

2017-09-01

Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better

Opportunities and Challenges for Repair of Macrovascular Disease using Circulating Blood-Derived Progenitor Cells

OpenAIRE

Loeken, Mary R.

2014-01-01

There are currently few solutions for diabetic vascular disease that involve repair of damaged tissues. The manuscript by Porat, et al., suggests a possible method to use a patient’s own circulating blood cells to provide progenitors to repair damaged vascular tissues.

Inhibition of homologous recombination repair in irradiated tumor cells pretreated with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin

International Nuclear Information System (INIS)

Noguchi, Miho; Yu, Dong; Hirayama, Ryoichi; Ninomiya, Yasuharu; Sekine, Emiko; Kubota, Nobuo; Ando, Koichi; Okayasu, Ryuichi

2006-01-01

In order to investigate the mechanism of radio-sensitization by an Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), we studied repair of DNA double strand breaks (DSBs) in irradiated human cells pre-treated with 17-AAG. DSBs are thought to be the critical target for radiation-induced cell death. Two human tumor cell lines DU145 and SQ-5 which showed clear radio-sensitization by 17-AAG revealed a significant inhibition of DSB repair, while normal human cells which did not show radio-sensitization by the drug indicated no change in the DSB repair kinetics with 17-AAG. We further demonstrated that BRCA2 was a novel client protein for Hsp90, and 17-AAG caused the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for homologous recombination (HR) pathway of DSB repair. Our data demonstrate for the first time that 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing

Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Three-year report, February 1, 1981-September 30, 1983

International Nuclear Information System (INIS)

Evans, H.H.

1983-01-01

Mutant strains were selected which are deficient in various DNA repair pathways and these were studied with regard to (1) the nature of the defect in repair, and (2) the mutability and transformability of the defective cells by various agents as compared to the wild type parental cells. Lightly mutagenized wild-type cells were infected with irradiated herpes simplex virus (HSV). Cells which repair HSV are lysed so the surviving population is enriched in repair-deficient cells. Six strains which survived two rounds of infection were characterized with respect to their radiosensitivity

Aromatic hydrocarbons upregulate glyceraldehyde-3-phosphate dehydrogenase and induce changes in actin cytoskeleton. Role of the aryl hydrocarbon receptor (AhR)

International Nuclear Information System (INIS)

Reyes-Hernandez, O.D.; Mejia-Garcia, A.; Sanchez-Ocampo, E.M.; Castro-Munozledo, F.; Hernandez-Munoz, R.; Elizondo, G.

2009-01-01

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and β-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with β-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.

Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F

International Nuclear Information System (INIS)

Hayakawa, H.; Ishizaki, K.; Yagi, T.; Takebe, H.; Inoue, M.; Sekiguchi, M.; Kyoto Univ.

1981-01-01

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair. (orig.)

A Preliminary Study: Human Fibroid Stro-1+/CD44+ Stem Cells Isolated From Uterine Fibroids Demonstrate Decreased DNA Repair and Genomic Integrity Compared to Adjacent Myometrial Stro-1+/CD44+ Cells.

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Prusinski Fernung, Lauren E; Al-Hendy, Ayman; Yang, Qiwei

2018-01-01

Although uterine fibroids (UFs) continue to place a major burden on female reproductive health, the mechanisms behind their origin remain undetermined. Normal myometrial stem cells may be transformed into tumor-initiating stem cells, causing UFs, due to unknown causes of somatic mutations in MED12, found in up to 85% of sporadically formed UFs. It is well established in other tumor types that defective DNA repair increases the risk of such tumorigenic somatic mutations, mechanisms not yet studied in UFs. To examine the putative cause(s) of this stem cell transformation, we analyzed DNA repair within stem cells from human UFs compared to those from adjacent myometrium to determine whether DNA repair in fibroid stem cells is compromised. Human fibroid (F) and adjacent myometrial (Myo) stem cells were isolated from fresh tissues, and gene expression relating to DNA repair was analyzed. Fibroid stem cells differentially expressed DNA repair genes related to DNA double- (DSBs) and single-strand breaks. DNA damage was measured using alkaline comet assay. Additionally, DNA DSBs were induced in these stem cells and DNA DSB repair evaluated (1) by determining changes in phosphorylation of DNA DSB-related proteins and (2) by determining differences in γ-H2AX foci formation and relative DNA repair protein RAD50 expression. Overall, F stem cells demonstrated increased DNA damage and altered DNA repair gene expression and signaling, suggesting that human F stem cells demonstrate impaired DNA repair. Compromised F stem cell DNA repair may contribute to further mutagenesis and, consequently, further growth and propagation of UF tumors.

Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

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Nolan, N.L.; Kidwell, W.R.

1982-04-01

Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

International Nuclear Information System (INIS)

Nolan, N.L.; Kidwell, W.R.

1982-01-01

Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37 0 C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of γ-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of γ-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels

Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, August 1, 1977-October 31, 1980

International Nuclear Information System (INIS)

Evans, H.H.

1980-10-01

The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. More specifically, mutant strains will be selected which are deficient in various DNA repair pathways. These strains will be studied with regard to (1) the nature of the defect in repair, and (2) the mutability and transformability of the defective cells by various agents as compared to the wild type parental cells. The results to date include progress in the following areas: (1) determination of optimum conditions for growth and maintenance of cells and for quantitative measurement of various cellular parameters; (2) investigation of the effect of holding mutagenized cells for various periods in a density inhibited state on survival and on mutation and transformation frequencies; (3) examination of the repair capabilities of BHK cells, as compared to repair-proficient and repair-deficient human cells and excision-deficient mouse cells, as measured by the reactivation of Herpes simplex virus (HSV) treated with radiation and ethylmethane sulfonate (EMS); (4) initiation of host cell reactivation viral sucide enrichment and screening of survivors of the enrichment for sensitivity to ionizing radiation; and (5) investigation of the toxicity, mutagenicity, and carcinogenicity of various metabolites of 4-nitroquinoline-1-oxide (4-NQO)

The effect of 2-[(aminopropyl)amino] ethanethiol (WR 1065) on radiation-induced DNA damage and repair and cell progression in V79 cells

International Nuclear Information System (INIS)

Grdina, D.J.; Nagy, B.

1986-01-01

The radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) was investigated with respect to its ability to affect radiation-induced DNA damage and repair in V79 cells. At a concentration of 4mM, WR 1065 protected against the formation of single strand breaks (SSB), when present during irradiation. The protector appeared, however, to inhibit the subsequent postirradiation repair or rejoining of SSB. While repair was complete within 24h, the protector reduced the rate of repair by a factor of 3. This inhibitory effect on the rate of repair did not correlate with either measured differences in cell survival or mutagenesis. WR 1065 present in the growth medium inhibited the progression of cells through S-phase, and cell-doubling time following a 3h exposure to the protector was increased from 11 to 18h. These data are consistent with the property of thiols to inhibit DNA polymerase activity. It was concluded that, while the presence of WR 1065 during irradiation reduced SSB-DNA damage, its effect on the subsequent rejoining of these breaks could not be correlated with its observed effect on protecting against radiation-induced mutagenesis. (author)

DNA demethylation by 5-aza-2-deoxycytidine treatment abrogates 17 beta-estradiol-induced cell growth and restores expression of DNA repair genes in human breast cancer cells.

