Search

Chronic wound healing disorders that occur as a result of a sulfur mustard (SM) exposure present a particular challenge. These chronic wounds are similar to other chronic wounds. In the past, it has been shown that mesenchymal stem cells (MSC) play an important role in the healing of chronic wounds. An important property to support wound healing is their ability to migrate. However, we were able to show that SM leads to a reduction in MSC migration even at low concentrations. Currently, exposed MSCs are still able to differentiate. Further alterations are not known. The current investigation therefore focused onto the question how SM affects MSC. Material & methods The effect of SM on MSC was investigated. Here, the alkylation of DNA was considered, and DNA adducts were quantified over a period of 48 h. The modification of the nuclei under the influence of SM was analyzed as well as proliferation of the cells by immunohistochemical staining with Ki-67 and quantification. For the quantification of the apoptosis rate, antibodies against cleaved Caspase-3, 8, and apoptosis inducing factor (AIF) were used. The senescence analysis was performed after histological staining against β-galactosidase. Quantifications were carried out by using the TissueQuest System and the software TissueFAX. Results SM exposure of MSC results in a dose dependent formation of nuclear DNA adducts. 4 h after exposure the cells display a decreasing concentration of DNA adducts. This process is accompanied by a change of nuclei shape but without an increase of apoptosis induction. In parallel the number of cells undergoing senescence increases as a function of the SM concentration. Discussion SM exposure of MSC leads to adduct formation on chromosomal DNA. These DNA adducts can be reduced without MSC are undergoing apoptosis. This indicates an active DNA damage response (DDR) pathway in combination with the formation of persistent nuclear DNA damage foci. This process is accompanied by a reduced capability of proliferation and a transition into the senescent state. [ABSTRACT FROM AUTHOR]

The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent. [ABSTRACT FROM AUTHOR]

It is well established that inflammation leads to the creation of potent DNA damaging chemicals, including reactive oxygen and nitrogen species. Nitric oxide can react with glutathione to create S-nitrosoglutathione (GSNO), which can in turn lead to S-nitrosated proteins. Of particular interest is the impact of GSNO on the function of DNA repair enzymes. The base excision repair (BER) pathway can be initiated by the alkyl-adenine DNA glycosylase (AAG), a monofunctional glycosylase that removes methylated bases. After base removal, an abasic site is formed, which then gets cleaved by AP endonuclease and processed by downstream BER enzymes. Interestingly, using the Fluorescence-based Multiplexed Host Cell Reactivation Assay (FM-HCR), we show that GSNO actually enhances AAG activity, which is consistent with the literature. This raised the possibility that there might be imbalanced BER when cells are challenged with a methylating agent. To further explore this possibility, we confirmed that GSNO can cause AP endonuclease to translocate from the nucleus to the cytoplasm, which might further exacerbate imbalanced BER by increasing the levels of AP sites. Analysis of abasic sites indeed shows GSNO induces an increase in the level of AP sites. Furthermore, analysis of DNA damage using the CometChip (a higher throughput version of the comet assay) shows an increase in the levels of BER intermediates. Finally, we found that GSNO exposure is associated with an increase in methylation-induced cytotoxicity. Taken together, these studies support a model wherein GSNO increases BER initiation while processing of AP sites is decreased, leading to a toxic increase in BER intermediates. This model is also supported by additional studies performed in our laboratory showing that inflammation in vivo leads to increased large-scale sequence rearrangements. Taken together, this work provides new evidence that inflammatory chemicals can drive cytotoxicity and mutagenesis via BER imbalance. [ABSTRACT FROM AUTHOR]

This study has shown for the first time that deuterium oxide (D2O) enhances expression of the E. coli ada-regulon induced by alkylating compounds, N-nitroso-N-methylurea (NMU) and methyl methanesulfonate (MMS). To compare the induction of ada-regulon in deuterated and in non-deuterated (control) cultures of E. coli, a biosensor based on the E. coli strain K12 MG1655 (pAlkA-lux) luminescing as a result of activating the alkA gene promoter in response to DNA alkylation was used. Depending on the D2O concentration from 5 to 10% in the pre-deuterium medium, these alkylating compounds at a concentration of 0.005 M induced expression of the alkA gene from 1.3 to 5 times higher as compared to induction in nondeuterated culture. It is suggested that deuterium can be one of the main factors in stabilizing the bond between the promoter and the alkylated Ada protein, leading to increased transcription of the ada-regulon. [ABSTRACT FROM AUTHOR]

Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. Toward this goal, I detected nascent DNA mismatches as a proxy for mutagenesis and simultaneously followed gene expression dynamics in single Escherichia coli cells using microfluidics. This general microscopy-based approach revealed the real-time dynamics of mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating the creation of DNA mismatches to the chronology of the underlying molecular processes. By avoiding population averaging, I discovered cell-to-cell variation in mutagenesis that correlated with heterogeneity in the expression of alternative responses to DNA damage. Pulses of mutagenesis are shown to arise from transient DNA repair deficiency. Constitutive expression of DNA repair pathways and induction of damage tolerance by the SOS response compensate for delays in the activation of inducible DNA repair mechanisms, together providing robustness against the toxic and mutagenic effects of DNA alkylation damage. [ABSTRACT FROM AUTHOR]

Rationale: Since extraterrestrial organic matter in meteorites is a very complex mixture that is hard to ionize due to its association with minerals, in situ analysis of polar organic compounds has never been performed. In addition, when studying powdered samples, spatial information of organic compounds is lost. Methods: In situ molecular analysis and chemical imaging of polar organic compounds were performed on a meteorite surface by desorption electrospray ionization coupled with high‐resolution mass spectrometry (DESI‐HRMS) using an Orbitrap mass spectrometer. Results: Many CHN compounds, including alkylated pyridine and imidazole homologues, were identified from the complex peaks by HRMS using a spray of electrically charged methanol with a spatial resolution of approximately 50 μm. The same alkylated homologues have the same spatial distribution in the meteorite matrix, while alkylpyridines occur in a different location from alkylimidazoles. Conclusions: The compound distribution suggests a different source for each compound series or a chromatographic separation effect associated with fluid movement in the meteorite parent body. The DESI‐HRMS imaging will further our understanding of organic compound distribution with respect to mineral and water interactions in meteorites. [ABSTRACT FROM AUTHOR]

Background We studied the expression of some major proteins involved in cell-cycle regulation and DNA repair, the roles of which are not well known in pancreatic ductal adenocarcinoma (PDAC), but which have a significant impact on carcinogenesis of many other cancers. Methods We immunohistochemically assessed expression levels of the cell-cycle regulators Rb1, p16 and cyclin-dependent kinase 4 (CDK4), and the DNA repair enzymes O6-methylguanine-DNA-alkyltransferase (MGMT) and flap endonuclease-1 (FEN1) separately in malignant tissue and benign tissue from resection margins in 102 cases of PDAC. Nearly all (95.1%) patients had undergone pancreaticoduodenectomy. Results The studied proteins showed wide but somewhat variable expression in both benign and malignant pancreatic tissues. Strong CDK4 expression in islets of Langerhans predicted poor relapse-free survival (RFS) (HR 2.874; 95% CI 1.261–6.550; p = .012) and within T3–4 tumors CDK4 expression in adenocarcinoma cells also predicted poor disease-free survival (DFS) (RR 2.148; 95% CI 1.081–4.272; p = .029). Strong MGMT expression was associated in N1 patients with weak local relapse-free survival (RFS), DFS and overall survival; all significantly in Cox regression analysis. FEN1 was also an independent predictor of decreased DFS (in the whole study population) and worse RFS (in the patients with T3–4 tumors). Conclusions Major cell-cycle regulator also have predictive significance, but further studies are required to evaluate this. [ABSTRACT FROM AUTHOR]

Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by theDNAreplication machinery in human cells.Wefound that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T3A and T3G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymeraseη or polymeraseζ, but not polymeraseκ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrate the roles of TLS polymerases in replicative bypass of these lesions in human cells. [ABSTRACT FROM AUTHOR]

