Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra-and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 µg of DNA. This sensitivity may allow the method to be used for clinical applications.

We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.

The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative. We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion. Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium. In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C. The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site. Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage. Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions. SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations. We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase. Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo.

(trans-R, R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a recently approved platinum analogue for use in the chemotherapy of metastatic colorectal cancer. Like many cytotoxic drugs, oxaliplatin exerts its antitumor effects by covalent modification of DNA. We report an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair. We determined the apparent rate of oxaliplatin adduction to salmon sperm DNA. The oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by HPLC-AMS. Cultured platinum-sensitive testicular (833K) and platinum-resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells were incubated with a subpharmacological concentration of oxaliplatin (0.2 microM). Both cellular and DNA radiocarbon contents in the drug-sensitive testicular cells had approximately twice the area under the curve as compared to the more platinum-resistant cell lines, implying that differential accumulation of the drug may be responsible for the sensitivity of cancer cells to platinum treatment. The lowest concentration of radiocarbon measured was approximately 1+/-0.1 amol/microg of DNA, when assaying 1 microg of DNA. This sensitivity for measuring oxaliplatin-DNA adducts is the highest reported to date. The sensitivity offered by this method may be applicable to other DNA-damaging drugs, metabolisms studies, and diagnostics development.

Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2 -deoxyguanosine (8-oxodG), which may initiate diseases related to aging and carcinogenesis. Kinetic measurement of 8-oxodG metabolism and repair in cells has been hampered by poor assay sensitivity and by difficulty characterizing the flux of oxidized nucleotides through the relevant metabolic pathways. We report here the development of a sensitive and quantitative approach to characterizing the kinetics and metabolic sources of 8-oxodG in MCF-7 human breast cancer cells by accelerator mass spectrometry. We observed that [ 14 C]8-oxodG at medium concentrations of up to 2 pmol/ml was taken up by MCF-7 cells, phosphorylated to mono-, di-, and triphosphate derivatives, and incorporated into DNA. Oxidative stress caused by exposure of the cells to 17␤-estradiol resulted in a reduction in the rate of [ 14 C]8-oxodG incorporation into DNA and an increase in the ratio of 8-oxodG monophosphate (8-oxodGMP) to 8-oxodG triphosphate (8-oxodGTP) in the nucleotide pool. 17␤-Estradiolinduced oxidative stress up-regulated the nucleotide pool cleansing enzyme MTH1 and possibly other Nudix-related pyrophosphohydrolases. These data support the conclusion that 8-oxodGTP is formed in the nucleotide pool by both 8-oxodG metabolism and endogenous reactive oxygen species. The metabolism of 8-oxodG to 8-oxodGTP, followed by incorporation into DNA is a mechanism by which the cellular presence of this oxidized nucleoside can lead to mutations.DNA repair ͉ nucleoside metabolism ͉ oxidative stress ͉ breast cancer

The first organic artificial proteinase based on synthetic materials is obtained by building active sites on the backbone of poly(ethylenimine) (PEI). The active site comprising three salicylate residues is prepared by preassemblage of three molecules of a salicylate derivative with Fe(III) ion followed by crosslinkage of the salicylates with PEI and demetalation of the resulting polymer. Proteinase activity of the artificial enzyme is demonstrated with hydrolytic cleavage of γ-globulin (Gbn). Both the heavy (50 kDa) and the light (25 kDa) chains of Gbn are effectively cleaved into peptides smaller than 5 kDa. The optimum activity of the artificial proteinase is manifested at pH 5-7. The half-life for cleavage of the two chains of Gbn by the artificial proteinase is ∼1 h at pH 7 and 50 °C when the concentration of the artificial active site is 0.4-1 mM. The activity (at pH 7) of the artificial proteinase prepared with PEI is comparable to that (at pH 9) of a catalytic antibody elicited by a joint hybridoma and combinatorial antibody library approach and to that (at pH < 3) achieved by the intramolecular carboxyl group in the hydrolysis of N-methyl maleamic acid. Thus, the approach reported here is useful for designing active sites of artificial enzymes.

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APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a K m of 9.32 μM and a V max of 1.39 μmol min −1 mg −1 . The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.squamous carcinoma | systemic inflammatory response syndrome T he programmed death of dangerous cells, either infected or transformed, has critical importance for the survival of the multicellular organism and therefore is also of great medical relevance. APIP, Apaf-1 interacting protein, was initially identified as an inhibitor of apoptotic cell death induced by hypoxia/ ischemia and cytotoxic drugs (1). Recently APIP was also shown to inhibit pyroptosis, an inflammatory form of cell death, induced by Salmonella infection (2). Thus, APIP has been implicated in two major types of programmed cell death: apoptosis and pyroptosis. In apoptosis, APIP inhibits the mitochondrial pathway involving caspase-9 but not the receptor pathway involving caspase-8 (1, 3). In pyroptosis, APIP's inhibitory function was recently revealed in a functional genetic screen for the SNP associated with increased caspase-1-mediated cell death in response to Salmonella infection (2) and subsequently confirmed by cell viability assays (2, 4). Intriguingly, other SNPs near APIP were found in patients suffering from systemic inflammatory response syndrome (2), which further implicates APIP in inflammation.Distinct from its inhibitory role in the programmed cell death, APIP was recently shown to act as an enzyme in the methionine salvage pathway (2, 4). The amino acid sequence of human APIP exhib...