Dietary flavonoids bind to mono-ubiquitinated annexin A1 in nuclei, and inhibit chemical induced mutagenesis.

In order to investigate the mechanisms of anti-mutagenic action by dietary flavonoids, we investigated if they inhibit mutation of the thymidine kinase (tk) gene in L5178Ytk(±) lymphoma cells. Silibinin, quercetin and genistein suppressed mutation of the tk gene induced in L5178Ytk(±) lymphoma cells by methyl methanesulfonate (MMS) and As(3+). Flavone and flavonol were less effective. To establish that mutation of the tk gene in L5178Ytk(±) lymphoma cells by MMS and As(3+) is mediated through mono-ubiquitinated annexin A1, L5178Ytk(±) lymphoma cells were treated with annexin A1 anti-sense oligonucleotide. The treatment reduced mRNA as well as protein levels of annexin A1, and suppressed mutation of the tk gene. Nuclear extracts from L5178Ytk(±) lymphoma cells catalyzed translesion DNA synthesis with an oligonucleotide template containing 8-oxo-guanosine in an annexin A1 dependent manner. This translesion DNA synthesis was inhibited by the anti-mutagenic flavonoids, silibinin, quercetin and genistein, in a concentration dependent manner, but only slightly by flavone and flavonol. Because these observations implicate involvement of annexin A1 in mutagenesis, we examined if flavonoids suppress nuclear annexin A1 helicase activity. Silibinin, quercetin and genistein inhibited ssDNA binding, DNA chain annealing and DNA unwinding activities of purified nuclear mono-ubiquitinated annexin A1. Flavone and flavonol were ineffective. The apparent direct binding of anti-mutagenic flavonoids to the annexin A1 molecule was supported by fluorescence quenching. Taken together, these findings illustrate that nuclear annexin A1 may be a novel and productive target protein of prevention for DNA damage induced gene mutation, ultimately conferring cancer chemoprevention.

Ubiquitination and SUMOylation of annexin A1 and helicase activity.

BACKGROUND: While annexin A1 in nuclei is proposed to be involved in cell transformation, its functions remain poorly understood. Since annexin A1 has the consensus motif, ¹6°LKRD, for SUMOylation as well as Ks, acceptors for ubiquitination that regulates localization and functions of proteins, we investigated SUMOylation and ubiquitination of annexin A1. METHODS: SUMOylation and ubiquitination of bovine annexin A1 were biochemically tested in vitro by purified proteins, and were confirmed by cell experiments with L5178 lymphoma cells. Effects of the modifications on DNA helicase activity were measured by ssDNA binding activity and by dsDNA unwinding activity. RESULTS: SUMOylation of annexin A1 was catalyzed by Ubc9, while its ubiquitination was by Rad6-Rad 18. Ubiquitinated annexin A1 had higher affinity for damaged DNA, and promoted in vitro translesion DNA synthesis by Pol ß. In mouse lymphoma L5178Y tk(+/-) cells, levels of SUMOylated annexin A1 decreased by DNA damaging agents, MMS or As³, whereas those of ubiquitinated annexin A1 increased under the same conditions. CONCLUSION: These observations suggest but do not necessarily prove that ubiquitinated annexin A1 in nuclei may be involved in DNA damage response, while SUMOylated annexin A1 functions in proliferation-differentiation. SIGNIFICANCE: Ubiquitination of annexin A1 may play an important role in mutagenesis, an initial step of cell transformation.

Carcinogenic heavy metals, As3+ and Cr6+, increase affinity of nuclear mono-ubiquitinated annexin A1 for DNA containing 8-oxo-guanosine, and promote translesion DNA synthesis.

To elucidate the biological roles of mono-ubiquitinated annexin A1 in nuclei, we investigated the interaction of purified nuclear mono-ubiquitinated annexin A1 with intact and oxidatively damaged DNA. We synthesized the 80mer 5'-GTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCA-3' (P0G), and four additional 80mers, each with a selected single G in position 14, 30, 37 or 48 replaced by 8-oxo-guanosine (8-oxo-G) to model DNA damaged at a specific site by oxidation. Nuclear mono-ubiquitinated annexin A1 was able to bind oligonucleotides containing 8-oxo-G at specific positions, and able to anneal damaged oligonucleotide DNA to M13mp18 in the presence of Ca(2+) or heavy metals such as As(3+) and Cr(6+). M13mp18/8-oxo-G-oligonucleotide duplexes were unwound by nuclear annexin A1 in the presence of Mg(2+) and ATP. The binding affinity of nuclear annexin A1 for ssDNA was higher for oxidatively damaged oligonucleotides than for the undamaged oligonucleotide P0G, whereas the maximal binding was not significantly changed. The carcinogenic heavy metals, As(3+) and Cr(6+), increased the affinity of mono-ubiquitinated annexin A1 for oxidatively damaged oligonucleotides. Nuclear mono-ubiquitinated annexin A1 stimulated translesion DNA synthesis by Pol ß. Nuclear extracts of L5178Y tk(+/-) lymphoma cells also promoted translesion DNA synthesis in the presence of the heavy metals As(3+) and Cr(6+). This DNA synthesis was inhibited by anti-annexin A1 antibody. These observations do not prove but provide strong evidence for the hypothesis that nuclear mono-ubiquitinated annexin A1 is involved in heavy metal promoted translesion DNA synthesis, thereby exhibiting the capacity to increase the introduction of mutations into DNA.