DNA oligonucleotides containing site-specific N7-guanine monoadducts of cisplatin, diepoxybutane, and epichlorohydrin were used as templates for DNA synthesis by two bacterial DNA polymerases and human polymerase β. These polymerases were able to bypass the lesions effectively, although the efficiency was decreased, with inhibition increasing with the size of the lesion. Fidelity of incorporation was essentially unaltered, suggesting that N7-guanine monoadducts do not significantly contribute to the mutational spectra of these agents. [ABSTRACT FROM AUTHOR]

A recyclable Ruthenium tris [4,4′- bis (dinonylmethyl)-2,2′-bipyridine] (Ru[(DNM) 2 bpy] 3 2+ ) photocatalyst (PC) was synthesized. Hexane-phase-selective solubility allowed its simple and efficient separation from reaction products via solvent extraction. The excellent catalytic activity and recoverability were demonstrated in batch and flow perfluoroalkylation reactions of coumarin under visible-light irradiation. High reaction rates and easy reusability of the catalyst make this approach attractive for large-scale applications. [ABSTRACT FROM AUTHOR]

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13-18 nucleotides 5' and 6-7 nucleotides 3' to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism. [ABSTRACT FROM AUTHOR]

The characteristic that discriminates gasoline from other ignitable liquids is that it contains high-octane blending components. This study elaborates on the idea that the presence of gasoline in fire debris samples should be based on the detection of known high-octane blending components. The potential of the high-octane blending component alkylate as a characteristic feature for gasoline detection and identification in fire debris samples is explored. We have devised characteristic features for the detection of alkylate and verified the presence of alkylate in a large collection of gasoline samples from petrol stations in the Netherlands. Alkylate was detected in the vast majority of the samples. It is demonstrated that alkylate can be detected in fire debris samples that contain traces of gasoline by means of routine GC-MS methods. Detection of alkylate, alongside other gasoline blend components, results in a more solid foundation for gasoline detection and identification in fire debris samples. [ABSTRACT FROM AUTHOR]

Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) is an alkylating agent that causes severe damages to the skin, eyes, and the respiratory system. DNA alkylation is one of the most critical lesions that could lead to monoadducts and cross-links, as well as DNA strand breaks. In response to these adducts, cells initiate a series of reactions to recruit specific DNA repair pathways. The main DNA repair pathways in human cells, which could be involved in the DNA SM-induced DNA damages, are base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ). There is, thus, a need for a short review to clarify which damage caused by SM is repaired by which repair pathway. Increasing our knowledge about different DNA repair mechanisms following SM exposure would lay the first step for developing new therapeutic agents to treat people exposed to SM. In this review, we describe the major DNA repair pathways, according to the DNA adducts that can be caused by SM. [ABSTRACT FROM AUTHOR]

Cisplatin (CDDP) isan anti-cancerDNA alkylating chemotherapeutic agent act against a variety of tumors.The present study aimed to evaluate the possible protective effects of green tea on the physiological parameters in mice chronically treated with CDDP. Four groups of mice were examined: a control mice saline PBS solution (group I), mice treated with CDDP (group II), mice treated with CDDP and green tea (group III) and normal mice treated with green tea (group IV). All animals were treated for successively five days and killed one week after the last treatment. The results recorded that CDDP treatment significantly decreasedthe levels of white blood cells (WBCs), red blood cell distribution width (RDW) and lymphocytes count. Also, CDDP increased the hepatocytes oxidative stress which characterized by increasing prooxidants xanthine oxidase (XO), thiobarbituric acid-reactive substances (TBARS) and decreasing of antioxidants glutathione peroxidase (GPx). As a result, hepatocytes injury took place that characterized with serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) andalkaline phosphatase (ALP) activities. The treatment of mice with green tea to CDDP group (group III) or mice treated with gr een tea alone group (group IV) successfully normalized the physiological parameters in form returning WBCs, RDW and lymphocytes counts to normal levels and decreased the hepatocytes oxidative stress which characterized by decreasing Prooxidants (OX, TBARS) and increasing of antioxidants GPx reflected by significant decrease in the serum activities of AST, ALTand(ALP) activities. [ABSTRACT FROM AUTHOR]

