DNA Repair-deficient Chinese Hamster Ovary Research Articles

The effect of the rs1799931 G857A (G286E) polymorphism on N-acetyltransferase 2-mediated carcinogen metabolism and genotoxicity differs with heterocyclic amine exposure.

Human N-acetyltransferase 2 (NAT2) is subject to genetic polymorphism in human populations. In addition to the reference NAT2*4 allele, two genetic variant alleles (NAT2*5B and NAT2*7B) are common in Europe and Asia, respectively. NAT2*5B possesses a signature rs1801280 T341C (I114T) single-nucleotide polymorphism (SNP), whereas NAT2*7B possesses a signature rs1799931 G857A (G286E) SNP. NAT2 alleles possessing the T341C (I114T) or G857A (G286E) SNP were recombinant expressed in yeast and tested for capacity to catalyze the O-acetylation of the N-hydroxy metabolites of heterocyclic amines (HCAs). The T341C (I114T) SNP reduced the O-acetylation of N-hydroxy-2-amino-3-methylimidazo [4,5-f] quinoline (N-OH-IQ), N-hydroxy-2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (N-OH-MeIQx) and N-hydroxy- 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (N-OH-PhIP), whereas the G857A (G286E) SNP reduced the O-acetylation of N-OH-IQ and N-OH-MeIQx but not N-OH-PhIP. The G857A (G286E) SNP significantly (p < 0.05) reduced apparent Km toward N-OH-PhIP but did not significantly (p > 0.05) affect apparent Vmax. Cultures of DNA repair-deficient Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B alleles were incubated with various concentrations of IQ, MeIQx or PhIP and double-stranded DNA damage and reactive oxygen species (ROS) were measured. Transfection with human CYP1A2 did not significantly (p > 0.05) increase HCA-induced DNA damage and ROS over un-transfected cells. Additional transfection with NAT2*4, NAT2*5B or NAT2*7B allele increased both DNA damage and ROS. The magnitude of the increases was both NAT2 allele- and substrate-dependent showing the same pattern as observed for the O-acetylation of the N-hydroxylated HCAs suggesting that both are mediated via NAT2-catalyzed O-acetylation. The results document the role of NAT2 and its genetic polymorphism on the O-acetylation and genotoxicity of HCAs.

Effect of N-acetyltransferase 2 genetic polymorphism on 4,4'-methylenebis(2-chloroaniline)-induced genotoxicity and oxidative stress.

4,4'-Methylenebis(2-chloroaniline) or MOCA is an aromatic amine used primarily in polyurethane and rubber industry. MOCA has been linked to hepatomas in animal studies while limited epidemiologic studies reported the association of exposure to MOCA and urinary bladder and breast cancer. We investigated MOCA-induced genotoxicity and oxidative stress in DNA repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human metabolizing enzymes CYP1A2 and N-acetyltransferase 2 (NAT2) variants as well as in rapid, intermediate, and slow NAT2 acetylator cryopreserved human hepatocytes. N-acetylation of MOCA was highest in UV5/1A2/NAT2*4 followed by UV5/1A2/NAT2*7B and UV5/1A2/NAT2*5B CHO cells. Human hepatocytes showed a NAT2 genotype-dependent response with highest N-acetylation in rapid acetylators followed by intermediate and slow acetylators. MOCA induced higher levels of mutagenesis and DNA damage in UV5/1A2/NAT2*7B compared to UV5/1A2/NAT2*4 and UV5/1A2/NAT2*5B cells (p < 0.0001). MOCA also induced higher levels of oxidative stress in UV5/1A2/NAT2*7B cells. MOCA caused concentration-dependent increase in DNA damage in cryopreserved human hepatocytes (linear trend p < 0.001) which was NAT2 genotype dependent i.e., highest in rapid acetylators, lower in intermediate acetylators, and lowest in slow acetylators (p < 0.0001). Our findings show that N-acetylation and genotoxicity of MOCA is NAT2 genotype dependent and suggest that individuals possessing NAT2*7B are at higher risk to MOCA-induced mutagenicity. DNA damage, and oxidative stress. They confirm significant differences in genotoxicity between the NAT2*5B and NAT2*7B alleles, both of which are associated with slow acetylator phenotype.

Identification and characterization of potent, selective, and efficacious inhibitors of human arylamine N-acetyltransferase 1.

