Title
ゲノムのメチル化変動に着目した肝発がん物質に共通する
発がん機序に関する研究( 本文(Fulltext) )
Author(s)
水上, さやか
Report No.(Doctoral
Degree)
博士(獣医学) 甲第497号
Issue Date
2018-03-13
Type
博士論文
Version
ETD
URL
http://hdl.handle.net/20.500.12099/75220
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。䝀䝜䝮䛾䝯䝏䝹ኚື䛻╔┠䛧䛯⫢Ⓨ䛜䜣≀㉁䛻
ඹ㏻䛩䜛Ⓨ䛜䜣ᶵᗎ䛻㛵䛩䜛◊✲
2017 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ⋇་Ꮫ◊✲⛉
䠄ᮾி㎰ᕤᏛ䠅
Ỉୖ㻌 䛥䜔䛛
䝀䝜䝮䛾䝯䝏䝹ኚື䛻╔┠䛧䛯⫢Ⓨ䛜䜣≀㉁䛻
ඹ㏻䛩䜛Ⓨ䛜䜣ᶵᗎ䛻㛵䛩䜛◊✲
1
┠
┠㻌 ḟ
ᗎㄽ ... 3 ➨1 ❶㻌 䝷䝑䝖䜈䛾⫢Ⓨ䛜䜣≀㉁ᢞ䛻䜘䜚 DNA 㐣䝯䝏䝹䛸㑇ఏᏊⓎ⌧ῶᑡ䛾䜏䜙䜜䜛㻌 㑇 ఏᏊ䛾᥈⣴䛸⫢Ⓨ䛜䜣䛸䛾㛵㐃ᛶ䛾᳨ウ ⥴ゝ ... 8 ᮦᩱ䛚䜘䜃᪉ἲ ... 11 ⤖ᯝ ... 20 ⪃ᐹ ... 24 ᑠᣓ ... 30 ➨2 ❶㻌 TMEM70 䛾䝷䝑䝖⫢Ⓨ䛜䜣㐣⛬䛻䛚䛡䜛ᶵ⬟ ⥴ゝ ... 33 ᮦᩱ䛚䜘䜃᪉ἲ ... 36 ⤖ᯝ ... 46 ⪃ᐹ ... 56 ᑠᣓ ... 61 ➨3 ❶㻌 UBE2E2 䛾䝷䝑䝖⫢Ⓨ䛜䜣㐣⛬䛻䛚䛡䜛ᶵ⬟ ⥴ゝ ... 64 ᮦᩱ䛚䜘䜃᪉ἲ ... 67 ⤖ᯝ ... 762 ⪃ᐹ ... 82 ᑠᣓ ... 88 ⥲ ⥲ྜ⪃ᐹ ... 90 ⤖ㄽ ... 94 ㅰ㎡ ... 95 ᘬ⏝ᩥ⊩ ... 96 せ᪨ ... 110 Abstract ... 114 ᅗ⾲ ... 119
3 ᗎ ᗎ㻌 ㄽ Ꮫ≀㉁䛾Ⓨ䛜䜣ᛶホ౯䛿䠈Ꮫ≀㉁䛾Ᏻᛶホ౯䛻䛚䛔䛶ᴟ䜑䛶㔜せ䛷䛒䜛䚹䛧䛛䛧 䛺䛜䜙䠈୍⯡ⓗ䛺Ⓨ䛜䜣ᛶヨ㦂䛿䠈ఱⓒ༉䜒䛾䛢䛳ṑ㢮ື≀䜢⏝䛔䛶㛗ᮇ㛫䛻Ώ䛳䛶⿕㦂 ≀㉁䜢ᢞ䛩䜛䛯䜑䠈ከ䛺㛫䛸㈝⏝䜢せ䛩䜛䚹䛣䛾䛣䛸䛛䜙䠈▷ᮇ㛫䛷ྜ⌮ⓗ䛻Ⓨ䛜䜣ᛶ 䜢ண 䛧ᚓ䜛ᣦᶆ䛾☜❧䛜ồ䜑䜙䜜䛶䛔䜛䚹ᡤᒓ◊✲ᐊ䛾ඛ⾜◊✲䛻䛚䛔䛶䠈」ᩘ✀㢮䛾 Ⓨ䛜䜣≀㉁䜢28 ᪥㛫䝷䝑䝖䛻ᑐ䛧㐃⥆⤒ཱྀᢞ䛧䛯㝿䛻䠈Ⓨ䛜䜣䛾ᶆⓗ⣽⬊䛻䛚䛔䛶䠈⣽⬊ ࿘ᮇṆ䜢ྵ䜐⣽⬊࿘ᮇไᚚ␗ᖖ䜢ᫎ䛩䜛䜘䛖䛺㛵㐃ศᏊ䛾Ⓨ⌧ኚື䜢ぢฟ䛧䛶䛔䜛 [36, 65, 76]䚹⣽⬊࿘ᮇṆ䛜⣽⬊⪁䛻≉ᚩⓗ䛺⌧㇟䛷䛒䜛䛣䛸䜢⪃៖䛩䜛䛸 [7]䠈ඛ⾜◊ ✲䛷ᚓ䜙䜜䛯⤖ᯝ䛿䠈㐃⥆ⓗ䛺Ⓨ䛜䜣≀㉁䛾่⃭䛻䜘䜚䠈⣽⬊⪁䛻㝗䜛⣽⬊䛜ቑຍ䛧䛯䛣 䛸䜢ᫎ䛧䛶䛔䜛䛸⪃䛘䜙䜜䜛䚹୍⯡ⓗ䛻⣽⬊⪁䛿䝔䝻䝯䜰䛾▷⦰䜔䠈䛜䜣㑇ఏᏊ䛾άᛶ 䛜ᘬ䛝㔠䛸䛺䜚, ⣽⬊࿘ᮇṆ䛜㐍⾜䛧䛶䛔䛟㐣⛬䛷䛒䜛䛸䛥䜜䜛 [21]䚹䛣䛾䜘䛖䛺䠈䛜䜣㑇ఏ Ꮚ䛜ㄏᑟ䛩䜛⪁䛿䠈p53 䛚䜘䜃⥙⭷ⱆ⣽⬊⭘⺮ⓑ㉁䜢䝁䞊䝗䛩䜛㑇ఏᏊ䛾䜘䛖䛺⣽⬊࿘ᮇ 䝏䜵䝑䜽䝫䜲䞁䝖䛻ാ䛟䛜䜣ᢚไ㑇ఏᏊ䛾άᛶ䜢䛩䜛䛯䜑, ⭘⒆ᢚไ䛾ᶵ⬟䜒᭷䛩䜛 [12]䚹䛥䜙䛻䠈䛜䜣㑇ఏᏊ Ras 䛻䜘䛳䛶ㄏⓎ䛥䜜䛯ቑṪᛶኚ䛻䛚䛔䛶䠈⪁⣽⬊䛿䠈๓䛜䜣 ኚ䛷䛿ㄆ䜑䜙䜜䜛䜒䛾䛾, 㐍ᒎ䛧䛯⭘⒆⤌⧊ෆ䛷䛿ㄆ䜑䜙䜜䛺䛟䛺䛳䛯䛸䛾ሗ࿌䛜䛒䜛 [11]䚹䛣䜜䜙䛾ሗ࿌䛛䜙䠈⪁⣽⬊䛾୍㒊䛾⣽⬊㞟ᅋ䛻䛚䛔䛶⪁䛾◚⥢䛜㉳䛣䜚䠈䛜䜣 䜈䛾㐨䜢㎺䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䜛䚹
4
䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺㑇ఏᏊⓎ⌧ಟ㣭䛿䠈䝀䝜䝮㓄ิ䛻ኚ䜢䛘䛪䛻䠈䝀䝜䝮䛾ಟ㣭䜔ᰁ Ⰽయ䜽䝻䝬䝏䞁䛾ኚ䜢䛸䛧䛶䠈ᶆⓗ㑇ఏᏊ䛾Ⓨ⌧䛻ᙳ㡪䜢䛘䜛 [73]䚹䛣䜜䜙䛾ಟ㣭䛿 DNA 䛾䝅䝖䝅䞁ሷᇶ䛾䝯䝏䝹䠈䝠䝇䝖䞁⺮ⓑ㉁䛾 (⬺) 䜰䝉䝏䝹䜔䝯䝏䝹䛺䛹䛾⩻ヂᚋ ಟ㣭䠈microRNA 䛸䜀䜜䜛 noncoding RNA 䛻䜘䜛㌿ᚋ䛾㑇ఏᏊⓎ⌧ㄪ⠇䛺䛹䛜ྵ䜎䜜䜛 [58]䚹䛣䛾䛖䛱䠈DNA 䛾䝯䝏䝹䛿䠈ከ⣽⬊⏕≀䛾ṇᖖ䛺Ⓨ⏕ẁ㝵䛻䛚䛔䛶ᗈ䛟ㄆ䜙䜜䛶䛚䜚䠈 䜎䛯䠈ከ䛟䛾ᝈ䛾ᙧᡂ䛻䛚䛔䛶㔜せ䛺ᙺ䜢ᢸ䛖䛣䛸䛜䜟䛛䛳䛶䛝䛶䛔䜛 [6, 52]䚹䝅䝖䝅䞁 䛾ḟ䛻䜾䜰䝙䞁䛜୪䜆2 ሷᇶ㓄ิ䛷䛒䜛 CpG 䛿䠈䛻㑇ఏᏊ䝥䝻䝰䞊䝍䞊㡿ᇦ䜒䛧䛟䛿䛭䛾 ㏆ഐ䛻㧗⃰ᗘ䛻Ꮡᅾ䛧䠈CpG 䜰䜲䝷䞁䝗䛸䜀䜜䛶䛔䜛䚹CpG 䜰䜲䝷䞁䝗䛾䝯䝏䝹䛿䠈㌿ᅉ Ꮚ䛾䝥䝻䝰䞊䝍䞊㡿ᇦ䜈䛾⤖ྜ䜢┤᥋䛒䜛䛔䛿㛫᥋ⓗ䛻㜼ᐖ䛧䠈䜽䝻䝬䝏䞁ᵓ㐀䛾จ㞟䜢ㄏ ᑟ䛧䜔䛩䛟䛧䛶⤖ྜ䜢ไ㝈䛧䠈㑇ఏᏊⓎ⌧䜢ᢚไ䛩䜛 [50]䚹ὀ┠䛩䜉䛝Ⅼ䛸䛧䛶䠈㑇ఏᏊ䛾䝯 䝏䝹≧ែ䛜䠈ፉ⣽⬊䜈ᘬ䛝⥅䛜䜜䜛䛸䛔䛖Ⅼ䛜ᣲ䛢䜙䜜䜛 [27]䚹 䛣䛾䝀䝜䝮䛻䛚䛡䜛 DNA 䝯䝏䝹␗ᖖ䛿ᵝ䚻䛺䛜䜣䛷ሗ࿌䛥䜜䛶䛚䜚䠈䛜䜣ᢚไ㑇ఏᏊ䛾Ⓨ⌧ᢚไ䜢⏕䛨䜛䛣䛸䛷䠈⭘ ⒆䛾ቑṪ䜔㐍ᒎ䛻᭷䛺ᙧ㉁⋓ᚓ䛻ᐤ䛩䜛䛸⪃䛘䜙䜜䜛 [33]䚹䛩䛺䜟䛱䠈Ⓨ䛜䜣≀㉁䛾 ᢞ䛻䜘䛳䛶䠈䝥䝻䝰䞊䝍䞊㡿ᇦ䛻䝯䝏䝹ಟ㣭䛜✚䛩䜛㑇ఏᏊ䛿䠈Ⓨ䛜䜣㐣⛬᪩ᮇ 䛾㔜せ䛺㘽䜢ᥱ䛳䛶䛔䜛ྍ⬟ᛶ䛜䛒䜛䚹௨ୖ䛾䛣䛸䛛䜙䠈ᮏ◊✲䛷䛿䠈Ⓨ䛜䜣≀㉁ᢞ 䛻䜘䜚䠈๓䛜䜣ኚ䛺䛹䛾ቑṪᛶኚ䜢ᙧᡂ䛩䜛๓䛾ᮇ䛛䜙 DNA 䝯䝏䝹䛸䛔䛖䜶䝢䝆䜵 䝛䝔䜱䝑䜽䛺ಟ㣭䛜⏕䛨䛶䠈Ⓨ䛜䜣䛻᭷䛺ᙧ㉁䜢⋓ᚓ䛧䛯⣽⬊㞟ᅋ䛜⏕䛨䜛䜒䛾䛸௬ᐃ䛧䠈 㐣䝯䝏䝹䛸Ⓨ⌧ῶᑡ䛾䜏䜙䜜䜛㑇ఏᏊ䛜䠈⣽⬊⪁䛾◚⥢䛻㉳ᅉ䛩䜛Ⓨ䛜䜣ᛶ䛾▷ᮇண
5 ᣦᶆ䛸䛺䜚䛖䜛䛸⪃䛘䠈ḟ䛾୕❶❧䛶䛾◊✲䜢ᐇ䛧䛯䚹 ➨ 1 ❶䛷䛿䠈▷ᮇ㛫䛾⫢Ⓨ䛜䜣≀㉁ᢞ䛻䜘䜚䠈⫢⮚䛻䛚䛔䛶㐣䝯䝏䝹䛾䜏䜙䜜䜛㑇ఏ Ꮚ䜢㑅ฟ䛧䠈⫢Ⓨ䛜䜣㐣⛬䛻䜶䞁䝖䝸䞊䛩䜛䛯䜑䛾ᶵᗎ䜢䛘䜛ศᏊᣦᶆ䜢☜❧䛩䜛䛣䛸䜢┠ ⓗ䛻ᐇ㦂䜢⾜䛳䛯䚹䛣䛾┠ⓗ䛾䛯䜑䛻䠈⫢Ⓨ䛜䜣≀㉁䛷䛒䜛 thioacetamide (TAA) 䜢䝷䝑䝖䜈 28 ᪥㛫⤒ཱྀᢞ䛧䠈⫢⮚䛻䛚䛡䜛 DNA 䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲䜢ᐇ䛧䛯䚹㐣䝯䝏䝹 䛚䜘䜃㑇ఏᏊ䛾Ⓨ⌧ῶᑡ䜢᳨ド䛧䛯ᚋ䠈䝷䝑䝖䛾ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䠈⫢Ⓨ䛜䜣≀ ㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚ㄏⓎ䛥䜜䛯⫢Ⓨ䛜䜣ቑṪᛶኚ䛻䛚䛡䜛䠈 ೃ⿵ศᏊ䛾Ⓨ⌧ศᕸ䜢᳨ウ䛧䛯䚹 ➨2 ❶䛷䛿䠈➨ 1 ❶䛷ᚓ䜙䜜䛯ೃ⿵ศᏊ䛾䛖䛱䠈䝭䝖䝁䞁䝗䝸䜰⺮ⓑ㉁䛷 ATP ྜᡂ㓝⣲䛾⏕
ྜᡂ䛻㛵䛩䜛䛸䛥䜜䜛transmembrane protein 70 (TMEM70) 䛻ὀ┠䛧䛯䚹TMEM70 䛾Ⓨ䛜 䜣㐣⛬䛻䛚䛡䜛ᶵ⬟䜢᫂䜙䛛䛻䛩䜛䛯䜑䠈䝷䝑䝖䛾ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䠈glutathione
S-transferase placental form (GST-P䠈䝷䝑䝖䛾⫢⮚๓䛜䜣ኚ䝬䞊䜹䞊) 㝧ᛶ(+)ኚ␗⫢⣽⬊ᕢ
䛻䛚䛡䜛TMEM70 䛾Ⓨ⌧ῶᑡ䛸䠈䛜䜣⣽⬊䛻≉ᚩⓗ䛺௦ㅰኚ䛸䛾㛵㐃䜢᳨ウ䛧䛯䚹䛥䜙䛻 䝨䝹䜸䜻䝅䝋䞊䝮ቑṪᅉᏊάᛶཷᐜయ (PPAR) α 䜰䝂䝙䝇䝖䛻䜘䜚ㄏⓎ䛥䜜䛯 GST-P 㝜ᛶ(–) ኚ␗⫢⣽⬊ᕢ䠈䝷䝑䝖䛾Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶⏥≧⭢䠈⭤⬔䠈๓⫶䠈⭢⫶䠈ᑠ⭠䛺䛔䛧⭠䛻 ᙧᡂ䛥䜜䛯ቑṪᛶኚ䛻䛚䛡䜛 TMEM70 䛾㛵䛾᭷↓䜢᳨ウ䛧䛯䚹䜎䛯䠈䛜䜣⣽⬊䛻䛚䛔 䛶㓟ⓗ䝸䞁㓟䛛䜙ゎ⢾⣔䜈௦ㅰ䛜䝅䝣䝖䛩䜛䛸䛔䛖䛄Warburg ຠᯝ䛅䛻㛵䜟䜛ศᏊኚື䛜 Ⓨ䛜䜣≀㉁ᢞ᪩ᮇ䛛䜙㉳䛣䜛䛛䜢᳨ド䛩䜛䛯䜑䠈䝷䝑䝖䜈⫢Ⓨ䛜䜣≀㉁䜢28 ᪥䛺䛔䛧 90 ᪥
6
㛫ᢞ䛧䠈⣽⬊ෆ௦ㅰ㛵㐃㑇ఏᏊ䛾Ⓨ⌧䜢⤒ⓗ䛻᳨ウ䛧䛯䚹
➨3 ❶䛷䛿䠈➨ 1 ❶䛷ᚓ䜙䜜䛯ೃ⿵ศᏊ䛾䛖䛱䠈䝴䝡䜻䝏䞁-䝥䝻䝔䜰䝋䞊䝮ศゎ䛻㛵䛩䜛
ubiquitin-conjugating enzyme E2E 2 (UBE2E2) 䛻ὀ┠䛧䛯䚹UBE2E2 䛾Ⓨ䛜䜣㐣⛬䛻䛚䛡䜛