A density functional theory study is performed to understand the effect of discrete water molecules during Az+ion formation in nitrogen mustards. A comparative study in gas phase, and implicit and explicit solvation models of three drug molecules (mustine, chlorambucil and melphalan) is reported. Noteworthy changes in the structure and C–N stretching frequencies of the transition states have been observed in the presence of explicit water molecules. Presence of explicit water molecules reduces the positive charge around the tricyclic Az+ring, and hence stabilising it. Both activation energy and rate constants are seen to be significantly affected in the presence of discrete water molecules. [ABSTRACT FROM PUBLISHER]

[Display omitted] • Based on native guanine, a group of G-analogues are obtained by fluorophore replacement and ring extension. • The efficient fluorescence is quenched when xG paired with the complementary adduct O4-meT. • The fluorescence quenching is attributed to intermolecular charge transfer character. DNA alkylation is caused by long-term exposure of cells to the environmental and endogenous alkylating agents, which can also lead to DNA mutations and therefore trigger some cancers. Since O4-methylthymidine (O4-meT), mismatched with guanine (G), is the most common but not easily repaired alkylated nucleoside, monitoring O4-meT can help to effectively reduce the occurrence of carcinogenesis. In this work, the modified G-analogues are selected as the fluorescence probe to monitor the existence of O4-meT according to its pairing characteristics. The photo-physical properties of considered G-analogues formed by ring expansion or addition of fluorophores were studied in detail. It is found that, compared with natural G, the absorption peaks of these fluorescence analogues are red-shifted (>55 nm) and the luminescence is enhanced by π-conjugation. Especially, the xG has a large Stokes shift (65 nm) with fluorescence insensitive to natural cytosine (C) and retains efficient emission after pairing, while it is sensitive to O4-meT and the quenching phenomenon occurs due to the excited state intermolecular charge transfer. Accordingly, the xG can be used as a fluorescent probe to identify the O4-meT in solution. In addition, the direct use of deoxyguanine fluorescent analogue for monitoring O4-meT was evaluated by the effects of ligating deoxyribose on absorption and fluorescence emission. [ABSTRACT FROM AUTHOR]

In this work, novel potential anthraquinone-temozolomide (TMZ) antitumor hybrids N -(2-((9,10-dioxo-9,10-dihydroanthracen-1-yl)amino)ethyl)−3-methyl-4-oxo-3,4-dihydroimidazo [5, 1-d][1,2,3,5]tetrazine-8-carboxamide (C-1) and 2-(9,10-dioxo-9,10-dihydroanthracen-1-yl)amino) ethyl-3-methyl-4-oxo-3,4-dihydroimidazo[5,1- d ][1,2,3,5]tetrazine-8-carboxylate (C - 9) were designed and synthesized successfully. The electrochemical behaviors of C - 1 (C - 9) involved the reversible processes of 9,10-anthraquinone ring, the irreversible reduction and oxidation processes of TMZ ring. Electrochemical biosensors were constructed with ct DNA, poly (dG) and poly (dA) modifying the surface of glassy carbon electrode (GCE) to evaluate the DNA oxidative damage caused by the interaction of C - 1 (C - 9) with DNA. Anthracycline skeleton and TMZ ring in C - 1 (C - 9) could exhibit bifunctional effects with both intercalating and alkylation modes toward DNA strands. The DNA biosensor had good practicability in mouse serum. The results of gel electrophoresis further demonstrated that C - 1 (C - 9) could effectively intercalated into ct DNA and disrupt plasmid conformation. Finally, anthraquinone-TMZ hybrid C-1 possessed high cytotoxicity toward A549 and GL261 cells, which could be a novel and optimal candidate for the clinic antitumor treatment. [Display omitted] • Two novel potential antitumor hybrids C-1 and C-9 were designed and synthesized. • C-1 (C-9) could intercalate and alkylate toward DNA strands. • C-1 (C-9) had the potency of cleaving and unwinding DNA strands. • C-1 (C-9) could intercalate into DNA base pairs with a high affinity constant. • C-1 possessed high cytotoxicity toward A549 and GL261 cells. [ABSTRACT FROM AUTHOR]