Carcinogenic heavy metals replace Ca2+ for DNA binding and annealing activities of mono-ubiquitinated annexin A1 homodimer.

Mono-ubiquitinated annexin A1 was purified from rat liver nuclei. The homodimer form of mono-ubiquitinated annexin A1 was able to unwind dsDNA in a Mg(2+)- and ATP-dependent manner, and to anneal ssDNA in a Ca(2+)-dependent manner. Phospholipids decreased the concentration of Ca(2+) required for maximal annealing activity. Heavy metals such as As(3+), Cr(6+), Pb(2+) and Cd(2+) substituted for Ca(2+) in the ssDNA binding and annealing activities of annexin A1. While these metals inhibited the unwinding of dsDNA by nuclear annexin A1 in the presence of Mg(2+) and ATP, they enhanced dsDNA-dependent ATPase activity of annexin A1. Heavy metals may have produced dsDNA, a substrate for the DNA unwinding reaction, via the DNA annealing reaction. DNA synthesomes were isolated from L5178Y tk(+/-) mouse lymphoma cells in exponential growth, and were found to contain helicase activities. The As(3+)- or Cr(6+)-induced increases in ssDNA binding activity of DNA synthesomes were reduced by a mono-specific anti-annexin A1 antibody, but not by anti-Ig antibody. Anti-annexin A1 antibody also blocked the inhibitory and stimulatory effects of As(3+) or Cr(6+) towards DNA unwinding and annealing activities of DNA synthesomes. Based on these observations, it can be concluded that the effects of heavy metals on DNA annealing and unwinding activities are mediated, at least in substantial part, through actions of the mono-ubiquitinated annexin A1 homodimer.

Reversal of methylprednisolone effects in allergen-exposed female BALB/c mice.

A high percentage of asthma is associated with aeroallergen exposures. Glucocorticoids such as methylprednisolone represent a major method for managing chronic asthma. However, studies suggested that corticosteroid therapy might have the potential to stimulate rather than inhibit adaptive immune inflammatory reactions, raising concerns about possible adverse reactions due to excessive repeated methylprednisolone treatment. Therefore, a murine model of allergen-induced inflammation was characterized and used to investigate the effects of repeated intraperitoneal (ip) and transnasal treatments with methylprednisolone (0-20 mg/kg body weight) and cyclosporin A (20 mg/kg body weight). Sensitized BALB/c female mice were exposed daily to ovalbumin (OVA) aerosols for up to 5 d with 24-h postexposure analyses for airway responses to methacholine aerosols and inflammatory cell recoveries by bronchoalveolar lavage (BAL) and tissue collagenase dispersion. Although increased tissue neutrophils, lymphocytes, monocytes, and macrophages reached maximal levels after 2 daily OVA exposures, recoverable eosinophil numbers continued to rise over the 5-d period. Daily ip treatments with a 5-mg/kg body weight dose of methylprednisolone diminished both OVA-induced airway responses to methacholine and inflammatory-cell accumulations to levels comparable to those observed with cyclosporin A. However, treatments with higher doses of methylprednisolone reversed this anti-inflammatory effect, indicated by a return to untreated levels of OVA-induced eosinophil recovery. A similar biphasic response in eosinophil recoveries was observed using daily transnasal methylprednisolone treatments that correlated with a concomitant fall and rise in BAL interleukin-13. These results supported the hypothesis that repeated high-steroid treatments might activate rather than suppress allergen-induced immune responses.

Cigarette smoke, inflammation, and lung injury: a mechanistic perspective.

