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Study of genome stability maintenance to avoid cancer

松野 悠介 Yusuke Matsuno 東京理科大学 DOI:info:doi/10.20604/00003671

2022.06.17

概要

本論文の第1章では、本研究の全般的な序論を述べた。がんは多くの人の死因となる疾患である。がん化過程は多段階からなり、『各段階は、その段階の突彼に関わる変異が誘導されて、この変異細胞がクローン進化に至ることで進行する』と考えられている。この過程では、初期に細胞が無秩序に増殖する能力を獲得し、さらに、これらが浸潤・転移する能力を獲得することで様々な臓や組織の機能低下に至らしめ、最終的に、治療抵抗性の能力の獲得に至る。近年、このがん化過程の進行に関わる変異細胞は、ゲノム不安定性に伴って誘導されることが示された。実際、一部の小児腫瘍を除いて、殆どのがんは、比較的高齢で、ゲノム不安定性を伴って発症している。がんで認められるゲノム不安定性は、主に染色体不安定性(CIN:chromosomal instability)が誘導されるものと、マイクロサテライト不安定性(MSI: microsatellite instability)が誘導されるものに大別され、通常、どちらか一方が認められる。近年の研究から、『DNA 複製ストレス(DNA 複製を阻害する様々な要因)に起因して生じた DNA 二重鎖切断(DSB: DNA double-strand break)が引き金となり、ゲノム不安定性が誘導される」とが明確になった。実際、様々な DNA 修復能の欠損下で、ゲノム不安定性の誘導リスクが上昇し、この背景で“がんのリスクも上昇することが知られている。しかし、実際の殆どのヒトのがんは、DNA 修復能に遺伝的な異常が無い背景で、ゲノム不安定性を伴って発がんに至っている。現段階で、DNA 修復能の正常な背景で、どの様に DNA損傷を修復できない状態に陥り、ゲノム不安定性の誘導に至るのか、正常細胞におけるゲノム不安定性のリスク影響には不明な点が多い。また、これまで一般に、『変異は“DNA 複製エラーによってランダムに導入される』と考えられ、このため、『がんは、不運にも複製過程で“がんドライバー遺伝子”が変異した場合に発症する』と捉えられてきた。しかし、この点は、Peto のパラドックス(ランダムに入る変異に起因して発がんに至ると考えた場合、巨大動物で発がん率が高いと考えられるが、その様な相関はない)など、矛盾点も明確で、現段階で、がんドライバー変異の誘導機構についても不明な点が多い。そこで本研究では、マウス脂仔由来線維芽細胞(MER)の不死化モデルを基盤として、ゲノム不安定性のリスク影響を解析し、ゲノムの安定性・不安定性の制御メカニズムの解明に挑戦することとした。特に、ゲノム不安定性と変異導入との関係、それに伴うクローン進化への影響、さらに、発がんに与える影響を明確にすることを目指した。

