Study of mitochondria-associated diseases by pathogenic mitochondrial DNA replication delay

• Project/Area Number: 25461573
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Pediatrics
• Research Institution: Chiba Cancer Center (Research Institute)
• Principal Investigator: Koshikawa Nobuko 千葉県がんセンター(研究所), がん遺伝創薬研究室, 主席研究員 (90260249)
• Co-Investigator(Kenkyū-buntansha): WATANABE Takayoshi 千葉県がんセンター研究所, がん遺伝創薬研究室, 研究員 (60526060) ; NAGASE Hiroki 千葉県がんセンター研究所, がん遺伝創薬研究室, 研究所長 (90322073)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2013: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 病因性ミトコンドリアDNA / ミトコンドリア病 / PIポリアミド / ミットコンドリア病 / MELAS / ミトコンドリアDNA / mtDNA異常 / cybrid / PIポリアミドFITC修飾 / PI ポリアミド / mtDNA 変異 / 疾患特異的mtDNA変異

Outline of Final Research Achievements

Mitochondrial DNA (mtDNA) mutation 3243 A>G is one of the causes of MELAS. We designed Pyrrole/Imidazole polyamide (PIP) in order to inhibit this gene expression (PIP2). HeLamt3243 cybrids have 3243A>G mutation, at different rates. A cybrid was treated with FITC labeled PIP2. Mitochondria were stained by FITC and were visible as dots, so were mtDNAs also stained. But FITC (equal PIP2) was eliminated from mitochondria 48 hours after treatment. In order to extend the residence time of PIP in mitochondria, triphenylphosphonium (TPP) was conjugated with fluorochrome and was introduced to the cybrid with 3243A>G mutation. Mitochondria were stained by TPP conjugated fluorochrome and kept it’s fluorescence for at least 48 hours on mitochondria. It is suggested that TPP conjugated compound reached mitochondria and remained bound for at least 48 hours post treatment. Then PIP conjugated TPP was synthesized (PIP-TPP). A cybrid with 3243 A>G mutation is being treated with PIP-TPP.

Research Products

• [Journal Article] Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole-imidazole polyamide conjugate. (2014)
  • Author(s): Hiraoka K, Inoue T, Taylor RD, Watanabe T, Koshikawa N, Yoda H, Shinohara K, Takatori A, Sugimoto H, Maru Y, Denda T, Fujiwara K, Balmain A, Ozaki T, Bando T, Sugiyama H, Nagase H.
  • Journal Title: Nat. Commun. Volume: 6 Issue: 1 Pages: 6706-6706
  • DOI: 10.1038/ncomms7706
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] A novel alkylating pyrrole-imidazole polyamide, KR12, specifically recognized mutant KRAS genes and potently induces cell death (2015)
  • Author(s): K Hiraoka, T Inoue, H Yoda, A Takatori, T Watanabe, N Koshikawa, T Ozaki, H Nagase
  • Organizer: America Association for Cancer Research Annual Meeting 2015
  • Place of Presentation: アメリカ合衆国
  • Year and Date: 2015-04-22
  • Int'l Joint Research
• [Presentation] KRAS mutation specific alkylating pyrrole-imidazole polyamide (KR12) suppresses mutant KRAS expression and inhibits tumor growth by showing accumulation in KRAS mutant xenografts (2015)
  • Author(s): T Inoue,K Hiraoka, Y Suzuki, H Yoda, A Takatori, N Koshikawa, T Ozaki, H Nagase
  • Organizer: America Association for Cancer Research Annual Meeting 2015
  • Place of Presentation: アメリカ合衆国
  • Year and Date: 2015-04-22
  • Int'l Joint Research