Facile Enzymatic Synthesis of Base J-Containing Oligodeoxyribonucleotides and an Analysis of the Impact of Base J on DNA Replication in Cells

<div><p>We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.</p></div

ESI-MS and MS/MS characterizations of d(ATGGCGJGCTAT) (‘J’ represents base J): (A) Negative-ion ESI-MS; (B) The product-ion spectrum of the [M–3H]^3– ion (<i>m/z</i> 1283.8).

<p>ESI-MS and MS/MS characterizations of d(ATGGCGJGCTAT) (‘J’ represents base J): (A) Negative-ion ESI-MS; (B) The product-ion spectrum of the [M–3H]^3– ion (<i>m/z</i> 1283.8).</p

Liquid Chromatography-Tandem Mass Spectrometry for the Quantification of Tobacco-Specific Nitrosamine-Induced DNA Adducts in Mammalian Cells

Quantification of DNA lesions constitutes one of the main tasks in toxicology and in assessing health risks accompanied by exposure to carcinogens. Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and <i>N</i>′-nitrosonornicotine (NNN) can undergo metabolic transformation to give a reactive intermediate that pyridyloxobutylates nucleobases and phosphate backbone of DNA. Here, we reported a highly sensitive method, relying on the use of nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS), for the simultaneous quantifications of <i>O</i>⁶-[4-(3-pyridyl)-4-oxobut-1-yl]-2′-deoxyguanosine (<i>O</i>⁶-POBdG) as well as <i>O</i>²- and <i>O</i>⁴-[4-(3-pyridyl)-4-oxobut-1-yl]-thymidine (<i>O</i>²-POBdT and <i>O</i>⁴-POBdT). By using this method, we measured the levels of the three DNA adducts with the use of 10 μg of DNA isolated from cultured mammalian cells exposed to a model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). Our results demonstrated, for the first time, the formation of <i>O</i>⁴-POBdT in naked DNA and in genomic DNA of cultured mammalian cells exposed with NNKOAc. We also revealed that the levels of the three lesions increased with the dose of NNKOAc and that <i>O</i>²-POBdT and <i>O</i>⁴-POBdT could be subjected to repair by the nucleotide excision repair (NER) pathway. The method reported here will be useful for investigations about the involvement of other DNA repair pathways in the removal of these lesions and for human toxicological studies in the future

A Quantitative Mass Spectrometry-Based Approach for Assessing the Repair of 8‑Methoxypsoralen-Induced DNA Interstrand Cross-Links and Monoadducts in Mammalian Cells

Interstrand cross-links (ICLs) are highly toxic DNA lesions that block transcription and replication by preventing strand separation. ICL-inducing agents were among the earliest and are still the most widely used forms of chemotherapeutic drugs. Because of the repair of DNA ICLs, the therapeutic efficacy of the DNA cross-linking agents is often reduced by the development of chemoresistance in patients. Thus, it is very important to understand how various DNA ICLs are repaired. Such studies are currently hampered by the lack of an analytical method for monitoring directly the repair of DNA ICLs in cells. Here we report a high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method, together with the isotope dilution technique, for assessing the repair of 8-methoxypsoralen (8-MOP)-induced DNA ICLs, as well as monoadducts (MAs), in cultured mammalian cells. We found that, while there were substantial decreases in the levels of ICL and MAs in repair-competent cells 24 h after 8-MOP/UVA treatment, there was little repair of 8-MOP-ICLs and -MAs in xeroderma pigmentosum, complementation group A-deficient human skin fibroblasts and excision repair cross-complementing rodent repair deficiency, complementation group 1-deficient Chinese hamster ovary cells over a 24 h period. This result provided unequivocal evidence supporting the notion that the 8-MOP photoadducts are substrates for nucleotide excision repair in mammalian cells. This is one of the first few reports about the application of LC–MS/MS for assessing the repair of DNA ICLs. The analytical method developed here, when combined with genetic manipulation, will also facilitate the assessment of the roles of other DNA repair pathways in removing these DNA lesions, and the method can also be generally applicable for investigating the repair of other types of DNA ICLs in mammalian cells

Chemical Structures of 2′-deoxynucleosides containing 5hmU, 5hmC, base J and Glc-5hmC.

<p>Chemical Structures of 2′-deoxynucleosides containing 5hmU, 5hmC, base J and Glc-5hmC.</p

<i>In-vivo</i> replication studies of base J and 5hmU in HEK293T cells.

