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A Novel High-Through-Put Assay for Screening of Pro-Apoptotic Drugs

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Abstract

Screening for anti-cancer substances iscommonly conducted using viability assays.An inherent problem with this approach isthat all compounds that are toxic andgrowth inhibitory, irrespective ofmechanism of action, will score positive.It would be beneficial to be able to screenfor compounds that specifically induceapoptosis. We here describe an ELISA-assaybased on a monoclonal antibody (M30) whichrecognizes a neo-epitope on cytokeratin 18exposed after cleavage by caspases duringapoptosis. We show that this assay detectsapoptosis in epithelial cells and that thesensitivity is sufficient for screening inthe 96-well format. We used the M30-ELISAassay to screen 500 low molecular weightcompounds from a chemical library from theNational Cancer Institute and identified 16drugs with strong pro-apoptotic activity,suggesting that the assay is a useful toolfor discovery of pro-apoptotic drugs.

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References

  1. Twentyman PR, Luscombe M: A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer 56:279–285, 1987

    Google Scholar 

  2. Larsson R, Nygren P: Pharmacological modification of multidrug resistance (MDR) in vitro detected by a novel fluorometric microculture cytotoxicity assay. Reversal of resistance and selective cytotoxic actions of cyclosporin A and verapamil on MDR leukemia T-cells. Int J Cancer 46:67–72, 1990

    Google Scholar 

  3. Viktorsson K, Heiden T, Molin M, Akusjärvi G, Linder S, Shoshan MC: Increased apoptosis and increased clonogenic survival of 12V-RAS transformed rat fibroblasts in response to cisplatin. Apoptosis 5:355–367, 2000

    Google Scholar 

  4. Kaufmann SH, Earnshaw WC: Induction of apoptosis by cancer chemotherapy. Exp Cell Res 256:42–49, 2000

    Google Scholar 

  5. Costantini P, Jacotot E, Decaudin D, Kroemer G: Mitochondrion as a novel target of anticancer chemotherapy. J Natl Cancer Inst 92:1042–1053, 2000

    Google Scholar 

  6. Caulin C, Salvesen GS, Oshima RG: Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 138:1379–1394, 1997

    Google Scholar 

  7. Ku NO, Liao J, Omary MB: Apoptosis generates stable fragments of human type I keratins. J Biol Chem 272:33197–33203, 1997

    Google Scholar 

  8. MacFarlane M, Merrison W, Dinsdale D, Cohen GM: Active caspases and cleaved cytokeratins are sequestered into cytoplasmic inclusions in TRAIL-induced apoptosis. J Cell Biol 148:1239–1254, 2000

    Google Scholar 

  9. Leers_MP, Björklund V, Bergman T, Tribbick G, Persson B, Björklund P, Ramaekers FC, Björklund B, Nap MHJ, Schutte B: Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187:567–572, 1999

    Google Scholar 

  10. Runnebaum IB, Nagarajan M, Bowman M, Soto D, Sukumar S: Mutations in p53 as potential molecular markers for human breast cancer. Proc Natl Acad Sci USA 88:10657–10661, 1991

    Google Scholar 

  11. Zhou J-N, Linder S: Expression of CDK inhibitor genes in immortalized and carcinoma derived breast cell lines. Anticancer Res 16:1931–1936, 1996

    Google Scholar 

  12. Vanhamme L, Szpirer C: Transforming activity of the human mammary line HBL100 DNA is associated with SV40 large T antigen genetic information integrated in its genome. Carcinogenesis 9:653–655, 1988

    Google Scholar 

  13. Rydlander L, Ziegler E, Bergman T, Schoberl E, Steiner G, Bergman AC, Zetterberg A, Marberger M, Bjorklund P, Skern T, Einarsson R, Jornvall H: Molecular characterization of a tissue-polypeptide-specific-antigen epitope and its relationship to human cytokeratin Eur J Biochem 241:309–314, 1996

    Google Scholar 

  14. Janicke RU, Sprengart ML, Wati MR, Porter AG: Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem 273:9357–9360, 1998

    Google Scholar 

  15. Ross WE, Bradley MO: DNA double-stranded breaks in mammalian cells after exposure to intercalating agents. Biochim Biophys Acta 654:129–134, 1981

    Google Scholar 

  16. Tewey KM, Chen GL, Nelson EM, Liu LF: Intercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:9182–9187, 1984

    Google Scholar 

  17. Ohashi M, Sugikawa E, Nakanishi N: Inhibition of p53 protein phosphorylation by 9-hydroxyellipticine: a possible anticancer mechanism. Jpn J Cancer Res 86:819–827, 1995

    Google Scholar 

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Hägg, M., Bivén, K., Ueno, T. et al. A Novel High-Through-Put Assay for Screening of Pro-Apoptotic Drugs. Invest New Drugs 20, 253–259 (2002). https://doi.org/10.1023/A:1016249728664

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  • DOI: https://doi.org/10.1023/A:1016249728664

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