Identification of minimal residual cells after the treatment of leukemia cells by the imaging of mRNA

2008 Fiscal Year Final Research Report

• Project/Area Number: 19590552
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Laboratory medicine
• Research Institution: Hamamatsu University School of Medicine
• Principal Investigator: TAKESHITA Akihiro Hamamatsu University School of Medicine, 医学部, 准教授 (00242769)
• Co-Investigator(Kenkyū-buntansha): TAKESHITA Kaori 浜松医科大学, 医学部附属病院, 医員 (30397393) ; MAEKAWA Masato 浜松医科大学, 医学部, 教授 (20190291)
• Project Period (FY): 2007 – 2008
• Keywords: 白血病 / 微少残存病変 / mRNA / flow cytometry

Research Abstract

特定の白血病細胞の分画の検出と分離を施行するため、細胞内のmRNAの染色を試みた。QUAL/FRET(quenched autoligating/fluorescence resonance energy transfer)法を改良し、蛍光probeの導入技術や反応時間の調整など多方面の改良を施行した。適切なdonor probe とacceptor probeを設定し、energy transferにて得られた特異的蛍光を検出しようとした。標的分子(mRNA)をWT1とし、白血病細胞の同定と分離を生細胞の状態で行う可能性を模索した。作成されたprobeの細胞膜の透過性を一時的に高めるためstreptlysin O(SLO)を使用した。SLOの至適濃度の決定に関しては細胞や試薬の状態などによる再現性の低下を防止するため、probe導入前にSLOの処理濃度の最適化実験を行い、flow cytometryにて検討した。培養細胞では細胞内蛍光の増加を認め、WT1が特異的に検出できると考えられた。しかし、WT1の発現が本来非常に弱いとされている正常のリンパ球における蛍光がprobe量の増加とともに増加し、non-specificな蛍光をより低下させる必要性がでてきた。Non-specificな蛍光の原因を検討するため、分子量を変えたprobeを設計して細胞内に導入した。またprobeが核内へ移行することも考えられたため、probeに接着させる蛍光物質の量を変化させ検討している。