Temozolomide (TMZ) is the standard first line treatment for malignant glioma, reaching “blockbuster” status in 2010, yet it remains the only drug in its class. The main constraints on the clinical effectiveness of TMZ therapy are its requirement for active DNA mismatch repair (MMR) proteins for activity, and inherent resistance through O6-methyl guanine-DNA methyl transferase (MGMT) activity. Moreover, acquired resistance, due to MMR mutation, results in aggressive TMZ-resistant tumour regrowth following good initial responses. Much of the attraction in TMZ as a drug lies in its PK/PD properties: it is acid stable and has 100% oral bioavailability; it also has excellent distribution properties, crosses the blood-brain barrier, and there is direct evidence of tumour localisation. This review seeks to unravel some of the mysteries of the imidazotetrazine class of compounds to which TMZ belongs. In addition to an overview of different synthetic strategies, we explore the somewhat unusual chemical reactivity of the imidazotetrazines, probing their mechanisms of reaction, examining which attributes are required for an active drug molecule and reviewing the use of this combined knowledge towards the development of new and improved anti-cancer agents.

Cells have evolved mechanisms that allow them to adapt to changes in their intracellular and extracellular milieu to maintain homeostasis. This is frequently referred to as the SUMO Stress Response (SSR), which is thought to promote cell survival by altering the sumoylation status of key components of stress response pathways. Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II. [Extracted from the article]

Thiamine (vitamin B1) is an essential nutrient acting mainly as an enzymatic cofactor on diverse cell processes. It has been reported that vitamin B1 has a significant role in the signaling pathways related to the response to adverse environmental conditions (chemical and physical). The objectives of this study were to evaluate the antimutagenic potential of vitamin B1 in front of DNA-alkylating agents in the presence/absence of ogt and ada repairing genes in Salmonella typhimurium strains and against damage induced by ultraviolet light type C in Escherichia coli strains mutated at the uvrABC system and recBCD enzymes. For S. typhimurium , an antimutagenesis test (Ames test) was performed using strains deficient in one or both genes (YG7100 ada − / ogt + , YG7104 ada + /og t − , YG7108 ada − / ogt − ). For E. coli , mutated strains (K-12 derived strains Hfr H180 uvrB + / recA + , W3110 uvrB + / recA − and ATCC®8739 uvrB − / recA + ) were exposed to UV-C light at different time intervals, with and without vitamin B1. Our results showed that thiamine is an antimutagen against methyl-N-nitro-N-nitrosoguanidine or ethyl-N-nitro-N-nitrosoguanidine only when the ogt gene is present. While for E. coli , the presence of vitamin B1 increased the survival rate, implying an antimutagenesis independent of uvrABC repairing system and recBCD enzymes. [ABSTRACT FROM AUTHOR]

O6-DNA-alkyl-guanine-DNA-alkyl-transferases (OGTs) are evolutionarily conserved, unique proteins that repair alkylation lesions in DNA in a single step reaction. Alkylating agents are environmental pollutants as well as by-products of cellular reactions, but are also very effective chemotherapeutic drugs. OGTs are major players in counteracting the effects of such agents, thus their action in turn affects genome integrity, survival of organisms under challenging conditions and response to chemotherapy. Numerous studies on OGTs from eukaryotes, bacteria and archaea have been reported, highlighting amazing features that make OGTs unique proteins in their reaction mechanism as well as post-reaction fate. This review reports recent functional and structural data on two prokaryotic OGTs, from the pathogenic bacterium Mycobacterium tuberculosis and the hyperthermophilic archaeon Sulfolobus solfataricus, respectively. These studies provided insight in the role of OGTs in the biology of these microorganisms, but also important hints useful to understand the general properties of this class of proteins. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis, an asaccharolytic Gram-negative oral anaerobe, is a major pathogen associated with adult periodontitis, a chronic infective disease that a significant percentage of the human population suffers from. It preferentially utilizes dipeptides as its carbon source, suggesting the importance of dipeptidyl peptidase (DPP) types of enzyme for its growth. Until now DPP IV, DPP5, 7 and 11 have been extensively investigated. Here, we report the characterization of DPP III using molecular biology, biochemical, biophysical and computational chemistry methods. In addition to the expected evolutionarily conserved regions of all DPP III family members, PgDPP III possesses a C-terminal extension containing an Armadillo (ARM) type fold similar to the AlkD family of bacterial DNA glycosylases, implicating it in alkylation repair functions. However, complementation assays in a DNA repair-deficient Escherichia coli strain indicated the absence of alkylation repair function for PgDPP III. Biochemical analyses of recombinant PgDPP III revealed activity similar to that of DPP III from Bacteroides thetaiotaomicron, and in the range between activities of human and yeast counterparts. However, the catalytic efficiency of the separately expressed DPP III domain is ~1000-fold weaker. The structure and dynamics of the ligand-free enzyme and its complex with two different diarginyl arylamide substrates was investigated using small angle X-ray scattering, homology modeling, MD simulations and hydrogen/deuterium exchange (HDX). The correlation between the experimental HDX and MD data improved with simulation time, suggesting that the DPP III domain adopts a semi-closed or closed form in solution, similar to that reported for human DPP III. The obtained results reveal an atypical DPP III with increased structural complexity: its superhelical C-terminal domain contributes to peptidase activity and influences DPP III interdomain dynamics. Overall, this research reveals multifunctionality of PgDPP III and opens direction for future research of DPP III family proteins. [ABSTRACT FROM AUTHOR]

