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Highlights: [•] The repair kinetics of two common cyclic DNA adducts derived lipid peroxidation, Acr-dG and HNE-dG, in human colon cells was studied. [•] This study is the first to measure DNA repair rates by both DNA repair synthesis and LC–MS/MS methods. [•] Acr-dG, like HNE-dG, is repaired by NER pathway, but it is repaired at a much slower rate compared to HNE-dG. [•] HNE-dG can inhibit the repair of Acr-dG if both are present in the same DNA. [ • ] These results provide an explanation for the higher levels of Acr-dG than HNE-dG observed in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Acrolein (Acr), a hazardous air pollutant, reacts readily with deoxyguanosine (dG) in DNA to produce cyclic 1, N 2-propanodeoxyguanosine adducts (Acr-dG). Studies demonstrate that these adducts are detected in vivo and may play a role in mutagenesis and carcinogenesis. In the study described here, a quantitative 32P-postlabeling/solid-phase extraction/HPLC method was developed by optimizing the solid-phase extraction and the 32P-postlabeling conditions for analysis of Acr-dG in DNA samples with a detection limit of 0.1fmol. It was found that Acr-dG can form as an artifact during the assay. Evidence obtained from mass spectrometry indicates that the Acr in water used in the assay is a likely source of artifact formation of Acr-dG. The formation of Acr-dG as an artifact can be effectively blocked by adding glutathione (GSH) to the DNA sample to be analyzed. In addition, Acr-dG was detected as a contaminant in the commercial dG and dT 3′-monophosphate samples. Finally, this method was used to detect Acr-dG in calf thymus and human colon HT29 cell DNA with an excellent linear quantitative relationship. [Copyright &y& Elsevier]

Abstract: Novel 5-aza-ellipticine derivatives were synthesized and tested as antitumor agents. The new compounds were prepared more readily than the analogous ellipticine derivatives, which are known to be potent anti-tumor agents Although the novel 5-aza-ellipticine derivatives are not as biologically active as their corresponding ellipticine analogues, the new compounds represent a new, readily accessible class of heteroaromatic catalytic inhibitors of topoisomerase II and possible anti-tumor agents. [Copyright &y& Elsevier]

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient) and state 2 (active, effector binding enabled). Here, we use 31P NMR to delineate the differences in state 1 and state 2 populations present in WT and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analog GppNHp (guanosine-5'-[(β,γ)-imido] triphosphate). Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RAS-binding domain and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small-molecule inhibitor, BBO-8956, which is effective against both GDP- and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs state 1-state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1-RAS-binding domain is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action for this novel and active-conformation inhibitor. [ABSTRACT FROM AUTHOR]

Previous studies showed that 7-(1′,2′-dihydroxyheptyl)-substituted etheno DNA adducts are products of reactions with the epoxide of (E)-4-hydroxy-2-nonenal, an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1′,2′-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a 32P-postlabeling/HPLC method and an isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method, demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid with deoxyadenosine, supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E, and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are afflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that although the survival of LEC rats was increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA, and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats. [ABSTRACT FROM AUTHOR]

Our previous studies revealed that the nonapeptide fragment of HLA-DR molecule, located in the β chain 164–172 with the VPRSGEVYT sequence, suppresses the immune responses. The sequence is located on the exposed molecule loop, therefore it may be involved in the interactions with other proteins. We suggested that the loop may serve as a functional epitope on the HLA class II surface for intermolecular binding, and that possible mechanism of biological action of the synthesized peptides is associated with interfering of adhesion of HLA class II molecules to their coreceptors. It has been postulated that oligomerization of the coreceptors is required for stable binding to class II HLA. Based on the crystal dimeric structure of HLA-DR molecules, we designed, and synthesized molecules able to induce the putative coreceptors dimerization. The synthesized series of compounds consisted of two VPRSGEVYT sequences linked through their C-termini by spacers of different length: (VPRSGEVYTGn)2K-NH2 (n=4–6). The results demonstrate that the dimerization of the nonapeptide fragment of HLA-DR results in enhanced immunosuppressory properties. [Copyright &y& Elsevier]

A comparative study of thermodynamic and kinetic aspects of Cu(II) and Ni(II) binding at the N-terminal binding site of human and bovine serum albumins (HSA and BSA, respectively) and short peptide analogues was performed using potentiometry and spectroscopic techniques. It was found that while qualitative aspects of interaction (spectra and structures of complexes, order of reactions) could be reproduced, the quantitative parameters (stability and rate constants) could not. The N-terminal site in HSA is much more similar to BSA than to short peptides reproducing the HSA sequence. A very strong influence of phosphate ions on the kinetics of Ni(II) interaction was found. This study demonstrates the limitations of short peptide modelling of Cu(II) and Ni(II) transport by albumins. [ABSTRACT FROM AUTHOR]

The acrolein derived cyclic 1,N 2 -propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125 μM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116 + ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA. [ABSTRACT FROM AUTHOR]

