Localization of DNA Topoisomerase II on High Salt Extract Chromatin Fraction of Mouse Embry Fibroblast Cells Treated with Bezo(a)pyrene and Doxorubicin

Lee, D G; Kim, J G; Jung, I C; Cho, M Y

Abstract

This study was conducted to determine if there exists a DNA topoisomerase II participating in the cleave and rejoining of DNA in the nuclear fraction of a mouse embryo fibroblast, C3H/10T1/2 cells to measure the expression of c-myc gene in the cells cultivated with benzo(a)pyrene (BP) and doxorubicin (Dx), together or alone, by means of Northern blot hybridization, and to observe changes of DNA topoisomerase II activity in chromatin subfraction extracted with high salt solution, and to confirm the existence of DNA topoisomerase II by Western blot immunodetection using the DNA topoisomerase II-antibody from the rabbit. The results are summarized as follows: 1. There was a region in the nuclear fraction of the C3H/10T1/2 cell showing a strong DNA topoisomerase II activity, which was included in the fraction insoluble to high salt (2M NaCl) solution. 2. In in vitro experiments, the relaxation activity of DNA topoisomerase II in pBR 322 DNA was increased by treatment with different concentration (1, 4, 10microM) of BP similar results were obserbed with BP/Dx treatment, although there was some mobility shift. 3. In Northern blot analysis, c-myc gene expression was increased in C3H/10T1/2 cell treated BP or Dx, but it decreased in cells treated with BP and Dx together. 4. Centrifugation of 2M NaCl chromatin extraction of C3H/10T1/2 cell treated with BP, Dx or BP/Dx yielded two components, DNA-S and DNA-P. In case of BP treated, the topoisomerase activity was exist in both fraction but the group with Dx was activity in DNA-S only when the enzyme activity was determined in the presence of Dx, it was only higher than that of BP in DNA-S fraction. 5. The Western blot immunodetection of the above fractions indicated that DNA topoisomerase II existed in DNA-S and DNA-P fractions for the BP treated group but Dx trated group was in DNA-S fraction only. However, the amount of enzyme in the DNA-S of BP/Dx treated group were increased to compare with Dx treated group. These results indicated that the high salt insoluble fraction was representative region to topoisomerase II activity, also the DNA-S fraction by the action of Dx may play an important role in the function of a gene.