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance. [ABSTRACT FROM AUTHOR]

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer. [ABSTRACT FROM AUTHOR]

DNA alkylating agents form the first line of cancer chemotherapy. They not only kill cells but also behave as potential carcinogens. MNU, a DNA methylating agent, is well known to induce mammary tumours in rodents. However, the mechanism of tumorigenesis is not well understood. Our study reports a novel role played by DNA-dependent protein kinase (DNA-PK) in methylation damage-induced transformation using three-dimensional breast acinar cultures. Here, we report that exposure of breast epithelial cells to MNU inhibited polarisation at the basolateral domain, increased dispersal of the Golgi at the apical domain and induced an epithelial-to-mesenchymal transition (EMT)-like phenotype as well as invasion. This altered Golgi phenotype correlated with impaired intracellular trafficking. Inhibition of DNA-PK resulted in almost complete reversal of the altered Golgi phenotype and partial rescue of the polarity defect and EMT-like phenotype. The results confirm that methylation damage-induced activation of DNA-PK is a major mechanism in mediating cellular transformation. [ABSTRACT FROM AUTHOR]

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove–sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions. [ABSTRACT FROM AUTHOR]

Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae , examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution. [ABSTRACT FROM AUTHOR]

Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O 6 -chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide. [ABSTRACT FROM AUTHOR]

In the present study, synthesis and antineoplastic activity of phenylacetic acid and benzoic acid nitrogen mustard conjugates of various steroidal oximes are reported for the first time. The conjugation was achieved through a more stable oxime-ester linkage and the resulting newly synthesized conjugates were evaluated in vitro on various human cancer cell lines for cytotoxicity. The extent of their alkylating activity was investigated by the in vitro colorimetric 4-( p -nitrobenzyl)pyridine (NBP) assay. The 17 E -steroidal oxime-benzoic acid mustard ester 3β-acetoxy-17 E -[ p -( N , N- bis(2-chloroethyl)amino)]benzoyloxyimino-androst-5-ene ( 8 ) emerged as the most potent conjugate having significant cytotoxicity on most of the NCI 60-cell lines. Outstanding growth inhibition was observed on the IGROV1 ovarian cancer cell line with GI 50 = 0.937 µM. In general, the D-ring derived androstene oxime-nitrogen mustard conjugates were found to possess better antineoplastic activity over a variety of cancer cells in comparison to those derived from other rings of the steroid skeleton. [ABSTRACT FROM AUTHOR]

