Down-regulation of survivin suppresses uro-plasminogen activator through transcription factor JunB

Survivin, a member of the inhibitors of apoptosis protein family, is expressed during development and in various human cancers. However, the clinical relevance of survivin in cancer is still a matter of debate. Genes induced by hepatocyte growth factor (HGF) were screened using cDNA microarray technology in the stomach cancer cell lines, NUGC3 and MKN28. The levels of JunB, survivin, and uro-plasminogen activator (uPA) were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up regulation of JunB, survivin, and uPA was inhibited by pre-treatment with a MEK inhibitor (PD 98059). HGF-induced up-regulation of uPA was repressed by survivin knockdown. HGF enhanced the binding activity of JunB to the survivin promoter in control cells, but not in the JunB-shRNA cells. Transfection with survivin- shRNA resulted in a decrement of cell proliferation, as determined with MTT assays. In an in vitro invasion assay, significantly fewer cells transfected with survivin shRNA than control cells were able to invade across a Matrigel membrane barrier. In conclusion, survivin appeared to play an important role in the up-regulation of uPA induced by HGF via JunB and might contribute to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.     

Extent of formation of a dimeric adenine photoproduct in polynucleotides and DNA

Quantum yields are reported for the formation of a dimeric adenine photoproduct, A = A, in adenine homopolymers and DNA irradiated at 254 nm. The A = A content of irradiated samples was assayed by using reversed-phase HPLC to isolate the 4,6-diamino-5-guanidinopyrimidine (DGPY) which is produced from A = A on acid hydrolysis. Acid hydrolysates derived from DNA radiolabelled with [14C] 2'-deoxyadenosine were spiked with unlabelled DGPY before fractionation on HPLC and the recovered material was further purified by chromatography on Sephadex G-10 followed by co-crystallization with DGPY sulphate. Although A = A is formed with a relatively high quantum yield of 1.6 X 10(-3) mol einstein-1 in single-stranded poly(dA) the photoaddition reaction is strongly quenched in base-paired poly(dA).poly(dT) and undetectable in poly(rA).poly(dT). Respective quantum yields of 6 X 10(-5) and 9 X 10(-6) were estimated for the formation of A = A in single- and double-stranded E. coli DNA implying that the photoproduct has very limited biological significance. From studies with d(ApG), d(GpA), ApG, GpA, d(A)20 and d(A4G)4 it is concluded that adjacent guanine and adenine bases do not form a photoadduct analogous to A = A and also that guanine residues have no local or long-range quenching effect on photodimerization within A-A doublets.     

The sugar conformation governs (6-4) photoproduct formation at the dinucleotide level

The LNA dinucleotide mimic of TpT whose two-sugar puckers are locked in the C3'-endo conformation selectively produces the corresponding cyclobutane pyrimidine dimer under 254 nm irradiation. In the natural series (TpT) the sugar puckers are in a major C2'-endo sugar conformation and the (6-4) photoproduct is also produced. Consequently, this study demonstrates that the C2'-endo conformation of the sugar pucker is necessary for (6-4) photoproduct formation.     

Monitoring photoproduct formation and photobleaching by fluorescence spectroscopy has the potential to improve PDT dosimetry with a verteporfin-like photosensitizer

In current clinical practice, photodynamic therapy (PDT) is carried out with prescribed drug doses and light doses as well as fixed drug-light intervals and illumination fluence rates. This approach can result in undesirable treatment outcomes of either overtreatment or undertreatment because of biological variations between different lesions and patients. In this study, we explore the possibility of improving PDT dosimetry by monitoring drug photobleaching and photoproduct formation. The study involved 60 mice receiving the same drug dose of a novel verteporfin-like photosensitizer, QLT0074, at 0.3 mg/kg body weight, followed by different light doses of 5, 10, 20, 30, 40 or 50 J/cm2 at 686 nm and a fluence rate of 70 mW/cm2. Photobleaching and photoproduct formation were measured simultaneously, using fluorescence spectroscopy. A ratio technique for data processing was introduced to reliably detect the photoproduct formed by PDT on mouse skin in vivo. The study showed that the QLT0074 photoproduct is stable and can be reliably quantified. Three new parameters, photoproduct score (PPS), photobleaching score (PBS) and percentage photobleaching score (PBS%), were introduced and tested together with the conventional dosimetry parameter, light dose, for performance on predicting PDT-induced outcome, skin necrosis. The statistical analysis of experimental results was performed with an ordinal logistic regression model. We demonstrated that both PPS and PBS improved the prediction of skin necrosis dramatically compared to light dose. PPS was identified as the best single parameter for predicting the PDT outcome.     