Science.gov (United States)

Singh, Kamaleshwar P; Treas, Justin; Tyagi, Tulika; Gao, Weimin

2012-03-01

Prolonged exposure to elevated levels of estrogen is a risk factor for breast cancer. Though increased cell growth and loss of DNA repair capacity is one of the proposed mechanisms for estrogen-induced cancers, the mechanism through which estrogen induces cell growth and decreases DNA repair capacity is not clear. DNA hypermethylation is known to inactivate DNA repair genes and apoptotic response in cancer cells. Therefore, the objective of this study was to determine the role of DNA hypermethylation in estrogen-induced cell growth and regulation of DNA repair genes expression in breast cancer cells. To achieve this objective, the estrogen-responsive MCF-7 cells either pretreated with 5-aza-2-deoxycytidine (5-aza-dC) or untreated (as control) were exposed to 17 beta-estradiol (E2), and its effect on cell growth and expression of DNA repair genes were measured. The result revealed that 5-aza-dC abrogates the E2-induced growth in MCF-7 cells. An increased expression of OGG1, MSH4, and MLH1 by 5-aza-dC treatment alone, suggest the DNA hypermethylation as a potential cause for decreased expression of these genes in MCF-7 cells. The decreased expression of ERCC1, XPC, OGG1, and MLH1 by E2 alone and its restoration by co-treatment with 5-aza-dC further suggest that E2 reduces the expression of these DNA repair genes potentially through promoter hypermethylation. Reactivation of mismatch repair (MMR) gene MLH1 and abrogation of E2-induced cell growth by 5-aza-dC treatment suggest that estrogen causes increased growth in breast cancer cells potentially through the inhibition of MMR-mediated apoptotic response. In summary, this study suggests that estrogen increases cell growth and decreases the DNA repair capacity in breast cancer cells, at least in part, through epigenetic mechanism. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Induced DNA repair pathway in mammalian cells

International Nuclear Information System (INIS)

Overberg, R.

1985-01-01

The survival of cultured rat kangaroo cells (PtK-2) and human xeroderma pigmentosum cells incubated with 5 μM cycloheximide subsequent to ultraviolet irradiation is lower than that of cells incubated without cycloheximide. The drop in survival is considerably larger than that produced by incubation of unirradiated cells with cycloheximide. The phenomenon was also observed when PtK-2 cells were incubated with emetine, another protein synthesis inhibitor, or with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, a RNA synthesis inhibitor. PtK cells which received a preliminary UV treatment followed by an incubation period without cycloheximide and then a second irradiation and 24 hour incubation with cycloheximide, survived the effects of the second irradiation better than cells which were incubated in the presence of cycloheximide after the first and second UV irradiation. The application of cycloheximide for 24 hours after UV irradiation of PtK cells resulted in one-half as many 6-thioguanine resistant cells as compared to the number of 6-thioguanine resistant cells found when cycloheximide was not used. These experiments indicate that a UV-inducible cycloheximide-sensitive DNA repair pathway is present in PtK and xeroderma pigmentosum cells, which is error-prone in PtK cells

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

Phage T4 endonuclease V stimulates DNA repair replication in isolated nuclei from ultraviolet-irradiated human cells, including xeroderma pigmentosum fibroblasts

International Nuclear Information System (INIS)

Smith, C.A.; Hanawalt, P.C.

1978-01-01

The repair mode of DNA replication has been demonstrated in isolated nuclei from uv-irradiated human cells. Nuclei are incubated in a mixture containing [ 3 H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The 3 H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is uv-dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg 2+ -dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote uv-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms

Kinetics of repair of DNA single-strand breaks in cultured mammalian cells

International Nuclear Information System (INIS)

Vexler, F.B.; Eidus, L.Kh.; Vexler, A.M.

1984-01-01

Postirradiation treatment of Chinese hamster cells with cysteamine (MEA), caffeine-benzoate (CB) and caffeine sharply inhibits the repair of DNA single-strand breaks in the first five minutes. This inhibition is reversible since removing of the agent leads immediately to the resumption of the repair. The rate of the repair is decreased with prolongation of treatment and increasing concentration of the modifying agent. The efficiency of the substances studied depends not only on their concentration in the medium. For MEA and CB, which are weak electrolytes, it is also pH-dependent. This is explained by the theory of dissociation of weak electrolytes and their distribution between the cell and medium. It is shown that intracellular concentration of the substances is the most important factor determining their efficiency. All the three substances exert practically the same effect when compared at equal intracellular concentration. The above presented data serve as evidence for the existence of an unspecific mechanism of the effect of the substances studied. (author)

Identification of Drugs that Regulate Dermal Stem Cells and Enhance Skin Repair

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Sibel Naska

2016-01-01

Full Text Available Here, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on skin-derived precursors (SKPs, a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated five such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKP self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy.

Excision repair of bulky lesions in the DNA of mammalian cells

International Nuclear Information System (INIS)

Setlow, R.B.; Grist, E.

1980-01-01

The report examines the process of excision repair of pyrimidine dimers from uv-irradiated and chemically challenged human cells. It is shown by means of a sensitive endonuclease assay that the amount of excision observed depends upon the isotope used to label cells, and that XP heterozygotes are between normals and XPs

An Inducible, Isogenic Cancer Cell Line System for Targeting the State of Mismatch Repair Deficiency

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Bailis, Julie M.; Gordon, Marcia L.; Gurgel, Jesse L.; Komor, Alexis C.; Barton, Jacqueline K.; Kirsch, Ilan R.

2013-01-01

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells. PMID:24205301

DNA repair and cancer

International Nuclear Information System (INIS)

Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha

2012-01-01

DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

International Nuclear Information System (INIS)

Intine, R.V.; Rainbow, A.J.

1990-01-01

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair

Investigations on the carbon contaminations on the alkali cells of DPAL with hydrocarbon buffer gas

Science.gov (United States)

Li, Zhiyong; Tan, Rongqing; Wang, Yujie; Ye, Qing; Bian, Jintian; Huang, Wei; Li, Hui; Han, Gaoce

2017-10-01

Diode pumped alkali laser (DPAL) with hydrocarbon buffer gases has the features of low threshold and high efficiency. The chemical reaction between alkali and hydrocarbon gases affects the life time of DPAL. In this paper, a method based on Fourier transform infrared spectroscopy and Lambert-Beer law is adopted to find a safe temperature at which DPAL runs for a long term. A theoretical model is established to figure out ways to reduce the peak temperature in the cell window. The results indicates that 170 °C is a safe temperature. Although the absorbance of the cell window to the pump light and alkali laser is lower, there is temperature increase. Small light-transmitting area and air blowing on the windows can reduce the peak temperature effectively. Cooling the cell window is essential and critical in a long-term running DPAL.

