Taj Mohammad

IL-12 Prevents the Inhibitory Effects of cis-Urocanic Acid on Tumor Antigen Presentation by Langerhans Cells: Implications for Photocarcinogenesis

UV radiation induces skin cancer primarily by its DNA-damaging properties, but also by its capacity to suppress the immune system. The photoisomer of urocanic acid (UCA), cis-UCA, is an important mediator of UV-induced immunosuppression and is involved in the inhibition of tumor immunity. The immunomodulatory cytokine IL-12 is known to counteract many of the immunosuppressive effects of UV radiation, including UV-induced immune tolerance. In this study, we addressed whether IL-12 also reverts the immunosuppressive activities of cis-UCA. Cis-UCA inhibits the ability of Langerhans cells to present tumor Ags for primary and secondary tumor immune responses. IL-12 treatment completely prevented the suppression by cis-UCA. IL-12 also protected mice from cis-UCA-induced suppression of contact hypersensitivity responses. To study the effects of cis-UCA on Ag-processing and Ag-presenting function in vitro, Langerhans cells were treated with UCA isomers and incubated with OVA or OVA peptide323–339 before exposure to OVA-specific transgenic T cells. Cis-, but not trans-UCA suppressed Ag presentation, which was completely reversed upon addition of IL-12. Since these findings suggest that cis-UCA may play an important role in photocarcinogenesis by inhibiting a tumor immune response, mice were chronically UVB irradiated to induce skin cancer. Whereas all mice in the control groups developed tumors, mice treated with a mAb with specificity for cis-UCA showed a significantly reduced tumor incidence. These data strongly indicate the importance of cis-UCA during photocarcinogenesis and support the concept of counteracting cis-UCA as an alternative strategy to prevent UV-induced skin cancer, possibly via the application of IL-12.

Urocanic Acid Photochemistry and Photobiology

Urocanic acid (2-propenoic acid, 3-[ lH-imidazol-4(5)-yl], UA)? is one of the smallest molecules to have stimulated global interest among biologists, environmentalists, photochemists, photobiologists, medicinal chemists and immunologists. Its history can be traced back more than a century to when it was first found in the urine of dogs (1). Interest in the molecule remained dormant until the middle of the century but rekindled in the late 1940s when it was detected in animal skin and sweat. This led to the proposal that UA acts as a natural sunscreen, possibly as a specific photoprotecting agent for DNA because of the overlap of the UA and DNA absorption spectra (2). Since 1983 there has been an explosive growth in UA research, primarily as a consequence of the proposal by De Fabo and Noonan (3) that the cis photoisomer (cUA) of trans-UA (tUA) could be responsible for the phenomenon of photoimmunosuppression. In a recent literature survey we found that about 25 research papers have appeared every year over the last decade involving UA. Several comprehensive reviews of the molecules photobiology, photochemistry and photophysics have appeared (4-

Photochemical covalent binding of urocanic acid to polynucleic acids

Photolysis of E-[ring-2-14C]urocanic acid (UA) with native or denatured calf thymus DNA leads to covalent binding of the radiolabel to the nucleic acid. A similar observation is made upon photolysis of the labeled UA with the polyribonucleotides, in which case a strong preference is observed for binding to poly[U]. DNA or poly[U], which had been reacted with UA and purified by dialysis and multiple precipitations, releases UA upon further irradiation with 254 nm light (as expected for cyclobutane adducts). Quantum efficiencies for binding of the UA to native DNA have been measured at 308 and 266 nm and are 0.30 x 10(-5) and 1.3 x 10(-4), respectively, at comparable reactant concentrations. The large increase at the shorter wavelength (where DNA absorption is more competitive) is taken as evidence for the primary role of a DNA excited state in initiating the binding reaction(s).

Photoinduced cross-linking of RNA by cis-Rh(phen)2Cl2+ and cis-Rh(phen)(phi)Cl2+: A new family of light activatable nucleic acid cross-linking agents

The metal complexes, cis-Rh(phen)2Cl2+ and its more hydrophobic analog cis-Rh(phen)(phi)Cl2+, have been shown to photocross-link the 120-base phi29-encoded pRNA. Primer extension on the cis-Rh(phen)(phi)Cl2(+)-photocross-linked RNA revealed that guanines are responsible for the interstrand cross-links.

PHOTOSENSITIZED INACTTVATION OF INFECTIOUS DNA BY UROCANIC ACID, INDOLEACRYLIC ACID AND RHODIUM COMPLEXES

Naked, infectious single‐stranded (ss) and double‐stranded (ds) DNA from phages SI3 and G4 were irradiated with 308 nm UV radiation in the absence and presence of several photobiologically active compounds: E‐ and Z‐urocanic acid (E‐ and Z‐UA), their methyl esters (E‐ and Z‐MU), E‐ and Z‐indoleacrylic acid (E‐ and Z‐IA), cis‐dichlorobis(1,10‐phenanthroline)rhodium(III) chloride (cDCBPR) and tris(1,10‐phenanthroline)rhodium (III) perchlorate (TPR). E‐urocanic acid protects against cyclobutane pyrimidine dimer formation in ssDNA but concomitantly photosensitizes the formation of other lesions that inactivate ssDNA. Z‐urocanic acid also protects ssDNA against such dimerization but without the associated sensitized damage. The methyl ester isomers behave similarly. There is no such differential activity observed for the IA isomers, both of which sensitize the inactivation of ssDNA. Photostationary state mixtures of both UA and IA efficiently sensitize the inactivation of dsDNA, and cDCBPR strongly protects ssDNA from UV damage, while TPR is a significant sensitizer. Both of these metal complexes sensitize the inactivation of dsDNA slightly. For all compounds, cyclobutane pyrimidine dimers were the predominant lethal lesions produced by sensitization of the dsDNA, but they were not the major lethal lesions created by sensitization of the ssDNA. In the case of dsDNA, both UA and IA created pyrimidine dimers with a high degree of potential for mutagenesis, as determined by an assay that monitors the frequency of mutations following the spontaneous deamination of cytosine in photodimers.