Research Products

• [Journal Article] Effective blood utilization via system for massive blood transfusion, including cardiovascular operation in local areas (2009)
  • Author(s): Takeshita A, Asai T, Murakami M, Fujihara H, Ishizuka T, Nakai S, Yamada C, Suzumura T, Uchiyama Y, Maekawa M, Shigeno K, Washiyama N, Yamashita K, Unno N, Shinjo K
  • Journal Title: Jpn J Transfusion and Cell Therapy 55 Pages: 64-67
• [Journal Article] Pharmacokinetics of arsenic species in Japanese patients with relapsed or refractory acute promyelocytic leukemia treated with arsenic trioxide (2007)
  • Author(s): Fujisawa S, Ohno R, Shigeno K, Sahara N, Nakamura S, Naito K, Kobayashi M, Shinjo K, Takeshita A, Suzuki Y, Hashimoto H, Kinoshita K, Shimoya M, Kaise T, Ohnishi K
  • Journal Title: Cancer Chemother Pharmacol 54 Pages: 485-493
• [Journal Article] A randomized study with or without intensified maintenance chemotherapy in patients with acute promyelocytic leukemia who have become negative for PML-RARα transcript after consolidation therapy : The Japan Adult Leukemia Study Group(JALSG)APL97 study (2007)
  • Author(s): Asou N, KIshimoto Y, Kiyoi H, Okada M, Kawai Y, Tsuzuki M, Horikawa K, Matsuda M, Shinagawa K, Ohtake S, NishimuraM, Takahashi M, Yagasaki F, Takeshita A, Kimura Y, Iwanaga M, Naoe T, Ohno R
  • Journal Title: Blood 110 Pages: 59-66
• [Journal Article] Phase I Trial of FLAGM with High Doses of Cytosine Arabinoside for Relapsed, Refractory Acute Myeloid Leukemia : Study of the Japan Adult Leukemia Study Group(JALSG) (2007)
  • Author(s): Miyawaki S, Kawai Y, Takeshita A, Komatsu N, Usui N, Arai Y, Ishida F, Morii T, Kano Y, Ogura M, Doki N, Ohno R
  • Journal Title: Int J Heamatol 86 Pages: 343-347
• [Journal Article] Phase I/II study of humanized anti-CD33 antibody conjugated with calicheamicin, gemtuzumab ozogamicin, in relapsed or refractory acute myeloid leukemia : Final results of Japanese multi-center cooperative study
  • Author(s): Kobayashi Y, Naito K, Takeshita A, Morishima Y, Ogura M, Besso H, Hotta T, Mitani K, Takeuchi H, Miyawaki S, Naoe T, Ohno R.
  • Journal Title: Int J Hematol (in press)
• [Journal Article] CMC544(inotuzumab ocogamicin), an ati-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells
  • Author(s): Takeshita A, Yamakage N, Shinjo K, Ono T, Hirano I, Nakamura S, Shigeno K, Tobita T, Maekawa M, Kiyoi H, Naoe T, Ohnishi K, Sugimoto Y, Ohno R
  • Journal Title: Leukemia (in press)
• [Journal Article] CMC-544(inotuzumab ozogamicin)shows less effect on multidrug resistant cells : analyses in cell lines and cells from patients with B-cell chronic lymphocytic leukemia and lymphoma
  • Author(s): Takeshita A, Shinjo K, Yamakage N, Ono T, Hirano I, Matsui H, Nakamura S, Shigeno K, Tobita T, Maekawa M, Kiyoi H, Naoe T, Ohnishi K, Sugimoto Y, Ohno R
  • Journal Title: Br J Haematol (in press)
• [Presentation] Cell cycle features and quantitative alteration of target molecules of malignant B cells treated with CMC544 alone or in combination with rituximab (2007)
  • Author(s): Yamakage N, Takeshita A, ShinjoK, Ono T, Hirano I, Okinaka K, Nakamura S, Shigeno K, Maekawa M, Ohnishi K, Ohno R
  • Organizer: Annual Meeting of American Society of Hematology, Sanfrancisco, USA
  • Place of Presentation: Atranta, USA, Dec
  • Year and Date: 20070000
• [Presentation] Reduc ed effect of CMC544 on P-glycoprotein po sitive malignant B cells and its restoration by multidrug resistance modifiers (2007)
  • Author(s): Takeshita A, Shinjo K, YamakageN, O no T, Hirano I, Okinaka K, Matsui H, Nakamura S, Shigeno K, Maekawa M, Ohnishi K , Sugimoto Y , Ohno R
  • Organizer: Annual Meeting of American Society of Hematology, Sanfrancisco, USA
  • Place of Presentation: Atranta, USA, Dec
  • Year and Date: 20070000
• [Presentation] The usefulness of the expected elevation value of serum albumin before replacement therapy
  • Author(s): Fujihara H, Takeshita K, Nakai S, Yamada C, Suzumura T, Uchiyama S, Takeshita A.
  • Organizer: Annual Meeting of AABB Montreal
  • Place of Presentation: Canada
• [Presentation] A Randomized Trial Comparing Individualized Vs. Non-Individualized Treatment for Elderly Acute Myeloid Leukemia : JALSG GML200 Study
  • Author(s): Wakita A, Ohtake S, Takada S, Yagasaki F, Komatsu H, Miyazaki Y, Kubo K, Kimura Y, Takeshita A, Adachi Y, Kiyoi H, Yamaguchi T, Yoshida M, Ohnishi K, Miyawaki S, Naoe T, Ueda R, Ohno R.
  • Organizer: Annual Meeting of American Society of Hematology
  • Place of Presentation: Sanfrancisco, USA
• [Book] Clinical Significance of P-Glycoprotein in Acute Leukemia and a Strategy to Overcome Drug Resistance
  • Author(s): Motoji T, Motomura S, Wang Y, Tsuji K, Takanashi M, Shiozaki H, Miyawaki S, Asou N, Takeshita A, Saburi Y, Ohno R, Mizoguchi H
  • Total Pages: 123-151
  • Publisher: Frontiers in Cancer Res