Quantitative Methods for Measuring Repair Rates and Innate-Immune Cell Responses in Wounded Mouse Skin

Directory of Open Access Journals (Sweden)

Zhi Li

2018-02-01

Full Text Available In skin wounds, innate-immune cells clear up tissue debris and microbial contamination, and also secrete cytokines and other growth factors that impact repair process such as re-epithelialization and wound closure. After injury, there is a rapid influx and efflux of immune cells at wound sites, yet the function of each innate cell population in skin repair is still under investigation. Flow cytometry is a valuable research tool for detecting and quantifying immune cells; however, in mouse back skin, the difficulty in extracting immune cells from small area of skin due to tissue complexity has made cytometric analysis an underutilized tool. In this paper, we provide detailed methods on the digestion of lesion-specific skin without disrupting antigen expression followed by multiplex cell staining that allows for identification of seven innate-immune populations, including rare subsets such as group-3 innate lymphoid cells (ILC3s, by flow-cytometry analysis. Furthermore, when studying the functions of immune cells to tissue repair an important metric to monitor is size of the wound opening. Normal wounds close steadily albeit at non-linear rates, while slow or stalled wound closure can indicate an underlying problem with the repair process. Calliper measurements are difficult and time-consuming to obtain and can require repeated sedation of experimental animals. We provide advanced methods for measuring of wound openness; digital 3D image capture and semi-automated image processing that allows for unbiased, reliable measurements that can be taken repeatedly over time.

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

Energy Technology Data Exchange (ETDEWEB)

Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra, E-mail: rr228@georgetown.edu

2015-05-15

Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

International Nuclear Information System (INIS)

Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra

2015-01-01

Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination

Repair of X-ray-induced single-strand breaks by a cell-free system

International Nuclear Information System (INIS)

Seki, Shuji; Ikeda, Shogo; Tsutui, Ken; Teraoka, Hirobumi

1990-01-01

Repair of X-ray-induced single-strand breaks of DNA was studied in vitro using an exonuclease purified from mouse ascites sarcoma (SR-C3H/He) cells. X-ray-dose-dependent unscheduled DNA synthesis was primed by the exonuclease. Repair of X-ray-induced single-strand breaks in pUC19 plasmid DNA was demonstrated by agarose gel electrophoresis after incubating the damaged DNA with the exonuclease, DNA polymerase (Klenow fragment of DNA polymerase I or DNA polymerase β purified from SR-C3H/He cells), four deoxynucleoside triphosphates, ATP and DNA ligase (T4 DNA ligase or DNA ligase I purified from calf thymus). The present results suggested that the exonuclease is involved in the initiation of repair of X-ray-induced single-strand breaks in removing 3' ends of X-ray-damaged DNA. (author)

Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

DEFF Research Database (Denmark)

Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

2008-01-01

cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...

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

Relevance of DNA repair pathways on ascorbic acid effects on Echerichia Coli K-12 cells

International Nuclear Information System (INIS)

Slyus, M.A. van; Oliveira, R.L.B. da C.; Felzenszwalb, I.; Gomes, R.A.; Menck, C.F.

1985-01-01

Inactivation kinetics were performed with repair proficient and deficient Escherichia coli K-12 cells treated with oxidized solutions of ascorbic acid. The repair pathways controlled by the recA and uvrA gene products are essential for cell survival to the treatment. However, SOS chromotest result indicates that the SOS functions are only induced at high and toxic concentrations of the drug. Moreover, single strand breaks in DNA from treated cells are detected, demonstrating genome damage promoted by oxidized solutions of ascorbate. (M.A.C.) [pt

Polycyclic aromatic hydrocarbons modulate cell proliferation in rat hepatic epithelial stem-like WB-F344 cells

International Nuclear Information System (INIS)

Chramostova, Katerina; Vondracek, Jan; Sindlerova, Lenka; Vojtesek, Borivoj; Kozubik, Alois; Machala, Miroslav

2004-01-01

Although many polycyclic aromatic hydrocarbons (PAHs) are recognized as potent mutagens and carcinogens, relatively little is known about their role in the tumor promotion. It is known that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce release of rat hepatic oval epithelial cells from contact inhibition by a mechanism possibly involving the aryl hydrocarbon receptor (AhR) activation. Many PAHs are AhR ligands and are known to act as transient inducers of AhR-mediated activity. In this study, effects of 19 selected PAHs on proliferation of confluent rat liver epithelial WB-F344 cells were investigated. Non-mutagens that are weak activators or nonactivators of AhR-mediated activity had no effect on cell proliferation. Relatively strong or moderate AhR ligands with low mutagenic potencies, such as benzofluoranthenes, benz[a]anthracene, and chrysene, were found to increase cell numbers, which corresponded to an increased percentage of cells entering S-phase. Strong mutagens, including benzo[a]pyrene and dibenzo[a,l]pyrene, increased a percentage of cells in S-phase without inducing a concomitant increase in cell numbers. The treatment with mutagenic PAHs was associated with an increased DNA synthesis and induction of cell death, which corresponded with the activation of p53 tumor suppressor. Apoptosis was blocked by pifithrin-α, the chemical inhibitor of p53. Both weakly and strongly mutagenic PAHs known as AhR ligands were found to induce significant increase of cytochrome P4501A activity, suggesting a presence of functional AhR. The results of the present study seem to suggest that a release from contact inhibition could be a part of tumor promoting effects of AhR-activating PAHs; however, the genotoxic effects of some PAHs associated with p53 activation might interfere with this process

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: caffeine sensitive and caffeine resistant mechanism

International Nuclear Information System (INIS)

Fujiwara, Y.; Tatsumi, M.

1976-01-01

Replicative bypass repair of UV damage to DNA was studied in a wide variaty of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthesized after irradiation with 10 J/m 2 to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionally, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimodine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative bypassing became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability

Repair of full-thickness articular cartilage defect using stem cell-encapsulated thermogel.

