Identification of Differentially Expressed Genes by Exposure of Methylmercury in Neuroblastoma Cell Line Using Suppression Subtractive Hybridization (SSH)

  • Kim, Youn-Jung (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology) ;
  • Ryu, Jae-Chun (Cellular and Molecular Toxicology Laboratory, Korea Institute of Science and Technology)
  • 발행 : 2006.03.31

초록

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. This study, using of suppression subtractive hybridization (SSH) method, was peformed to identify differentially expressed genes by MeHg in SH-SY5Y human neuroblastoma cell line. We prepared to total RNA from SH-SY5Y cells treated with solvent (DMSO) and $6.25\;{\mu}M\;(IC_{50})$ MeHg and performed forward and reverse SSH. Differentially expressed cDNA clones were screened by dot blot, sequenced and confirmed that individual clones indeed represent differentially expressed genes with real time RT-PCR. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as ubiquitous environmental pollutants.

키워드

참고문헌

  1. U.S. Environmental Protection Agency. Mercury Study Report to Congress, Volume V: Health effects of mercury and mercury compounds. EPA-452/R-97- 007. Washington, DC: U.S. Environmental Protection Agency; (1997)
  2. Choi, B.H. The effects of methylmercury on the developing brain. Prog. Neurobiol. 32, 447-470(1989) https://doi.org/10.1016/0301-0082(89)90018-X
  3. Castoldi, A.F., Coccini, T., Ceccatelli, S. & Manzo, L. Neurotoxicity and molecular effects of methylmercury. Brain Res. Bull. 55, 197-203 (2001) https://doi.org/10.1016/S0361-9230(01)00458-0
  4. Vogel, D.G., Margolis, R.L. & Mottet, N.K. The effects of methylmercury binding to microtubules. Toxicol. Appl. Pharmacol. 80, 473-486 (1985) https://doi.org/10.1016/0041-008X(85)90392-8
  5. Naganuma, A. et al. Overexpression of manganesesuperoxide dismutase prevents methylmercury toxicity in HeLa cells. Life Sci. 62, 157-161 (1998) https://doi.org/10.1016/S0024-3205(98)00037-X
  6. Kim, Y.J., Chai, Y.G & Ryu, J.C. Selenoprotein W as molecular target of methylmercury in human neuronal cells is down-regulated by GSH depletion. Biochem. Biophys. Res. Commun. 330(4), 1095-1102 (2005) https://doi.org/10.1016/j.bbrc.2005.03.080
  7. Liang, P. & Pardee, A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967-971 (1992) https://doi.org/10.1126/science.1354393
  8. Sokolov, B.P. & Prockop, D.J. A rapid and simple PCR-based method for isolation of cDNAs from differentially expressed genes. Nucl. Acids Res. 22, 4009 -4015 (1994) https://doi.org/10.1093/nar/22.19.4009
  9. Lisitsyn, N. & Wigler, M. Cloning the differences between two complex genomes. Science 259, 946- 951 (1993) https://doi.org/10.1126/science.8438152
  10. Akopian, A.N. & Wood, J.N. Peripheral nervous system-specific genes identified by subtractive cDNA cloning. J. Biol. Chem. 270, 21264-21270(1995) https://doi.org/10.1074/jbc.270.36.21264
  11. Deleersnijder, W. et al. Isolation of markers for chondro- osteogenic differentiation using cDNA library subtraction. Molecular cloning and characterization of a gene belonging to a novel multigene family of integral membrane proteins. J. Biol. Chem. 271, 19475-19482 (1996) https://doi.org/10.1074/jbc.271.32.19475
  12. Butte, A. The use and analysis of microarray data. Nat. Rev. Drug Discov. 1, 951-960 (2002) https://doi.org/10.1038/nrd961
  13. Diatchenko, L. et al. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025-6030 (1996)
  14. Von Stein, O.D., Thies W.G. & Hofmann, M. A high throughput screening for rarely transcribed differentially expressed genes. Nucl. Acids Res. 25, 2598- 2602 (1997) https://doi.org/10.1093/nar/25.13.2598
  15. Goering, P.L. & Fisher, B.R. Metals and stress proteins. In: Goyer, R.A. & Cherian, M.G. editors. Toxicology of Metals: Biochemical Aspects. Berlin: Springer-Verlag; 229-266 (1995)
  16. Papaconstantinou, A.D. et al. Mercury, cadmium, and arsenite enhance heat shock protein synthesis in chick embryos prior to embryotoxicity. Birth Defects Res. B. Dev. Reprod. Toxicol. 68(6), 456-464 (2003) https://doi.org/10.1002/bdrb.10044
  17. Brkljacic, J., Milutinovic, D.V., Dundjerski, J. & Matic, G. Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70. J. Biochem. Mol. Toxicol. 18(5), 257-260 (2004) https://doi.org/10.1002/jbt.20032
  18. Verlinden, L. et al. Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1, 25-dihydroxyvitamin D3. J. Biol. Chem. 280(45), 37319-37330 (2005) https://doi.org/10.1074/jbc.M503587200
  19. Land, J.M, Booth, R.F, Berger, R. & Clark, J.B. Development of mitochondrial energy metabolism in rat brain. Biochem. J. 164(2), 339-348 (1977) https://doi.org/10.1042/bj1640339
  20. Booth, R.F., Patel, T.B. & Clark, J.B. The development of enzymes of energy metabolism in the brain of a precocial (guinea pig) and non-precocial (rat) species. J. Neurochem. 34(1), 17-25 (1980) https://doi.org/10.1111/j.1471-4159.1980.tb04616.x
  21. Nakai, N. et al. The abundance of mRNAs for pyruvate dehydrogenase kinase isoenzymes in brain regions of young and aged rats. Life Sci. 68(5), 497-503 (2000) https://doi.org/10.1016/S0024-3205(00)00947-4
  22. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63 (1983) https://doi.org/10.1016/0022-1759(83)90303-4