ᶵ⬟䜢᫂䜙䛛䛻䛩䜛䛯䜑䠈䝷䝑䝖䛾ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䠈GST-P+ቑṪᛶኚ䛻䛚䛡 䜛䠈UBE2E2 䛜㛵㐃䛩䜛䝴䝡䜻䝏䞁䛾ᶆⓗ⺮ⓑ㉁䛾Ⓨ⌧ศᕸ䜢᳨ウ䛧䛯䚹䛥䜙䛻䠈 PPARα 䜰䝂䝙䝇䝖䛻䜘䜚ㄏⓎ䛥䜜䛯 GST-P–ኚ␗⫢⣽⬊ᕢ䠈䝷䝑䝖䛾Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶⏥≧⭢䠈⭤ ⬔䠈๓⫶䠈⭢⫶䠈ᑠ⭠䛺䛔䛧⭠䛻ᙧᡂ䛥䜜䛯ቑṪᛶኚ䛻䛚䛡䜛UBE2E2 䛾㛵䛾᭷↓䜢 ᳨ウ䛧䛯䚹䜎䛯䠈UBE2E2 䛚䜘䜃㛵㐃ศᏊ䛜䠈⣽⬊⪁䛾◚⥢䛸䛜䜣䛻㛵䛩䜛䛛᳨ド䛩 䜛䛯䜑䠈䝷䝑䝖䜈⫢Ⓨ䛜䜣≀㉁䜢᭱㛗 90 ᪥㛫ᢞ䛧䠈UBE2E2 䛚䜘䜃䝴䝡䜻䝏䞁㛵㐃 ⺮ⓑ㉁䛻䛴䛔䛶䠈⫢⮚䛚䛡䜛Ⓨ⌧ศᕸ䜢ゎᯒ䛧䛯䚹᭱ᚋ䛻䠈Ⓨ䛜䜣䜲䝙䝅䜶䞊䝅䝵䞁ฎ⨨䛺䛧 䛻䠈90 ᪥㛫䛾 TAA ᢞ䛻䜘䜚ㄏⓎ䛥䜜䛯 GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛔䛶䜒䠈UBE2E2 䛸㛵㐃 ศᏊ䛾Ⓨ⌧ศᕸ䜢᳨ウ䛧䛯䚹
7 ➨ ➨1 ❶
䝷䝑䝖䜈䛾⫢Ⓨ䛜䜣≀㉁ᢞ䛻䜘䜚DNA 㐣䝯䝏䝹䛸㑇ఏᏊⓎ⌧ῶᑡ䛾䜏䜙䜜䜛
8 ⥴ ⥴㻌 ゝ 䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺㑇ఏᏊⓎ⌧ಟ㣭䛿䠈㑇ఏᏊ㓄ิ䛻ኚ䜢䛘䛪䛻䠈䝀䝜䝮䛾ಟ㣭䜔 ᰁⰍయ䜽䝻䝬䝏䞁䛾ኚ䜢䛸䛧䛶䠈ᶆⓗ㑇ఏᏊ䛾Ⓨ⌧䛻ᙳ㡪䜢䛘䜛 [73]䚹䛣䜜䜙䛾ಟ㣭 䛿DNA 䛾䝅䝖䝅䞁ሷᇶ䛾䝯䝏䝹䠈䝠䝇䝖䞁⺮ⓑ㉁䛾 (⬺)㻌 䜰䝉䝏䝹䜔䝯䝏䝹䛺䛹䛾⩻ヂ
ᚋಟ㣭䠈 microRNA 䛸䜀䜜䜛 noncoding RNA 䛻䜘䜛㌿ᚋ䛾Ⓨ⌧ㄪ⠇䛺䛹䛜ྵ䜎䜜䜛 [58]䚹䛣䛾䛖䛱䠈DNA 䛾䝯䝏䝹䛿䠈ከ⣽⬊⏕≀䛾ṇᖖ䛺Ⓨ⏕ẁ㝵䛻䛚䛔䛶ᗈ䛟ㄆ䜑䜙䜜䛶䛚 䜚䠈䜎䛯䠈ከ䛟䛾ᝈ䛾ᙧᡂ䛻䛚䛔䛶㔜せ䛺ᙺ䜢ᢸ䛖䛣䛸䛜䜟䛛䛳䛶䛝䛶䛔䜛 [6, 52]䚹䝅䝖䝅 䞁䛾ḟ䛻䜾䜰䝙䞁䛜୪䜆2 ሷᇶ㓄ิ䛷䛒䜛 CpG 䛿䠈䛻㑇ఏᏊ䝥䝻䝰䞊䝍䞊㡿ᇦ䜒䛧䛟䛿䛭 䛾㏆ഐ䛻㧗⃰ᗘ䛻Ꮡᅾ䛧䠈CpG 䜰䜲䝷䞁䝗䛸䜀䜜䛶䛔䜛䚹CpG 䜰䜲䝷䞁䝗䛾䝯䝏䝹䛿䠈㌿ ᅉᏊ䛾䝥䝻䝰䞊䝍䞊㡿ᇦ䜈䛾⤖ྜ䜢┤᥋䛒䜛䛔䛿㛫᥋ⓗ䛻㜼ᐖ䛧䠈䜽䝻䝬䝏䞁ᵓ㐀䛾จ㞟䜢 ㄏᑟ䛧䜔䛩䛟䛧䛶⤖ྜ䜢ไ㝈䛧䠈㑇ఏᏊ䛾Ⓨ⌧䜢ᢚไ䛩䜛 [50]䚹㔜せ䛺Ⅼ䛸䛧䛶䠈㑇ఏᏊ䛾䝯 䝏䝹≧ែ䛜䠈ፉ⣽⬊䜈ᘬ䛝⥅䛜䜜䜛䛸䛔䛖Ⅼ䛜ᣲ䛢䜙䜜䜛 [27]䚹 䛣䛾䝀䝜䝮䛻䛚䛡䜛 DNA 䝯䝏䝹␗ᖖ䛿ᵝ䚻䛺䛜䜣䛷ሗ࿌䛥䜜䛶䛚䜚䠈䛜䜣ᢚไ㑇ఏᏊ䛾Ⓨ⌧ᢚไ䜢⏕䛨䜛䛣䛸䛷䠈⭘ ⒆䛾ቑṪ䜔㐍ᒎ䛻᭷䛺ᙧ㉁⋓ᚓ䛻㛵䛩䜛ྍ⬟ᛶ䛜䛒䜛 [33]䚹䛩䛺䜟䛱䠈Ⓨ䛜䜣≀㉁䛾 ᢞ䛻䜘䛳䛶䠈䝥䝻䝰䞊䝍䞊㡿ᇦ䛻䝯䝏䝹ಟ㣭䛜✚䛩䜛㑇ఏᏊ䛿䠈Ⓨ䛜䜣㐣⛬᪩ᮇ 䛾㔜せ䛺㘽䜢ᥱ䜛ྍ⬟ᛶ䛜䛒䜛䚹 ⣽⬊⪁䛿䠈䝔䝻䝯䜰䛾▷⦰䜔䛜䜣㑇ఏᏊ䛾άᛶ䛜ᘬ䛝㔠䛸䛺䜚䠈⣽⬊࿘ᮇṆ䛜㐍⾜
9 䛧䛶䛔䛟㐣⛬䛷䛒䜛 [21]䚹䛣䛾䜘䛖䛺䠈䛜䜣㑇ఏᏊ䛜ㄏᑟ䛩䜛⪁䛿䠈p53 䛚䜘䜃⥙⭷ⱆ⣽⬊ ⭘⺮ⓑ㉁䜢䝁䞊䝗䛩䜛㑇ఏᏊ䛾䜘䛖䛺⣽⬊࿘ᮇ䝏䜵䝑䜽䝫䜲䞁䝖䛻ാ䛟䛜䜣ᢚไ㑇ఏᏊ䛾άᛶ 䜢䛩䜛䛯䜑䠈⭘⒆ᢚไ䛾ᶵ⬟䜒᭷䛩䜛 [12]䚹䛥䜙䛻䠈䛜䜣㑇ఏᏊ Ras 䛻䜘䛳䛶ㄏⓎ䛥䜜䛯 ቑṪᛶኚ䛻䛚䛔䛶䠈⪁⣽⬊䛿䠈๓䛜䜣ኚ䛷䛿ㄆ䜑䜙䜜䜛䜒䛾䛾䠈㐍ᒎ䛧䛯⭘⒆⤌⧊ෆ 䛷䛿ㄆ䜑䜙䜜䛺䛟䛺䛳䛯䛸䛾ሗ࿌䛜䛒䜛 [11]䚹䛩䛺䜟䛱䠈⣽⬊⪁䛛䜙⬺䛧䛯⣽⬊㞟ᅋ䛜䠈䛜 䜣䜈䛾㐨䜢㎺䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䜛䚹 ᡤᒓ◊✲ᐊ䛾ඛ⾜◊✲䛻䛚䛔䛶䠈⣽⬊ቑṪ䜢ஹ㐍䛩䜛Ⓨ䛜䜣≀㉁䜢 28 ᪥㛫䝷䝑䝖䛻ᑐ䛧 㐃⥆⤒ཱྀᢞ䛧䛯㝿䛻䠈Ⓨ䛜䜣ᶆⓗ⣽⬊䛻䛚䛔䛶䠈G2ᮇ䛻䛚䛡䜛ubiquitin D 䛾␗ᖖⓎ⌧䛜 ⏕䛨䠈M ᮇ䛾䝇䝢䞁䝗䝹䝏䜵䝑䜽䝫䜲䞁䝖ᶵ⬟䛾◚⥢䛸䜰䝫䝖䞊䝅䝇䛾㉳䛣䛳䛶䛔䜛䛣䛸䠈ຍ䛘䛶 G1/S 䛚䜘䜃 G2/M ᮇ䛻䛚䛡䜛⣽⬊࿘ᮇṆ䛾㉳䛣䛳䛶䛔䜛䛣䛸䜢ぢฟ䛧䛶䛔䜛 [65, 76]䚹䛥䜙䛻䠈 ⫢⣽⬊䛻ቑṪάᛶஹ㐍䛾䜏䜙䜜䜛⫢Ⓨ䛜䜣≀㉁ᢞ䛻䜘䜚䠈G1/S ᮇ䛻䛚䛡䜛䝏䜵䝑䜽䝫䜲䞁䝖 ᶵ⬟䛾◚⥢䜢♧၀䛩䜛Rbl2 䛾Ⓨ⌧ῶᑡ䠈⥙⭷ⱆ⣽⬊⭘⺮ⓑ㉁䛾ศゎಁ㐍䜢♧၀䛩䜛 Mdm2 䛾Ⓨ⌧ቑຍ䜢ぢฟ䛧䠈ᢞ28 ᪥㛫ᚋ䛻䜏䜙䜜䜛⣽⬊ቑṪஹ㐍䛻䛣䜜䜙䛾ศᏊ䛜㛵䛩䜛ྍ ⬟ᛶ䛜♧၀䛥䜜䛯 [36]䚹ྠ◊✲䛻䛚䛔䛶䠈Mdm2 䛾Ⓨ⌧䛻క䛳䛶 p21WAF1/CIP1䛾Ⓨ⌧ቑຍ䜒 䜏䜙䜜䛶䛚䜚䠈DNA യᐖᛂ⟅䜢ᫎ䛩䜛䜒䛾䛸⪃䛘䜙䜜䛯䚹p21WAF1/CIP1䛚䜘䜃 Mdm2 䛾Ⓨ⌧ ቑຍ䛿䠈䛸䜒䛻⪁⣽⬊䛷ሗ࿌䛥䜜䛶䛔䜛 [39, 53]䚹䛣䜜䜙䛾䛣䛸䛛䜙䠈ඛ⾜◊✲䛻䛚䛡䜛⤖ᯝ 䛿䠈䝷䝑䝖䜈䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛾 28 ᪥㛫ᢞ䛻䜘䜚䠈⣽⬊⪁䛻 㝗䜛⣽⬊䛜ቑຍ䛧䛯䛣䛸䜢♧䛩䜒䛾䛸⪃䛘䜙䜜䛯䚹
10 TAA 䛿᪤▱䛾⫢Ⓨ䛜䜣≀㉁䛷䠈䛢䛳ṑ㢮䛾⫢Ⓨ䛜䜣◊✲䛻ከ䛟⏝䛔䜙䜜䛶䛔䜛 [54]䚹 TAA 䛾៏ᛶⓗ䛺ᢞ䛻䜘䜚䠈⫢⣽⬊䛻␗ᩘయ䛺䛔䛧ಸᩘయ䛾ᙧᡂ䜢ᫎ䛩䜛䠈ᕧ᰾䛺䛔䛧 ⣽⬊⫧䛜⏕䛨䠈⣽⬊࿘ᮇไᚚ␗ᖖ䛜㉳䛣䜛䛣䛸䛜♧၀䛥䜜䛶䛔䜛 [69]䚹䛣䛾㇟䛿䠈⫢⣽ ⬊䛻䛚䛔䛶ᰁⰍయᏳᐃᛶ䜢⏕䛨䠈⭘⒆Ⓨ⏕䜢ᘬ䛝㉳䛣䛩ྍ⬟ᛶ䛜䛒䜛 [78]䚹ඛ⾜◊✲䛻 䛚䛔䛶䜒䠈TAA 䜢䝷䝑䝖䜈 28 ᪥㛫ᢞᚋ䠈⫢⣽⬊䛻⣽⬊ቑṪ䛾ஹ㐍䛸ྛᮇ䛻䛚䛡䜛⣽ ⬊࿘ᮇṆ䛾㉳䛣䜛䛣䛸䜢☜ㄆ䛧䛶䛔䜛 [36, 65, 76]䚹௨ୖ䛾䛣䛸䛛䜙䠈Ⓨ䛜䜣≀㉁ᢞ 䛻䜘䜚䠈Ⓨ䛜䜣㐣⛬䛻᭷䛺ᙧ㉁䜢⋓ᚓ䛧䛯⣽⬊㞟ᅋ䛻䛚䛔䛶䠈DNA 䝯䝏䝹䛸䛔䛖䜶䝢䝆䜵 䝛䝔䜱䝑䜽䛺ಟ㣭䛜⏕䛨䜛䛾䛷䛒䜜䜀䠈㐣䝯䝏䝹䛸Ⓨ⌧ῶᑡ䛾䜏䜙䜜䜛㑇ఏᏊ䛜䠈⣽⬊⪁ 䛾◚⥢䛻㉳ᅉ䛩䜛Ⓨ䛜䜣ᛶ䛾▷ᮇண ᣦᶆ䛸䛺䜚䛖䜛䛸⪃䛘䛯䚹 ᮏ❶䛷䛿䠈▷ᮇ㛫䛾⫢Ⓨ䛜䜣≀㉁䛾ᢞ䛻䜘䜚䠈⫢⮚䛻䛚䛔䛶㐣䝯䝏䝹䛾䜏䜙䜜䜛 㑇ఏᏊ䜢㑅ฟ䛧䠈⫢Ⓨ䛜䜣㐣⛬䛻䜶䞁䝖䝸䞊䛩䜛䛯䜑䛾ᶵᗎ䜢䛘䜛ศᏊᣦᶆ䜢☜❧䛩䜛䛣䛸 䜢┠ⓗ䛻ᐇ㦂䜢⾜䛳䛯䚹䛣䛾┠ⓗ䛾䛯䜑䛻䠈 TAA 䜢䝷䝑䝖䜈 28 ᪥㛫⤒ཱྀᢞ䛧䠈⫢⮚䛻 䛚䛡䜛 DNA 䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲ᐇ䛧䛯䚹㐣䝯䝏䝹䛚䜘䜃㑇ఏᏊ䛾Ⓨ⌧ῶᑡ䜢᳨ド䛧 䛯ᚋ䠈䝷䝑䝖䛾ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䠈⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛾 Ⓨ䛜䜣ಁ㐍䛷ㄏⓎ䛥䜜䛯⫢Ⓨ䛜䜣ቑṪᛶኚ䛻䛚䛡䜛䠈ೃ⿵ศᏊ䛾Ⓨ⌧ศᕸ䜢᳨ウ䛧䛯䚹
11 ᮦ
ᮦᩱ䛚䜘䜃᪉ἲ
Ꮫ≀㉁
TAA䠈β-naphthoflavone (BNF) 䛚䜘䜃 phenobarbital (PB) 䛿ග⣧⸆ᕤᴗᰴᘧ♫ ( 㜰䠈᪥ᮏ) 䜘䜚㉎ධ䛧䛯䚹Piperonyl butoxide (PBO) 䛿 Acros Organics (Geel, Belgium) 䜘䜚䠈 N-diethylnitrosamine (DEN) 䛿ᮾிᡂᕤᴗᰴᘧ♫ (ᮾி䠈᪥ᮏ) 䜘䜚䠈fenbendazole (FB) 䛿 Sigma-Aldrich Japan K.K. (ᮾி䠈᪥ᮏ) 䜘䜚㉎ධ䛧䛯䚹 ౪ヨື≀ 㻌 䛩䜉䛶䛾ᐇ㦂䛻䛚䛔䛶䠈5 㐌㱋䛾㞝ᛶ F344 䝷䝑䝖䜢᪥ᮏ䜶䝇䜶䝹䝅䞊䜘䜚㉎ධ䛧䠈 ᗘ 23 ± 2°C䠈‵ᗘ 55 ± 15%䠈 ↷᫂䝃䜲䜽䝹 12 㛫᫂/12 㛫ᬯ᮲௳䠈⢊ᮎ CRF-1 (䜸䝸䜶䞁䝍 䝹㓝ẕᕤᴗᰴᘧ♫, ᮾி) 䛚䜘䜃㣧ᩱỈ䛾⮬⏤⤥Ỉୗ䛷㣫⫱䛧䠈1 㐌㛫䛾㥆ᮇ㛫䛾ᚋ䠈 ␗ᖖ䛜ㄆ䜑䜙䜜䛺䛛䛳䛯ື≀䜢ᐇ㦂䛻౪䛧䛯䚹 ᐇ㦂䝕䝄䜲䞁 ᮏᐇ㦂䛸䛧䛶䠈4 䛴䛾ື≀ᐇ㦂䜢ᐇ䛧䛯䚹
12 ᐇ ᐇ㦂1 1 㐌㛫䛾㥆ᮇ㛫䛾ᚋ䠈䝷䝑䝖䜢ྛ⩌ 10 ༉䛾 2 ⩌䛻ศ䛡䠈↓ฎ⨨ᑐ↷⩌䛻䛿ᇶ♏㣫ᩱ䜢 䛘䠈TAA ⩌䛻䛿Ⓨ䛜䜣⏝㔞䛷䛒䜛 400 ppm 䛾⏝㔞䛷ΰ㣵䛻䜘䜚 28 ᪥㛫ᢞ䛧䛯 [3]䚹 ヨ㦂ᮇ㛫୰䛔䛪䜜䛾⩌䛾ື≀䜒䠈㣧ᩱỈ䛿⮬⏤⤥Ỉୗ䛷㣫⫱䛧䛯䚹ᐇ㦂ᮇ㛫⤊ᚋ䠈䛩䜉 䛶䛾ື≀䛿CO2/O2㯞㓉ୗ䛷ᚋ㟼⬦䛛䜙䛾ᨺ⾑䛻䜘䜚Ᏻᴦẅ䛧䠈⫢⮚䜢ฟ䛧䠈㔜㔞䜢 ᐃ 䛧䛯䚹⫢⮚䛿㑇ఏᏊⓎ⌧ゎᯒ⏝䛸䛧䛶 1 mm ゅ䛾⣽∦䛻䛧䠈ᾮయ❅⣲䛻ᾐₕ䛧䛶⤖䛧䠈䌦 80䉝䛻䛶ಖᏑ䛧䛯䚹 ᐇ㦂2 䛚䜘䜃 3 㥆ᮇ㛫䛾ᚋ䠈⫢୰ᮇⓎ䛜䜣ᛶヨ㦂ἲ䜢ཧ⪃䛻䠈䝷䝑䝖⫢ẁ㝵Ⓨ䛜䜣ᛶヨ㦂䜢ᐇ䛧䛯 [32, 60]䚹䛩䜉䛶䛾ື≀䛻ᑐ䛧䛶䠈䜲䝙䝅䜶䞊䝍䞊䛸䛧䛶 DEN 䛾༢ᅇ⭡⭍ෆᢞ (200 mg/kg య㔜) 䜢⾜䛔䠈䛭䛾 2 㐌㛫ᚋ䛻䠈ᐇ㦂 2 䛷䛿ྛ⩌ 12 ༉䛾 4 ⩌䛻䠈ᐇ㦂 3 䛷䛿ྛ⩌ 15 ༉䛾 3 ⩌䛻ศ䛡䛯䚹ᐇ㦂 2 䛷䛿䠈ᇶ♏㣫ᩱ䛾䜏 (DEN-alone ⩌)䠈400 ppm TAA 䛾ΰ㣵ᢞ
(DEN+TAA ⩌)䠈5000 ppm BNF 䛾ΰ㣵ᢞ (DEN+BNF ⩌) 䛺䛔䛧 15,000 ppm PBO 䛾ΰ㣵 ᢞ (DEN+PBO) 䜢 6 㐌㛫⾜䛳䛯䚹ᐇ㦂 3 䛷䛿䠈ᇶ♏㣫ᩱ䛾䜏 (DEN-alone ⩌)䠈3500 ppm FB 䛾ΰ㣵ᢞ (DEN+FB ⩌) 䛺䛔䛧 500 ppm PB 䛾ΰ㣵ᢞ (DEN+PB) 䜢 8 㐌㛫⾜䛳䛯䚹 ⏝㔞タᐃ䛻䛚䛔䛶䛿䠈㐣ཤ䛾ሗ࿌䛻䛚䛔䛶䝷䝑䝖䛻䛚䛔䛶⫢Ⓨ䛜䜣ಁ㐍స⏝䛜ㄆ䜑䜙䜜䛯⏝ 㔞䜢⏝䛔䛯 [22, 38, 44, 61]䚹DEN ᢞ 3 㐌㛫ᚋ䛻䠈䛩䜉䛶䛾ື≀䛻 2/3 㒊ศ⫢ษ㝖䜢ᐇ