Science.gov (United States)

Zhang, Yanbo; Zhang, Jin; Chang, Fei; Xu, Weiguo; Ding, Jianxun

2018-07-01

Cartilage defect repair by hydrogel-based tissue engineering is becoming one of the most potential treatment strategies. In this work, a thermogel of triblock copolymer poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) was prepared as scaffold of bone marrow mesenchymal stem cells (BMMSCs) for repair of full-thickness articular cartilage defect. At first, the copolymer solution showed a reversible sol-gel transition at physiological temperature range, and the mechanical properties of such thermogel were high enough to support the repair of cartilage. Additionally, excellent biodegradability and biocompatibility of the thermogel were demonstrated. By implanting the BMMSC-encapsulated thermogel into the full-thickness articular cartilage defect (5.0 mm in diameter and 4.0 mm in depth) in the rabbit, it was found that the regenerated cartilage integrated well with the surrounding normal cartilage and subchondral bone at 12 weeks post-surgery. The upregulated expression of glycosaminoglycan and type II collagen in the repaired cartilage, and the comparable biomechanical properties with normal cartilage suggested that the cell-encapsulated PLGA-PEG-PLGA thermogel had great potential in serving as the promising scaffold for cartilage regeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

Fate and degradation of petroleum hydrocarbons in stormwater bioretention cells

Science.gov (United States)

LeFevre, Gregory Hallett

This dissertation describes the investigation of the fate of hydrocarbons in stormwater bioretention areas and those mechanisms that affect hydrocarbon fate in such systems. Seventy-five samples from 58 bioretention areas were collected and analyzed to measure total petroleum hydrocarbon (TPH) residual and biodegradation functional genes. TPH residual in bioretention areas was greater than background sites but low overall (hydrocarbon biodegradation. Field soils were capable of mineralizing naphthalene, a polycyclic aromatic hydrocarbon (PAH) when incubated in the laboratory. In an additional laboratory investigation, a column study was initiated to comprehensively determine naphthalene fate in a simulated bioretention cell using a 14C-labeled tracer. Sorption to soil was the greatest sink of naphthalene in the columns, although biodegradation and vegetative uptake were also important loss mechanisms. Little leaching occurred following the first flush, and volatilization was insignificant. Significant enrichment of naphthalene degrading bacteria occurred over the course of the experiment as a result of naphthalene exposure. This was evident from enhanced naphthalene biodegradation kinetics (measured via batch tests), significant increases in naphthalene dioxygenase gene quantities, and a significant correlation observed between naphthalene residual and biodegradation functional genes. Vegetated columns outperformed the unplanted control column in terms of total naphthalene removal and biodegradation kinetics. As a result of these experiments, a final study focused on why planted systems outperform unplanted systems was conducted. Plant root exudates were harvested from hydroponic setups for three types of plants. Additionally, a solution of artificial root exudates (AREs) as prepared. Exudates were digested using soil bacteria to create metabolized exudates. Raw and metabolized exudates were characterized for dissolved organic carbon, specific UV absorbance

DNA strand breaks, repair, and survival in x-irradiated mammalian cells

International Nuclear Information System (INIS)

Dugle, D.L.; Gillespie, C.J.; Chapman, J.D.

1976-01-01

The yields of unrepairable single- and double-strand breaks in the DNA of x-irradiated Chinese hamster cells were measured by low-speed neutral and alkaline sucrose density gradient sedimentation in order to investigate the relation between these lesions and reproductive death. After maximal single-strand rejoining, at all doses, the number of residual single-strand breaks was twice the number of residual double-strand breaks. Both double-strand and unrepairable single-strand breaks were proportional to the square of absorbed dose, in the range 10-50 krad. No rejoining of double-strand breaks was observed. These observations suggest that, in mammalian cells, most double-strand breaks are not repairable, while all single-strand breaks are repaired except those that are sufficiently close on complementary strands to constitute double-strand breaks. Comparison with cell survival measurements at much lower doses suggests that loss of reproductive capacity corresponds to induction of approximately one double-strand break

Effect of an aminothiol (WR-1065) on radiation-induced mutagenesis and cytotoxicity in two repair-deficient mammalian cell lines

International Nuclear Information System (INIS)

Grdina, D.J.; Nagy, B.; Meechan, P.J.

1991-01-01

WR-2721 and its free thiol WR-1065 have been found to effectively protect against radiation- and/or chemotherapy-induced mutagenesis, transformation and carcinogenesis. With respect to the antimutagenic effect, WR-1065 significantly reduced the frequency of HGPRT mutants even when it was administered up to three hours following exposure of cells to radiation. The mechanisms of action most often attributed to these agents include their ability to scavenge free radicals, enter into chemical repair processes through the donation of hydrogen atoms, and induce intracellular hypoxia by means of auto-oxidative processes. Although evidence exists for each of these processes, none is sufficiently satisfactory to account for the post-irradiation protection of WR-1065 against mutation induction in mammalian cells. The most elegant work describing the role of aminothiols on cellular enzymatic repair processes has focused on well-characterized repair-proficient and -deficient bacterial and yeast cell systems. Protection against radiation-induced cytotoxicity by the aminothiol cysteamine was absent in E. coli cell lines that were characterized as having genetically defective repair systems. Until recently, such studies could not be effectively performed with mammalian cells. However, with the isolation and characterization of rodent cell lines deficient in their ability to repair DNA damage, it is now possible to investigate the role of cell-mediated repair systems on aminothiol radioprotection. Specifically, the authors have investigated the effects of WR-1065 on radiation-induced mutagenesis and cytotoxicity in cell lines EM9 and xrs-5, which are defective in DNA single-strand break (SSB) and double-strand break (DSB) rejoining, respectively. Corresponding parental repair-proficient cell lines, AA8 and K1, were also studied for comparative purposes. 26 refs., 5 figs., 2 tabs

Multiple effects of fluorescent light on repair of ultraviolet-induced DNA lesions in cultured goldfish cells

International Nuclear Information System (INIS)

Uchida, Nobuhiro; Mitani, Hiroshi; Shima, Akihiro

1995-01-01

It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cyclohexamide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH. (author)

Multiple effects of fluorescent light on repair of ultraviolet-induced DNA lesions in cultured goldfish cells

Energy Technology Data Exchange (ETDEWEB)

Uchida, Nobuhiro; Mitani, Hiroshi; Shima, Akihiro [Tokyo Univ. (Japan). Lab. of Radiation Biology

1995-01-01

It is known that fluorescent light illumination prior to UV irradiation (FL preillumination) of cultured fish cells increases photorepair (PR) ability. In the present study, it was found that FL preillumination also enhanced UV resistance of logarithmically growing cells in the dark. This enhancement of UV resistance differs from induction of PR because it was not suppressed by cyclohexamide (CH) and it occurred immediately after FL preillumination. The effects of FL preillumination on repair of UV-induced DNA lesions in the dark were examined by an endonuclease-sensitive site assay to measure the repair of cyclobutyl pyrimidine dimers, and by enzyme-linked immunosorbent assay to quantitate the repair of (6-4) photoproducts. It was found that excision repair ability for (6-4) photoproducts in the genome overall was increased by FL preillumination. Moreover, a decrease in (6-4) photoproducts by FL illumination immediately after UV irradiation of the cells was found, the decrement being enhanced by FL preillumination with or without CH. (author).

Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendants.

Directory of Open Access Journals (Sweden)

Bret R Adams

2010-04-01

Full Text Available The DNA double-strand break (DSB is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ is dominant. We have characterized the DNA damage response (DDR and quality of DNA double-strand break (DSB repair in human embryonic stem cells (hESCs, and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF was used as a surrogate for DSB repair. The resolution of gamma-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR, showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ] in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.

Role of repair saturation in the response of plateau-phase Chinese hamster ovary cells

International Nuclear Information System (INIS)

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

1987-01-01

Two repair rates are seen in split-dose experiments on starved plateau-phase CHO cells. It has been assumed that this indicates two different processes repairing two distinct types of sublethal damage. However results of experiments at different dose levels are not consistent with models that assume that the damage is entirely sublethal. Another hypothesis that has been considered is the saturation of a repair mechanism having a limited pool of repair enzymes. Such saturation phenomena have been observed in biochemical repair studies and have thus formed the basis for a model of cellular response, which was shown to be capable of producing dose response curves in good agreement with experimental observations. This model can be extended to account for both dose-rate and split-dose effects

Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury.

Science.gov (United States)

Arthur-Farraj, Peter J; Morgan, Claire C; Adamowicz, Martyna; Gomez-Sanchez, Jose A; Fazal, Shaline V; Beucher, Anthony; Razzaghi, Bonnie; Mirsky, Rhona; Jessen, Kristjan R; Aitman, Timothy J

2017-09-12

Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Efficiency of repair of pyrimidine dimers and psoralen monoadducts in normal and xeroderma pigmentosum human cells

International Nuclear Information System (INIS)

Cleaver, J.E.; Charles, W.C.; Kong, S.H.

1984-01-01

Repair of DNA damage produced by ultraviolet light or 5-methylisopsoralen in normal and xeroderma pigmentosum human cells involves many similar steps. Aphidicolin and cytosine arabinoside block repair of both kinds of damage with similar efficiency, indicating that DNA polymerase α has a major role in repair for these lesions. In xeroderma pigmentosum cells of various complementation groups, the relative efficiency of excision repair for both ultraviolet- and 5-methylisopsoralen-induced damage was group A< C< D, indicating a close resemblance between both kinds of lesions in relation to the repair deficiencies in these groups. At high doses, the maximum rate of repair of damage by ultraviolet light was about twice that for methylisopsoralen damage, possibly because ultraviolet-induced damage forms a substrate that is more readily recognized and excised than that of the psoralen adducts. Differences in the structural distortions to DNA caused by these kinds of damage could be detected using single strand specific nucleases which excised dimers but not 5-MIP adducts from double strand DNA. (author)

Decreased cell survival and DNA repair capacity after UVC irradiation in association with down-regulation of GRP78/BiP in human RSa cells

International Nuclear Information System (INIS)

Zhai Ling; Kita, Kazuko; Wano, Chieko; Wu Yuping; Sugaya, Shigeru; Suzuki, Nobuo

2005-01-01

In contrast to extensive studies on the roles of molecular chaperones, such as heat shock proteins, there are only a few reports about the roles of GRP78/BiP, an endoplasmic reticulum (ER) stress-induced molecular chaperone, in mammalian cell responses to DNA-damaging stresses. To investigate whether GRP78/BiP is involved in resistance to a DNA-damaging agent, UVC (principally 254 nm in wavelength), we established human cells with down-regulation of GRP78/BiP by transfection of human RSa cells with antisense cDNA for GRP78/BiP. We found that the transfected cells showed higher sensitivity to UVC-induced cell death than control cells transfected with the vector alone. In the antisense-cDNA transfected cells, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4) photoproducts) in vivo and DNA synthesis activity of whole cell extracts to repair UVC-irradiated plasmids in vitro were remarkably decreased compared with those in the control cells. Furthermore, the antisense-cDNA transfected cells also showed slightly higher sensitivity to cisplatin-induced cell death than the control cells. Cisplatin-induced DNA damage is primarily repaired by nucleotide excision repair, like UVC-induced DNA damage. The present results suggest that GRP78/BiP plays a protective role against UVC-induced cell death possibly via nucleotide excision repair, at least in the human RSa cells tested

Cell-Based Meniscus Repair and Regeneration: At the Brink of Clinical Translation?

Science.gov (United States)

Korpershoek, Jasmijn V.; de Windt, Tommy S.; Hagmeijer, Michella H.; Vonk, Lucienne A.; Saris, Daniel B. F.

2017-01-01

Background: Meniscus damage can be caused by trauma or degeneration and is therefore common among patients of all ages. Repair or regeneration of the menisci could be of great importance not only for pain relief or regaining function but also to prevent degenerative disease and osteoarthritis. Current treatment does not offer consistent long-term improvement. Although preclinical research focusing on augmentation of meniscal tear repair and regeneration after meniscectomy is encouraging, clinical translation remains difficult. Purpose: To systematically evaluate the literature on in vivo meniscus regeneration and explore the optimal cell sources and conditions for clinical translation. We aimed at thorough evaluation of current evidence as well as clarifying the challenges for future preclinical and clinical studies. Study Design: Systematic review. Methods: A search was conducted using the electronic databases of MEDLINE, Embase, and the Cochrane Collaboration. Search terms included meniscus, regeneration, and cell-based. Results: After screening 81 articles based on title and abstract, 51 articles on in vivo meniscus regeneration could be included; 2 additional articles were identified from the references. Repair and regeneration of the meniscus has been described by intra-articular injection of multipotent mesenchymal stromal (stem) cells from adipose tissue, bone marrow, synovium, or meniscus or the use of these cell types in combination with implantable or injectable scaffolds. The use of fibrochondrocytes, chondrocytes, and transfected myoblasts for meniscus repair and regeneration is limited to the combination with different scaffolds. The comparative in vitro and in vivo studies mentioned in this review indicate that the use of allogeneic cells is as successful as the use of autologous cells. In addition, the implantation or injection of cell-seeded scaffolds increased tissue regeneration and led to better structural organization compared with scaffold

N-acetylcysteine normalizes the urea cycle and DNA repair in cells from patients with Batten disease.

Science.gov (United States)

Kim, June-Bum; Lim, Nary; Kim, Sung-Jo; Heo, Tae-Hwe

2012-12-01

Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Copyright © 2012 John Wiley & Sons, Ltd.