第2章では、放射線のゲノム不安定性のリスク影響と、その誘導機構について述べた。放射線ばく露ば“ゲノム不安定性を伴うがん”のリスク要因である。そこで、まず、放射線照射の影響をリスク促進モデルとして解析した。MERに1Gyまたは10 Gyのガンマ線を照射したところ、いずれの条件でも増殖停止が誘導された。その後、1Gyの照射を受けた MERは、通常の半分程度の期間で不死化が誘導された。ガンマ線照射の有無によらず、不死化した MEF には染色体の転座や欠失などのゲノム不安定性が見られた。ガンマ線照射を受けた MER に現れる影響を調べるため、yH2AXを指標として DNA損傷状態を解析した結果、『ガンマ線で直接に生じたDSBは数時間以内に修復されるが、その後、“DNA複製ストレスに起因したDSB”が生じる」とが明確になった。これらの結果から、ガンマ線照射に伴う MERの不死化は、“DNA 複製ストレスに起因したDSB” を引き金としたゲノム不安定化により誘導されていることが示唆された。驚いたことに、この“ゲノム不安定性と不死化”への影響は、広い線量域(0.25-2 Gy)と線量率域(1.39-909 mGy/min) で認められた。これらの条件において、ガンマ線で直接に生じた DSB の数は、照射した線量や線量率に相関して増加したが、一方で、複製ストレスに起因したDSB の数は、照射した線量や線量率に依存せず同程度であった。さらに、ゲノム異常への影響を全グノムシークエンスにより解析したところ、ゲノム再編などの“染色体の構造異常(DSB の修復エラーで誘導される)”の導入には、放射線ばく露の有無で変化が現れないことが分かった。これらのことは、放射線ばく露の直接のリスク影響は、『“DNA 複製ストレスに起因した DSB”の蓄積に伴うゲノム不安定性リスクの高い細胞状態”の誘導』で、『このリスクには“放射線で直接に生じた損傷”は関係ない」ことを示唆している。さらに、放射線の照射・非照射に関わらず、『クローン進化した細胞では大規模な“染色体の構造異常”と“塩基置換変異”が誘導されており、これらの誘導には強く相関が認められる』とが分かった。以上から、『ゲノム不安定性の誘導過程では“大規模な変異誘導”を伴うため、変異細胞のクローン進化の誘導に至る』こと、『放射線ばく露による直接のリスクは、ゲノム不安定性リスクの高い細胞状態(“DNA 複製ストレスに起因した DSB”の蓄積した状態)の誘導である」とが明確になった。これは、『放射線で“直接に生じるDNA損傷”とは異なり、“DNA 複製ストレスに起因したDSB”は広い線量域と線量率域で同程度誘導されるため』であることが示された。

第3章では、ポリフェノールのゲノム安定性維持への影響について述べた。これまでに、『ポリフェノールの摂取による“がんの予防効果”は様々な動物モデルで報告されている』どから、これらを調べたところ、『予防効果の認められた報告では“ゲノム不安定性を伴って発症するがん”が対象となっている』とを見出した。そこで、ここでは、ポリフェノールによる“がん予防効果”の作用点は“ゲノム安定性の促進との可能性を考えて解析した。MEFを生理学的濃度(2.5 HM)のレスベラトロール存在下で培養すると、ゲノム安定性が維持され、不死化が抑制されることが分かった。この背景では、増殖停止期に現れる“DNA 複製ストレスに起因したDSBの蓄積”と“H2AXの発現”が抑制されており、ゲノム不安定性リスクの低い細胞状態を示していた。レスベラトロール添加後の細胞状態を解析すると、H2AXの発現量が一過的に上昇し、これに伴って DSB 数の減少が見られたことから、“DNA 複製ストレスに起因した DSB”の修復が誘導される結果、“ゲノム安定性の増強効果”が現れていることが分かった。同様の“ゲノム安定性を促進する効果”は、レスベラトロールやクロロゲン酸など、複数のポリフェノールの投与で共通して現れる効果であることが分かった。また、DNA 修復欠損マウスモデルにおいてポリフェノールを含んだ食餌を与えると、寿命延伸効果と腫瘍抑制効果が現れた。これらの結果から、レスベラトロールやその類似ポリフェノールはゲノム安定性保持効果、がん予防効果を示すことが示唆された。

第4章では、本研究の全般的な結論を述べた。本研究により、ゲノム不安定性の誘導過程では変異誘導を伴うため、結果的に“変異細胞のクローン進化”の誘導に至ることが明確になった。さらに、本研究からは、『放射線ばく露の場合の様に、ゲノム不安定性リスクの上昇によってがんのリスクが上昇する』のに対し、逆に、『ポリフェノール類の摂取・投与の影響として現れる様に、ゲノム安定性の促進によって“変異細胞のクローン進化誘導のリスクが抑制される(がんのリスクが抑制される)」と考えられることが示された。これらの知見は、がんの発生や予防に関する今後の研究に買献するものであると考えられる。

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