Number Of Apurinic/apyrimidinic Sites Research Articles

Abstract C60: Exposure of low doses of quercetin on DNA oxidation in Pectobacterium carotovorum KD 100 and Agrobacterium tumefaciens GV 3103.

Abstract Oxidative stress may be a key factor in the onset of certain diseases, including cancer. Oxy-radicals play important roles in the initiation, promotion, and progression of carcinogenesis. During the course of DNA excision and repair of oxidized, deaminated or alkylated bases apurinic/apyrimidinic (AP) or abasic sites are formed. The flavonoid quercetin was investigated for its ability to inhibit the oxidation of plasmid from Pectobacterium carotovorum KD 100 and Agrobacterium tumefaciens GV 3103 using the Fenton's chemistry. Utilizing a Colorimetric Assay Kit (Oxford Biomedical Research #FR09, Oxford, MI) for DNA damage quantification, AP sites were measured for plasmid following incubation with low doses of quercetin (1, 2, 2.5, 3, 3.5 µM) or without for 12 or 24 hr. With the increasing concentrations of quercetin the number of AP sites in each sample significantly decreased more for Pectobacterium carotovorum KD 100 compared to Agrobacterium tumefaciens GV 3103 in a time- and dose-dependent manner in comparison to their respective controls for the 12 and 24 hr incubation periods. The differences in decrease in AP sites can be linked to structural differences between the two bacterial strains and/or decreased oxidation for each sample with increased quercetin aliquots. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C60. Citation Format: William Y. Boadi, Damitea Johnson, Korsi Dumenyo. Exposure of low doses of quercetin on DNA oxidation in Pectobacterium carotovorum KD 100 and Agrobacterium tumefaciens GV 3103. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C60.

Blood Lipid Levels and Injury to DNA in Obese Children

Obesity condition implies a significant health risk, and its association to other factors as dislipidemia and metabolic syndrome is the basis of several chronic non-communicable diseases. All of these elements have been lately associated to the impairment of DNA structure, which represents a critical menace to cellular function. The study of damage to DNA in obese children should be the basis for the administration of physical activity in early stages of life, due to its role as a major antioxidant. PURPOSE: To determine DNA damage and blood lipid levels in sedentary obese children. METHODS: Nine sedentary obese boys, 9.5 ± 1.3 years of age, 53.4 ± 8.4 kg of body weight, 140.3 ± 5.9 cm of height and 27.8 ± 3.4 kg/m2 of body mass index (BMI) were included. Not clinical evidence of other disease was detected. Oxidative damage to DNA was determined by the number of apurinic-apyrimidinic sites (APS) × 105. Plasma lipid levels by colorimetric-enzymatic procedure were measured after a 12 hour fast. Lipids were correlated to APS and were also compared with other reported values in some studies with obese children. RESULTS: Children's BMI was higher than 97 percentile of CDC charts and classified as severe obesity according to National Institute of Health in Mexico (INSP, 2006). The number of detected APS was 4.6 ± 4.4 × 105. Blood lipid levels were as follows: 189.3 ± 29.6 mg·dl-1 of total cholesterol (TC), 82.4 ± 35.7 mg·dl-1 of triglycerides (TG), 117.3 ± 23 mg·dl-1 of LDL-cholesterol (LDL) and 50.4 ± 11.5 mg·dl-1 of HDL-cholesterol (HDL). Values of TC, TG, LDL and HDL were adjusted by Z score according to those described in studies with obese children in Great Britain, Mexico and USA. TC, TG, and LDL were higher, while HDL was lower than those reported for children in the same range of age. No correlation between APS and lipids was observed. CONCLUSIONS: Oxidative damage in DNA, evidenced by the number of APS, and high blood lipid levels found in sedentary obese children may suppose an increased possibility for early development of chronic non-communicable diseases. More studies must be done to understand any association between these factors and should be the basis to perform physical activity programs which can change such risk, due to its antioxidant action.

