Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Identification of Novel Regulators of Apoptosis Using a High-Throughput Cell-based Screen

  • Park, Kyung Mi (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kang, Eunju (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Jeon, Yeo-Jin (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Nayoung (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Nam-Soon (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yoo, Hyang-Sook (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yeom, Young Il (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Soo Jung (Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2006.10.27
  • Accepted : 2007.01.31
  • Published : 2007.04.30
⟨ Previous Next ⟩

Abstract

High-throughput subcellular imaging is a powerful tool for investigating the function of genes. In order to identify novel regulators of apoptosis we transiently transfected HeLa cells with 938 hypothetical genes of unknown function, and captured their nuclear images with an automated fluorescence microscope. We selected genes that induced greater than 3-fold increase in the percentage of apoptotic nuclei compared with vector-transfected cells. The full-length genes C10orf61, MGC 26717, and FLJ13855 were identified as candidate proapoptotic genes, and their apoptotic effects were confirmed by DNA fragmentation ELISAs and Western blotting for caspase-7 and PARP. We conclude that a subcellular image-based apoptotic screen is useful for identifying genes with proapoptotic activity.

Keywords

Acknowledgement

Supported by : Korea Research Institute of Bioscience and Biotechnology

References

  1. Albayrak, T. and Grimm, S. (2003) A high-throughput screen for single gene activities: isolation of apoptosis inducers. Biochem. Biophys. Res. Commun. 304, 772−776
  2. Gasparri, F., Mariani, M., Sola, F., and Galvani, A. (2004) Quantification of the proliferation index of human dermal fibroblast cultures with the $ArrayScan^{TM}$ high-content screening reader. J. Biomol. Screen. 9, 232−242
  3. Hutchin, M. E., Kariapper, M. S., Grachtchouk, M., Wang, A., Wei, L., et al. (2005) Sustained hedgehog signaling is required for basal cell carcinoma proliferation and survival: conditional skin tumorigenesis recapitulates the hair growth curve. Genes Dev. 19, 214−223
  4. Kim, N-S., Hahn, Y., Oh, J. H., Lee, J. Y., Oh, K. J., et al. (2004) Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags. Genomics 83, 1024−1045
  5. Kittler, R., Putz, G., Pelletier, L., Poser, I., Heninger, A.-K., et al. (2004) An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division. Nature 432, 1036−1040
  6. Koenig-Hoffman, K., Bonin-Debs, A., Boche, I., Beate, G., Gnirke, A., et al. (2005) High throughput functional genomics: identification of novel genes with tumor suppressor phenotypes. Int. J. Cancer 113, 434−439
  7. Kolfschoten, I. G., van Leeuwen, B., Berns, K., Mullenders, J., Beijersbergen, R. L., et al. (2005) A genetic screen identifies a PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 121, 849−858
  8. Lovborg, H., Nygren, P., and Larsson, R. (2004) Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828. Mol. Cancer Ther. 3, 521−526
  9. MacKeigan, J. P., Murphy, L. O., and Blenis, J. (2005) Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat. Cell Biol. 7, 591−600
  10. Mannherz, O., Mertens, D., Hahn, M., and Lichter, P. (2006) Functional screening for proapoptotic genes by reverse transfection cell array technology. Genomics 87, 665−672
  11. Oh, J. H., Sohn, H. Y., Kim, J-M., Kim, Y. S., and Kim, N. S. (2004) Construction of multi-purpose vectors pCNS and pCNS-D2 are suitable for collection and functional study for large-scale cDNAs. Plasmid 51, 217−226
  12. Reiter, J. F. and Skarnes, W. C. (2006) Tectonic, a novel regulator of the hedgehog pathway required for both activation and inhibition. Genes Dev. 20, 22−27
  13. Shkumatava, A. and Neumann, C. J. (2005) Shh directs cellcycle exit by activating p57Kip2 in the zebrafish retina. EMBO Rep. 6, 563−569
  14. Westbrook, T. F., Martin, E. S., Schlabach, M. R., Leng, Y., Liang, A. C., et al. (2005) A genetic screen for candidate tumor suppressors identifies REST. Cell 121, 837−848
  15. Yang, Y. and Yu, X. (2003) Regulation of apoptosis: the ubiquitous way. FASEB J. 17, 790−799