Level Of DNA Repair Synthesis Research Articles

Deficiency in Incisions Produced by XPF at the Site of a DNA Interstrand Cross-Link in Fanconi Anemia Cells

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.

Inhibitory effect of berberine on tert-butyl hydroperoxide-induced oxidative damage in rat liver.

Berberine, a main protoberberine component of Coptidis Rhizoma, was studied for the mechanism of its inhibitory effects on the tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity and lipid peroxidation in rat liver. In the preliminary study, berberine expressed an antioxidant property by its capacity for quenching the free radicals of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Further investigations were conducted using t-BHP-induced cytotoxicity in rat primary hepatocytes and hepatotoxicity in rats to evaluate the antioxidative bioactivity of berberine. The results in rat primary hepatocytes demonstrated that berberine, at the concentrations of 0.01-1.0 mM, significantly decreased the leakage of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and the formation of malondialdehyde (MDA) induced by 30 min treatment of t-BHP (1.5 mM). Berberine also attenuated the t-BHP-induced depletion of glutathione (GSH) and induced a high level of DNA repair synthesis. The in vivo study showed that the intraperitoneal pretreatment with berberine (0.5 and 5 mg/kg) for 5 days before a single dose of t-BHP (0.1 mmol/kg) significantly lowered the serum levels of hepatic enzyme markers (ALT and aspartate aminotransferase) and reduced oxidative stress in the liver. The histopathological evaluation of the livers revealed that berberine reduced the incidence of liver lesions, including hepatocyte swelling, leukocyte infiltrations, and necrosis induced by t-BHP. These results lead us to speculate that berberine may play a chemopreventive role via reducing oxidative stress in living systems.

Functional Retroviral Vector for Gene Therapy of Xeroderma Pigmentosum Group D Patients

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder characterized by an increased frequency of skin cancer following minimal sunlight exposure. Cells isolated from XP patients are also hypersensitive to UV rays and UV-like chemicals. This sensitivity is directly related to a defect in the early steps of nucleotide excision repair (NER) of damaged DNA. No efficient treatment is available for this disease and skin cancer prevention can only be achieved by strict avoidance of sunlight exposure. Thus, we are developing a model for gene therapy in XP, particularly for patients belonging to group D. We report here the construction of a retroviral vector (LXPDSN) containing the XPD (ERCC2) cDNA, which fully complements the DNA repair deficiency of primary skin fibroblasts. Efficient integration, mRNA synthesis, and protein expression of the XPD gene were obtained in all LXPDSN-transduced XP-D fibroblasts tested. Full correction of the DNA repair defect was observed with all DNA repair assays used, such as an increased survival after UV-radiation of the transduced cells, a normal level of DNA repair synthesis (UDS), and the reactivation of a UV-irradiated reporter vector. This retroviral vector will be used to modify keratinocytes genetically to produce repair proficient reconstituted skin for engraftment to XP patients.

Reduced DNA repair response of cells from carcinogen-induced rat liver altered foci exposed to chemical carcinogens in culture.

Cells were cultured from rat livers containing enzyme-altered foci induced by N-2-fluorenylacetamide and the level of DNA repair synthesis in response to several carcinogens was compared in gamma-glutamyl transpeptidase-positive foci cells and in enzyme-negative hepatocytes from the same livers. The level of unscheduled DNA synthesis elicited in foci cells by either the activation-dependent carcinogens N-2-fluorenylacetamide and diethylnitrosamine or the activation-independent carcinogens N-methyl-N'-nitro-N-nitrosoguanidine and 1-methyl-1-nitrosourea was significantly lower than that in hepatocytes. The results suggest that the repair of DNA damage by these altered cells is abnormal.

Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate.

AbstractPretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5‐20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0‐25 J/m2. The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV‐induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV‐induced repair synthesis by reducing endogenous levels of thymidine.

An apparent correlation between the inhibition of induced ornithine decarboxylase activity by gamma radiation and the capacity for DNA repair synthesis in normal and ataxia telangiectasia human fibroblasts: no correlation with cell survival.

Exposure of normal human fibroblasts (F107) in stationary phase to gamma radiation inhibited the appearance of induced ornithine decarboxylase (ODC) activity. Skin fibroblasts derived from two ataxia telangiectasia (AT) patients (F184 and F182) displayed a similar response. The level of DNA repair synthesis was also similar in the three cell strains. Fibroblasts from another apparently normal donor (F196) were very sensitive to inhibition of induced ODC activity by gamma radiation and were also deficient in radiation-induced DNA repair synthesis. However, the two strains derived from normal donors displayed the same degree of cellular sensitivity towards X-rays, whereas the two AT strains showed the typical hypersensitivity to the cytotoxic effect of X-irradiation. The results suggest a possible correlation between the inhibition of induced ODC activity by gamma radiation and the extent of DNA repair synthesis at high radiation doses, but there is no correlation between these two parameters and cellular survival at low radiation doses.

