Time-Resolved FRET -Based Approach for Antibody Detection - A New Serodiagnostic Concept

Peer reviewe

Adenomatous Polyposis Coli-Mediated Accumulation of Abasic DNA Lesions Lead to Cigarette Smoke Condensate-Induced Neoplastic Transformation of Normal Breast Epithelial Cells

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5′-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients

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Not AvailableNot AvailablePoint pollution of pesticides originating from the washing of spraying machines could be controlled by biobed system and it is in use in temperate countries. The biobed system is yet to be established in tropical countries. An indigenous biobed system was prepared using local resources like rice straw, farm yard manures (FYM) and paddy field soil to suit the tropical climate. Lowermost 3 cm layer of the biobed system was filled with rice husk biochar to prevent leaching of pesticides from the system. This model system was tested with high doses of imidacloprid (178 mg/column), a commonly used pesticide against number of insect-pests in different crops, for its degradation. The bio-mix trapped a major part of the imidacloprid on the top most layer of the biobed column and only a very small part of imidacloprid recovered from the leachate. The biobed system could degrade 70.13% of applied imidacloprid within 15 days of the experiment and only 5.27% of the total pesticide recovered 90 days after incubation. Addition of biochar layer adsorbed imidacloprid from the outgoing leachate from the biobed column. Biomixture boosted microbial activity more particularly fungal population, which might be responsible for imidacloprid degradation. Microbial biomass carbon, and soil enzymes indicated faster dissipation of imi- dacloprid from the top layer of the biobed. This simple but efficient biobed system using local resources can fulfill the need of the small and marginal farmers of Asian countries for pesticide decontamination.Not Availabl

Effect of NSC666715 and PFTα on TMZ-induced levels of apoptosis-related proteins.

<p>The HCT116 cells were pretreated with different concentrations of PFTα and 50 μM NSC666715 for 2 h and then with 500 μM TMZ alone or in combination for an additional 48 h. Cells were harvested and the cellular lysates were prepared and processed for Western blot analysis. The Western blot analysis data are representative of two different experiments.</p

TMZ-induced senescence in HCT116 cells is dependent upon the p53/p21 pathway.

<p>HCT116 cells with or without p53 and p21 expression were treated with 500 μM TMZ for 48 h, and then processed for SA-βgal staining. <b>Panel A</b> shows the involvement of p53 and p21 in TMZ-induced senescence. <b>Panel B</b> represents quantitative analysis of the data, which are presented as mean ± SE of four different estimations. * = Significantly different than the untreated control (p<0.05).</p

Effect of TMZ on senescence in HCT116 cells.

<p>HCT116 cells were treated with different concentrations of TMZ for 48 h and then processed for SA-βgal staining. <b>Panel A</b> shows SA-βgal staining. <b>Panel B</b> represents the quantitative analysis of SA-βgal staining. Data are presented as mean ± SE of four different estimations. * = Significantly different than the untreated control (p<0.05).</p

Effect of PFTα on the cell cycle phases of HCT116 cells treated with TMZ and NSC666715 either alone or in combination.

<p>Cells were treated with different concentrations of drugs as indicated for 48 h. Cell cycle status and apoptosis were examined by FACS analysis. The percent distribution of cells in the G₀/G₁, S, G₂/M and sub-G₁ (apoptotic) phases of the cell cycle were established on the basis of the corresponding DNA content of the histograms. At least 10,000 cells per sample were considered in the gated regions for calculations. Data are presented as mean ± SE of three different estimations.</p><p>^§ = Significantly different than the control (p <0.05).</p><p>Effect of PFTα on the cell cycle phases of HCT116 cells treated with TMZ and NSC666715 either alone or in combination.</p

NSC666715 enhances TMZ-induced senescence in HCT116 cells.

<p>The HCT116 cells were pretreated with different concentrations of NSC666715 for 2 h followed by treatment with 250 μM of TMZ for an additional 48 h. After treatment, the cells were processed for SA-βgal staining. <b>Panel A</b> shows SA-βgal staining. <b>Panel B</b> represents the quantitative analysis of SA-β gal staining. Data are presented as mean ± SE of four different estimations. * and ^# = Significantly different than the untreated control or the 10, 25, 50 and 100 μM NSC666715 treated groups, respectively, (p<0.05).</p

NSC666715 and its analogs block Pol-β-directed LP-BER activity.

<p>The LP-BER was determined using an <i>in vitro</i> reconstituted assay system. <b><i>Panel A</i></b> shows the experimental protocol. The assay details are given in Materials and Methods. <b><i>Panel B</i></b> shows an autoradiogram of LP-BER, which is representative of three different experiments. Lane 1 shows ³²P-labeled 63-mer F-DNA and Lane 2 shows the 23-mer product after APE1 incision. Lane 3 shows Pol-β-mediated strand-displacement synthesis. Lane 4 shows Pol-β-mediated strand-displacement synthesis stimulated by Fen1 (Lane 4). Lane 5 shows the complete repair of the 63-mer F-DNA through the LP-BER pathway in the presence of APE1, Pol-β, Fen1 and DNA ligase I. Lanes 6–9, 10–13, 14–17, 18–21 and 22–25 show the effect of different concentrations of the Pol-β inhibitors NSC 661073, 666713, 666715, 666717 and 666719 on LP-BER activity.</p