13 䛧䛯䚹DEN ᢞ 8 㐌㛫ᚋ (ᐇ㦂 2) 䛺䛔䛧 10 㐌㛫ᚋ (ᐇ㦂 3) 䛻䠈䛩䜉䛶䛾ື≀䜢ᐇ㦂 1 䛸 ྠᵝ䛾᪉ἲ䛻䛶Ᏻᴦẅ䛧䠈⫢⮚䜢ฟ䛧䠈㔜㔞䜢 ᐃ䛧䛯䚹⫢⮚䛿⤌⧊Ꮫⓗ᳨⣴䛚䜘䜃ච ⤌⧊Ꮫⓗ᳨⣴䛾䛯䜑䠈1 ༉ᙜ䛯䜚 2 ⟠ᡤ䛾⫢⮚䛾䝇䝷䜲䝇䜢సᡂ䛧䠈4%䝟䝷䝩䝹䝮䜰䝹䝕䝠 䝗⁐ᾮ䛻䛶ᅛᐃ䜢⾜䛳䛯䚹 ᐇ ᐇ㦂4 ᐇ㦂4 䛷䛿䠈⫢⭘⒆ᛶኚ䜢ᙧᡂ䛩䜛䛣䛸䜢┠ⓗ䛻䠈ᐇ㦂 2 䛚䜘䜃 3 䛸ྠᵝ䛻䝷䝑䝖⫢ẁ 㝵Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶䠈10 ༉䛾䝷䝑䝖䛻 0.3% TAA 䜢 30 㐌㛫㣧Ỉᢞ䛧䛯䚹ᢞ᪉ἲ䛿䠈 ᩥ⊩ሗ䜢䜒䛸䛻㑅ᢥ䛧䛯 [19]䚹DEN ᢞ 32 㐌㛫ᚋ䛻䠈䛩䜉䛶䛾ື≀䜢ᐇ㦂 1 䛸ྠᵝ䛾᪉ ἲ䛻䛶Ᏻᴦẅ䛧䠈⫢⮚䜢ฟ䛧䠈㔜㔞䜢 ᐃ䛧䛯䚹⫢⮚䛿⤌⧊Ꮫⓗ᳨⣴䛚䜘䜃ච⤌⧊Ꮫ ⓗ᳨⣴䛾䛯䜑䠈ᐇ㦂2 䛚䜘䜃 3 䛸ྠᵝ䛾᪉ἲ䛻䛶ᅛᐃ䜢⾜䛳䛯䚹 䛩䜉䛶䛾ື≀ᐇ㦂ィ⏬䛿䠈ᅜ❧Ꮫἲேᮾி㎰ᕤᏛ䛾ື≀ᐇ㦂⌮ጤဨ䛻ᥦฟ䛧 䛶ᢎㄆ䜢ཷ䛡䠈ື≀䛾ྲྀ䜚ᢅ䛔䛻䛴䛔䛶䜒䠈ྠᏛ䛾ᐇ㦂ື≀ᣦ㔪䛚䜘䜃ື≀ᐇ㦂䛾㐺ṇ 䛺ᐇ䛻ྥ䛡䛯䜺䜲䝗䝷䜲䞁 (᪥ᮏᏛ⾡㆟䠈2006ᖺ6᭶1᪥) 䜢㑂Ᏺ䛧䛯䚹 DNA 䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲ゎᯒ DNA 䝯䝏䝹䛾⥙⨶ⓗゎᯒ䜢⾜䛖䛯䜑䠈ᐇ㦂 1 䛻䛚䛔䛶䠈↓ฎ⨨ᑐ↷⩌䛚䜘䜃 TAA ⩌䛾 ⫢⮚䛛䜙DNeasy Tissue and Blood Kit (Qiagen, Hilden, Germany) 䜢⏝䛔䛶䝀䝜䝮 DNA 䜢ᢳ
14
ฟ䛧䛯䚹䛥䜙䛻䠈䝀䝜䝮DNA 䜢ไ㝈㓝⣲ Mse I (New England Biolabs Inc., MA, USA) 䜢⏝䛔 䛶ษ᩿ᚋ䠈QIAquick PCR Purification Kit (Qiagen) 䜢⏝䛔䛶⢭〇䛧䛯䚹⇕ኚᛶᚋ䠈᩿∦ DNA 䛿 Input (䝁䞁䝖䝻䞊䝹) 䛚䜘䜃ᢠ 5-methyl cytidine ᢠయ (mouse monoclonal, clone 33D3, IgG; Abcam, Cambridge, UK) 䜢⏝䛔䛯චỿ㝆䛻䜘䜚⃰⦰䛧䛯䝯䝏䝹 DNA (IP DNA) 䛻 ศ䛡䛯䚹Input 䛚䜘䜃 IP DNA 䛿䠈䛥䜙䛻 WGA2 Kit (Sigma, St. Louis, MO, USA) 䜢⏝䛔䛶ቑ ᖜ䛧䛯ᚋ䠈QIAquick PCR Purification Kit (Qiagen) 䜢⏝䛔䛶⢭〇䛧䠈NimbleGen Dual–color DNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany) 䜢⏝䛔䛶䝀䝜䝮 DNA 䛾䝷
䝧䝹䜢⾜䛳䛯䚹䝷䝧䝹䛥䜜䛯 DNA 䝃䞁䝥䝹䛿䠈᪤▱䛾 CpG 䜰䜲䝷䞁䝗䛚䜘䜃䝥䝻䝰䞊䝍
䞊㡿ᇦ䜢⥙⨶䛩䜛385,000 䝥䝻䞊䝤䜢ᦚ㍕䛧䛯 CpG Island Plus Promoter Array (Meth 385K Catalog Arr, Roche NimbleGen, Inc. Madison, WI, USA) 䜢⏝䛔䛶䝝䜲䝤䝸䝎䜲䝊䞊䝅䝵䞁䛧䠈 Innoscan 700 (Innopsys, Carbonne, France) 䛷䝇䜻䝱䞁䛧䛯䚹
Innoscan700 (Innopsys, Carbonne, France) 䛻䛶䝅䜾䝘䝹䝕䞊䝍䜢ྲྀ䜚㎸䜏䠈ᐃ㔞䛚䜘䜃ṇ つᚋ䠈ChipMonk Ver. 1.2.3 (Babraham Institute, Cambridge, UK) 䛻䛶㑇ఏᏊ䝰䝏䞊䝣䛾᳨
⣴䜢⾜䛳䛯䚹ಶ䚻䛾CpG 䝃䜲䝖䛾䝯䝏䝹ẚ⋡䛿 IP DNA/input DNA 䛾䝅䜾䝘䝹ẚ⋡䛛䜙⟬
ฟ䛧䛯䚹TAA ⩌䛻䛚䛔䛶㌿㛤ጞⅬ䜘䜚ୖὶ䛻䛚䛔䛶㐣䝯䝏䝹䛜䜏䜙䜜䠈IP DNA/input DNA 䝅䜾䝘䝹ẚ⋡䛜 1.3 ௨ୖ䛸䛺䜛㑇ఏᏊ䛾䛖䛱䠈↓ฎ⨨ᑐ↷⩌䛻䛚䛔䛶䛿 IP DNA/input DNA 䛾䝅䜾䝘䝹ẚ⋡䛜 1 ௨ୗ䛸䛺䜛㑇ఏᏊ䜢㑅ฟ䛧䛯䚹
15 Real-time RT-PCR
ᐇ㦂1 䛷↓ฎ⨨ᑐ↷⩌䛚䜘䜃 TAA ⩌䛾⫢⮚䛛䜙 RNA 䜢ᢳฟ䛧䠈DNA 䝯䝏䝹䝬䜲䜽䝻䜰
䝺䜲ゎᯒ䛻䜘䛳䛶㑅ฟ䛥䜜䛯㑇ఏᏊ䛻䛴䛔䛶䠈real-time RT-PCR 䛻䜘䜚 mRNA Ⓨ⌧䛾ᐃ㔞ゎ ᯒ䜢ᐇ䛧䛯䚹䛩䛺䜟䛱䠈TAA ⩌䛚䜘䜃↓ฎ⨨ᑐ↷⩌䜢ᑐ㇟䛻䠈RNeasy Mini Kit (Qiagen) 䜢⏝䛔䛶ᢳฟ䛧䛯2 μg 䛾⥲ RNA 䜘䜚䠈SuperScript™ II Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) 䛻䛶 1 ᮏ㙐 cDNA 䜢ྜᡂ䛧䛯 (n=6/⩌)䚹Real-time PCR 䛿䠈 Power SYBR® Green PCR Master Mix 䛚䜘䜃 StepOnePlus™ Real-Time PCR System (Thermo
Fisher Scientific) 䜢⏝䛔䠈〇㐀ඖ䛾䝥䝻䝖䝁䞊䝹䛻ᚑ䛳䛶ᐇ䛧䛯䚹PCR 䝥䝷䜲䝬䞊䛿 Primer Express V3.0 (Thermo Fisher Scientific) 䜢⏝䛔䛶タィ䛧䠈Table 1-1 䛻♧䛧䛯䚹ྛ㑇ఏᏊ䛾 mRNA Ⓨ⌧㔞䛿䠈↓ฎ⨨ᑐ↷⩌䛷䛾Ⓨ⌧್䛻ᑐ䛩䜛┦ᑐ್䛸䛧䛶ồ䜑䠈ෆᅉᛶ䝁䞁䝖䝻䞊䝹 䛸䛧䛶Actb 䛾᳨㔞⥺䜢ồ䜑 2-ΔΔC Tἲ [40] 䛻䛶⟬ฟ䛧䛯䚹 䝯 䝯䝏䝹≉␗ⓗᐃ㔞PCR Ogfod3䠈Hist1h2aa䠈Ube2e2䠈Chd6䠈Slk䠈Zfp638䠈Mgarp䠈Tmem70 䛚䜘䜃 Yipf6 䛾 10 㑇ఏ Ꮚ䛻䛴䛔䛶䝯䝏䝹≉␗ⓗᐃ㔞 PCR 䜢ᐇ䛧䛯䚹ᐇ㦂 1 䛻䛚䛔䛶ᢳฟ䛧䛯䝀䝜䝮 DNA
(n=5/⩌) 䜢䠈Bioruptor™ (UCD-250; Cosmo Bio Co., Ltd., Tokyo, Japan) 䛻䛶᩿∦䛧䠈᩿
∦DNA 䛾୍㒊䛿 Input (䝁䞁䝖䝻䞊䝹) DNA 䛸䛧䛶⏝䜎䛷䌦20°C 䛻ಖᏑ䛧䛯䚹ṧ䜚䛾᩿
16
Mountain View, CA, USA) 䜢⏝䛔䛶䝯䝏䝹 CpG ⤖ྜ⺮ⓑ㉁䜢⤖ྜ䛥䛫䛯☢Ẽ䝡䞊䝈䛸ᐊ
䛷1 㛫ᛂ䛥䛫䠈䝯䝏䝹 DNA 䜢⃰⦰䛧䛯䚹Input 䛚䜘䜃䝯䝏䝹⃰⦰ DNA 䝃䞁䝥䝹䛿䠈
䝯䝏䝹≉␗ⓗᐃ㔞PCR 䛻⏝䛔䛯䚹䝥䝷䜲䝬䞊䛿䠈㌿㛤ጞⅬ䜘䜚ୖὶ䛾 CpG 䛜㇏ᐩ䛺㡿ᇦ
䜢ᶆⓗ䛸䛧䛶䠈Methyl Primer Express software ver. 1.0 䛺䛔䛧 Primer Express software ver. 3.0 (Thermo Fisher Scientific) 䜢⏝䛔䛶タィ䛧䛯 (Table 1-2)䚹ྛ㑇ఏᏊ䛾䝯䝏䝹ẚ⋡䛿䠈ẚ㍑
CTἲ䜢䜒䛸䛻䠈䝯䝏䝹⃰⦰DNA 䝃䞁䝥䝹䛾 CT್䛸 Input DNA 䝃䞁䝥䝹䛾 CT್䛾ẚ䛛䜙
⟬ฟ䛧䛯䚹
䝟
䝟䜲䝻䝬䞊䜽䝅䞊䜽䜶䞁䝅䞁䜾ゎᯒ
Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘䜃 Slk 䛾 4 㑇ఏᏊ䛻㛵䛧䛶䠈ᶆⓗ㓄ิ䛻䛚䛡䜛䝯䝏䝹 CpG 䛾ྜ䜢 PyroMark Q24 pyrosequencing system (Qiagen) 䛻䛶᳨ウ䛧䛯 (n=4/⩌) 䚹ᐇ㦂 1 䛾䝀䝜䝮 DNA 䝃䞁䝥䝹䜢 EpiTect Fast DNA Bisulfite Kit (Qiagen) 䜢⏝䛔䛶䝞䜲䝃䝹䝣䜯䜲䝖
ኚ䛧䠈䛣䛾䛖䛱10 ng 䜢䝔䞁䝥䝺䞊䝖䛸䛧䛶䝡䜸䝏䞁䝥䝷䜲䝬䞊䜢⏝䛔䛯 PCR 䜢⾜䛳䛯䚹䛣䜜
䜙䛾㐣⛬䛿䠈Qiagen PyroMark PCR Kit (Qiagen) 䜢⏝䛔䛶⾜䛔䠈95°C 䛷 15 ศ㛫䜲䞁䜻䝳䝧䞊 䝖ᚋ䠈94°C 䛷 30 ⛊㛫䠈56°C 䛷 30 ⛊㛫䠈72°C 䛷 30 ⛊㛫䛾ᕤ⛬䜢 45 ᅇ⧞䜚㏉䛧䠈72°C 䛷 10
ศ㛫ఙ㛗ᛂ䜢⾜䛳䛯䚹䝡䜸䝏䞁 PCR ⏘≀䛿䠈䝅䞊䜽䜶䞁䝅䞁䜾ᛂ䜢 PyroMark Gold
Q24 Reagents (Qiagen) 䜢⏝䛔䛶⾜䛔䠈PyroMark Q24 (Qiagen) 䛻䛶ゎᯒ䛧䛯䚹ቑᖜ⏝ PCR 䝥䝷䜲䝬䞊䛚䜘䜃䝅䞊䜿䞁䝇⏝䝥䝷䜲䝬䞊䛿䠈Pyrosequencing Assay Design Software V2.0