Ultraviolet-induced DNA-repair synthesis in lymphocytes from patients with chronic lymphatic leukemia

Ultraviolet-induced DNA-repair synthesis was studied in peripheral lymphocytes from 10 patients with chronic lymphatic leukemia (CLL). An ultraviolet-dose curve was established for each patient and compared with curves for healthy individuals. The same level of DNA-repair synthesis was recorded in CLL and normal lymphocytes. This contrasts with a report from another laboratory. Evidence is presented of the necessity of inhibiting replicative DNA-synthesis with hydroxyurea in order to adequately estimate the DNA-repair synthesis.

Deoxyribonucleic acid repair synthesis: a new factor for defining high blood pressure.

1. The level of DNA repair synthesis has been compared in males diagnosed as having hypertension (diastolic blood pressure greater than or equal to 100 mmHg), males with slightly elevated blood pressure (greater than 95th percentile corrected for age) and males with normal or subnormal blood pressure (less than or equal to 30th percentile corrected for age). 2. The hypertensive males and the males with elevated blood pressure could not be distinguished from each other with respect to the levels of chemically induced repair synthesis, but both were significantly increased over the induced repair synthesis values of individuals with normal or low blood pressure. Since it has been shown previously that lymphocytes with high repair synthesis values also have increased levels of both carcinogen-DNA binding and chromosomal aberrations (Nordén, Scherstén, Thulin, Pero, Bryngelsson & Mitelman, 1975), the biological significance of this variable appears to be of value in discriminating between normal and high blood pressure groups.

Organ-Specific DNA Damage Induced in Mice by the Organotropic Carcinogens 4-Nitroquinoline 1-Oxide and DimethyInitrosamine

The extent of DNA fragmentation induced in lung, kidney, and liver of mice injected with the chemical carcinogens 4-nitroquinoline 1-oxide (4NQO), dimethylnitrosamine (DMN) and the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO) was estimated by the alkaline sucrose gradient technique. A floating of minced lung tissue pieces in the alkaline lysing solution on top of the gradients afforded a gentle method of lung DNA extraction. This technique minimized mechanical shearing of lung DNA and permitted comparisons to be made with liver and kidney DNA sedimentation patterns. The extent of DNA damage induced by 4NQO followed the order: lung, kidney, liver, while that induced by DMN followed the order: liver, kidney, lung. The sites of greatest DNA damage appeared to correlate with sites of high levels of DNA repair synthesis and the sites of tumor induction. No DNA damage was induced by the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO).

Reduced DNA repair capacity and increased cytotoxicity following split doses of the mutagen 4-nitroquinoline-1-oxide in cultured human cells

Cultured human fibroblasts were exposed to single doses of 4-nitroquinoline-1-oxide (4NQO) and to two equimolar doses of 4NQO at intervals varying from 0.5 to 12 h. DNA repair synthesis as measured by an unscheduled uptake of tritium-labelled thymidine ([3H]TdR), cell survival as estimated by the clone-forming capacity, and frequency of chromosome aberrations were used as endpoints. Cells respond with a reduced level of DNA repair synthesis when the second 4NQOO dose (5.10−7 or 1.10−7M) is given within 3 h of the first 4QNO dose. If the interval between the two doses is 5 h or more, the level of DNA repair synthesis which is induced by the second 4NQO dose is comparable to that following a single 60-min 4QNO application. In this 3-h period the cultured cells show an increased sensitivity to the lethal effect and chromosome-damaging action of the second 4NQO dose. The reduced period of DNA repair capacity seems to increase the mutagenic effect of the chemical carcinogen.

Reduced DNA repair capacity and increased cytotoxicity following split doses of the mutagen 4-nitroquinoline-1-oxide in cultured human cells

Cultured human fibroblasts were exposed to single doses of 4-nitroquinoline-1-oxide (4NQO) and to two equimolar doses of 4NQO at intervals varying from 0.5 to 12 h. DNA repair synthesis as measured by an unscheduled uptake of tritium-labelled thymidine ([3H]TdR), cell survival as estimated by the clone-forming capacity, and frequency of chromosome aberrations were used as endpoints. Cells respond with a reduced level of DNA repair synthesis when the second 4NQOO dose (5.10−7 or 1.10−7M) is given within 3 h of the first 4QNO dose. If the interval between the two doses is 5 h or more, the level of DNA repair synthesis which is induced by the second 4NQO dose is comparable to that following a single 60-min 4QNO application. In this 3-h period the cultured cells show an increased sensitivity to the lethal effect and chromosome-damaging action of the second 4NQO dose. The reduced period of DNA repair capacity seems to increase the mutagenic effect of the chemical carcinogen.