Propofol promotes spinal cord injury repair by bone marrow mesenchymal stem cell transplantation

Science.gov (United States)

Zhou, Ya-jing; Liu, Jian-min; Wei, Shu-ming; Zhang, Yun-hao; Qu, Zhen-hua; Chen, Shu-bo

2015-01-01

Propofol is a neuroprotective anesthetic. Whether propofol can promote spinal cord injury repair by bone marrow mesenchymal stem cells remains poorly understood. We used rats to investigate spinal cord injury repair using bone marrow mesenchymal stem cell transplantation combined with propofol administration via the tail vein. Rat spinal cord injury was clearly alleviated; a large number of newborn non-myelinated and myelinated nerve fibers appeared in the spinal cord, the numbers of CM-Dil-labeled bone marrow mesenchymal stem cells and fluorogold-labeled nerve fibers were increased and hindlimb motor function of spinal cord-injured rats was markedly improved. These improvements were more prominent in rats subjected to bone marrow mesenchymal cell transplantation combined with propofol administration than in rats receiving monotherapy. These results indicate that propofol can enhance the therapeutic effects of bone marrow mesenchymal stem cell transplantation on spinal cord injury in rats. PMID:26487860

Modeling base excision repair in Escherichia coli bacterial cells

International Nuclear Information System (INIS)

Belov, O.V.

2011-01-01

A model describing the key processes in Escherichia coli bacterial cells during base excision repair is developed. The mechanism is modeled of damaged base elimination involving formamidopyrimidine DNA glycosylase (the Fpg protein), which possesses several types of activities. The modeling of the transitions between DNA states is based on a stochastic approach to the chemical reaction description

Stem Cells and Gene Therapy for Cartilage Repair

Directory of Open Access Journals (Sweden)

Umile Giuseppe Longo

2012-01-01

Full Text Available Cartilage defects represent a common problem in orthopaedic practice. Predisposing factors include traumas, inflammatory conditions, and biomechanics alterations. Conservative management of cartilage defects often fails, and patients with this lesions may need surgical intervention. Several treatment strategies have been proposed, although only surgery has been proved to be predictably effective. Usually, in focal cartilage defects without a stable fibrocartilaginous repair tissue formed, surgeons try to promote a natural fibrocartilaginous response by using marrow stimulating techniques, such as microfracture, abrasion arthroplasty, and Pridie drilling, with the aim of reducing swelling and pain and improving joint function of the patients. These procedures have demonstrated to be clinically useful and are usually considered as first-line treatment for focal cartilage defects. However, fibrocartilage presents inferior mechanical and biochemical properties compared to normal hyaline articular cartilage, characterized by poor organization, significant amounts of collagen type I, and an increased susceptibility to injury, which ultimately leads to premature osteoarthritis (OA. Therefore, the aim of future therapeutic strategies for articular cartilage regeneration is to obtain a hyaline-like cartilage repair tissue by transplantation of tissues or cells. Further studies are required to clarify the role of gene therapy and mesenchimal stem cells for management of cartilage lesions.

Integration of direct carbon and hydrogen fuel cells for highly efficient power generation from hydrocarbon fuels

Energy Technology Data Exchange (ETDEWEB)

Muradov, Nazim; Choi, Pyoungho; Smith, Franklyn; Bokerman, Gary [Florida Solar Energy Center, University of Central Florida, 1679 Clearlake Road, Cocoa, FL 32922-5703 (United States)

2010-02-15

In view of impending depletion of hydrocarbon fuel resources and their negative environmental impact, it is imperative to significantly increase the energy conversion efficiency of hydrocarbon-based power generation systems. The combination of a hydrocarbon decomposition reactor with a direct carbon and hydrogen fuel cells (FC) as a means for a significant increase in chemical-to-electrical energy conversion efficiency is discussed in this paper. The data on development and operation of a thermocatalytic hydrocarbon decomposition reactor and its coupling with a proton exchange membrane FC are presented. The analysis of the integrated power generating system including a hydrocarbon decomposition reactor, direct carbon and hydrogen FC using natural gas and propane as fuels is conducted. It was estimated that overall chemical-to-electrical energy conversion efficiency of the integrated system varied in the range of 49.4-82.5%, depending on the type of fuel and FC used, and CO{sub 2} emission per kW{sub el}h produced is less than half of that from conventional power generation sources. (author)

Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage

Energy Technology Data Exchange (ETDEWEB)

Little, J.B.; Ueno, A.M.; Dahlberg, W.K.

1989-07-01

We have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1-..beta..-D-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9-..beta..-D-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy.

Science.gov (United States)

Hengel, Sarah R; Spies, M Ashley; Spies, Maria

2017-09-21

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA-damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small-molecule binding determinates. In this review, we discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small-molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease, and WRN DNA helicase. Copyright © 2017 Elsevier Ltd. All rights reserved.

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

Energy Technology Data Exchange (ETDEWEB)

Dean, S.W.

1989-07-01

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.

Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells

International Nuclear Information System (INIS)

Dean, S.W.

1989-01-01

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, ∼35% of cross-links in an episomally maintained Epstein-Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA. (author)

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

International Nuclear Information System (INIS)

Sedliakova, M.; Slezarikova, V.; Brozmanova, J.; Masek, F.; Bayerova, V.

1980-01-01

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7, proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival. We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP + ). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells given with potentially lethal doses of UV irradiation. (orig.)

Degradation and repair of DNA from rat hepatoma cells after treatments with γ-rays and N-methyl-N-nitrosourea

International Nuclear Information System (INIS)

Zakrzhevskaya, D.G.; Kulagina, T.P.; Petrov, S.I.; Fomenko, L.A.; Gaziev, A.I.

1977-01-01

It has been shown, that DNA single-strand breaks induced in the cells of ascite hepatoma with γ-rays and metylnitrosourea (MNM) are effectively repaired. DNA two-strand breaks of hepatoma cells, treated with MNM are effectively repaired in situ as well. Only insignificant part of two-strand gamma-induced breaks in DNA of these cells is repaired during postirradiation period. Under combined effect of gamma rays and MNM on hepatoma cells a delay of DNA reparation and its further degradation as well as inhibition of nonplanned DNA synthesis and the suppression of DNA-polymerase 1 activity are observed

The Acid-Secreting Parietal Cell as an Endocrine Source of Sonic Hedgehog During Gastric Repair

Science.gov (United States)

Engevik, Amy C.; Feng, Rui; Yang, Li

2013-01-01

Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Despite its known function in tissue regeneration, the role of Shh secreted from the gastric epithelium during tissue repair in the stomach remains unknown. Here we tested the hypothesis that Shh secreted from the acid-secreting parietal cell is a fundamental circulating factor that drives gastric repair. A mouse model expressing a parietal cell-specific deletion of Shh (PC-ShhKO) was generated using animals bearing loxP sites flanking exon 2 of the Shh gene (Shhflx/flx) and mice expressing a Cre transgene under the control of the H+,K+-ATPase β-subunit promoter. Shhflx/flx, the H+,K+-ATPase β-subunit promoter, and C57BL/6 mice served as controls. Ulcers were induced via acetic acid injury. At 1, 2, 3, 4, 5, and 7 days after the ulcer induction, gastric tissue and blood samples were collected. Parabiosis experiments were used to establish the effect of circulating Shh on ulcer repair. Control mice exhibited an increased expression of Shh in the gastric tissue and plasma that correlated with the repair of injury within 7 days after surgery. PC-ShhKO mice showed a loss of ulcer repair and reduced Shh tissue and plasma concentrations. In a parabiosis experiment whereby a control mouse was paired with a PC-ShhKO littermate and both animals subjected to gastric injury, a significant increase in the circulating Shh was measured in both parabionts. Elevated circulating Shh concentrations correlated with the repair of gastric ulcers in the PC-ShhKO parabionts. Therefore, the acid-secreting parietal cell within the stomach acts as an endocrine source of Shh during repair. PMID:24092639

