Studies on the Nuclei Adduction and Expression of c-myc Gene by Benzo(a)pyrene and Doxorubicin in Human NC-37 Cells

사람 NC-37 세포에서 Benzo(a)pyrene과 Doxorubicin에 의한 Nuclei내전과 c-myc 유전자의 발현에 대한 연구

  • 김호찬 (고신대학교 의학부 신경정신과학교실) ;
  • 정인철 (고신대학교 의학부 생화학교실) ;
  • 조무연 (고신대학교 의학부 생화학교실)
  • Published : 1998.08.01

Abstract

Formation of adduct was studied in benzo(a)pyrene(BP)- and doxorubicin(Dx)-treated human NC-37 cells and isolated nuclei. Major adducts formed were determined by fluorescence absorption spectrophotometery and DNA-lin-ked protein assay. When isolated nuclei were exposed to carcinogens BP and DMBA, and anticancer drugs m-AMSA, ellipticine and Dx, varying degrees of adduct formation occured between DNA-protein complex and these drugs. When the mixture was centrifuged 1.7 M sucrose solution, binding BP and DMBA appeared to be similar between the sediment and the supernatant. When the sediment was centrifuged again with 0.35% polymin-P, the amount of BP bound was 2-fold greater in the protein(1077$\pm$55cpm) than in DNA fraction (470$\pm$20cpm), whereas that of DMBA was 1.6-fold greater in the DNA than in protein fraction. In the case of m-AMSA, ellipticine and Dx, the amount of binding was slightly greater in supernatant than in sediment in centrifugation with 1.7 M sucrose, and more than 3 times greater in the DNA- than in protein- fraction in centrifugation with 0.35% polymin P. DNA fractions which associated with a subset of nonhistone chromosomal protein were isolated from NC-37 cells exposed to $^{3}$H-BP and $^{14}$C-Dx. They were separated into two distince components DNA-S and DNA-P by centrifugation with 2M Nacl chromatin extraction. The results indicated that the amount of $^{3}$H-BP bound was 6.0-fold greater in DNA-P as compared with DNA-S, while that of $^{14}$C-Dx binding appreaed to be 6.2-fold greater in DNA-S than in DNA-P fraction. When $^{3}$H-BP binding wasdetermined in the presence of cold Dx, the amount of binding was reduced only in the DNA-P fraction, indicating that the interaction between DNA and protein is decreased. Gene expression by these drugs, BP treated cells were increased to compare with nomal cells but reduced by treatment with BP-Dx. These results suggest that the protein moiety which tightly bound to DNA-P fraction may play an important role in the regulation of gene expression.