Arylamine N-acetyltransferase 1 (NAT1) plays a pivotal role in the metabolism of carcinogens and is a drug target for cancer prevention and/or treatment. A protein-ligand virtual screening of 2 million chemicals was ranked for predicted binding affinity towards the inhibition of human NAT1. Sixty of the five hundred top-ranked compounds were tested experimentally for inhibition of recombinant human NAT1 and N-acetyltransferase 2 (NAT2). The most promising compound 9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester (compound 10) was found to be a potent and selective NAT1 inhibitor with an in vitro IC50 of 0.75µM. Two structural analogs of this compound were selective but less potent for inhibition of NAT1 whereas a third structural analog 1,2-dihydroxyanthraquinone (a compound 10 hydrolysis product also known as Alizarin) showed comparable potency and efficacy for human NAT1 inhibition. Compound 10 inhibited N-acetylation of the arylamine carcinogen 4-aminobiphenyl (ABP) both in vitro and in DNA repair-deficient Chinese hamster ovary (CHO) cells in situ stably expressing human NAT1 and CYP1A1. Compound 10 and Alizarin effectively inhibited NAT1 in cryopreserved human hepatocytes whereas inhibition of NAT2 was not observed. Compound 10 caused concentration-dependent reductions in DNA adduct formation and DNA double-strand breaks following metabolism of aromatic amine carcinogens beta-naphthylamine and/or ABP in CHO cells. Compound 10 inhibited proliferation and invasion in human breast cancer cells and showed selectivity towards tumorigenic versus non-tumorigenic cells. In conclusion, our study identifies potent, selective, and efficacious inhibitors of human NAT1. Alizarin's ability to inhibit NAT1 could reduce breast cancer metastasis particularly to bone.

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLβ, but did not disrupt the interactions with PARP-1, LIG3α and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLβ interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLβ, PARP-1, LIG3α, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLβ, PARP-1, LIG3α and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants—excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression—exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLβ-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks.

DNA repair deficiency and acetaldehyde-induced chromosomal alterations in CHO cells

Induction of chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) by acetaldehyde (AA) was evaluated in parental and different DNA repair-deficient Chinese hamster ovary (CHO) cell lines to elucidate the mechanisms involved in the protection against AA-induced chromosome damage. Cell lines employed included the parental (AA8), nucleotide excision repair (UV4, UV5, UV61), base excision repair (EM9), homologous recombination repair (HRR) (irs1SF, 51D1)-deficient and Fanconi-like (KO40) ones. The ranking of different cell lines for sensitivity to induction of CAs by AA was 51D1 > irs1SF > KO40 > UV4 > V33-EM9-AA8 > UV61-UV5 in a descending order. Cells deficient in HRR were most sensitive followed by Fanconi anaemia like (KO40) suggesting these pathways, especially HRR is very important for the repair of AA-induced lesions. These observations also suggest that interstrand cross links are primary biologically relevant DNA lesions induced by AA for induction of CAs. Only marginal differences were found between the cell lines for induction of SCEs. The possible mechanisms involved in AA-induced chromosomal alterations are discussed.

The Novel Poly(ADP-Ribose) Polymerase Inhibitor, AG14361, Sensitizes Cells to Topoisomerase I Poisons by Increasing the Persistence of DNA Strand Breaks

Poly(ADP-ribose) polymerase (PARP) inhibitors enhance DNA topoisomerase I (topo I) poison-induced cytotoxicity and antitumor activity in vitro and in vivo, but the mechanism has not been defined. We investigated the role of PARP-1 in the response to topo I poisons using PARP-1-/- and PARP-1+/+ mouse embryonic fibroblasts and the potent PARP-1 inhibitor, AG14361 (Ki < 5 nmol/L). PARP-1-/- mouse embryonic fibroblasts were 3-fold more sensitive to topotecan than PARP-1+/+ mouse embryonic fibroblasts (GI50, 21 and 65 nmol/L, respectively). AG14361 caused a >3-fold sensitization of PARP-1+/+ cells to topotecan compared with a <1.4-fold sensitization in PARP-1-/- cells. In human leukemia K562 cells, AG14361 caused a 2-fold sensitization to camptothecin-induced cytotoxicity. AG14361 did not affect the cellular activity of topo I as determined by measurement of cleavable complexes and topo I relaxation activity, showing that sensitization was not due to topo I activation. In contrast, repair of DNA following camptothecin removal, normally very rapid, was significantly retarded by AG14361, resulting in a 62% inhibition of repair 10 minutes after camptothecin removal. This led to a 20% increase in the net accumulation of camptothecin-induced DNA strand break levels in cells coexposed to AG14361 for 16 hours. We investigated the DNA repair mechanism involved using a panel of DNA repair-deficient Chinese hamster ovary cells. AG14361 significantly potentiated camptothecin-mediated cytotoxicity in all cells, except the base excision repair-deficient EM9 cells. Therefore, the most likely mechanism for the potentiation of topo I poison-mediated cytotoxicity by AG14361 is via PARP-1-dependent base excision repair.