The development of remote repairing system, decontamination and in-cell remote inspection equipment

International Nuclear Information System (INIS)

Ishibashi, Yuzo; Toyoda, Osamu; Haginoya, Isao; Yamamoto, Ryuichi; Tanaka, Yasumasa

1993-01-01

PNC has been developing remote repair and inspection technologies for in-cell components in reprocessing Plants. In this report, several remote technologies such as remote dismantling and removal, decontamination, remote pipe maintenance and remote in-cell inspection equipment are described. (author)

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

International Nuclear Information System (INIS)

Rainbow, A.J.

1989-01-01

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D 0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus. (author)

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

Transient elevation of glycolysis confers radio-resistance by facilitating DNA repair in cells

International Nuclear Information System (INIS)

Bhatt, Anant Narayan; Chauhan, Ankit; Khanna, Suchit; Rai, Yogesh; Singh, Saurabh; Soni, Ravi; Kalra, Namita; Dwarakanath, Bilikere S

2015-01-01

Cancer cells exhibit increased glycolysis for ATP production (the Warburg effect) and macromolecular biosynthesis; it is also linked with therapeutic resistance that is generally associated with compromised respiratory metabolism. Molecular mechanisms underlying radio-resistance linked to elevated glycolysis remain incompletely understood. We stimulated glycolysis using mitochondrial respiratory modifiers (MRMs viz. di-nitro phenol, DNP; Photosan-3, PS3; Methylene blue, MB) in established human cell lines (HEK293, BMG-1 and OCT-1). Glucose utilization and lactate production, levels of glucose transporters and glycolytic enzymes were investigated as indices of glycolysis. Clonogenic survival, DNA repair and cytogenetic damage were studied as parameters of radiation response. MRMs induced the glycolysis by enhancing the levels of two important regulators of glucose metabolism GLUT-1 and HK-II and resulted in 2 fold increase in glucose consumption and lactate production. This increase in glycolysis resulted in resistance against radiation-induced cell death (clonogenic survival) in different cell lines at an absorbed dose of 5 Gy. Inhibition of glucose uptake and glycolysis (using fasentin, 2-deoxy-D-glucose and 3-bromopyruvate) in DNP treated cells failed to increase the clonogenic survival of irradiated cells, suggesting that radio-resistance linked to inhibition of mitochondrial respiration is glycolysis dependent. Elevated glycolysis also facilitated rejoining of radiation-induced DNA strand breaks by activating both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways of DNA double strand break repair leading to a reduction in radiation-induced cytogenetic damage (micronuclei formation) in these cells. These findings suggest that enhanced glycolysis generally observed in cancer cells may be responsible for the radio-resistance, partly by enhancing the repair of DNA damage

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.)

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

Science.gov (United States)

Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

2010-07-07

Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

Regulation of Innate Lymphoid Cells by Aryl Hydrocarbon Receptor

Science.gov (United States)

Li, Shiyang; Bostick, John W.; Zhou, Liang

2018-01-01

With striking similarity to their adaptive T helper cell counterparts, innate lymphoid cells (ILCs) represent an emerging family of cell types that express signature transcription factors, including T-bet+ Eomes+ natural killer cells, T-bet+ Eomes− group 1 ILCs, GATA3+ group 2 ILCs, RORγt+ group 3 ILCs, and newly identified Id3+ regulatory ILC. ILCs are abundantly present in barrier tissues of the host (e.g., the lung, gut, and skin) at the interface of host–environment interactions. Active research has been conducted to elucidate molecular mechanisms underlying the development and function of ILCs. The aryl hydrocarbon receptor (Ahr) is a ligand-dependent transcription factor, best known to mediate the effects of xenobiotic environmental toxins and endogenous microbial and dietary metabolites. Here, we review recent progresses regarding Ahr function in ILCs. We focus on the Ahr-mediated cross talk between ILCs and other immune/non-immune cells in host tissues especially in the gut. We discuss the molecular mechanisms of the action of Ahr expression and activity in regulation of ILCs in immunity and inflammation, and the interaction between Ahr and other pathways/transcription factors in ILC development and function with their implication in disease. PMID:29354125

Maintenance of DNA repair capacity in differentiating rat muscle cells in vitro

International Nuclear Information System (INIS)

Koval, T.M.; Kaufman, S.J.

1981-01-01

Unscheduled DNA synthesis was measured at several times during the differentiation of cultured rat skeletal muscle cells in response to exposures to 254 nm UV light. There was no change in the amount of repair DNA synthesis as the cells fuse and differentiate from postmitotic prefusion myoblasts to multinucleated contracting myotubes. (author)

Base excision repair activities differ in human lung cancer cells and corresponding normal controls

DEFF Research Database (Denmark)

Karahalil, Bensu; Bohr, Vilhelm A; De Souza-Pinto, Nadja C

2010-01-01

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway...... for the repair of oxidized modifications both in nuclear and mitochondrial DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non......-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three...

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

Mismatch repair deficient hematopoietic stem cells are preleukemic stem cells.

Directory of Open Access Journals (Sweden)

Yulan Qing

Full Text Available Whereas transformation events in hematopoietic malignancies may occur at different developmental stages, the initial mutation originates in hematopoietic stem cells (HSCs, creating a preleukemic stem cell (PLSC. Subsequent mutations at either stem cell or progenitor cell levels transform the PLSC into lymphoma/leukemia initiating cells (LIC. Thymic lymphomas have been thought to develop from developing thymocytes. T cell progenitors are generated from HSCs in the bone marrow (BM, but maturation and proliferation of T cells as well as T-lymphomagenesis depends on both regulatory mechanisms and microenvironment within the thymus. We studied PLSC linked to thymic lymphomas. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Following BM transplantation, we found that MSH2-/- BM cells from young mice are able to fully reconstitute multiple hematopoietic lineages of lethally irradiated wild-type recipients. However, all recipients developed thymic lymphomas within three and four months post transplantation. Transplantation of different fractions of BM cells or thymocytes from young health MSH2-/- mice showed that an HSC enriched fraction always reconstituted hematopoiesis followed by lymphoma development. In addition, lymphomas did not occur in thymectomized recipients of MSH2-/- BM. These results suggest that HSCs with DNA repair defects such as MSH2-/- are PLSCs because they retain hematopoietic function, but also carry an obligate lymphomagenic potential within their T-cell progeny that is dependent on the thymic microenvironment.