17 (Qiagen)㻌 䜢⏝䛔䛶タィ䛧䛯㻌 (Table 1-3)䚹 ⌮⤌⧊ᏛⓗᰁⰍ䛚䜘䜃ච⤌⧊ᏛᰁⰍ 㻌 䝟䝷䝣䜱䞁ໟᇙ䛧䛯⫢⮚䛿 3 μm 䛾ཌ䛥䛻ⷧษ䛧䛯ᚋ䠈䝦䝬䝖䜻䝅䝸䞁䞉䜶䜸䝆䞁 (HE) ᰁ Ⰽ䛚䜘䜃ච⤌⧊Ꮫⓗゎᯒ䛻౪䛧䛯䚹ᐇ㦂 4 䛾⫢⮚䛾ᶆᮏ䛷䛿䠈ほᐹ䛥䜜䛯䛩䜉䛶䛾ቑ Ṫᛶኚ䛻䛴䛔䛶 Thoolen 䜙䛾ศ㢮䛻ᚑ䛳䛶䠈ኚ␗⫢⣽⬊ᕢ䠈⫢⣽⬊⭢⭘䛺䛔䛧⫢⣽⬊⒴
䛻ศ㢮䛧䛯 [67]䚹ච⤌⧊Ꮫⓗゎᯒ䛿䠈Vectastain® Elite ABC Kit (Vector Laboratories Inc.,
Burlingame, CA, USA) 䛾 䝥 䝻 䝖 䝁 䞊 䝹 䛻 ᚑ 䛳 䛶 䝅 䜾 䝘 䝹 ᳨ ฟ 䜢 ⾜ 䛔 䠈 ච ᛂ 䛿 3,3′-diaminobenzidine/H2O2 䜢⏝䛔䛶ྍど䛧䛯ᚋ䠈䝦䝬䝖䜻䝅䝸䞁䛻䜘䜚ᑐẚᰁⰍ䛧䛯䚹ᐇ㦂
2 䛚䜘䜃 3 䛾⫢⮚ (n=10/⩌) 䛻䛴䛔䛶䛿䠈䝷䝑䝖๓䛜䜣ኚ䝬䞊䜹䞊䛾 GST-P [32, 60]䠈䝚䜽 䝺䜸䝋䞊䝮䜢ᵓᡂ䛩䜛histone H2A type 1-A (HIST1H2AA) [45]䠈䝭䝖䝁䞁䝗䝸䜰⺮ⓑ㉁䛷 ATP
ྜᡂ㓝⣲䛾⏕ྜᡂ䛻㛵䛩䜛 TMEM70 [10]䠈䝴䝡䜻䝏䞁-䝥䝻䝔䜰䝋䞊䝮ศゎ䛻㛵䛩䜛
UBE2E2 [59]䠈䛭䛧䛶䠈䜰䝫䝖䞊䝅䝇䛚䜘䜃⣽⬊ቑṪ䛾ㄏᑟ䛻㛵䜟䜛 STE20-like kinase (SLK) [2] 䛾ᢠయ䛻䛴䛔䛶䜒ච⤌⧊ᏛᰁⰍ䜢ᐇ䛧䛯䚹㈿ά᪉ἲ䛚䜘䜃ᢠయᕼ㔘᮲௳䛿 Table 1-4 䛻♧䛧䛯䚹ᐇ㦂 4 䛾⫢⮚䛷䛿䠈ᐇ㦂 2 䛚䜘䜃 3 䛸ྠᵝ䛾᪉ἲ䛷 GST-P䠈TMEM70
18 ච ච⤌⧊ᏛᰁⰍ䛾ᐃ㔞ゎᯒ 㻌 ᐇ㦂2 䛚䜘䜃 3 䛾⫢⮚䛻䛴䛔䛶䛿䠈┤ᚄ 200 μm ௨ୖ䛾 GST-P +ኚ␗⫢⣽⬊ᕢ䛾ᩘ䛚䜘 䜃㠃✚䜢ィ 䛧䛯䚹㠃✚䛾 ᐃ䛿䠈⏬ീゎᯒ䝋䝣䝖 WinROOF (୕㇂ၟ䠈ᮾி) 䜢⏝䛔䛯䚹 HIST1H2AA䠈TMEM70䠈UBE2E2 䛚䜘䜃 SLK 䛻䛴䛔䛶䛿䠈Ⓨ䛜䜣㐣⛬䛸㛵㐃䛩䜛䛾䛺䜙䜀 ๓䛜䜣ኚ䛷䛒䜛GST-P+ኚ␗⫢⣽⬊ᕢ䛸୍⮴䛧䛯Ⓨ⌧ῶᑡ䛜䜏䜙䜜䜛䛸௬ㄝ䜢❧䛶䠈࿘ᅖ䛾 ⫢⣽⬊䛻䛚䛡䜛Ⓨ⌧ᙉᗘ䛸ẚ㍑䛧䠈1 ಶయ䛒䛯䜚䛾⥲ GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛䠈ྛศᏊ
䛾Ⓨ⌧ῶᑡ (–) 䛩䜛 GST-P+ኚ␗⫢⣽⬊ᕢ (HIST1H2AA–ᕢ䠈TMEM70–ᕢ䠈UBE2E2–ᕢ䜎
䛯䛿SLK–ᕢ)㻌 䛾ྜ䜢ồ䜑䛯䚹ᐇ㦂 4 䛻䛚䛔䛶䛿䠈⭘⒆䛾㐍ᒎ䜈䛾㛵䛾᭷↓䜢᳨ウ䛩䜛
䛯䜑䠈ᐇ㦂2 䛚䜘䜃 3 䛾᪉ἲ䛸ྠᵝ䛻䠈GST-P+ኚ␗⫢⣽⬊ᕢ䠈⫢⣽⬊⭢⭘䛺䛔䛧⫢⣽⬊⒴䛻
䛚䛡䜛TMEM70 䜎䛯䛿 UBE2E2 䛾Ⓨ⌧䜢ほᐹ䛧䠈ྛቑṪᛶኚ䛻䛚䛡䜛 TMEM70–䜎䛯䛿
UBE2E2–䛾ྜ䜢ồ䜑䛯䚹 ⤫ィゎᯒ 㻌 ᐃ㔞䝕䞊䝍䛻䛴䛔䛶ᖹᆒ್䛚䜘䜃ᶆ‽೫ᕪ䜢ồ䜑䛯䚹Real-time RT-PCR䠈䝯䝏䝹≉␗ ⓗPCR 䛚䜘䜃䝟䜲䝻䝅䞊䜽䜶䞁䝅䞁䜾䛾⩌㛫ẚ㍑䛿䠈F ᳨ᐃ䛻䜘䜚➼ศᩓᛶ䛾☜ㄆ䜢⾜䛳 䛯䚹➼ศᩓ䛜ㄆ䜑䜙䜜䛯ሙྜ䛿 Student 䛾 t ᳨ᐃ䜢⾜䛔䠈➼ศᩓ䛜ㄆ䜑䜙䜜䛺䛛䛳䛯ሙྜ䛿 Aspin-Welch 䛾 t ᳨ᐃ䜢⾜䛳䛯䚹ᐇ㦂 2 䛚䜘䜃 3 䛾ච⤌⧊ᏛᰁⰍ䛾⤖ᯝ䛾䜘䛖䛺ከ⩌㛫 ẚ㍑䛿䠈Bartlett ᳨ᐃ䛷➼ศᩓ䜢☜ㄆ䛧䛯ᚋ䠈୍ඖ㓄⨨ศᩓศᯒ䜢⾜䛳䛯䚹᭷ពᕪ䛜ㄆ䜑䜙
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䜜䛯ሙྜ䛿 Dunnett ᳨ᐃ䜢⾜䛳䛯䚹Bartlett ᳨ᐃ䛷➼ศᩓ䛷䛺䛛䛳䛯ሙྜ䠈Steel ᳨ᐃ䜢⾜䛳
䛯䚹䛣䛾䠈䛩䜉䛶䛾ᢞ⩌䛷DEN-alone ⩌䛸ẚ㍑䛧䛯䚹ᐇ㦂 4 䛾ච⤌⧊ᏛᰁⰍ䛾⤖ᯝ
䛻䛴䛔䛶䛿䠈Fisher 䛾ṇ☜☜⋡᳨ᐃ䜢⾜䛔䠈GST-P+ቑṪᛶኚ (ኚ␗⫢⣽⬊ᕢ䠈⫢⣽⬊⭢
⭘䠈⫢⣽⬊⒴) 㛫䛷䛭䜜䛮䜜ẚ㍑䜢⾜䛳䛯䚹䛺䛚䠈䛩䜉䛶䛾᳨ᐃ䛻䛚䛔䛶᭷ពỈ‽ 5%௨ୗ 䜢᭷ពᕪ䛒䜚䛸䛧䛯䚹䛩䜉䛶䛾ゎᯒ䛿䠈⤫ィゎᯒ䜰䝗䜲䞁䝋䝣䝖 䜶䜽䝉䝹⤫ィ 2010 䜢⏝䛔䛶⾜ 䛳䛯 (ᰴᘧ♫♫ሗ䝃䞊䝡䝇䠈ᮾி䠈᪥ᮏ)䚹
20 ⤖ ⤖㻌 ᯝ DNA 䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲ゎᯒ䛚䜘䜃㌿Ⓨ⌧ゎᯒ ᐇ㦂1䛻䛚䛡䜛䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲ゎᯒ䛾⤖ᯝ䠈TAA⩌䛻䛚䛔䛶↓ฎ⨨ᑐ↷⩌䛻ẚ䜉䠈 1.3ಸ௨ୖ䛾ẚ⋡䛷㐣䝯䝏䝹䛜䜏䜙䜜䠈䛛䛴↓ฎ⨨ᑐ↷⩌䛷䛿䝯䝏䝹ẚ⋡䛾ῶᑡ䛺䛔䛧 䝯䝏䝹≧ែ䛻ኚ䛜䜏䜙䜜䛺䛛䛳䛯㑇ఏᏊ䛸䛧䛶47㑇ఏᏊ䛜㑅ฟ䛥䜜䛯 (Fig. 1-1䛚䜘䜃 Table 1-5)䚹 䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲䛷㑅ฟ䛥䜜䛯㑇ఏᏊ䛻䛴䛔䛶 real-time RT-PCR 䜢ᐇ䛧䛯⤖ᯝ䠈 Ogfod3䠈Hist1h2aa䠈Ube2e2䠈Chd6䠈Slk䠈Zfp638䠈Mgarp䠈Tmem70 䛚䜘䜃 Yipf6 䛜↓ฎ⨨ᑐ↷
⩌䛻ẚ䛧TAA ⩌䛷᭷ព䛻Ⓨ⌧ῶᑡ䛧䛯 (Fig. 1-2A)䚹
DNA 䝯䝏䝹᳨ドゎᯒ 䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲䛚䜘䜃 real-time RT-PCR 䛾⤖ᯝ䛛䜙㑅ฟ䛥䜜䛯㑇ఏᏊ䛻䛴䛔䛶䠈䝯 䝏䝹≉␗ⓗPCR 䛻䜘䜚㐣䝯䝏䝹䛾᳨ド䜢⾜䛳䛯⤖ᯝ䠈Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘 䜃 Slk 䛜↓ฎ⨨ᑐ↷⩌䛻ẚ䛧 TAA ⩌䛷᭷ព䛻䝯䝏䝹ẚ⋡䛾ቑຍ䛜䜏䜙䜜䛯䚹୍᪉䛷䠈 Ogfod3䠈Chd6䠈Zfp638䠈Mgarp 䛚䜘䜃 Yipf6 䛻䛴䛔䛶䛿䝯䝏䝹≧ែ䛻 2 ⩌㛫䛻ᕪ䛿ㄆ䜑䜙 䜜䛺䛛䛳䛯 (Fig. 1-2B)䚹 Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘䜃 Slk 䛻䛴䛔䛶䠈䛥䜙䛻ྛ CpG 䛻䛚䛡䜛䝯䝏䝹⛬ᗘ䜢
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ᐃ㔞䛩䜛䛯䜑䛻䝟䜲䝻䝬䞊䜽䝅䞊䜽䜶䞁䝅䞁䜾ゎᯒ䜢⾜䛳䛯䚹Hist1h2aa䠈Tmem70䠈Ube2e2 䛚
䜘䜃Slk 䛿䠈䛭䜜䛮䜜㛤ጞ䝁䝗䞁䛾䜰䝕䝙䞁䛛䜙ୖὶ 24–46 ␒┠䠈6–58 ␒┠䠈166–184 ␒┠䛺
䛔䛧372–406 ␒┠䛾ሷᇶ䛻䛴䛔䛶ゎᯒ䜢⾜䛳䛯䚹䝅䞊䜽䜶䞁䝇㡿ᇦ䛻ྵ䜎䜜䜛」ᩘ䛾 CpG 䝃
䜲䝖䛾䛖䛱䠈Hist1h2aa 䛿 4 ಶ䛾䛖䛱䛩䜉䛶䛷䠈Tmem70 䛿 5 ಶ䛾䛖䛱 1 ␒┠䛷䠈Ube2e2 䛿 3 ಶ
䛾䛖䛱1 ␒┠䛷䠈Slk 䛿 8 ಶ䛾䛖䛱 3 ␒┠䛷䠈↓ฎ⨨ᑐ↷⩌䛻ẚ㍑䛧䛶 TAA ⩌䛷᭷ព䛺䝯䝏 䝹ẚ⋡䛾ቑຍ䛜䜏䜙䜜䛯㻌 (Fig. 1-3)䚹 ⫢ ⫢⮚䛾ቑṪᛶኚ䛾⌮⤌⧊Ꮫⓗゎᯒ ᐇ㦂2 䛚䜘䜃 3 䛻䛚䛔䛶䠈䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛻䜘䜛 6 㐌 㛫䛺䛔䛧8 㐌㛫䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈GST-P+ኚ␗⫢⣽⬊ᕢ䛾ᩘ䛚䜘䜃㠃✚䛜DEN-alone ⩌䛸 ẚ㍑䛧䛶᭷ព䛻ቑຍ䛧䛯 (Table 1-6)䚹ᐇ㦂 4 䛷䛿䠈30 㐌㛫䛾 TAA ᢞ䛻䜘䜚䠈ከ䛟䛾⫢⣽⬊ ⭢⭘䛚䜘䜃⭢⒴ᕢ䛾ᙧᡂ䛜䜏䜙䜜䛯䚹ほᐹ䛧䛯ᶆᮏ䛷ㄆ䜑䜙䜜䛯ቑṪᛶኚ䛿䛩䜉䛶 GST-P 䛻㝧ᛶ䜢♧䛧䛶䛚䜚䠈䛭䛾ᩘ䛿ዲ㓟ᛶኚ␗⫢⣽⬊ᕢ䛜 123 ಶ䠈⫢⣽⬊⭢⭘䛜 81 ಶ䠈 ⫢⣽⬊⭢⒴䛜52 ಶ䛷䛒䛳䛯䚹 ⫢Ⓨ䛜䜣㐣⛬᪩ᮇ䛻䛚䛡䜛ච⤌⧊ᏛᰁⰍ ᐇ㦂 1 䛾ྛ✀ゎᯒ⤖ᯝ䛻䛚䛔䛶㑅ฟ䛥䜜䛯㑇ఏᏊ䠈Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘䜃 Slk 䛾⩻ヂ⏘≀䛻䛴䛔䛶䠈ᐇ㦂 2 䛚䜘䜃 3 䛾⫢⮚䜢⏝䛔䛶ච⤌⧊ᏛᰁⰍ䜢⾜䛳䛯䚹
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HIST1H2AA 䛿䠈DEN-alone ⩌䛷䛿 GST-P+ኚ␗⫢⣽⬊ᕢ䛻㛵ಀ䛫䛪䠈䜋䜌䛩䜉䛶䛾⫢⣽
⬊ 䛻 䛚 䛔 䛶 ᰾ 䛻 㝧 ᛶ 䜢 ♧ 䛧 䛯 䚹TAA 䛾 Ⓨ 䛜 䜣 ಁ 㐍 䛷 䛿 䠈 ⣽ ⬊ ㉁ 䛚 䜘 䜃 ᰾ 䛻 䛚 䛡 䜛
HIST1H2AA 䛾Ⓨ⌧䛜࿘ᅖ⫢⣽⬊䛻ẚ㍑䛧䛶䠈GST-P+ኚ␗⫢⣽⬊ᕢෆ䛷ῶᑡ䛧䛯 (Fig.