Keywords

References

  1. Cell v.20 Integration and excision of SV40 DNA from the chromosome of a transformed cell Botchan M.;Stringer J.;Mitchisno T;Sambrook J.
  2. Cold Spring Harbor Symp. Quant. Biol. v.44 Progenitor-cell populations can be infected by RNA tumor viruses but kansformation is dependent on the expression of specific diferentiated functions Bottinger D;Durban E.M.
  3. Biochem. Biophys. Res. Commun. v.70 The role of epidermal aryl hydrocarbon hydroxylase in the covalent binding of polycyclic hydrocarbons to DNA and its relationship to tumor initiation Buty S.G.;Thornpson S.;Slaga TJ
  4. Nature(London) New Biol. v.233 Acetylation of histone H4 and its role in chromatin structure and function Chahal S.S.;Matthews H.R.;Bradbury E.M.
  5. Cancer Res v.39 Metabolism and DNA binding of 3-methy1 cholanthrene Eastrnan A.;Bresnick E.
  6. Nucleic Acids Res. v.10 Benzo(a)pyrene groups bind preferentially to the DNA of active chromatin in human lung cells Arrand J.E.;Murray A.M.
  7. Carcinogen Mutagen v.1 Ehrenberg L.;Teratol S.O.G.
  8. Carcinogenesis v.3 Selective modification of nuclear proteins by polycyclic aromatic hydrocarbons and by benzo(a)pyrene diol epoxides Kootstra A.;MacLeod M.C.;Selkirk I.J.K.;Slaga T.J.
  9. Cancer Res. v.32 Binding of chemical carcinogens tn nuclear proteins of rat liver Jungmann R.A.;Schweppe J.S.
  10. Biochem. Biophys. Res. Commun. v.69 Binding of polycyclic hydrocarbons to nuclear components in vitro Vaught J.;Bresnick E.
  11. Int. J. Cancer v.23 A quantitative determination of the covalent binding of a series of golysyclic hydrocarbons to DNA in mouse skin Phillips D.H.;Grover P.L.;Sims P.
  12. Nucleic Acids Res. v.11 DNA topoisomerases from rat liver : physiological variations Duguet M.;Lavenot C.;Harper F.;Mirambeau G.;DeRecondo A.M.
  13. Cell v.41 DNA topoisomerase Ⅱ is required at the time of mitosis in yeast Holm C.;Goto T.;Wang J.;Botstein D.
  14. Cell v.18 Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells : Chromatin and mRNA interactions in the nuclear matrix Long B.H.;Huang C.Y.;Pogo A.O.
  15. Cancer Lett. v.72 Comparison of DNA adduct formation by means of synchronous scanning and by isotope tracers : in-vitro study on formation of DNA adducts in human lymphocytes exposed to benzo(a%pyrene) Clausen J.
  16. Cancer Res. v.54 Metabolic activation arrd carcinogen-DNA adduct detection in human Larynx Degawa M.;Stern S.J.;Martin M.V.;Geungerich F.P.;Fu P.P.;IIett K.F.
  17. Carcinogenesis v.7 Comparative DNA binding of 7, 12-dimethylbenz(a)anthracene and some of its metabolites in mouse epidermis in vivo as revealed by the 32P-postlabeling technique Schoepe K.B.;Friesel H.;Schurdak M.E.;Randerath K.;Hecker E.
  18. Cancer Lett v.56 Modulatory effect of crocetin on aflatoxin B1 cytotoxicity and DNA adduct formation in $C^3H$/IOTI/2 fibroblast cell Wang C.J.;Shiah H.S.;Lin JK
  19. Nucleic Acids Res. v.22 Formation of adriamycin-DNA adducts in vitro Cullinane C.;Cutts S.M.;Rosmalen A.V.;Phillips D.R.
  20. Nucleic Acids Res. v.23 Stability of adriamycin-induced DNA adducts and interstrand cross links Rosmalen A.V.;Cullinance C.;Cutts S.M.;Phillips D.R.
  21. Biochemistry v.25 Isolation intercalator dependent protein linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase Ⅱ Minford J.;Pommier Y.;Filipski J.;Kohn K.W.(et al.)
  22. Biochemistry v.18 Simple isolation of DNA hydrophobically complexed with presumed gene regulatory proteins Bekhor I;Mirell C.J.
  23. J. Biol. Chem. v.193 Protein measurement with the folin phenol reagent Lowry O.H.;Rosenbrough N.J.;Farr A.L.;Randall R.J.
  24. Biochemical J. v.62 A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid Buton K.
  25. Molecular cloning v.1 Maniatis T.;Fritsch E.F.;Sambrook J.
  26. J. Chromatography v.528 Reactivity and adduct formation of a polyaromatic hydrocarbon, 7-bromomethylben(a)anthracene with chromatin histone proteins Stacks P.C.
  27. J. Biol. Chem. v.250 Regulation of in vitro mRNA transcription by a fraction of chromosomal proteins Chiu J.F.;Tsai Y.-H.;Sakama K.;Hinilica L.S.
  28. Biochemistry v.20 Enrichment of selected active human gene sequences in the placental deoxyribonucleic acid fraction associated with tightly bound nonhistone chromosomal proteins Norman G.L.;Bekhor I.
  29. Science v.207 Distribution of active gene sequences : a subset associated with tightly bound chromosomal proteins Gates D.M.;Bekhor I.