Susceptibility of xeroderma pigmentosum cells to chromosome breakage by adenovirus type 12.

DNA repair-deficient microbial systems have attracted considerable attention because they offer an insight into the genetic control of sensitivity towards mutagens and carcinogens1. These studies have become highly relevant to man with Cleaver's discovery2 that cells of most patients with xeroderma pigmentosum (XP) have a reduced level of DNA repair synthesis following ultraviolet-irradiation or exposure to several chemical carcinogens3–6. The impaired repair capacity of XP cells seems to result in their greater sensitivity towards the lethal6–10 and chromosome-damaging action11–12 of those physical and chemical carcinogens which induce irreparable DNA alterations. On the other hand the behaviour of XP cells does not differ from that of controls when treated with several potent alkylating mutagens and carcinogens5,10,12,13.

Chromosome aberrations in xeroderma pigmentosum cells exposed to the carcinogens, 4-nitroquinoline-1-oxide and N-methyl-N'-nitrosoguanidine.

SummaryThe frequency of chromatid breaks and chromatid exchanges was estimated in cultured xeroderma pigmentosum (XP) cells and control cells exposed to the carcinogens 4-nitroquinoline-1-oxide (4NQO) and N-methyl-N′-nitro-nitrosoguanidine (MNNG). The level of DNA repair synthesis in the XP cells which were used in this study is about 21% of that found in cells of nonafflicted persons following exposure to 4NQO. The XP cells show an elevated sensitivity to the chromosome damaging effect of 4NQO. Doses of 4NQO, which have no effect on control cells, increased significantly the frequency of metaphase plates with chromosome aberrations in the XP cells over a 2 day period post-treatment. On the other hand the frequency of chromatid breaks and exchanges of XP cells resembled that found in control cells exposed to equimolar concentrations of MNNG. It appears likely, but unproven, that the different sensitivity of the XP cells towards 4NQO and MNNG is linked with their different capacity to repair 4NQO and MNNG ...

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens. The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and UV-irradiation. The XP cells showed ( 1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, ( 2) no unscheduled DNA synthesis following 6NQO and ( 3) a normal degree of DNA repair synthesis after treatment with MNNG. When the clone-forming capacity was examined the XP cells exhibited ( 1) a higher increased sensitivity to the various carcinogenic N-oxides, ( 2) no reduction in the clone formation following 6NQO and ( 3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG. The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.

The combined effect of chemical mutagen and virus on DNA repair, chromosome aberrations and neoplastic transformation.

The combined effect of chemical mutagen and oncogenic virus was examined by exposing cultured cells of Syrian hamsters to 4-nitroquinolin-1-oxide (4NQO) and human (AD12) or simian (SA7) adenovirus. The level of DNA repair synthesis, frequency of chromosome aberrations, and neoplastic transformation were used as parameters. The combined application of 4NQO and virus did not alter the level of DNA repair, had a cumulative effect on the frequency of metaphases with chromosome aberrations and produced an up to 23-fold enhancement of viral induced cell transformation.

Various Levels of DNA Repair Synthesis in Xeroderma Pigmentosum Cells exposed to the Carcinogens N-Hydroxy and N-Acetoxy-2-acetyl-aminofluorene

IN assessing environmental health hazards, the question has arisen of whether “safe”, “tolerable” or “permissible” levels of carcinogens, mutagens or teratogens can be derived by extrapolation of bioassays using rodents exposed for various periods to very high concentrations of chemicals or using cultured mammalian cell lines. Variations in susceptibility are only rarely taken into account, if at all and doses which seem to be harmless to the average person may be harmful to susceptible people. The reduced capacity to repair ultraviolet-induced DNA lesions in xeroderma pigmentosum (XP) cells may exemplify a mechanism leading to an elevated neoplastic transformation rate in man1–4. The question arises as to whether cells with deficient repair synthesis respond to chemical carcinogens in the same manner as cells with adequate repair systems. We report here the levels of DNA repair synthesis in XP cells of five patients exposed to the carcinogenic5,6 and mutagenic7 compounds N-acetoxy or N-hydroxy-2-acetyl-aminofluorene, which are ultimate and proximate carcinogenic forms of 2-acetylaminofluorene (AAF). We were particularly interested in comparing the different levels of DNA repair synthesis following ultraviolet irradiation with those following treatment with chemical carcinogens.