Exposure to environmental allergens is a major risk factor for asthma development. Allergens possess proteolytic activity that is capable of disrupting the airway epithelium. Although there is increasing evidence pointing to asthma as an epithelial disease, the underlying mechanism that drives asthma has not been fully elucidated. In this study, we investigated the direct DNA damage potential of aeroallergens on human bronchial epithelial cells and elucidated the mechanisms mediating the damage. Human bronchial epithelial cells, BEAS-2B, directly exposed to house dust mites (HDM) resulted in enhanced DNA damage, as measured by the CometChip and the staining of DNA double-strand break marker, γH2AX. HDM stimulated cellular reactive oxygen species production, increased mitochondrial oxidative stress, and promoted nitrosative stress. Notably, expression of nuclear factor erythroid 2-related factor 2-dependent antioxidant genes was reduced immediately after HDM exposure, suggesting that HDM altered antioxidant responses. HDM exposure also reduced cell proliferation and induced cell death. Importantly, HDM-induced DNA damage can be prevented by the antioxidants glutathione and catalase, suggesting that HDM-induced reactive oxygen and nitrogen species can be neutralized by antioxidants. Mechanistic studies revealed that HDM-induced cellular injury is NADPH oxidase (NOX)-dependent, and apocynin, a NOX inhibitor, protected cells from double-strand breaks induced by HDM. Our results show that direct exposure of bronchial epithelial cells to HDM leads to the production of reactive oxygen and nitrogen species that damage DNA and induce cytotoxicity. Antioxidants and NOX inhibitors can prevent HDM-induced DNA damage, revealing a novel role for antioxidants and NOX inhibitors in mitigating allergic airway disease. [ABSTRACT FROM AUTHOR]

Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in S. islandicus in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation. The former occurred immediately after drug treatment, leading to chromosomal DNA degradation that concurred with TopR1 degradation, followed by chromatin protein degradation and DNA-less cell formation. To gain a further insight into TopR1 function, the expression of the enzyme was reduced in S. islandicus cells using a CRISPR-mediated mRNA interference approach (CRISPRi) in which topR1 mRNAs were targeted for degradation by endogenous III-B CRISPR-Cas systems. We found that the TopR1 level was reduced in the S. islandicus CRISPRi cells and that the cells underwent accelerated genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo. [ABSTRACT FROM AUTHOR]

Alkylation chemotherapy is one of the most widely used systemic therapies for cancer. While somewhat effective, clinical responses and toxicities of these agents are highly variable. A major contributing factor for this variability is the numerous distinct lesions that are created upon alkylation damage. These adducts activate multiple repair pathways. There is mounting evidence that the individual pathways function cooperatively, suggesting that coordinated regulation of alkylation repair is critical to prevent toxicity. Furthermore, some alkylating agents produce adducts that overlap with newly discovered methylation marks, making it difficult to distinguish between bona fide damaged bases and so-called ‘epigenetic’ adducts. Here, we discuss new efforts aimed at deciphering the mechanisms that regulate these repair pathways, emphasizing their implications for cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM) is a chemical warfare agent which is cytotoxic in nature, and at the molecular level, SM acts as DNA alkylating agent leading to genotoxic and reproductive effects. Mostly, the exposed areas of the body are the main targets for SM; however, it also adversely affects various tissues of the body and ultimately exhibits long-term complications including genotoxic and reproductive effects, even in the next generations. The effect of SM on reproductive system is the reason behind male infertility. The chronic genotoxic and reproductive complications of SM have been observed in the next generation, such as reproductive hormones disturbances, testicular atrophy, deficiency of sperm cells, retarded growth of sperm and male infertility. SM exerts toxic effects through various mechanisms causing reproductive dysfunction. The key mechanisms include DNA alkylation, production of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide (NAD) depletion. However, the exact molecular mechanism of such long-term effects of SM is still unclear. In general, DNA damage, cell death and defects in the cell membrane are frequently observed in SM-exposed individuals. SM can activate various cellular and molecular mechanisms related to oxidative stress (OS) and inflammatory responses throughout the reproductive system, which can cause decreased spermatogenesis and impaired sperm quality via damage to tissue function and structure. Moreover, the toxic effects of SM on the reproductive system as well as the occurrence of male infertility among exposed war troopers in the late exposure phase is still uncertain. The chronic effects of SM exposure in parents can cause congenital defects in their children. In this review, we aimed to investigate chronic genotoxic and reproductive effects of SM and their molecular mechanisms in the next generations. [ABSTRACT FROM AUTHOR]

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation. [ABSTRACT FROM AUTHOR]