Extremely low frequency (ELF) magnetic fields enhance chemically induced formation of apurinic/apyrimidinic (AP) sites in A172 cells

Purpose: To detect the effects of extremely low frequency (ELF) magnetic fields, the number of apurinic/apyrimidinic (AP) sites in human glioma A172 cells was measured following exposure to ELF magnetic fields.Materials and methods: The cells were exposed to an ELF magnetic field alone, to genotoxic agents (methyl methane sulfonate (MMS) and hydrogen peroxide (H2O2)) alone, or to an ELF magnetic field with the genotoxic agents. After exposure, DNA was extracted, and the number of AP sites was measured.Results: There was no difference in the number of AP sites between cells exposed to an ELF magnetic field and sham controls. With MMS or H2O2 alone, the number of AP sites increased with longer treatment times. Exposure to an ELF magnetic field in combination with the genotoxic agents increased AP-site levels compared with the genotoxic agents alone.Conclusions: Our results suggest that the number of AP sites induced by MMS or H2O2 is enhanced by exposure to ELF magnetic fields at 5 millitesla (mT). This may occur because such exposure can enhance the activity or lengthen the lifetime of radical pairs.

Bcl2 Suppresses DNA Repair by Enhancing c-Myc Transcriptional Activity

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Transient adenoviral N-methylpurine DNA glycosylase overexpression imparts chemotherapeutic sensitivity to human breast cancer cells

Abstract In an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change, we have used adenoviral overexpression of N-methylpurine DNA glycosylase (MPG or ANPG/AAG) in breast cancer cells to study its ability to imbalance base excision repair (BER) and sensitize cancer cells to alkylating agents. Our results show that MPG-overexpressing cells are significantly more sensitive to the alkylating agents methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, methylnitrosourea, dimethyl sulfate, and the clinical chemotherapeutic temozolomide. Sensitivity is further increased through coadministration of the BER inhibitor methoxyamine, which covalently binds abasic or apurinic/apyrimidinic (AP) sites and makes them refractory to subsequent repair. Methoxyamine reduction of cell survival is significantly greater in cells overexpressing MPG than in control cells, suggesting a heightened production of AP sites that, if made persistent, results in increased cellular toxicity. We further explored the mechanism of MPG-induced sensitivity and found that sensitivity was associated with a significant increase in the number of AP sites and/or single-strand breaks in overexpressing cells, confirming a MPG-driven accumulation of toxic BER intermediates. These data establish transient MPG overexpression as a potential therapeutic approach for increasing cellular sensitivity to alkylating agent chemotherapy.

Induction of direct adducts, apurinic/apyrimidinic sites and oxidized bases in nuclear DNA of human HeLa S3 tumor cells by tetrachlorohydroquinone.

DNA damage induced by tetrachlorohydroquinone (Cl(4)HQ), the quinonoid metabolite of pentachlorophenol (PCP), was investigated in human HeLa S3 tumor cells. Formation of one major and two minor DNA adducts in cells treated with Cl(4)HQ (50-300 microM) was detected by (32)P-post-labeling assay and the adducts accumulated over the course of the experiment (0.5-2 h), with total adduct levels estimated to be 3-6 per 10(8) nucleotides. These adducts did not correspond to those derived from calf thymus DNA treated with tetrachloro-1,4-benzoquinone. Results from the apurinic/apyrimidinic (AP) sites assay indicated that the number of AP sites was 2-fold greater in cells exposed to Cl(4)HQ (300 microM) than the corresponding control. Further characterization of the AP sites confirmed that Cl(4)HQ induced predominantly (75%) putrescine-excisable AP sites in HeLa S3 cells. In parallel, the concentration of 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in cells treated with Cl(4)HQ for 0.5 and 2 h was increased 2- and 5-fold, respectively, compared with the control. The extent of oxidative DNA damage induced by Cl(4)HQ was approximately two orders of magnitude greater than those of direct DNA adducts. Overall, it appears that reactive oxygen species mediate the parallel formation of AP sites and 8-HO-dG in HeLa S3 cells following treatment with Cl(4)HQ and that the contribution of depurination/depyrimidination of direct DNA adducts is relatively insignificant compared with the formation of oxidized AP sites. We conclude that putrescine-excisable AP sites represent a major type of ROS-mediated oxidative DNA damage in cellular DNA induced by Cl(4)HQ and may play a role in PCP-induced clastogenicity in mammalian cells.

A method for detecting abasic sites in living cells: age-dependent changes in base excision repair.

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992) Biochemistry 31, 3703-3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP-DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA-HRP complex at each biotin residue, which is rapidly separated from free avidin-HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.

Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IR)-sensitive sites. Seven Fpg protein-sensitive sites per PBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.