Inflammation is a common feature in the pathogenesis of cigarette smoke-associated diseases. The recruitment of inflammatory cells into the lung following cigarette smoke exposure presents a risk of tissue damage through the release of toxic mediators, including proteolytic enzymes and reactive oxygen species. This review represents a toxicological approach to investigation of cigarette smoke-induced lung injury, with a focus on laboratory studies and an emphasis on inflammatory mechanisms. The studies discussed in this review analyze the role of inflammation and inflammatory mediators in the development of injury. In cases where information relating to cigarette smoke is limited, examples are taken from other models of lung injury applicable to cigarette smoke. The primary aim of the review is to summarize published work so as to permit (1) an evaluation of chronic lung injury and inflammatory responses in animal models, (2) a discussion of inflammatory mediators in the development of chronic injury, and (3) identification of immunological mechanisms of injury. These studies discuss the currently understood roles of cytokines, cell adhesion molecules, and oxidative stress in inflammatory reactions and lung injury. A role for lipocortin 1 (annexin 1), a naturally occurring defense factor against inflammation, is discussed because of the possibility that impaired synthesis and degradation of lipocortin 1 will influence immune responses in animals exposed to cigarette smoke either by augmenting T helper cell Th1 response or by shifting Th1 to Th2 response. While Th1 augmentation will increase the risk for development of emphysema, Th1 to Th2 shift will favor development of asthma.

Overexpression of Bcl-2 as a proxy redox stimulus to enhance activity of non-viral redox-responsive delivery vectors.

Redox-sensitive non-viral delivery systems exploit intracellular reducing environment to improve the efficacy of the delivery of nucleic acids by selectively releasing the cargo in the subcellular space. Bcl-2 overexpression is frequently observed in human cancers and is closely associated with increased resistance to chemotherapy and radiotherapy. One of the biochemical alterations accompanying Bcl-2 overexpression is the increase in cellular glutathione (GSH) levels. In this study, we hypothesize that such increase of GSH concentration will selectively enhance the transfection activity of redox-sensitive delivery systems in cells overexpressing Bcl-2. Transfection studies were conducted in MCF-7 mammary carcinoma cells and MCF-7 clones overexpressing Bcl-2. It was confirmed that Bcl-2 overexpression resulted in the expected increase in GSH concentration. Redox-sensitive complexes containing plasmid DNA, mRNA, antisense oligodeoxynucleotides, and siRNA exhibited selectively increased activity in cells overexpressing Bcl-2 compared to non-redox complexes. The effect of Bcl-2 overexpression on the selective enhancement of transfection was highly dependent on the type of the delivered nucleic acid, and was most pronounced for mRNA. This study shows that Bcl-2 overexpression can serve as a proxy redox stimulus to enhance the activity of all major classes of potential nucleic acid therapeutics, when delivered using redox-sensitive vectors.

Polyamine-based small molecule epigenetic modulators.

Chromatin remodelling enzymes such as the histone deacetylases (HDACs) and histone demethylases such as lysine-specific demethylase 1 (LSD1) have been validated as targets for cancer drug discovery. Although a number of HDAC inhibitors have been marketed or are in human clinical trials, the search for isoform-specific HDAC inhibitors is an ongoing effort. In addition, the discovery and development of compounds targeting histone demethylases are in their early stages. Epigenetic modulators used in combination with traditional antitumor agents such as 5-azacytidine represent an exciting new approach to cancer chemotherapy. We have developed multiple series of HDAC inhibitors and LSD1 inhibitors that promote the re-expression of aberrantly silenced genes that are important in human cancer. The design, synthesis and biological activity of these analogues is described herein.

DNA chain unwinding and annealing reactions of lipocortin (annexin) I heterotetramer: regulation by Ca(2+) and Mg(2+).

Lipocortin I-S100 (calcyclin) heterotetramer exhibited ATPase activity in the presence of dsDNA but not ssDNA. To demonstrate its helicase activity, an 80-mer polynucleotide complementary to the replication origin of M13mp18 was synthesized, and the oligonucleotide, (dC)(20), was ligated to either its 5'- or 3'- end for binding to lipocortin. Lipocortin I heterotetramer displaced chains of the partially Y-shaped duplexes with a dC-tail at either the 5'- or 3'- end. The chain displacement required ATP and Mg(2+). Nonhydrolyzable ATP analogues were not effective. Lipocortin I heterotetramer also catalyzed annealing of the polynucleotides to M13mp18. Ca(2+) and phospholipids but not ATP and Mg(2+) were essential for this reaction. Since the chain displacing and annealing reactions were inhibited by monospecific anti-lipocortin I or anti-S100 antibodies, the present observations suggest that the lipocortin I heterotetramer regulates unwinding and annealing of DNA by Mg(2+) (plus ATP) and Ca(2+) (and phospholipids), respectively.