<p>(A) Experimental procedures for determining the effects of 5hmU and base J on DNA replication in cells. (B) Representative PAGE gel image showing the restriction fragments. (C) The bypass efficiencies of base J and 5hmU in HEK293T cells. The data represent the mean and standard deviation of results from three independent replication experiments.</p

Quantitative Proteomic Analysis Revealed <i>N</i>′‑Nitrosonornicotine-induced Down-regulation of Nonmuscle Myosin II and Reduced Cell Migration in Cultured Human Skin Fibroblast Cells

The association of tobacco smoke with decreased cell motility and wound healing is well documented; however, the cellular mechanisms and specific toxic tobacco constituents responsible for this effect are not well understood. Tobacco-specific <i>N</i>-nitrosamines (TSNAs) are among the most important classes of carcinogens found in tobacco products. The TSNA <i>N</i>′-nitrosonornicotine (NNN) is present at relatively high levels in tobacco and its smoke, as well as second- and third-hand smoke. To investigate the cellular pathways that are perturbed upon NNN exposure, we employed a quantitative proteomic approach, utilizing stable isotope labeling by amino acids in cell culture and mass spectrometry, to assess the NNN-induced alteration of protein expression in GM00637 human skin fibroblast cells. With this approach, we were able to quantify 2599 proteins, 191 of which displayed significantly changed expression following NNN exposure. One of the main findings from our proteomic analysis was the down-regulation of six different subunits of myosin, particularly nonmuscle myosin II heavy chain, isoforms A, B, and C. In addition, we found the altered expression of several extracellular matrix proteins and proteins involved in cellular adhesion. Together, our quantitative proteomic results suggested that NNN exposure may interfere with fibroblast motility. An <i>in vitro</i> scratch wound assay result supported that NNN exposure reduced the ability of dermal fibroblast to migrate into the scratched area. The results from the present study offer novel insights into the cellular mechanisms of NNN toxicity and identify NNN as a specific tobacco constituent that contributes to decreased fibroblast migration

Quantitative Proteomic Analysis Revealed 4-(Methylnitrosamino)-1-(3-pyridinyl)-1-butanone-Induced Up-Regulation of 20S Proteasome in Cultured Human Fibroblast Cells

The tobacco-specific <i>N</i>-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK), is a well-known carcinogen. Although the ability of the metabolically activated form of NNK to generate DNA adducts is well established, little is known about the cellular pathways perturbed by NNK in its native state. In this study, we utilized stable isotope labeling by amino acid in cell culture (SILAC), together with mass spectrometry, to assess the perturbation of protein expression in GM00637 human skin fibroblast cells upon NNK exposure. With this approach, we were able to quantify 1412 proteins and 137 of them were with significantly altered expression following NNK exposure, including the up-regulation of all subunits of the 20S proteasome core complex. The up-regulation of the 20S core complex was also reflected by a significant increase in 20S proteasome activities in GM00637, IMR90, and MCF-7 cells upon NNK treatment. Furthermore, the β-adrenergic receptor (β-AR) antagonist propranolol could attenuate significantly the NNK-induced increase in proteasome activity in all the three cell lines, suggesting that up-regulation of the 20S proteasome may be mediated through the β-AR. Additionally, we found that NNK treatment altered the expression levels of other important proteins including mitochondrial proteins, cytoskeleton-associated proteins, and proteins involved in glycolysis and gluconeogenesis. Results from the present study provided novel insights into the cellular mechanisms targeted by NNK

Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric <i>O</i>²-, <i>N</i>3-, and <i>O</i>⁴-ethylthymidine (<i>O</i>²-, <i>N</i>3-, and <i>O</i>⁴-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf^–) of <i>Escherichia coli</i> DNA polymerase I. Our results showed that hPol η and Kf^– could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove <i>N</i>²-substituted 2′-deoxyguanosine lesions, hPol κ was able to bypass <i>O</i>²-EtdT, though it experienced great difficulty in bypassing <i>N</i>3-EtdT and <i>O</i>⁴-EtdT. yPol ζ was only modestly blocked by <i>O</i>⁴-EtdT, but the polymerase was strongly hindered by <i>O</i>²-EtdT and <i>N</i>3-EtdT. LC–MS/MS analysis of the replication products revealed that DNA synthesis opposite <i>O</i>⁴-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of <i>O</i>²-EtdT and <i>N</i>3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf^–, dTMP. Thus, our results suggested that <i>O</i>²-EtdT and <i>N</i>3-EtdT may also contribute to the AT → TA and AT → GC mutations observed in cells and tissues of animals exposed to ethylating agents

SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells

Humans are exposed to arsenic species through inhalation, ingestion, and dermal contact, which may lead to skin, liver, and bladder cancers as well as cardiovascular and neurological diseases. The mechanisms underlying the cytotoxic and carcinogenic effects of arsenic species, however, remain incompletely understood. To exploit the mechanisms of toxicity of As­(III), we employed stable isotope labeling by amino acids in cell culture (SILAC) together with LC/MS/MS analysis to quantitatively assess the As­(III)-induced perturbation of the entire proteome of cultured human skin fibroblast cells. Shotgun proteomic analysis on an LTQ-Orbitrap Velos mass spectrometer facilitated the quantification of 3880 proteins, 130 of which were quantified in both forward and reverse SILAC-labeling experiments and displayed significant alterations (>1.5 fold) upon arsenite treatment. Targeted analysis on a triple-quadrupole mass spectrometer in multiple-reaction monitoring (MRM) mode confirmed the quantification results of some select proteins. Ingenuity pathway analysis revealed the arsenite-induced alteration of more than 10 biological pathways, including the Nrf2-mediated oxidative stress response pathway, which is represented by the upregulation of nine proteins in this pathway. In addition, arsenite induced changes in expression levels of a number of selenoproteins and metallothioneins. Together, the results from the present study painted a more complete picture regarding the biological pathways that are altered in human skin fibroblast cells upon arsenite exposure