Analysis of dynamics of DNA damage repair protein DDB1 in oxidative stresses in N2a cells

• Project/Area Number: 15591241
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Psychiatric science
• Research Institution: Nara Medical University
• Principal Investigator: NAKAMURA Yu Nara Medical University, Psychiatry, Associate Professor, 医学部, 助教授 (70291440)
• Co-Investigator(Kenkyū-buntansha): KISHIMOTO Toshifumi Nara Medical University, Psychiatry, Professor, 医学部, 教授 (60201456) ; YOSHINO Hiroaki Nara Medical University, Psychiatry, Assistant Professor, 医学部, 助手 (10347560) ; MORI Toshio Nara Medical University, Psychiatry, Associate Professor, 医学部, 助教授 (10115280) ; SUGIURA Sigeki Nara Medical University, Psychiatry, Associate Professor, 医学部, 助教授 (40179130)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,300,000 (Direct Cost: ¥3,300,000); Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000); Fiscal Year 2003: ¥1,900,000 (Direct Cost: ¥1,900,000)
• Keywords: DNA damage / repair / DDB1 / oxidative stress / radiation / chaperon / fluorescent protein / UV / シャペロン

Research Abstract

We established a cell line derived from HEK293 (EGFP-DDB1-HEK293-1) stably expressing EGFP-fused DDB1. Both in EGFP-DDB1-HEK293-1 cells and in HEK293 cells, repair of UV-induced DNA damages (CPDs and 6-4PPs) was compared. As a result, there was no difference in the velocity of repair of them, indicating that massive expression of DDB1 did not affect the repair of them. On the other hand, there was no difference in the velocity of cell division in both cells. Translocation of fluorescently labeled DDB1 from cytoplasm to nucleus was on real time observed in the cells of EGFP-DDB1-HEK293-1 after oxidative stresses on the stage kept at 37℃ by means of fluorescence microscopy. As the oxidative stresses, we employed addition of H_2O_2. In living cells, intensity of local fluorescence was measured on real time, and dynamics of DDB1 was analyzed. Otherwise, 20 J/m^2 UV radiation and 1〜20gy ionizing X ray radiation were also compared in dynamics of DDB1. The mount of translocated fluorescent DDB1 was largest in case of ionizing X ray radiation, and the amount of translocated fluorescence was observed in maximum level 24 hours after damages. And fluorescent DDB1 was relocated to cytoplasm 48 hours after damages. These findings indicated that DDB1 reacted with a variety of DNA damages, and that DDB1 functioned as a chaperon-like protein that transported certain signals from cytoplasm to nucleus.
On the other hand, we failed to establish a cell line derived from SKN-MC neuroblastoma cells stably expressing EGFP-fused DDB1. It indicated that massive expression of full-length DDB1 might be harmful to SKN-MC neuroblastoma cells. Because DDB1 lack of its C-terminus region has no ability to bind to APP fragments, translocation of APP fragments to nucleus was expected to harm neuronal cells.

Research Products

• [Journal Article] In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies (2004)
  • Author(s): Iwamoto, T-A., Kobayashi, N., Imoto, K., Yamamoto, A., Nakamura, Y., Yamauchi, Y., Okumura, H., Tanaka, A., Hanaoka, F., Shibutani, S., Miyagawa, S., Mori, T.
  • Journal Title: DNA Repair (Amst) 3 Pages: 1475-1482
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dieters and (6-4) photoproducts (2004)
  • Author(s): Nishiwaki, Y., Kobayashi, N., Imoto, K., Iwamoto, T., Yamamoto, A., Nakamura, Y., Sarasin, A., Miyagawa, S., Mori, T.
  • Journal Title: J.Invest.Dermatol 122 Pages: 526-532
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies (2004)
  • Author(s): Iwamoto, T-A., Kobayashi, N., Imoto, K., Yamamoto, A., Nakamura, Y., Yamauchi, Y., Okumura, H., Tanaka, A., Hanaoka, F., Shibutani, S., Miyagawa, S., Mori, T.
  • Journal Title: DNA repair(Amst) 3 Pages: 1475-1482
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts (2004)
  • Author(s): Nishiwaki, Y., Kobayashi, N., Imoto, K., Iwamoto, T., Yamamoto, A., Nakamura, Y., Sarasin, A., Miyagawa, S., Mori, T.
  • Journal Title: J.Invest.Dermatol. 122 Pages: 526-532
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts (2004)
  • Author(s): Nishiwaki, Y., Kobayashi, N., Imoto, K., Iwamoto, T., Yamamoto, A., Nakamura, Y., Sarasin, A., Miyagawa, S., Mori, T.
  • Journal Title: J.Invest.Dermatol 122 Pages: 526-532
• [Publications] Imoto K: "The total amount of DNA damage determines ultraviolet-radiation-induced cytotoxicity after uniformor localized irradiation of human cells"J Invest Dermatol. 119. 1177-1182 (2002)
• [Publications] Nishiwaki Y: "Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts"J Invest Dermatol. (in press). (2004)