1-4A)䚹HIST1H2AA 䛾Ⓨ⌧ῶᑡ䛜䜏䜙䜜䜛 GST-P+ኚ␗⫢⣽⬊ᕢ (HIST1H2AA–/GST-P+ᕢ)
䛾ྜ䛿TAA ⩌䛻䛚䛔䛶 DEN-alone ⩌䛸ẚ㍑䛧䛶᭷ព䛻ቑຍ䛧䛯䚹୍᪉䠈TAA ௨እ䛾⫢Ⓨ
䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛿DEN-alone 䛸ྠ⛬ᗘ䛷䛒䛳䛯䚹 TMEM70 䛿䠈⣽⬊㉁䛷Ⓨ⌧䛜䜏䜙䜜䠈DEN-alone ⩌䛷䛿㒊ศ䛾 GST-P+ኚ␗⫢⣽⬊ᕢ 䛷䠈࿘ᅖ䛾⫢⣽⬊䛸Ⓨ⌧䛾⛬ᗘ䛿ྠ䛨䛷䛒䛳䛯䚹䛧䛛䛧䠈୍㒊䛾 GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛔 䛶࿘ᅖ䛾⫢⣽⬊䛻ẚ㍑䛧䛶Ⓨ⌧䛾ῶᑡ䛜䜏䜙䜜䛯 (Fig. 1-4B)䚹TAA 䜢䛿䛨䜑䛸䛧䛶䠈᳨ウ䜢 ⾜䛳䛯䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧 ⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛻䜘䜛Ⓨ䛜䜣 ಁ㐍䛻䜘䜚䠈 TMEM70–/GST-P+ᕢ䛾ྜ䛿䠈DEN-alone ⩌䛸ẚ㍑䛧䛶᭷ព䛻ቑຍ䛧䛯䚹
TMEM70 䛸ྠᵝ䛻䠈UBE2E2 䛾Ⓨ⌧䛿⫢⣽⬊䛾⣽⬊㉁䛷ㄆ䜑䜙䜜䠈DEN-alone ⩌䛷䛿
㒊ศ䛾 GST-P+ኚ␗⫢⣽⬊ᕢ䛷䠈࿘ᅖ䛾⫢⣽⬊䛸Ⓨ⌧䛾⛬ᗘ䛿ྠ⛬ᗘ䛷䛒䛳䛯䚹䛧䛛䛧䠈୍ 㒊䛾 GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛔䛶࿘ᅖ䛾⫢⣽⬊䛻ẚ㍑䛧䛶Ⓨ⌧䛾ῶᑡ䛜䜏䜙䜜䛯 (Fig. 1-4C)䚹TAA 䜢䛿䛨䜑䛸䛧䛶䠈᳨ウ䜢⾜䛳䛯䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍 䞊䛻䜘䜛Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈UBE2E2–/GST-P+ᕢ䛾ྜ䛿䠈DEN-alone ⩌䛸ẚ㍑䛧䛶᭷ព䛻ቑ ຍ䛧䛯 (Fig. 1-4C)䚹 SLK 䛿䠈⫢⣽⬊䛾⣽⬊㉁䛷ㄆ䜑䜙䜜䠈DEN-alone ⩌䛷䛿䜋䛸䜣䛹䛾 GST-P+ኚ␗⫢⣽⬊ᕢ
23 䛻䛚䛔䛶࿘ᅖ䛾⫢⣽⬊䛸Ⓨ⌧䛾⛬ᗘ䛿ྠ⛬ᗘ䛷䛒䛳䛯䚹䛧䛛䛧䠈䜟䛪䛛୍㒊䛾 GST-P+ኚ␗ ⫢⣽⬊ᕢ䛻䛚䛔䛶࿘ᅖ䛾⫢⣽⬊䛻ẚ㍑䛧䛶Ⓨ⌧䛾ῶᑡ䛜䜏䜙䜜䛯 (Fig. 1-4D)䚹TAA 䛻䜘䜛 Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈SLK–/GST-P+ᕢ䛾ྜ䛿䠈DEN-alone ⩌䛸ẚ㍑䛧䛶᭷ព䛻ቑຍ䛧䛯䚹䛣䜜 䛿䠈DEN+PBO ⩌䛻䛚䛔䛶䜒ྠᵝ䛻䜏䜙䜜䛯䛜䠈䛭䜜௨እ䛾⩌ (DEN+BNF䠈DEN+PB 䛚䜘 䜃DEN+FB ⩌) 䛷䛿䠈⤫ィᏛⓗ䛻᭷ព䛺ᕪ䛿ㄆ䜑䜙䜜䛺䛛䛳䛯 (Fig. 1-4D)䚹 TAA 䛻䛻䜘䜛⫢Ⓨ䛜䜣ಁ㐍䛷ᙧᡂ䛥䜜䛯⫢ቑṪᛶኚ䛻䛚䛡䜛ච⤌⧊ᏛᰁⰍ 㻌 ᐇ㦂4 䛻䛚䛔䛶ㄆ䜑䜙䜜䛯⫢ቑṪᛶኚ (ኚ␗⫢⣽⬊ᕢ䠈⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⭢⒴)
䛻䛚䛡䜛TMEM70 䛚䜘䜃 UBE2E2 䛾Ⓨ⌧䜢᳨ウ䛧䛯䚹TMEM70 䛚䜘䜃 UBE2E2 䛿䛸䜒䛻䠈
ᐇ㦂 2 䛚䜘䜃 3 䛸ྠᵝ䛻ቑṪᛶኚ࿘ᅖ䛾⫢⣽⬊䛻䛚䛔䛶䠈⣽⬊㉁䛻Ⓨ⌧䛜ㄆ䜑䜙䜜䛯
(Fig. 1-5)䚹GST-P+ቑṪᛶኚ䛻䛚䛔䛶䛿䠈TMEM70 䛚䜘䜃 UBE2E2 䛔䛪䜜䜒Ⓨ⌧ῶᑡ䛧䠈
⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⒴䛷䛿ኚ␗⫢⣽⬊ᕢ䛻ẚ䜉䛶㧗䛔㢖ᗘ䛷ቑṪᛶኚ䛻୍⮴䛧䛯 TMEM70 䛚䜘䜃 UBE2E2 䛾Ⓨ⌧ῶᑡ䛜䜏䜙䜜䛯 (Fig. 1-5)䚹
24 ⪃ ⪃㻌 ᐹ 㻌 ᮏ❶䛷䛿䠈㠀㑇ఏẘᛶ⫢Ⓨ䛜䜣≀㉁TAA 䜢䝷䝑䝖䜈 28 ᪥㛫ᢞᚋ䠈⫢⮚䛻䛚䛔䛶䝥 䝻䝰䞊䝍䞊㡿ᇦ䛻DNA 㐣䝯䝏䝹䛜䜏䜙䜜䜛㑇ఏᏊ䜢䠈䝯䝏䝹䝬䜲䜽䝻䜰䝺䜲䛻䜘䜚⥙⨶ⓗ 䛻ゎᯒ䛧䠈47 㑇ఏᏊ䜢㑅ฟ䛧䛯䚹䛣䛾䛖䛱䠈9 㑇ఏᏊ䛿 mRNA Ⓨ⌧䛾ῶᑡ䛜䜏䜙䜜䠈 Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘䜃 Slk 䛾 4 㑇ఏᏊ䛻䛴䛔䛶䛿䠈䝯䝏䝹䛾᳨ドゎᯒ䛻䜘䜚 䝥䝻䝰䞊䝍䞊㡿ᇦ䛻䛚䛡䜛㐣䝯䝏䝹䜢☜ㄆ䛩䜛䛣䛸䛜䛷䛝䛯䚹ᮏゎᯒ䛻䛚䛔䛶䠈䝯䝏䝹≉ ␗ⓗPCR 䛜䝟䜲䝻䝅䞊䜽䜶䞁䝅䞁䜾䛻ẚ㍑䛧䛶㧗ឤᗘ䛻 DNA 䝯䝏䝹䜢᳨ฟ䛩䜛䛣䛸䛜䛷䛝 䛯䛜䠈䛣䜜䛿䝯䝏䝹≉␗ⓗPCR 䛜䝯䝏䝹 DNA ⃰⦰䝃䞁䝥䝹䜢⏝䛔䛶䛔䜛䛣䛸䛻㉳ᅉ䛩䜛 䛸⪃䛘䜙䜜䛯䚹䜎䛯䠈ච⤌⧊ᏛᰁⰍ䛻䛚䛔䛶䛿䠈GST-P+␗⫢⣽⬊ᕢ䛸୍⮴䛧䛯 TMEM70 䛚䜘䜃UBE2E2 䛾Ⓨ⌧ῶᑡ䛜ㄆ䜑䜙䜜䛯䚹⥲ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛 TMEM70–/GST-P+ᕢ䛚 䜘䜃 UBE2E2–/GST-P+ᕢ䛾ྜ䛿䠈᳨ウ䛧䛯䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊 䝍䞊䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈ඹ㏻䛧䛶ቑຍ䛧䛯䚹䛥䜙䛻䠈㛗ᮇ䛻䛚䛡䜛TAA 䛾Ⓨ䛜䜣ಁ㐍䛻䜘䛳 䛶ᙧᡂ䛥䜜䛯⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⒴䛻䛚䛡䜛 TMEM70–/GST-P+䛚䜘䜃 UBE2E2– /GST-P+䛾ྜ䛿䠈ኚ␗⫢⣽⬊ᕢ䜘䜚䜒㧗䛛䛳䛯䚹୍᪉䠈HIST1H2AA 䛚䜘䜃 SLK 䛻㛵䛧䛶䜒䠈 TMEM70 䛚䜘䜃 UBE2E2 䛸ྠᵝ䛻 GST-P+ኚ␗⫢⣽⬊ᕢ䛸୍⮴䛧䛯Ⓨ⌧ῶᑡ䛜䜏䜙䜜䛯䚹䛧 䛛䛧䛺䛜䜙䠈Ⓨ䛜䜣ಁ㐍䛻䜘䜛 HIST1H2AA–/GST-P+ᕢ䛾ቑຍ䛿 TAA 䛻䛚䛔䛶䛾䜏䠈SLK– /GST-P+ᕢ䛾ቑຍ䜒 TAA 䛚䜘䜃 PBO 䛻䛚䛔䛶䛾䜏ㄆ䜑䜙䜜䠈䛶䛾Ⓨ䛜䜣ಁ㐍䛻䛚䛡䜛ඹ
25 ㏻ᛶ䛿䜏䜙䜜䛺䛛䛳䛯䚹ᮏ❶䛷䛿䠈㠀㑇ఏẘᛶ⫢Ⓨ䛜䜣≀㉁TAA 䛾䝷䝑䝖䜈䛾 28 ᪥㛫ᢞ 䛻䜘䜚䝥䝻䝰䞊䝍䞊㡿ᇦ䛻 DNA 㐣䝯䝏䝹䛚䜘䜃Ⓨ⌧ῶᑡ䛾䜏䜙䜜䜛㑇ఏᏊ䛸䛧䛶 Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘䜃 Slk 䜢ぢฟ䛩䛣䛸䛜䛷䛝䛯䚹䛥䜙䛻䠈Tmem70 䛚䜘䜃 Ube2e2 䛻䛴䛔䛶䛿␗䛺䜛⫢Ⓨ䛜䜣㐣⛬䛾᪩ᮇ䛛䜙㔜せ䛺ᙺ䜢ᢸ䛳䛶䛔䜛ྍ⬟ᛶ䛜♧၀䛥 䜜䛯䚹 㻌 Tmem70 䛿䠈䝭䝖䝁䞁䝗䝸䜰䛾 ATP ྜᡂ㓝⣲䛾⏕ྜᡂ䛻㛵䛩䜛䝭䝖䝁䞁䝗䝸䜰⭷ෆ⺮ⓑ㉁䜢䝁 䞊䝗䛩䜛䛣䛸䛜▱䜙䜜䛶䛔䜛 [23]䚹ATP ྜᡂ㓝⣲㞀ᐖ䛻㉳ᅉ䛧䠈ங㓟䜰䝅䝗䞊䝅䝇䠈ᚰ➽䝭䜸 䝟䝏䞊䠈⫢⭘䛾ཎᅉ䛸䛺䜛䝠䝖䛾䝭䝖䝁䞁䝗䝸䜰䛷䠈TMEM70 䛾ኚ␗䛜㧗㢖ᗘ䛷ሗ࿌䛥䜜䛶 䛔䜛 [10]䚹Ⓨ䛜䜣㐣⛬䛻䛚䛔䛶䛿 ATP ྜᡂ㓝⣲䛾Ⓨ⌧ῶᑡ䛿ゎ⢾⣔䛾ஹ㐍䛸ඹ䛻䠈ᵝ䚻䛺 ⒴䛷ሗ࿌䛥䜜䛶䛔䜛 [31, 79]䚹䜎䛯䠈ᐇ㦂ⓗ䛻䛿䠈⫢Ⓨ䛜䜣≀㉁ N-nitrosomorpholine 䜢䝷䝑䝖 䜈ᢞᚋ䠈ㄏⓎ䛥䜜䛯ኚ␗⫢⣽⬊ᕢ䛻䛚䛔䛶ゎ⢾⣔㓝⣲䛾 hexokinase䠈pyruvate kinase
isoenzyme M2 䛚䜘䜃 glucose-6-phosphate dehydrogenase 䛾Ⓨ⌧䛜ቑຍ䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶 䛔䜛 [13]䚹䛥䜙䛻䠈diethylnitrosamine 䜢䝷䝑䝖䛻ᢞ䛩䜛䛣䛸䛷ㄏⓎ䛥䜜䛯⫢⣽⬊⭘⒆䛻䛚䛔 䛶䠈ATP ྜᡂ㓝⣲䜢䛸䛩䜛䝭䝖䝁䞁䝗䝸䜰྾㙐」ྜయ䛾άᛶ䛜పୗ䛩䜛䛣䛸䛜▱䜙䜜䛶䛔䜛 [5]䚹䛣䜜䜙䛾ሗ࿌䛿䠈䛜䜣⣽⬊䛜㓟⣲䛾᭷↓䛻䜘䜙䛪 ATP ⏘⏕䜢ゎ⢾⣔䛻౫Ꮡ䛩䜛䠈㛗䛝䛻 Ώ䛳䛶ᥦၐ䛥䜜䛶䛝䛯䛄Warburg ຠᯝ䛅䜢䛡䜛䜒䛾䛸⪃䛘䜙䜜䜛 [72]䚹୍᪉䛷䠈䛜䜣⣽⬊䛸 䛿ᑐ↷ⓗ䛻䠈⪁⣽⬊䛷䛿䝭䝖䝁䞁䝗䝸䜰䛾DNA 䛚䜘䜃⺮ⓑ㉁䛜Ⓨ⌧ቑຍ䛩䜛䛣䛸䠈⪁⣽⬊ 䛜䜶䝛䝹䜼䞊⏘⏕䜢䝭䝖䝁䞁䝗䝸䜰䛾ATP ྜᡂ㓝⣲䛻౫Ꮡ䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [51]䚹䛧䛯