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

Methods for natural gas and heavy hydrocarbon co-conversion

Science.gov (United States)

Kong, Peter C [Idaho Falls, ID; Nelson, Lee O [Idaho Falls, ID; Detering, Brent A [Idaho Falls, ID

2009-02-24

A reactor for reactive co-conversion of heavy hydrocarbons and hydrocarbon gases and includes a dielectric barrier discharge plasma cell having a pair of electrodes separated by a dielectric material and passageway therebetween. An inlet is provided for feeding heavy hydrocarbons and other reactive materials to the passageway of the discharge plasma cell, and an outlet is provided for discharging reaction products from the reactor. A packed bed catalyst may optionally be used in the reactor to increase efficiency of conversion. The reactor can be modified to allow use of a variety of light sources for providing ultraviolet light within the discharge plasma cell. Methods for upgrading heavy hydrocarbons are also disclosed.

Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway

Science.gov (United States)

Weeden, Clare E.; Chen, Yunshun; Ma, Stephen B.; Hu, Yifang; Ramm, Georg; Sutherland, Kate D.; Smyth, Gordon K.

2017-01-01

Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC. PMID:28125611

Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair.

Science.gov (United States)

Shakhbazau, Antos; Kawasoe, Jean; Hoyng, Stefan A; Kumar, Ranjan; van Minnen, Jan; Verhaagen, Joost; Midha, Rajiv

2012-05-01

Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a lentiviral vector encoding NGF (NGF-SCs). Transplantation of NGF-SCs in a rat sciatic nerve transection/repair model led to significant increase of NGF levels 2weeks after injury and correspondingly to substantial improvement in axonal regeneration. Numbers of NF200, ChAT and CGRP-positive axon profiles, as well as the gastrocnemius muscle weights, were significantly higher in the NGF-Schwann cell group compared to the animals that received control SCs transduced with a lentiviral vector encoding GFP (GFP-SCs). Comparison with other models of NGF application signifies the important role of this neurotrophin during the early stages of regeneration, and supports the importance of developing combined gene and cell therapy for peripheral nerve repair. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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.

ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

Directory of Open Access Journals (Sweden)

Erhong Meng

Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

Fibroblast growth factor 2 and DNA repair involvement in the keratinocyte stem cells response to ionizing radiation

International Nuclear Information System (INIS)

Harfouche, L'Emira Ghida

2010-02-01

Keratinocyte stem cells (KSCs) from the human inter follicular epidermis are regarded as the major target to radiation during radiotherapy. We found herein that KSCs are more resistant to ionizing radiation than their direct progeny, and presented more rapid DNA damage repair kinetics than the progenitors. Furthermore, we provided evidence describing the effect of fibroblast growth factor 2 (FGF2) signaling on the ability of KSCs and progenitors to repair damaged DNA. Despite our knowledge of the fact, that FGF is an anti-apoptotic factor in multiple cell types, the direct link between DNA repair and FGF2 signaling has rarely been shown. Existence of such link is an important issue with implications not only to stem cell field but also to cancer therapy. (author)

DNA repair in neurons: So if they don't divide what's to repair?

International Nuclear Information System (INIS)

Fishel, Melissa L.; Vasko, Michael R.; Kelley, Mark R.

2007-01-01

Neuronal DNA repair remains one of the most exciting areas for investigation, particularly as a means to compare the DNA repair response in mitotic (cancer) vs. post-mitotic (neuronal) cells. In addition, the role of DNA repair in neuronal cell survival and response to aging and environmental insults is of particular interest. DNA damage caused by reactive oxygen species (ROS) such as generated by mitochondrial respiration includes altered bases, abasic sites, and single- and double-strand breaks which can be prevented by the DNA base excision repair (BER) pathway. Oxidative stress accumulates in the DNA of the human brain over time especially in the mitochondrial DNA (mtDNA) and is proposed to play a critical role in aging and in the pathogenesis of several neurological disorders including Parkinson's disease, ALS, and Alzheimer's diseases. Because DNA damage accumulates in the mtDNA more than nuclear DNA, there is increased interest in DNA repair pathways and the consequence of DNA damage in the mitochondria of neurons. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the BER pathway. Following the notion that the bulk of neuronal DNA damage is acquired by oxidative DNA damage and ROS, the BER pathway is a likely area of focus for neuronal studies of DNA repair. BER variations in brain aging and pathology in various brain regions and tissues are presented. Therefore, the BER pathway is discussed in greater detail in this review than other repair pathways. Other repair pathways including direct reversal, nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination and non-homologous end joining are also discussed. Finally, there is a growing interest in the role that DNA repair pathways play in the clinical arena as they relate to the neurotoxicity and neuropathy associated with cancer treatments. Among the numerous side effects of cancer treatments, major clinical effects

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

Design and rescue scenario of common repair equipment for in-vessel components in ITER hot cell

International Nuclear Information System (INIS)

Kakudate, Satoshi; Takeda, Nobukazu; Nakahira, Masataka; Shibanuma, Kiyoshi

2006-06-01

Transportation of the in-vessel components to be repaired in the ITER hot cell is carried by two kinds of transporters, i.e., overhead cranes and floor vehicles. The access area for repair operations in the hot cell is duplicated by these transporters. Clear sharing of the respective roles of these transporters with the minimum duplication is therefore useful for rationalization. The overhead cranes, which are independently installed in the respective cells in the hot sell, cannot pass through the components to be repaired between cells, i.e., receiving cell and refurbishment cell as an example. If the floor vehicle with simple mechanisms can cover the inaccessible area for the overhead cranes, a global transporter system in the hot cell will be simplified and the reliability will be increased. Based on this strategy, the overhead crane and floor vehicle concepts are newly proposed. The overhead crane has an adapter for change of the end-effectors, which can be easily changed, to grasp many kinds of components to be repaired. The floor vehicle, which is equipped with wheel mechanisms for transportation, is just to pass through the components between cells with only straight (linear) motion on the floor. The simple wheel mechanism can solve the spread of the dust, which is the critical issue of the original air bearing mechanism for traveling in the 2001 FDR design. Rescue scenarios and procedures in the hot cell are also studied in this report. The proposed rescue crane has major two functions for rescue operations of the hot cell facility, i.e., one for the overhead crane and the other for refurbishment equipment such as workstation for divertor repair. The rescue of the faulty overhead crane is carried out using the rescue tool installed on the rescue crane or directly traveled by pushing/pulling by the rescue crane after docking on the faulty overhead crane. For the rescue of the workstation, the rescue crane consists of a telescopic manipulator (maximum length