Photobiological properties of methylene violet.

The interaction of methylene violet (MV) and 4‐bromo‐methylene violet (BrMV) with calf thymus and super‐coiled φX174 phage RF I DNA is reported. Measurements employing UV‐visible absorption spectroscopy and equilibrium dialysis give evidence for the formation of complexes by each dye with DNA in the dark. They covalently bind to DNA, and MV nicks DNA, when the nucleic acid/dye mixtures are irradiated with visible light in a deoxygenated environment. Quantum efficiencies for singlet oxygen formation are 0.27 and 0.25 for MV and BrMV, respectively. A higher value (0.49) is observed for 4‐iodomethyIene violet (IMV).

Simultaneous Photoconjugation of Methylene Blue and cis-Rh(phen)2Cl2+ to DNA via a Synergistic Effect †

Irradiation of the red‐light absorbing dye, methylene blue (MB), in the presence of the metal complex, cis‐Rh(phen)2Cl2+ (BISPHEN), leads to irreversible photobinding of both reagents to DNA. Evidence from absorption and emission spectroscopy indicates that the dye is strongly complexed to the DNA at the concentrations used in the experiments and that this complex is unaffected by the presence of BISPHEN. The level of covalent binding is proportional to the absorbed light dose, with the quantum efficiency for covalent binding of BISPHEN to the DNA with 633 nm light equal to 3.5 × 10−4. Electrospray ionization mass spectrum of a mixture of DNA fragments created by enzymatic degradation of DNA isolated following irradiation indicates that purine adducts are formed with both BISPHEN and the dye. In addition, UV–Vis and high‐performance liquid chromatography analyses of the irradiated MB/BISPHEN/DNA mixture and isolated adducts show extensive conversion of the dye and metal complex to the corresponding N‐demethylated and aquated derivatives, respectively. Triplet quenchers for MB, for example oxygen and benzoquinone, inhibit both the photoconjugation and the photochemistry of BISPHEN. A mechanism for the synergistic interaction is proposed that involves photoconjugation of both partners to the DNA following oxidation and reduction via electron transfer between 1MB*/DNA and 3MB*/BISPHEN.

PHOTOLYTIC COVALENT BINDING OF INDOLEACRYLIC ACID TO DNA

Abstract— E‐β‐IndoI‐3‐ylacrylic acid (IA), radiolabeled at the 2 position with l+C, and calf thymus DNA have been irradiated with UV light (Λ > 280 nm) in phosphate buffer (pH 7.0). Re‐isolation of the DNA indicates covalent binding of IA at levels of up to 295 nmol IA/mg native DNA (0.097 IA/ base). Binding is observed for both native and heat denatured DNA, but is more efficient with the latter. Quantum efficiencies of 2.60 times 10−5 and 2.30 times 10 −4 mol IA bound to native DNA/mol photon absorbed have been measured at 308 and 266 nm, respectively. Studies with the four polyribonucleotides indicate a strong preference for binding to poly[U]. Photolysis of either untreated or enzymatically degraded labeled native DNA with 254 nm light leads to the reformation of IA, and a 2 + 2 photocy‐cloadduct of IA and thymidine has been isolated and characterized and matched by HPLC to a DNA derived adduct. Equilibrium dialysis studies provide no evidence for preassociation of IA to DNA.

Simultaneous determination of methylene violet, halogenated methylene violet and their photoproducts in the presence of DNA by high-performance liquid chromatography using an internal surface reversed-phase column

A simple and rapid isocratic high-performance liquid chromatographic method for the analysis of methylene violet, a neutral phenothiazine dye, in the presence of DNA has been developed. These chromatographic conditions are also applicable to its N-demethylated, bromo and iodo analogs. The method utilizes an internal surface reversed-phase column and a mobile phase consisting of 20% acetonitrile in 50 mM phosphate buffer (pH 7.0) and detection at 280 nm. Under these conditions all five dyes are well resolved from one another and from the faster migrating DNA. The effects of organic modifiers, ionic strength and pH of the buffer on the capacity factors of the dyes have been investigated. The method has successfully been applied to detect the photoproducts of methylene violet and its bromo analog in the presence of DNA without removing the biopolymer from the reaction mixture.

Photochemical covalent binding of p-methoxycinnamic acid to calf thymus DNA

Abstract Irradiation of E- p -[α- 14 C]methoxycinnamic acid and calf thymus DNA with uv light leads to incorporation of the radiolabel into the DNA. Sephadex chromatography confirms that the label incorporation is due to covalent bond formation. Photobinding is more efficient with denatured vs native DNA and with higher frequency light, e.g., overall quantum efficiencies at 308 nm for denatured and native DNA are 1.1 and 0.6 × 10 −5 , respectively, vs 22.0 and 8.0 × 10 −5 for 266 nm, respectively (values for 0.7 m M pMCA and ca. 0.7 mg/ml DNA). A comparative study with several polyribonucleotides shows the relative selectivity for binding to be poly(C) > poly (A) ⪢ poly(G). Photolysis of labeled DNA with 254-nm light leads to the release of both isomers of pMCA, evidence that at least a portion of the covalent binding is due to the formation of cyclobutane adducts. Equilibrium dialysis studies give no evidence for preassociation of pMCA and DNA.