26 䛜䛳䛶䠈ᮏ❶䛻䛚䛔䛶⫢Ⓨ䛜䜣≀㉁ TAA 䛾 28 ᪥㛫ᢞ䛻䜘䜚⫢⣽⬊䛻䛚䛔䛶䜏䜙䜜䛯䠈 Tmem70 䛾䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺㑇ఏᏊಟ㣭䛿䠈⣽⬊䛜⪁䛛䜙㐓⬺䛧䠈䛜䜣䜈ྥ䛛䛖䛯䜑 䛻ዲ㒔ྜ䛺⣽⬊⎔ቃ䜢ᩚ䛘䜛ୖ䛷㔜せ䛺ᙺ䠈䛴䜎䜚䠈䝭䝖䝁䞁䝗䝸䜰䛾 ATP ྜᡂ㓝⣲䛾㞀ᐖ 䜢䛧䛶㓟ⓗ䝸䞁㓟䛛䜙ゎ⢾⣔䜈䛾௦ㅰ䛾䝅䝣䝖䛻ᐤ䛩䜛䛸⪃䛘䜙䜜䛯䚹 UBE2E2䛿䠈 UBE2E䝣䜯䝭䝸䞊䛾䝯䞁䝞䞊䛷䛒䜛䠈䝴䝡䜻䝏䞁⤖ྜ㓝⣲䛾୍䛴䛷䛒䜚䠈 Cullin-RINGᆺE3䝴䝡䜻䝏䞁䝸䜺䞊䝊䛸༠ྠ䛧䠈䝃䜲䜽䝸䞁౫Ꮡᛶ䜻䝘䞊䝊䜲䞁䝠䝡䝍䞊䛷䛒䜛 p21WAF1/CIP1䛚䜘䜃c-MYC䛺䛹䛾䝴䝡䜻䝏䞁-䝥䝻䝔䜰䝋䞊䝮ศゎ䛻㛵䛩䜛䛣䛸䛜▱䜙䜜䛶䛔 䜛 [8, 59]䚹ᡤᒓ◊✲ᐊ䛾ඛ⾜◊✲䛻䛚䛔䛶䠈TAA䜢䝷䝑䝖䛻28᪥㛫ᢞ䛧䛯ᚋ䠈⫢⣽⬊䛻䛚 䛡䜛p21WAF1/CIP1䛾Ⓨ⌧䛾ቑຍ䛩䜛䛣䛸䜢ሗ࿌䛧䛶䛔䜛 [36]䚹p21WAF1/CIP1䛿p53䛾άᛶ䜢䛧 䛯⣽⬊࿘ᮇṆ䛻㛵䛩䜛䛣䛸䜢⪃៖䛩䜛䛸 [41]䠈TAAᢞ䛻䜘䜛䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺 UBE2E2䛾Ⓨ⌧ῶᑡ䛜䠈p21WAF1/CIP1䛾Ᏻᐃ䜢䛧䛶⣽⬊࿘ᮇṆ䠈䛴䜎䜚䛿⣽⬊⪁䛻㛵 䛩䜛䛣䛸䜢♧၀䛧䛶䛔䜛䚹⯆῝䛔䛣䛸䛻䠈ู䛾ඛ⾜◊✲䛻䛚䛔䛶䠈ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹 䜢⏝䛔䛶TAA䛾4㐌㛫䛾ಁ㐍䛻䜘䜚䠈p21WAF1/CIP1䛾Ⓨ⌧䛜ቑຍ䛩䜛GST-P+ኚ␗⫢⣽⬊ᕢ䛾ᩘ 䛜ቑ䛘䜛䛣䛸䜢☜ㄆ䛧䛶䛔䜛 [68]䚹䛧䛛䛧䛺䛜䜙䠈ྠ◊✲䛻䛚䛔䛶8㐌㛫䛾TAA䛻䜘䜛ಁ㐍䛷 䛿䠈4㐌㛫䛾ಁ㐍䛸䛿ᑐ↷ⓗ䛻p21WAF1/CIP1䛾Ⓨ⌧䛜ῶᑡ䛩䜛GST-P+ ኚ␗⫢⣽⬊ᕢ䛾ቑຍ䛜 䜏䜙䜜䠈⫢Ⓨ䛜䜣㐣⛬䛾᪩ᮇ䛻䛚䛡䜛⣽⬊⪁䛾◚⥢䛜♧၀䛥䜜䛯䚹ᮏ❶䛻䛚䛡䜛Ⓨ䛜䜣 ≀㉁䛺䛔䛧Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛻䜘䜛ಁ㐍ᮇ㛫䛿6䛺䛔䛧8㐌㛫䛷䛒䜚䠈UBE2E2䛾Ⓨ⌧ቑຍ 䛿p21WAF1/CIP1䛾Ᏻᐃ䛻㛵䛧䛺䛔ྍ⬟ᛶ䛜⪃䛘䜙䜜䛯䚹䜎䛯䠈c-MYC䛻㛵䛧䛶䛿䠈ඛ⾜◊
27 ✲䛻䛚䛔䛶䠈TAA䛻䜘䜛ಁ㐍ᚋ䛾GST-P+ ኚ␗⫢⣽⬊ᕢ䛷䠈c-MYC䛾Ⓨ⌧䛜࿘ᅖ⫢⣽⬊䛻 ẚ㍑䛧䛶ῶᑡ䛩䜛䛣䛸䜢ሗ࿌䛧䛶䛔䜛 [29]䚹䛧䛛䛧䠈ู䛾ඛ⾜◊✲䛻䛚䛔䛶䠈TAA䛻䜘䜛ಁ㐍 䛷䠈GST-P+ ኚ␗⫢⣽⬊ᕢෆ䛻䛚䛡䜛PCNA+ቑṪ⣽⬊䛜䠈ಁ㐍䜢⾜䜟䛺䛔GST-P+ ኚ␗⫢⣽ ⬊ᕢ䛻ẚ㍑䛧䛶ቑຍ䛩䜛䛣䛸䜢ぢฟ䛧䛶䛔䜛 [1]䚹c-MYC䛿䛭䛾ᶵ⬟䛸䛧䛶䠈⣽⬊࿘ᮇ䜢ಁ㐍 䛩䜛䛣䛸䛜▱䜙䜜䛶䛚䜚 [18]䠈ᮏ◊✲䛷ぢฟ䛧䛯⫢Ⓨ䛜䜣ಁ㐍䛻䜘䜛UBE2E2–/GST-P+ᕢ䛾ቑ
ຍ䛿䠈UBE2E2–䛺䛔䛧UBE2E2+ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛c-MYC䛾Ⓨ⌧䜢ẚ㍑䛩䜛ᚲせ䛿䛒䜛
䛜䠈c-MYC䛾Ᏻᐃ䜢䛧䛶Ⓨ䛜䜣㐣⛬䛻䛚䛡䜛⣽⬊ቑṪ䛾ஹ㐍䛻ᐤ䛩䜛ྍ⬟ᛶ䛜⪃䛘 䜙䜜䜛䚹䛧䛯䛜䛳䛶䠈ᮏ❶䛻䛚䛔䛶⫢Ⓨ䛜䜣≀㉁TAA䛾28᪥㛫ᢞ䛻䜘䜚⫢⣽⬊䛻䛚䛔䛶䜏 䜙䜜䛯䠈Ube2e2䛾䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺㑇ఏᏊಟ㣭䛸mRNA䛾Ⓨ⌧ῶᑡ䛿䠈䛭䛾䝴䝡䜻䝏䞁 ᶆⓗ⺮ⓑ㉁䛾Ᏻᐃ䜢䛧䠈๓䛜䜣ኚ䜢ᙧᡂ䛩䜛⫢⣽⬊䛾ቑṪஹ㐍䛻㛵䛩䜛ྍ⬟ᛶ䛜 ♧၀䛥䜜䛯䚹 ᮏ❶䛻䛚䛔䛶䠈TAA䛾㛗ᮇ㛫䛾Ⓨ䛜䜣ಁ㐍䛷ㄏⓎ䛥䜜䛯GST-P+ቑṪᛶኚ䛸୍⮴䛧䛶 TMEM70䛚䜘䜃UBE2E2䛔䛪䜜䜒Ⓨ⌧䛜ῶᑡ䛧䠈⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⒴䛷䛿ኚ␗⫢⣽ ⬊ᕢ䛻ẚ䜉䛶㧗䛔㢖ᗘ䛷ቑṪᛶኚ䛻୍⮴䛧䛯TMEM70䛚䜘䜃UBE2E2䛾Ⓨ⌧ῶᑡ䛜䜏䜙 䜜䛯䚹䛣䛾⤖ᯝ䛛䜙䠈TMEM70䛚䜘䜃UBE2E2䛜⭘⒆䛾Ⓨ⏕㐣⛬䛻㛵䛩䜛䛣䛸䜢♧၀䛥䜜䛯䚹 ⭘⒆䛾ᝏᛶ䛾㐣⛬䛻䛚䛔䛶䠈㏻ᖖ䠈ᛴ㏿䛻ቑṪ䛩䜛⭘⒆⤌⧊ෆ㒊䛚䜘䜃ᾐ₶䛜䜣⣽⬊䛿 ༑ศ䛺⾑ὶ䛜☜ಖ䛷䛝䛪䠈⤖ᯝ䠈ప㓟⣲≧ែ䛻䛥䜜䜛䚹ୖ㏙䛧䛯䜘䛖䛻䠈㓟ⓗ䝸䞁㓟䛾 ᢚไ䛸ゎ⢾⣔䛾ஹ㐍䛻䜏䜙䜜䜛⣽⬊ෆ௦ㅰ䛾䝅䝣䝖䛿ᵝ䚻䛺✀㢮䛾⒴䛷䜏䜙䜜䛶䛔䜛 [31,
28 79]䚹䛣䜜䛿䠈ᝏᛶ㌿ᮇ䛻䛚䛡䜛ప㓟⣲᮲௳ୗ䛷䜒䠈ゎ⢾⣔䜢䛧䛶䜶䝛䝹䜼䞊⏘⏕䜢ྍ⬟ 䛻䛩䜛䠈⭘⒆⣽⬊䛾㐺ᛂ䜢♧၀䛩䜛䜒䛾䛷䛒䜛䚹ᮏ❶䛾⤖ᯝ䛛䜙䠈TMEM70䛾Ⓨ⌧ῶᑡ䛿䠈 Ⓨ䛜䜣㐣⛬䛻䛚䛡䜛䜲䝧䞁䝖䛾୍䛴䛷䛒䜛௦ㅰ䛾䝅䝣䝖䛻㓟ⓗ䝸䞁㓟䛾ᢚไ䜢䛧䛶㛵 䛩䜛䛣䛸䛜♧၀䛥䜜䛯䚹୍᪉䛷䠈䝴䝡䜻䝏䞁-䝥䝻䝔䜰䝋䞊䝮⤒㊰䛿䠈ṇᖖ⣽⬊䛻䛚䛔䛶⒴㛵㐃 ⺮ⓑ㉁䛾ศゎ䜢䛩䜛䛣䛸䛷䠈䛜䜣䜢ᢚไ䛩䜛ᶵᗎ䛻㛵䜟䜛䛣䛸䛜䜟䛛䛳䛶䛔䜛 [37]䚹䛣䜜䛿䠈 䝴䝡䜻䝏䞁㓝⣲䛾Ⓨ⌧ῶᑡ䛜䠈䛜䜣⣽⬊䛻䜏䜙䜜䜛⒴㛵㐃⺮ⓑ㉁䛾㐣Ⓨ⌧䛻ᐤ䛩䜛䛣䛸 䜢♧၀䛧䛶䛚䜚䠈ᮏ❶䛻䜏䜙䜜䜛UBE2E2䛾Ⓨ⌧ῶᑡ䛿䠈䛜䜣䛻㛵㐃䛩䜛⒴㑇ఏᏊ⏘≀䛾 Ᏻᐃ䛻㛵䛩䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䛯䚹 䝁䜰䝠䝇䝖䞁䛾ᵓᡂ⺮ⓑ㉁䛷䛒䜛HIST1H2AA [45]䠈䜰䝫䝖䞊䝅䝇䛚䜘䜃⣽⬊ቑṪ䛾ㄏᑟ䛻 㛵䜟䜛SLK [2] 䛻㛵䛧䛶䛿䠈TMEM70䛚䜘䜃UBE2E2䛸␗䛺䜚䠈୍㒊䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧Ⓨ 䛜䜣䝥䝻䝰䞊䝍䞊䛻䛚䛔䛶䛾䜏䠈GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛Ⓨ⌧ῶᑡ䛜䜏䜙䜜䛯䚹㠀㑇 ఏẘᛶⓎ䛜䜣≀㉁䛻䜘䜛Ⓨ䛜䜣䝯䜹䝙䝈䝮䛿ከ✀ከᵝ䛷䠈䝅䝖䜽䝻䝮P450䛺䛔䛧㓟䝇䝖䝺䝇 䛾ㄏᑟ䠈䜼䝱䝑䝥⤖ྜ⣽⬊㛫䝁䝭䝳䝙䜿䞊䝅䝵䞁䛾ᢚไ䛺䛹䛜▱䜙䜜䛶䛔䜛 [24]䚹䛧䛯䛜䛳䛶䠈 ᮏ◊✲䛷᳨ウ䛧䛯⫢Ⓨ䛜䜣≀㉁䛚䜘䜃⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊㛫䛷䠈HIST1H2AA䛺䛔䛧SLK 䛾Ⓨ⌧䛻ඹ㏻ᛶ䛜䜏䜙䜜䛺䛛䛳䛯䛣䛸䛿䠈ྛ䚻䛾≀㉁䛾Ⓨ䛜䜣䝯䜹䝙䝈䝮䛾㐪䛔䜢ᫎ䛧䛯䜒 䛾䛸⪃䛘䜙䜜䛯䚹 ᮏ❶䛷䛿䠈䝷䝑䝖䜈䛾28᪥㛫䛾TAAᢞ䛻䜘䜚䠈⫢⮚䛻䛚䛔䛶䝥䝻䝰䞊䝍䞊㡿ᇦ䛾DNA㐣䝯 䝏䝹䛚䜘䜃㌿䛾ୗ᪉ไᚚ䛾䜏䜙䜜䜛㑇ఏᏊ䛸䛧䛶Hist1h2aa䠈Tmem70䠈Ube2e2䛚䜘䜃Slk
29 䜢ぢฟ䛧䛯䚹TMEM70䛚䜘䜃UBE2E2䛿䠈᳨ウ䛻䛧䛯䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛚䜘䜃⫢Ⓨ䛜䜣 䝥䝻䝰䞊䝍䞊䛻ඹ㏻䛧䛶䠈GST-P+ቑṪᛶኚ䛻୍⮴䛧䛯Ⓨ⌧ῶᑡ䛜䜏䜙䜜䠈๓䛜䜣ኚ䛚䜘 䜃⭘⒆ᛶኚ䛾ᙧᡂ䛻㛵䛩䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䛯䚹TMEM70䛾GST-P+ቑṪᛶኚ䛻䛚 䛡䜛Ⓨ⌧ῶᑡ䛿䠈㓟ⓗ䝸䞁㓟䛛䜙ゎ⢾⣔䜈䛾⣽⬊ෆ௦ㅰ䛾䝅䝣䝖䠈䛴䜎䜚䛿Ⓨ䛜䜣㐣⛬ 䛻䛚䛡䜛⣽⬊⪁䛾◚⥢䛻㛵䛩䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䛯䚹䜎䛯䠈UBE2E2䛾GST-P+ቑṪᛶ ኚ䛻䛚䛡䜛Ⓨ⌧ῶᑡ䛿䠈ຠ⋡ⓗ䛺⣽⬊ቑṪ䜢⾜䛖䛯䜑䠈c-MYC䛺䛹䛾䝴䝡䜻䝏䞁ᶆⓗ⺮ⓑ ㉁䛾Ᏻᐃ䛻㛵䛩䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䛯䚹
30 ᑠ ᑠ㻌 ᣓ ᮏ❶䛷䛿䠈⫢Ⓨ䛜䜣≀㉁TAA 䜢 28 ᪥㛫䝷䝑䝖䛻ᢞᚋ䠈䝯䝏䝺䞊䝅䝵䞁䝬䜲䜽䝻䜰䝺䜲䛻䜘䜚䠈 ⫢⮚䛻䛚䛔䛶䝥䝻䝰䞊䝍䞊㡿ᇦ䛻DNA 㐣䝯䝏䝹䛜䜏䜙䜜䜛㑇ఏᏊ䜢⥙⨶ⓗ䛻ゎᯒ䛧䛯䚹䛣 䛾⤖ᯝᚓ䜙䜜䛯47 㑇ఏᏊ䛾䛖䛱䠈Hist1h2aa䠈Tmem70䠈Ube2e2 䛚䜘䜃 Slk 䛿䝯䝏䝹≉␗ⓗ PCR 䛚䜘䜃䝟䜲䝻䝅䞊䜽䜶䞁䝅䞁䜾䛻䜘䜚㐣䝯䝏䝹䜢☜ㄆ䛧䠈real-time RT-PCR 䛻䜘䜚Ⓨ⌧ῶ ᑡ䜢☜ㄆ䛧䛯䚹㑅ฟ䛥䜜䛯4 㑇ఏᏊ䛾⩻ヂ⏘≀䛿䠈䝷䝑䝖䛾ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶䠈 TAA 䛾Ⓨ䛜䜣ಁ㐍䛻䜘䛳䛶ㄏⓎ䛥䜜䛯 GST-P+ኚ␗⫢⣽⬊ᕢ䛸୍⮴䛧䛶Ⓨ⌧ῶᑡ䛧䛯䚹䛣䛾䛖
䛱TMEM70 䛚䜘䜃 UBE2E2 䛿䠈᳨⣴䛧䛯 TAA 䜢ྵ䜐䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛚䜘䜃⫢Ⓨ䛜
䜣䝥䝻䝰䞊䝍䞊䛾Ⓨ䛜䜣ಁ㐍䛻䜘䛳䛶ㄏⓎ䛥䜜䛯 GST-P+ኚ␗⫢⣽⬊ᕢ䛸୍⮴䛧䛶Ⓨ⌧ῶᑡ 䛧䠈䛭䛾୍⮴⋡䛿ቑຍ䛧䛯䚹୍᪉䛷䠈HIST1H2AA 䛚䜘䜃 SLK 䛿䠈᳨ウ䜢⾜䛳䛯 TAA 䜢ྵ䜐 䛩䜉䛶䛾Ⓨ䛜䜣ಁ㐍ฎ⨨䛻ඹ㏻䛧䛯ᛂᛶ䛿䠈ᚓ䜙䜜䛺䛛䛳䛯䚹㛗ᮇ㛫䛾TAA 䛻䜘䜛Ⓨ䛜䜣 ಁ㐍䛻䜘䛳䛶ㄏⓎ䛥䜜䛯GST-P+ቑṪᛶኚ䛻୍⮴䛧䛶䠈TMEM70 䛚䜘䜃 UBE2E2 䛿Ⓨ⌧ῶ ᑡ䛧䠈䛣䛾୍⮴⋡䛿䠈ኚ␗⫢⣽⬊ᕢ䛻ẚ䜉䛶⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⭢⒴䛷㧗䛛䛳䛯䚹 TMEM70 䛿䝭䝖䝁䞁䝗䝸䜰䛾㓟ⓗ䝸䞁㓟䛻㛵䛧䠈UBE2E2 䛿⣽⬊࿘ᮇไᚚ⺮ⓑ㉁䛾ศゎ 䛻㛵䛩䜛䛣䛸䛜▱䜙䜜䛶䛔䜛䚹䛧䛯䛜䛳䛶䠈ᮏ❶䛷ぢฟ䛧䛯䠈䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺㑇ఏᏊ䛾 ୗ᪉ไᚚ䛿䠈⫢Ⓨ䛜䜣≀㉁䛾ᢞ28 ᪥ᚋ䛸䛔䛖᪩ᮇ䛛䜙䠈㓟ⓗ䝸䞁㓟䛛䜙ゎ⢾⣔䜈䛾௦ ㅰ䛾䝅䝣䝖䠈䛺䛔䛧⣽⬊ቑṪ䜢άᛶ䛩䜛⣽⬊࿘ᮇไᚚ䛾◚⥢䜢䛧䛶䛜䜣䛻ᐤ䛩䜛䛸
31 ⪃䛘䜙䜜䛯䚹
32 ➨ ➨2 ❶