The reaction of carboxylic acids with Baylis-Hillman reaction derived α-bromomethyl acrylic esters readily provide 2-(alkoxycarbonyl)allyl esters in good to excellent yields. These functionalized allyl esters have been evaluated for their cell proliferation inhibition properties against breast cancer (MDA-MB-231 and 4T1) and pancreatic cancer (MIAPaCa-2) cell lines to explore their potential as anticancer agents. Several of the synthesized derivatives exhibit good potency against all three cancer cell lines. Our structure activity relationship (SAR) studies on 2-carboxycarbonyl allyl esters indicate that substituted aromatic carboxylic acids provide enhanced activity compared to substituted aliphatic carboxylic acid analogs. Di- and tri-allyl esters derived from di-and tri-carboxylic acids exhibit higher inhibition of cell proliferation than mono esters. Further SAR studies indicate that the double bond in the 2-(alkoxycarbonyl)allyl ester is required for its activity, and there is no increase in activity with increased chain length of the alkoxy group. Two lead candidate compounds have been identified from the cell proliferation inhibition studies and their preliminary mechanism of action as DNA damaging agents has been evaluated using epifluorescence and western blot analysis. One of the lead compounds has been further evaluated for its systemic toxicity in healthy CD-1 mice followed by anticancer efficacy in a triple negative breast cancer MDA-MB-231 xenograft model in NOD-SCID mice. These two in vivo studies indicate that the lead compound is well tolerated in healthy CD-1 mice and exhibits good tumor growth inhibition compared to breast cancer drug doxorubicin. [ABSTRACT FROM AUTHOR]

The selective alkylation of a single meso-N atom of a corrolazine macrocycle is reported. Alkylation has a dramatic impact on the physicochemical properties of ReV(O)(TBP8Cz). New electron-transfer and hydrogen-atom-transfer reactivity is also seen for this complex, including one-electron reduction, which gives an air-stable 19π-electron aromatic radical complex. [ABSTRACT FROM AUTHOR]

The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC)2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC)2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Sulfur mustard (SM), a chemical warfare agent, causes DNA alkylation, which is believed to be the main cause of its toxicity. SM DNA adducts are commonly used to verify exposure to this vesicant. However, the required analytical state-of-the-art mass-spectrometry methods are complex, use delicate instruments, are not mobile, and require laboratory infrastructure that is most likely not available in conflict zones. Attempts have thus been made to develop rapid detection methods that can be used in the field. The analysis of SM DNA adducts (HETE-G) by immunodetection is a convenient and suitable method. For a diagnostic assessment, HETE-G levels must be determined in relation to the total DNA in the sample. Total DNA can be easily visualized by the use of fluorescent DNA dyes. This study examines whether SM and related compounds affect total DNA staining, an issue that has not been investigated before. After pure DNA was extracted from human keratinocytes (HaCaT cells), DNA was exposed to different S- and N-alkylating agents. Our experiments revealed a significant, dose-dependent decrease in the fluorescence signal of fluorescent dye-stained DNA after exposure to alkylating agents. After mass spectrometry and additional fluorescence measurements ruled out covalent modifications of ethidium bromide (EthBr) by SM, we assumed that DNA crosslinks caused DNA condensation and thereby impaired access of the fluorescent dyes to the DNA. DNA digestion by restriction enzymes restored fluorescence, a fact that strengthened our hypothesis. However, monofunctional agents, which are unable to crosslink DNA, also decreased the fluorescence signal. In subsequent experiments, we demonstrated that protons produced during DNA alkylation caused a pH decrease that was found responsible for the reduction in fluorescence. The use of an appropriate buffer system eliminated the adverse effect of alkylating agents on DNA staining with fluorescent dyes. An appropriate buffer system is thus crucial for DNA quantification with fluorescent dyes in the presence of alkylating compounds. [ABSTRACT FROM AUTHOR]

Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis. [ABSTRACT FROM AUTHOR]

Per-2,3-di-O-methyl- and per-2,3-di-O-allyl-β-cyclodextrin duplexes held by two disulfide bonds between their primary faces have been prepared. Permethylation significantly increased the solubility of the cyclodextrin duplexes in a wide range of solvents from water to chlorinated hydrocarbons. Per-2,3-di-O-methylated duplexes are able to form inclusion complexes with organic molecules in aqueous solutions, yet the stability constants are lower by 4–5 orders of magnitude as compared to analogous non-alkylatedβ-cyclodextrin duplexes. [ABSTRACT FROM AUTHOR]

DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS) by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA) involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analogue acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC) strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.