The DRAG test: an assay for detection of genotoxic damage.

A high throughput assay (the DRAG test) is described, which could be a useful tool for the detection of repairable DNA adducts, and which is based on the inhibition of the growth of DNA repair-deficient Chinese hamster ovary (CHO) cells. The cytotoxicity of a test substance towards DNA repair-deficient CHO cell lines is compared with the corresponding cytotoxicity in the parental wild-type CHO cell line (AA8). A more pronounced toxicity toward a DNA repair-deficient cell line is interpreted as being the consequence of its inability to repair the DNA adduct induced by the compound. (+)-7beta,8alpha-Dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, camptothecin, ethyl methanesulphonate and mitomycin C were used as reference substances, and the overall results indicate that the DRAG test could be useful in the screening of compounds for the production of repairable DNA adducts. The main advantages with the DRAG test are that it provides a relevant endpoint, it is rapid, it requires small amounts of the test item, and it permits a large number of compounds to be tested.

Bile salt/acid induction of DNA damage in bacterial and mammalian cells: Implications for colon cancer

Two bile salts, sodium chenodeoxycholate and sodium deoxycholate, induced a DNA repair response in the bacterium Escherichia coli. Similarly, a bile acid and a bile salt, chenodeoxycholic acid and sodium deoxycholate, induced DNA repair (indicated by unscheduled DNA synthesis) in human foreskin fibroblasts. Also, DNA repair-deficient Chinese hamster ovary (CHO) cells were found to be more sensitive than normal cells to killing by bile salts. In particular, mutant UV4 CHO cells, defective in DNA excision repair and DNA cross-link removal, were more sensitive to sodium chenodeoxycholate, and mutant EM9 CHO cells, defective in strand-break rejoining, were more sensitive to sodium deoxycholate than wild-type cells. These results indicate that bile salts/acid damage DNA of both bacterial and mammalian cells in vivo. Previous epidemiological studies have shown that colon cancer incidence correlates with fecal bile acid levels. The findings reported here support the hypothesis that bile salts/acids have an etiologic role in colon cancer by causing DNA damage.

Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids.

Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results that have been reported for human cells, UV irradiation of transfecting DNA did not stimulate the genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with the UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. However, transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. We conclude that the responses of recipient cells to UV-damaged transfecting plasmids depend both on the type of recipient cell and the characteristics of the genetic sequence used for transfection.

Genetic complementation of UV-induced DNA repair in Chinese hamster ovary cells by the denV gene of phage T4.

The denV gene of phage T4, encoding the pyrimidine dimer-specific DNA repair enzyme endonuclease V, has been introduced by DNA transfection into the UV-sensitive DNA repair-deficient Chinese hamster ovary (CHO) cell line UV5. Transformants were first selected for resistance to the antibiotic G418 conferred by the neo gene from Tn5 carried by the same plasmid. A majority of the isolated G418-resistant UV5 clones also showed an increased resistance to 254-nm UV light. One clone, designated I-A1, was found to have an intermediate level of colony-forming ability after UV irradiation when compared to UV5 and wild-type AA8 cells. A Southern blot showed that I-A1 carries a single integrated intact copy of the denV gene. Alkaline sucrose gradients revealed a dose-dependent appearance of breaks in the DNA of I-A1 cells following UV-irradiation, while unirradiated cells did not exhibit any significant breaks. Analysis of DNA repair by isopycnic sedimentation showed that DNA excision repair by I-A1 was at least equal to the level of repair in AA8 cells. These results show that the prokaryotic denV gene can restore UV repair capabilities in vivo to CHO UV5 cells defective in repair of UV-induced damage.

DNA Repair-Deficient Chinese Hamster Ovary Cells Exhibiting Differential Sensitivity to γ Rays under Aerobic and Hypoxic Conditions

The survival of the wild-type parent and two mutant lines of Chinese hamster cells, known to be defective in DNA repair, has been determined as a function of exposure to gamma rays under aerobic and hypoxic conditions. When compared to the wild-type line, one of the mutants selected for sensitivity to ethyl methyl sulfonate (EMS), and known to be defective in the repair of DNA strand breaks, exhibits a markedly enhanced sensitivity to aerobic irradiation but a reduced enhancement to hypoxic irradiation and thus an enhanced oxygen enhancement ratio (OER). In contrast, the other line, known to be defective in the incision step of excision repair, exhibits the reverse pattern of sensitivity and hence a reduced OER. The results are compared to findings in bacterial mutants and cells obtained from ataxia telangiectasia (AT) patients and heterozygotes.