33 ⥴ ⥴㻌 ゝ Ꮫ≀㉁䛾Ⓨ䛜䜣ᛶホ౯䛿䠈Ꮫ≀㉁䛾Ᏻᛶホ౯䛻䛚䛔䛶ᴟ䜑䛶㔜せ䛷䛒䜛䚹䛧䛛䛧 䛺䛜䜙䠈ᶆ‽ⓗ䛺Ⓨ䛜䜣ᛶヨ㦂䛿䠈ఱⓒ༉䜒䛾䛢䛳ṑ㢮ື≀䜢⏝䛔䛶㛗ᮇ㛫䛻Ώ䛳䛶⿕㦂 ≀㉁䜢ᢞ䛩䜛䛯䜑䠈ከ䛺㛫䛸㈝⏝䜢せ䛩䜛䚹ᡤᒓ◊✲ᐊ䛾ඛ⾜◊✲䛻䛚䛔䛶䠈」ᩘ✀ 㢮䛾Ⓨ䛜䜣ᶆⓗᛶ䜢ᑐ㇟䛸䛧䛯Ⓨ䛜䜣≀㉁䜢28 ᪥㛫䝷䝑䝖䜈㐃⥆⤒ཱྀᢞ䛧䠈Ⓨ䛜䜣ᶆⓗ⣽ ⬊䛻䛚䛔䛶䠈⣽⬊࿘ᮇṆ䜢ྵ䜐⣽⬊࿘ᮇไᚚ␗ᖖ䜢ᫎ䛩䜛䜘䛖䛺㛵㐃ศᏊ䛾Ⓨ⌧ኚື 䜢ぢฟ䛧䛶䛔䜛 [36, 65, 76]䚹⣽⬊࿘ᮇṆ䛜⣽⬊⪁䛻≉ᚩⓗ䛺⌧㇟䛷䛒䜛䛣䛸䜢⪃៖䛩䜛 䛸 [7]䠈ඛ⾜◊✲䛷ᚓ䜙䜜䛯⤖ᯝ䛿䠈㐃⥆ⓗ䛺Ⓨ䛜䜣≀㉁䛾่⃭䛻䜘䜚䠈⣽⬊⪁䛻㝗䜛⣽ ⬊䛜ቑຍ䛧䛯䛣䛸䜢ᫎ䛧䛶䛔䜛䛸⪃䛘䜙䜜䜛䚹➨1 ❶䛷䛿䠈▷ᮇ㛫䛾⫢Ⓨ䛜䜣≀㉁䛾ᢞ 䛻䜘䜚䠈⫢⮚䛻䛚䛔䛶㐣䝯䝏䝹ಟ㣭䛜⏕䛨䛶䠈ᶆⓗ⣽⬊䛾䛜䜣䜈䛾㘽䜢ᥱ䜛䛸⪃䛘䠈ᐇ㦂 䜢⾜䛳䛯 [43]䚹䛣䛾⤖ᯝ䠈䝷䝑䝖䜈Ⓨ䛜䜣≀㉁ TAA 䜢 28 ᪥㛫ᢞ䛧䠈⫢⮚䛻䛚䛔䛶㐣䝯䝏䝹 䛚䜘䜃Ⓨ⌧ῶᑡ䛾䜏䜙䜜䜛㑇ఏᏊ䛜㑅ฟ䛥䜜䛯䚹䛣䛾䛖䛱䠈Tmem70 䛾⩻ヂ⏘≀䛿䠈␗䛺䜛 ⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛻䜘䜛Ⓨ䛜䜣ಁ㐍䛷ㄏⓎ䛥䜜䛯 GST-P+ኚ␗⫢⣽ ⬊ᕢ䛻୍⮴䛧䛶Ⓨ⌧ῶᑡ䛧䛯䚹TAA 䛻䜘䜛㛗ᮇ㛫䛾Ⓨ䛜䜣ಁ㐍ᚋ䛻䛚䛔䛶䛿䠈ኚ␗⫢⣽⬊ 䛻ẚ䜉䛶㧗㢖ᗘ䛻⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⒴䛻䛚䛡䜛 TMEM70 䛾Ⓨ⌧ῶᑡ䛜ㄆ䜑䜙䜜䛯䚹 TMEM70 䛿䠈䝭䝖䝁䞁䝗䝸䜰䛾㓟ⓗ䝸䞁㓟䛻㛵䛩䜛䛣䛸䛜▱䜙䜜䛶䛚䜚 [23]䠈Tmem70 䛾
34 䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺Ⓨ⌧ῶᑡ䛿䠈Ⓨ䛜䜣≀㉁ᢞ䜟䛪䛛28 ᪥ᚋ䛸䛔䛖᪩ᮇ䛛䜙⏕䛨䠈㓟ⓗ 䝸䞁㓟䛾ᢚไ䜢䛧䛶Ⓨ䛜䜣䛻ᐤ䛩䜛ྍ⬟ᛶ䛜♧၀䛥䜜䛯䚹 TMEM70 䛿䝭䝖䝁䞁䝗䝸䜰⭷⺮ⓑ㉁䛷䛒䜚䠈ATP ྜᡂ㓝⣲㞀ᐖ䛻㉳ᅉ䛧䠈ங㓟䜰䝅䝗䞊䝅䝇䠈 ᚰ➽䝭䜸䝟䝏䞊䠈⫢⭘䛾ཎᅉ䛸䛺䜛䝠䝖䛾䝭䝖䝁䞁䝗䝸䜰䛻䛚䛔䛶䠈TMEM70 䛾Ḟᦆ䛜ㄆ䜑 䜙䜜䜛䛣䛸䛛䜙䠈TMEM70 䛿 ATP ྜᡂ㓝⣲䛾⏕ྜᡂ䛻㛵䛩䜛䛸⪃䛘䜙䜜䛶䛔䜛 [23]䚹⪁ ⣽⬊䛷䛿䠈䝭䝖䝁䞁䝗䝸䜰DNA 䛚䜘䜃䝭䝖䝁䞁䝗䝸䜰⺮ⓑ㉁䛾ቑຍ䛜㉳䛣䜛䛣䛸䠈ATP ྜᡂ㓝⣲䛻 ౫Ꮡ䛧䛯㓟ⓗ䝸䞁㓟䛻䜘䜚䜶䝛䝹䜼䞊⏘⏕䜢⾜䛖䛜▱䜙䜜䛶䛔䜛 [51]䚹䛧䛯䛜䛳䛶䠈䝷䝑 䝖䜈䛾Ⓨ䛜䜣≀㉁TAA 䛾 28 ᪥㛫ᢞᚋ䛻䠈⫢⮚䛻䛚䛔䛶䜏䜙䜜䛯 Tmem70 䛾䜶䝢䝆䜵䝛䝔 䜱䝑䜽䛺Ⓨ⌧ῶᑡ䛿䠈⣽⬊⪁䜢⬺䛧䛯⣽⬊㞟ᅋ䛾ቑຍ䜢ᫎ䛧䛯䜒䛾䛸⪃䛘䜙䜜䜛䚹䛣䜜䜎 䛷䛻TMEM70 䛾Ⓨ䛜䜣䛸䛾㛵䛻䛴䛔䛶䛾ሗ࿌䛿䛺䛔䛜䠈ATP ྜᡂ㓝⣲䛾Ⓨ⌧ῶᑡ䛿ᵝ䚻 䛺⒴䛻䛚䛔䛶ሗ࿌䛥䜜䛶䛔䜛 [31]䚹ṇᖖ⤌⧊䛻ẚ䜉䛶䠈⭘⒆⤌⧊䛿୍⯡ⓗ䛻䠈㓟⣲䛾᭷↓ 䛻㛵䜟䜙䛪䠈⢾䛾ྲྀ䜚㎸䜏䛚䜘䜃ங㓟䛾ྜᡂ䛜ቑຍ䛩䜛䠈䛔䜟䜖䜛䛄Warburg ຠᯝ䛅䛸䜀䜜 䜛⌧㇟䛜ㄆ䜑䜙䜜䜛䚹䛜䜣⣽⬊䛾⢾Ⴔዲᛶ䜢⏝䛧䠈ᨺᑕᛶྠඖ⣲䜢䛧䛯䝤䝗䜴⢾ 2-deoxy-2(18F)-fluoro-D-glucose 䜢⏝䛔䛯䠈㝧㟁Ꮚ᩿ᒙᙳἲ䛻䜘䜛䠈䛜䜣デ᩿䛜⮫ᗋⓗ䛻 ⾜䜟䜜䛶䛔䜛 [16]䚹ᐇ㦂ⓗ䛻䛿䠈䝷䝑䝖䜈Ⓨ䛜䜣≀㉁ N-nitrosomorpholine 䜢ᢞ䛧䠈ㄏⓎ䛥 䜜䛯 GST-P+ኚ␗⫢⣽⬊ᕢ䛸୍⮴䛧䛶ゎ⢾⣔㓝⣲䛾Ⓨ⌧䛜ቑຍ䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [13]䚹䛴䜎䜚䠈TAA 䛾ᢞᚋ䛻䜏䜙䜜䛯䠈䜶䝢䝆䜵䝛䝔䜱䝑䜽䛺ไᚚ䜢䛧䛯 TMEM70 䛾
35 Ⓨ⌧ῶᑡ䛿䠈⣽⬊⪁䛾◚⥢䜢㉳䛣䛧䠈ATP ྜᡂ㓝⣲㞀ᐖ䛻䜘䜛㓟ⓗ䝸䞁㓟䛛䜙ゎ⢾⣔ 䜈䛾௦ㅰ䛾䝅䝣䝖䛸䛔䛖䠈Ⓨ䛜䜣㐣⛬䛻᭷䛺⎔ቃᙧᡂ䛻ᐤ䛩䜛ྍ⬟ᛶ䛜䛒䜛䚹 ᮏ❶䛷䛿䠈䝷䝑䝖ẁ㝵⫢Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䠈GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛 TMEM70 䛾Ⓨ⌧ῶᑡ䛸௦ㅰ䛾䝅䝣䝖䛸䛾㛵㐃ᛶ䜢᳨ウ䛧䛯䚹䛥䜙䛻䠈PPARα 䜰䝂䝙䝇䝖䛻䜘䜚ㄏⓎ䛥䜜䛯 GST-P–ኚ␗⫢⣽⬊ᕢ䠈䝷䝑䝖䛾Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶⏥≧⭢䠈⭤⬔䠈๓⫶䞉⭢⫶䠈ᑠ⭠䛺䛔䛧 ⭠䛻ᙧᡂ䛥䜜䛯ቑṪᛶኚ䛻䛚䛡䜛 TMEM70 䛾㛵䛾᭷↓䜢᳨ウ䛧䛯䚹᭱ᚋ䛻䠈 䛄Warburg ຠᯝ䛅䛻䜏䜙䜜䜛ศᏊኚື䛜Ⓨ䛜䜣≀㉁ᢞ᪩ᮇ䛛䜙㉳䛣䜛䛸⪃䛘䠈䛥䜙䛻䝷䝑䝖䜈 ⫢Ⓨ䛜䜣≀㉁䜢28 ᪥䛺䛔䛧 90 ᪥㛫ᢞ䛧䠈⣽⬊ෆ௦ㅰ㛵㐃㑇ఏᏊ䛾Ⓨ⌧䜢⤒ⓗ䛻᳨ウ 䛧䛯䚹
36 ᮦ
ᮦᩱ䛚䜘䜃᪉ἲ
Ꮫ≀㉁
TAA䠈BNF䠈PB䠈catechol䠈promethazine (PMZ) 䛚䜘䜃 methylcellulose 400 䛿ග⣧⸆ᕤ ᴗ ᰴ ᘧ ♫ 䜘 䜚 ㉎ ධ 䛧 䛯 䚹 PBO 䛿 Acros Organics (Geel, Belgium) 䜘 䜚 䠈 N-bis(2-hydroxypropyl) nitrosamine (DHPN) 䛿䝘䜹䝷䜲䝔䝇䜽 (ி㒔䠈᪥ᮏ) 䜘䜚㉎ධ䛧䛯䚹 Methapyrilene hydrochloride (MP) 䠈 FB 䠈 sulfadimethoxine sodium salt (SDM) 䛚 䜘 䜃 azoxymethane (AOM) 䛿䠈 Sigma-Aldrich Japan K.K.䜘䜚㉎ධ䛧䛯䚹DEN䠈clofibrate (CF)䠈 bis(2-ethylhexyl) phthalate (DEHP) 䠈 N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) phenylethyl isothiocyanate (PEITC) 䛚䜘䜃 N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)䛿䠈 ᮾிᡂᕤᴗᰴᘧ♫䜘䜚㉎ධ䛧䛯䚹 ౪ヨື≀ 䛩䜉䛶䛾ᐇ㦂䛻䛚䛔䛶䠈5 㐌㱋䛾㞝ᛶ F344 䝷䝑䝖䜢᪥ᮏ䜶䝇䜶䝹䝅䞊䜘䜚㉎ධ䛧䠈 ᗘ 23 ± 2°C䠈‵ᗘ 55 ± 15%䠈 ↷᫂䝃䜲䜽䝹 12 㛫᫂/12 㛫ᬯ᮲௳䠈⢊ᮎ CRF-1 (䜸䝸䜶䞁䝍䝹㓝 ẕᕤᴗᰴᘧ♫) 䛚䜘䜃㣧ᩱỈ䛾⮬⏤⤥Ỉୗ䛷㣫⫱䛧䠈1 㐌㛫䛾㥆ᮇ㛫䛾ᚋ䠈␗ᖖ䛜ㄆ 䜑䜙䜜䛺䛛䛳䛯ື≀䜢ᐇ㦂䛻౪䛧䛯䚹
37 ᐇ ᐇ㦂䝕䝄䜲䞁 ᮏᐇ㦂䛸䛧䛶䠈7 䛴䛾ື≀ᐇ㦂䜢ᐇ䛧䛯 (Table 2-1) 䚹 ᐇ㦂1 䛚䜘䜃 2 TMEM70 Ⓨ⌧ῶᑡ䛸 GST-P+ኚ␗⫢⣽⬊ᕢ䛸䛾㛵㐃䜢᳨ウ䛩䜛䛯䜑䠈⫢୰ᮇⓎ䛜䜣ᛶヨ 㦂ἲ䜢ཧ⪃䛻䠈䝷䝑䝖⫢ẁ㝵Ⓨ䛜䜣ᛶヨ㦂䜢ᐇ䛧䛯 [32, 60]䚹1 㐌㛫䛾㥆ᮇ㛫ᚋ䠈䛩䜉 䛶䛾ື≀䛻ᑐ䛧䛶䠈䜲䝙䝅䜶䞊䝍䞊䛸䛧䛶DEN 䛾༢ᅇ⭡⭍ෆᢞ (200 mg/kg య㔜) 䜢⾜䛔䠈 䛭䛾2 㐌㛫ᚋ䛻䠈ᐇ㦂 1 䛷䛿ྛ⩌ 12 ༉䛾 5 ⩌䛻䠈ᐇ㦂 2 䛷䛿ྛ⩌ 15 ༉䛾 3 ⩌䛻ศ䛡䛯䚹
ᐇ㦂1 䛷䛿䠈ᇶ♏㣫ᩱ䛾䜏 (DEN-alone ⩌)䠈400 ppm TAA 䛾ΰ㣵ᢞ (DEN+TAA ⩌)䠈 1000 ppm MP 䛾ΰ㣵ᢞ (DEN+MP ⩌)䠈5000 ppm BNF 䛾ΰ㣵ᢞ (DEN+BNF ⩌) 䛺䛔 䛧15,000 ppm PBO 䛾ΰ㣵ᢞ (DEN+PBO) 䜢 6 㐌㛫⾜䛳䛯䚹ᐇ㦂 3 䛷䛿䠈ᇶ♏㣫ᩱ䛾䜏 (DEN-alone ⩌)䠈3500 ppm FB 䛾ΰ㣵ᢞ (DEN+FB ⩌) 䛺䛔䛧 500 ppm PB 䛾ΰ㣵ᢞ (DEN+PB) 䜢 8 㐌㛫⾜䛳䛯䚹⏝㔞タᐃ䛻䛚䛔䛶䛿䠈㐣ཤ䛾ሗ࿌䛻䛚䛔䛶䝷䝑䝖䛻䛚䛔䛶⫢Ⓨ 䛜䜣ಁ㐍స⏝䛜ㄆ䜑䜙䜜䛯⏝㔞䜢⏝䛔䛯 [3, 22, 38, 44, 47, 61]䚹DEN ᢞ 3 㐌㛫ᚋ䛻䠈䛩䜉 䛶䛾ື≀䛻2/3 㒊ศ⫢ษ㝖䜢ᐇ䛧䛯䚹DEN ᢞ 8 㐌㛫ᚋ (ᐇ㦂 1) 䛺䛔䛧 10 㐌㛫ᚋ (ᐇ 㦂 2) 䛻䠈ᐇ㦂ᮇ㛫⤊ᚋ䠈䛩䜉䛶䛾ື≀䛿 CO2/O2㯞㓉ୗ䛷ᚋ㟼⬦䛛䜙䛾ᨺ⾑䛻䜘䜚Ᏻ ᴦẅ䛧䠈⫢⮚䜢ฟ䛧䠈㔜㔞䜢 ᐃ䛧䛯䚹⫢⮚䛿⤌⧊Ꮫⓗ᳨⣴䛚䜘䜃ච⤌⧊Ꮫⓗ᳨⣴䛾 䛯䜑䠈1 ༉ᙜ䛯䜚 2 ⟠ᡤ䛾⫢⮚䛾䝇䝷䜲䝇䜢సᡂ䛧䠈4%䝟䝷䝩䝹䝮䜰䝹䝕䝠䝗⁐ᾮ䛻䛶ᅛᐃ䜢⾜
38 䛳䛯䚹 ᐇ ᐇ㦂3 ᐇ㦂3 䛷䛿䠈⫢⭘⒆ᛶኚ䛻䛚䛡䜛 TMEM70 䛾ᙺ䜢᳨ウ䛩䜛䛯䜑䛻䠈ᐇ㦂 1 䛚䜘䜃 2 䛸ྠᵝ䛻䝷䝑䝖⫢ẁ㝵Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶䠈10 ༉䛾䝷䝑䝖䛻 0.3% TAA 䜢 30 㐌㛫㣧Ỉᢞ 䛧䛯䚹ᢞ᪉ἲ䛿䠈ᩥ⊩ሗ䜢䜒䛸䛻㑅ᢥ䛧䛯 [19]䚹DEN ᢞ 32 㐌㛫ᚋ䛻䠈䛩䜉䛶䛾ື ≀䜢ᐇ㦂1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶Ᏻᴦẅ䛧䛯䚹䜎䛯䠈ゎ๗䠈䛩䜉䛶䛾ື≀䛛䜙⫢⮚䜢 ฟ䛧䠈ᐇ㦂1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶ᅛᐃ䛧䛯䚹ື≀䝃䞁䝥䝹䛿䛩䜉䛶➨ 1 ❶䛾ᐇ㦂 4 䛻 䛶⏝䛔䛯䜒䛾䛸ྠ䛨䜒䛾䜢⏝䛧䛯䚹 ᐇ㦂4 ᐇ㦂4 䛷䛿䠈GST-P–ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛TMEM70 䛚䜘䜃⣽⬊ෆ௦ㅰ㛵㐃ศᏊ䛾Ⓨ⌧ ศᕸ䜢᳨ウ䛩䜛䛯䜑䠈ᐇ㦂2 䛚䜘䜃 3 䛸ྠᵝ䛻䝷䝑䝖⫢ẁ㝵Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛶䠈ྛ⩌ 15 ༉䛾3 ⩌䛻ศ䛡䠈Ⓨ䛜䜣ಁ㐍ᮇ䛻ᇶ♏㣫ᩱ䛾䜏 (DEN-alone ⩌)䠈3000 ppm CF 䛾ΰ㣵ᢞ
(DEN+CF ⩌) 䛺䛔䛧 12,500 ppm DEHP 䛾ΰ㣵ᢞ (DEN+ DEHP) 䜢 13 㐌㛫⾜䛳䛯䚹⏝ 㔞タᐃ䛻䛚䛔䛶䛿䠈㐣ཤ䛾ሗ࿌䛷䝷䝑䝖䛻䛚䛔䛶⫢Ⓨ䛜䜣ಁ㐍స⏝䛜ㄆ䜑䜙䜜䛯⏝㔞䜢⏝䛔 䛯 [56]䚹DEN ᢞ 15 㐌㛫ᚋ䛻䠈䛩䜉䛶䛾ື≀䜢ᐇ㦂 1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶Ᏻᴦẅ䛧