The origin of stark splitting in the initial photoproduct state of MbCO

Ligand migration and binding in heme proteins have been measured by X-ray diffraction and time-resolved spectroscopy of photoproduct intermediates. In myoglobin (Mb), internal cavities serve as docking sites for carbon monoxide (CO) ligands. In these sites, the CO ligands display characteristic infrared (IR) stretching bands due to interactions with the local electrical field. In the primary docking site, a CO can reside in two opposite orientations, characterized by a doublet of infrared bands, B1 at approximately 2130 and B2 at approximately 2120 cm-1. To assign these bands to the specific orientations, we have reexamined the effects of mutating His64 and Val68 on the infrared stretching bands associated with the B1 and B2 photoproduct states. Wild-type, H64L, V68F, and H64L-V68F MbCO were selected for experimental and theoretical analyses. Fourier transform infrared (FTIR) spectroscopy and density functional theory (DFT) calculations were used to interpret the effects of the electrostatic environment on the B state bands. The imidazole side chain of His64 appears to be the primary cause of the observed Stark splitting. The high-frequency B1 band is assigned to the CO orientation in which the carbon (white atom) is directed toward the heme iron and the Nepsilon-H proton of His64. At low temperatures, CO molecules in the opposite orientational conformer, B2 with the O atom (red) toward His64, first rotate by 180 degrees into the more stable B1 state and then rebind.     

Electrospray ionization mass spectrometry probing of formation and recognition of the G‐quadruplex in the proximal promoter of the human vascular endothelial growth factor gene

The formation of the G‐quadruplex of the vascular endothelial growth factor (VEGF) gene was probed by electrospray ionization mass spectrometry (ESI‐MS). It found that cations (K⁺ and NH), CH₃OH and pH influence significantly the formation of the G‐quadruplex structure. Additionally, a perylene derivative (P3) and polydatin (P4) have shown to be potential G‐quadruplex binding agents with structurally specific recognition. Copyright © 2010 John Wiley & Sons, Ltd.     

The role played by an iodine-containing promoter in the formation of active polycrystalline silver catalyst surface

The programmed temperature desorption method was used to study the interaction of oxygen with the surface of a polycrystalline silver catalyst promoted with iodine. Ethyl iodide almost did not interact with the unoxidized surface of silver. The adsorption of C₂H₅I on the oxidized catalyst surface resulted in the formation of two adsorbed iodine forms, silver iodide and iodine deeply dissolved in subsurface silver crystal lattice layers. The character of oxygen adsorption from the iodine-containing surface of the catalyst was determined by the amount and form of adsorbed iodine. In the presence of a iodine-containing promoter, the concentration of oxide-like oxygen sharply decreased, and the amount of strongly bound atomically adsorbed oxygen responsible for the selective transformation of ethylene glycol into glyoxal increased.     

Direct H atom abstraction from spore photoproduct C-6 initiates DNA repair in the reaction catalyzed by spore photoproduct lyase: evidence for a reversibly generated adenosyl radical intermediate

Spore photoproduct (SP) lyase, which catalyzes the direct reversal of SP (5-thyminyl-5,6-dihydrothymine) to thymine monomers, is the only identified nonphotoactivatable pyrimidine dimer lyase. Unlike DNA photolyase, SP lyase does not contain a flavin cofactor and does not require light for activation. Instead, preliminary studies point to the presence of an iron-sulfur cluster in SP lyase and the requirement for S-adenosylmethionine (AdoMet) for catalytic activity, suggesting that SP lyase belongs to the growing group of iron-sulfur cluster and AdoMet-dependent radical enzymes. Here we provide evidence for the role of AdoMet as a reversible deoxyadenosyl radical generator, which initiates repair by hydrogen atom abstraction from C-6 of SP. Reaction of 6-(3)H-SP, but not methyl-(3)H-SP, with SP lyase and AdoMet results in transfer of (3)H to AdoMet, while no tritiated 5'-deoxyadenosine is observed. When 5'-tritiated AdoMet is used in the reaction with unlabeled SP, transfer of (3)H into the repaired thymine monomers is observed. These results point to the reversible generation of a 5'-deoxyadenosyl radical intermediate, which reacts directly with the DNA lesion to initiate a radical-mediated beta-scission. We also demonstrate that AdoMet is a catalytic cofactor that is not consumed during turnover. Together, these results support a novel radical-based mechanism for the repair of UV-induced DNA damage.