Acrolein (Acr) is a ubiquitous environmental pollutantfound incigarette smoke and automobile exhaust. It can also be produced endogenouslyby oxidation of polyunsaturated fatty acids. The Acr-derived 1,N2-propanodeoxyguanosine (Acr-dG) adducts inDNA are mutagenic lesions that are potentially involved in human cancers.In this study, monoclonal antibodies were raised against Acr-dG adductsand characterized using ELISA. They showed strong reactivity and specificitytoward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N2-propanodeoxyguanosines, and weak or no reactivity toward1,N6-ethenodeoxyadenosine and 8-oxo-deoxyguanosine.Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay fordetecting these adducts directly in cells; second, a highly sensitivedirect ELISA assay for measuring Acr-dG in cells and tissues usingonly 1 μg of DNA without DNA digestion and sample enrichment;and third, a competitive ELISA for better quantitative measurementof Acr-dG levels in DNA samples. The assays were validated using Acr-treatedHT29 cell DNA samples or calf thymus DNA, and the results were confirmedby LC–MS/MS-MRM. An immunohistochemical assay was also developedto detect and visualize Acr-dG in HT29 cells as well as in human oralcells. These antibody-based methods provide useful tools for the studiesof Acr-dG as a cancer biomarker and of the molecular mechanisms bywhich cells respond to Acr-dG as a ubiquitous DNA lesion. [ABSTRACT FROM AUTHOR]

Acrolein (Acr) is a major component in cigarette smoke and a ubiquitous environmental pollutant. It is also formed as a product of lipid peroxidation. Following ring closure via the Michael addition, Acr modifies deoxyguanosine (dG) in DNA by forming cyclic 1,N2-propanodeoxyguanosine adducts (OHPdG). The reactions of Acr with dG yield, depending on the direction of ring closure, two regioisomers, α- and γ-OHPdG, in approximately equal amounts. However, previous 32P-postlabeling studies showed that the γ isomers were detected predominantly in the DNA of rodent and human tissues. Because of the potential differential biological activity of the isomeric OHPdG adducts, it is important to confirm and study the chemical basis of the regioselective formation of γ isomers in vivo. In this study, it is confirmed that γ-OHPdG adducts are indeed the major isomers formed in vivo as evidenced by a LC-MS/MS method specifically developed for Acr-derived dG adducts. Furthermore, we have shown that the formation of γ-isomers is increased in the presence of amino-containing compounds, including amino acids, proteins, and cell lysates. A product of Acr and arginine that appears to mediate the regioselective formation of γ isomers was identified, but its structure was not fully characterized due to its instability. This study demonstrates that intracellular amino-containing compounds may influence the regiochemistry of the formation of OHPdG adducts and reveals a mechanism for the preferential formation of γ-OHPdG by Acr in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Hemiasterlin (Hem) and dolastatin (Dol) are marine natural products which are cytotoxic for cancer cells. Hem, a tripeptide, and Dol, a hexapeptide, were conjugated with linkers (L) to the universal BB agonist DPhe-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2(BA1) and the effects of the Hem-BB and Dol-BB conjugates investigated on NCI-H1299 lung cancer cells. Hem-LA-BA1 and Hem-LB-BA1 inhibited specific (125I-Tyr4)BB binding to NCI-H1299 cells, which have BB2 receptors (R), with IC50 values of 15 and 25 nM, respectively. Addition of Hem-LA-BA1 and Hem-LB-BA1 to Fura-2 AM loaded cells containing BB2R, caused elevated cytosolic Ca2+. In a growth assay, Hem-LA-BA1 and Hem-LB-BA1 inhibited the proliferation of NCI-H1299 cells. Dol-succinamide (Dols)-LD-BA1 and Dols-LE-BA1 bound with high affinity to NCI-H1299 cells and elevated cytosolic Ca2+, but did not inhibit the proliferation of NCI-H1299 cells. Also, Hem-LA-BA1 inhibited 125I-DTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2 (BA2) binding to Balb/3T3 cells transfected with BB1R or BB2R as well as with BRS-3 with IC50 values of 130, 8, and 540 nM, respectively. These results show that Hem-BB conjugates are cytotoxic for cancer cells containing BB2R. [Copyright &y& Elsevier]

The interactions of proteins with reactive oxygen species (ROS) may result in covalent modifications of amino acid residues in proteins, formation of protein–protein cross-linkages, and oxidation of the protein backbone resulting in protein fragmentation. In an attempt to elucidate the products of the metal-catalyzed oxidation of the human (H) and mouse (M) (1–10H), (1–10M), (1–16H) and (1–16M) fragments of β-amyloid peptide, the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) methods and Cu(II)/H2O2 as a model oxidizing system were employed. Peptide solution (0.50 mM) was incubated at 37 °C for 24 h with metal:peptide:H2O2 molar ratio 1:1:1 for the (1–16H), (1–16M) fragments, and 1:1:2 for the (1–10H), (1–10M) peptides in phosphate buffer, pH 7.4. Oxidation targets for all peptide studied are the histidine residues coordinated to the metal ions. For the (1–16H) peptide are likely His13 and/or His14, and for the (1–16M) fragment His6 and/or His14, which are converted to 2-oxo-His. Metal-binding residue, the aspartic acid (D1) undergoes the oxidative decarboxylation and deamination to pyruvate. The cleavages of the peptide bonds by either the diamide or α-amidation pathways were also observed. [Copyright &y& Elsevier]