䛯䚹䜎䛯䠈ゎ๗䠈䛩䜉䛶䛾ື≀䛛䜙⫢⮚䜢ฟ䛧䠈ᐇ㦂1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶ᅛᐃ䛧
39 ᐇ ᐇ㦂5 ᐇ㦂 5 䛷䛿䠈䝷䝑䝖䛾⏥≧⭢䠈⭤⬔䠈๓⫶䛺䛔䛧⭢⫶䛻ᙧᡂ䛥䜜䛯ቑṪᛶኚ䛻䛚䛡䜛䠈 TMEM70 䛚䜘䜃⣽⬊ෆ௦ㅰ㛵㐃ศᏊ䛾Ⓨ⌧ศᕸ䜢᳨ウ䛩䜛䛯䜑䠈ẁ㝵Ⓨ䛜䜣䝰䝕䝹䜢 ⏝䛔䛯Ⓨ䛜䜣ಁ㐍ᐇ㦂䜢⾜䛳䛯䚹䛩䜉䛶䛾ື≀䜢㥆ᮇ㛫ᚋ䠈ྛ⩌12 ༉䛾 6 ⩌䛻ศ䛡䛯䚹 ⏥≧⭢䜢ᶆⓗ䛸䛩䜛ᐇ㦂䛻⏝䛔䜛ື≀䛿䠈2800 mg/kg 䛾 DHPN 䜢༢ᅇ⓶ୗᢞ䛧䠈䛭䛾 1 㐌㛫ᚋ䛛䜙䠈 1500 ppm 䛾 SDM 䜢 4 㐌㛫㣧Ỉᢞ (DHPN + SDM ⩌)䠈䛺䛔䛧㣧ᩱỈ 䛷⥔ ᣢ䛧䛯 (DHPN-alone) 䚹SDM 䛿䠈ẁ㝵Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛯Ⓨ䛜䜣ಁ㐍 13 㐌㛫ᚋ䛻⏥ ≧⭢ℐ⬊ୖ⓶⣽⬊⒴䜢ㄏⓎ䛩䜛⏝㔞䜢ᢞ⏝㔞䛸䛧䛶タᐃ䛧䛯 [34]䚹⭤⬔䜢ᶆⓗ䛸䛩䜛ᐇ 㦂䛻⏝䛔䜛ື≀䛿䠈500 ppm 䛾 BBN 䜢 4 㐌㛫㣧Ỉᢞ䛧䠈䛭䛾ᚋ䠈ᇶ♏㣫ᩱ䛾䜏 (BBN-alone ⩌) 䛺䛔䛧 1000 ppm 䛾 PEITC 䛾ΰ㣵ᢞ (BBN + PEITC, 11 ༉) 䜢 8 㐌㛫⾜ 䛳䛯䚹PEITC 䛿䠈ẁ㝵Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛯Ⓨ䛜䜣ಁ㐍 32 㐌㛫ᚋ䛻⭤⬔䛻⛣⾜ୖ⓶⒴䜢 ㄏⓎ䛩䜛⏝㔞䜢ᢞ⏝㔞䛸䛧䛶タᐃ䛧䛯 [26]䚹㻌 ๓⫶䛚䜘䜃⭢⫶䜢ᶆⓗ䛸䛩䜛ᐇ㦂䛻⏝䛔䜛 ື≀䛿䠈150 mg/kg 䛾 MNNG 䜢༢ᅇᙉไ⤒ཱྀᢞ䛧䠈䛭䛾 1 㐌㛫ᚋ䜘䜚ᇶ♏㣫ᩱ䛾䜏 (MNNG -alone ⩌) 䛺䛔䛧䠈8000 ppm 䛾 catechol 䛾ΰ㣵ᢞ (MNNG + CC) 䜢 8 㐌㛫⾜䛳 䛯䚹Catechol 䛿䠈ẁ㝵Ⓨ䛜䜣䝰䝕䝹䜢⏝䛔䛯Ⓨ䛜䜣ಁ㐍 51 㐌㛫ᚋ䛻๓⫶䛚䜘䜃⭢⫶䛻䛜 䜣䜢ㄏⓎ䛩䜛⏝㔞䜢ᢞ⏝㔞䛸䛧䛶タᐃ䛧䛯 [71]䚹ᐇ㦂ᮇ㛫⤊ᚋ䠈䛩䜉䛶䛾ື≀䜢ᐇ㦂 1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶Ᏻᴦẅ䛧䛯䚹䜎䛯䠈ゎ๗䠈䛩䜉䛶䛾ື≀䛛䜙Ⓨ䛜䜣ᶆⓗ⮚ჾ䜢
40 ฟ䛧䠈㔜㔞 ᐃᚋ䠈ᐇ㦂1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶ᅛᐃ䛧䛯䚹ື≀䝃䞁䝥䝹䛿䛩䜉䛶ඛ ⾜◊✲䛻䛚䛔䛶䠈㇂ྜ䜙䛜⏝䛔䛯䜒䛾䛸ྠ䛨䜒䛾䜢⏝䛧䛯 [66]䚹 ᐇ ᐇ㦂6 ᐇ㦂6䛷䛿䠈䝷䝑䝖䛾⤖⭠䛺䛔䛧༑ᣦ⭠䛻ᙧᡂ䛥䜜䛯ቑṪᛶኚ䛻䛚䛡䜛䠈TMEM70䛚䜘 䜃⣽⬊ෆ௦ㅰ㛵㐃ศᏊ䛾Ⓨ⌧ศᕸ䜢᳨ウ䛩䜛䛯䜑䠈27༉䛾䝷䝑䝖䜢⏝䛔䠈AOMㄏⓎⓎ䛜䜣 ᐇ㦂䜢ᐇ䛧䛯䚹㥆ᮇ㛫ᚋ䠈15 mg/kg䛾AOM䛾⓶ୗᢞ䜢㐌1ᅇ3㐌㛫 (ィ3ᅇ) ⾜䛔䠈29 㐌䛛䜙35㐌㛫⥔ᣢ䛧䛯䚹ᐇ㦂ᮇ㛫⤊ᚋ䠈䛩䜉䛶䛾ື≀䛿䜲䝋䝣䝹䝷䞁㯞㓉ୗ䛷ᚋ㟼⬦䛛 䜙䛾ᨺ⾑䛻䜘䜚Ᏻᴦẅ䛧䠈⤖⭠䛚䜘䜃༑ᣦ⭠䜢ฟᚋ䠈10%୰ᛶ⦆⾪䝩䝹䝬䝸䞁⁐ᾮ䛻䛶 ᅛᐃ䜢⾜䛳䛯䚹ື≀䝃䞁䝥䝹䛿䛩䜉䛶ඛ⾜◊✲䛻䛚䛔䛶䠈⳥ᆅཎ䜙䛜⏝䛔䛯䜒䛾䜢⏝䛧䛯 [35]䚹 ᐇ㦂7 ᢞẘᛶヨ㦂䛾ᯟ⤌䜏䛷䠈䝷䝑䝖䜈䛾᭱㛗90 ᪥㛫䛾Ⓨ䛜䜣≀㉁ᢞ䛻䜘䜛䠈Tmem70 䛚䜘䜃⣽⬊ෆ௦ㅰ㛵㐃㑇ఏᏊ䛾Ⓨ⌧䜢᳨ウ䛩䜛䛯䜑䠈䝷䝑䝖䜢ྛ⩌10 ༉䛾 4 ⩌䛻ศ䛡䛯䚹ᇶ ♏㣫ᩱ䛾䜏 (↓ฎ⨨ᑐ↷⩌)䠈400 ppm 䛾 TAA 䛾ΰ㣵ᢞ (TAA ⩌)䠈1000 ppm 䛾 MP ΰ 㣵ᢞ (MP ⩌)䠈䛺䛔䛧 0.5% methylcellulose 䜢⁐፹䛸䛩䜛 100 mg/kg 䛾 PMZ 䛾ᙉไ⤒ཱྀᢞ (PMZ) 䜢 28 ᪥䛺䛔䛧 90 ᪥㛫⾜䛳䛯䚹TAA 䛚䜘䜃 MP 䛿⫢Ⓨ䛜䜣≀㉁䛸䛧䛶䠈PMZ 䛿㠀
41 Ⓨ䛜䜣⫢ẘᛶ≀㉁䛸䛧䛶㑅ᢥ䛧䛯䚹TAA 䛚䜘䜃 MP 䛿䠈Ⓨ䛜䜣⏝㔞䜢ᢞ⏝㔞䛸䛧䛶タᐃ䛧 䛯 [3, 47]䚹PMZ 䛿䠈䝷䝑䝖䜈䛾 13 㐌㛫䛾ᢞ䛻䜘䜚⫢ẘᛶ䛜䜏䜙䜜䜛⏝㔞䜢ᢞ⏝㔞䛸 䛧䛶タᐃ䛧䛯 [46]䚹ᐇ㦂ᮇ㛫⤊ᚋ䠈䛩䜉䛶䛾ື≀䜢ᐇ㦂 1 䛚䜘䜃 2 䛸ྠᵝ䛾᪉ἲ䛻䛶Ᏻᴦ ẅ䛧䛯䚹䜎䛯䠈ゎ๗䠈䛩䜉䛶䛾ື≀䛛䜙⫢⮚䜢ฟ䛧䠈㔜㔞䜢 ᐃᚋ䠈㑇ఏᏊⓎ⌧ゎᯒ⏝䛸 䛧䛶1 mm ゅ䛾⣽∦䛻䛧䠈ᾮయ❅⣲䛻ᾐₕ䛧䛶⤖䛧䌦80䉝䛻䛶ಖᏑ䛧䛯䚹 䛩䜉䛶䛾ື≀ᐇ㦂ィ⏬䛿䠈ᅜ❧Ꮫἲேᮾி㎰ᕤᏛ䛾ື≀ᐇ㦂⌮ጤဨ䛻ᥦฟ䛧 䛶ᢎㄆ䜢ཷ䛡䠈ື≀䛾ྲྀ䜚ᢅ䛔䛻䛴䛔䛶䜒䠈ྠᏛ䛾ᐇ㦂ື≀ᣦ㔪䛚䜘䜃ື≀ᐇ㦂䛾㐺ṇ 䛺ᐇ䛻ྥ䛡䛯䜺䜲䝗䝷䜲䞁 (᪥ᮏᏛ⾡㆟䠈2006ᖺ6᭶1᪥) 䜢㑂Ᏺ䛧䛯䚹 ⌮⤌⧊ᏛⓗᰁⰍ䛚䜘䜃ච⤌⧊ᏛᰁⰍ 㻌 䝟䝷䝣䜱䞁ໟᇙ䛧䛯⫢⮚䛿3 μm 䛾ཌ䛥䛻ⷧษ䛧䛯ᚋ䠈HE ᰁⰍ䛚䜘䜃ච⤌⧊Ꮫⓗゎ
ᯒ䛻౪䛧䛯䚹ච⤌⧊Ꮫⓗゎᯒ䛿䠈Vectastain® Elite ABC Kit (Vector Laboratories Inc.) 䛾䝥
䝻䝖䝁䞊䝹䛻ᚑ䛳䛶䝅䜾䝘䝹᳨ฟ䜢⾜䛔䠈චᛂ䛿 3,3′-diaminobenzidine/H2O2 䜢⏝䛔䛶
ྍど䛧䛯ᚋ䠈䝦䝬䝖䜻䝅䝸䞁䛻䜘䜚ᑐẚᰁⰍ䛧䛯䚹
ᐇ㦂1 䛚䜘䜃 2 䛾⫢⮚䛾ᶆᮏ䛷䛿䠈GST-P+⫢⮚๓䛜䜣ኚ䛻䛚䛡䜛䠈TMEM70 䛸⣽⬊ෆ
௦ㅰ㛵㐃ศᏊ䛸䛾㛵ಀᛶ䜢᳨ウ䛩䜛䛯䜑䠈GST-P䠈TMEM70䠈ATP synthase, H+ transporting,
42
(HSP60)䠈solute carrier family 2, facilitated glucose transporter member 1 (GLUT1)䠈䛺䛔䛧 glucose-6-phosphate dehydrogenase (G6PD)㻌 䛻䛴䛔䛶ච⤌⧊ᏛᰁⰍ䜢ᐇ䛧䛯䚹ATPB
䛿䠈䝭䝖䝁䞁䝗䝸䜰ATP ྜᡂ㓝⣲䜢ᵓᡂ䛩䜛䝨䝥䝏䝗䛷 ATP ྜᡂ㓝⣲䛾ᣦᶆ䛸䛧䛶䠈HSP60 䛿䝭 䝖䝁䞁䝗䝸䜰䛾䝬䞊䜹䞊䛸䛧䛶䠈GLUT1 䛚䜘䜃 G6PD 䛿⢾䛾ྲྀ䜚㎸䜏䛚䜘䜃ゎ⢾⣔㓝⣲䛾ᣦᶆ 䛸䛧䛶ྛ䚻㑅ᢥ䛧䛯䚹ᐇ㦂3 䛾⫢⮚䛾ᶆᮏ䛷䛿䠈GST-P+⭘⒆ᛶኚ䛻䛚䛡䜛䠈TMEM70 䛚䜘 䜃⣽⬊ෆ௦ㅰ㛵㐃ศᏊ䛾Ⓨ⌧ศᕸ䜢᳨ウ䛩䜛䛯䜑䛻䠈GST-P䠈TMEM70䠈ATPB䠈GLUT1 䛺 䛔䛧G6PD 䛾ච⤌⧊ᏛᰁⰍ䜢ᐇ䛧䛯䚹ᐇ㦂 4 䛻䛚䛔䛶䛿䠈PPARα 䜰䝂䝙䝇䝖䛾Ⓨ䛜䜣 ಁ㐍䛷ㄏⓎ䛥䜜䛯 GST-P–ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛䠈TMEM70 䛚䜘䜃⣽⬊ෆ௦ㅰ㛵㐃ศᏊ䛾 Ⓨ⌧ศᕸ䜢᳨ウ䛩䜛䛯䜑䛻䠈GST-P䠈TMEM70䠈ATPB䠈GLUT1 䛺䛔䛧 G6PD 䛾ච⤌⧊ ᏛᰁⰍ䜢ᐇ䛧䛯䚹ᐇ㦂5 䛷䛿⏥≧⭢䠈⭤⬔䛚䜘䜃⫶䛾ቑṪᛶኚ䠈䛚䜘䜃ᐇ㦂 6 䛷䛿⤖⭠ 䛚䜘䜃༑ᣦ⭠䛾ቑṪᛶኚ䛻䛚䛡䜛TMEM70 䛚䜘䜃⣽⬊ෆ௦ㅰ㛵㐃ศᏊ䛾Ⓨ⌧ศᕸ䜢 ᳨ウ䛩䜛䛯䜑䛻䠈GST-P䠈TMEM70䠈ATPB䠈GLUT1 䛺䛔䛧 G6PD 䛾ච⤌⧊ᏛᰁⰍ䜢ᐇ 䛧 䛯 䚹ᐇ㦂 1 䛾⫢⮚䛾ᶆᮏ䛷 䛿䠈䛥䜙䛻䠈TMEM70–䛸䛺䜛 GST-P+㻌 ኚ ␗⫢⣽ ⬊ᕢ (TMEM70–/GST-P+ᕢ) 䛚䛡䜛䠈ྛ௦ㅰ㛵㐃ศᏊ䛾Ⓨ⌧䛾⤌䜏ྜ䜟䛫䛻䜘䜚䠈⣽⬊ቑṪ䠈䜰䝫 䝖䞊䝅䝇䠈䛺䛔䛧ᵝ䚻䛺ᶵ⬟䛻㛵䜟䜛㌿ᅉᏊ䛾Ⓨ⌧䜢ච⤌⧊Ꮫⓗ䛻᳨ウ䛩䜛䛯䜑 䠈 proliferating cell nuclear antigen (PCNA)䠈cleaved caspase 3䠈䛺䛔䛧䝸䞁㓟 c-MYC 䛾ච⤌ ⧊ᏛᏛᰁⰍ䜢ᐇ䛧䛯䚹
43 ච ච⤌⧊ᏛᰁⰍ䛾ᐃ㔞ゎᯒ 㻌 ᐇ㦂1䠈2 䛚䜘䜃 7 䛾⫢⮚䛻䛴䛔䛶䛿䠈┤ᚄ 200 μm ௨ୖ䛾 GST-P+ኚ␗⫢⣽⬊ᕢ䛾ᩘ䛚 䜘䜃㠃✚䜢ィ 䛧䛯䚹㠃✚䛾 ᐃ䛿䠈⏬ീゎᯒ䝋䝣䝖WinROOF (୕㇂ၟ) 䜢⏝䛔䛯䚹ᐇ㦂 1 䛚䜘䜃2 䛿䠈TMEM70䠈ATPB䠈GLUT1䠈G6PD 䛚䜘䜃 HSP60 䛻䛴䛔䛶䠈GST-P+ኚ␗⫢⣽⬊ ᕢ䛻䛚䛡䜛Ⓨ⌧䜢䠈࿘ᅖ䛾⫢⣽⬊䛻䛚䛡䜛Ⓨ⌧ᙉᗘ䛸ẚ㍑䛧䛶䠈Ⓨ⌧ቑຍ (+)䠈ኚ䛺䛧 (±) 䛒䜛䛔䛿Ⓨ⌧ῶᑡ (–) 䛷ホ౯䛧䛯䚹䛥䜙䛻䠈1 ಶయ䛒䛯䜚䛾⥲ GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛
TMEM70–䠈ATPB–䠈HSP60+䠈HSP60+/–䠈HSP60–䠈GLUT1+䛚䜘䜃 G6PD+䛾ྜ䜢ồ䜑䛯䚹ᐇ
㦂3 䛷䛿䠈TMEM70䠈ATPB䠈GLUT1 䛚䜘䜃 G6PD 䛾ቑṪᛶኚ䛻䛚䛡䜛Ⓨ⌧䜢䠈࿘ᅖ䛾⫢
⣽⬊䛻䛚䛡䜛Ⓨ⌧ᙉᗘ䛸ẚ㍑䛧䛶䠈Ⓨ⌧ቑຍ (+) 䛺䛔䛧Ⓨ⌧ῶᑡ (–) 䛷ホ౯䛧䛯䚹䛥䜙䛻ኚ
␗⫢⣽⬊ᕢ䠈⫢⣽⬊⭢⭘䛺䛔䛧⫢⣽⬊⒴䛾ྛቑṪᛶኚ䛻䛚䛡䜛 TMEM70–䠈ATPB–䠈
GLUT1+䛚䜘䜃 G6PD+䛾ྜ䜢ồ䜑䛯䚹ᐇ㦂1 䛾 DEN+TAA䠈DEN+MP 䛚䜘䜃 DEN+PBO
⩌䛻㛵䛧䛶䛿䠈䛩䜉䛶䛾 TMEM70–/GST-P+ᕢ䛻䛚䛡䜛䠈ATPB–, GLUT1+䛚䜘䜃 G6PD+䛾
ྜ 䜢 ồ 䜑 䛯 䚹 ḟ 䛻 䠈TMEM70–/GST-P+ᕢ 䛻 㛵 䛧 䛶 䠈ATPB+/–ᕢ 䠈ATPB–ᕢ 䠈GLUT1+ᕢ 䠈
GLUT1+/–ᕢ䠈G6PD+ᕢ䛺䛔䛧G6PD+/–ෆ䛾PCNA+⣽⬊䠈cleaved caspase 3+⣽⬊䛚䜘䜃᰾ෆ䝸
䞁㓟c-MYC+⣽⬊䜢ィ 䛧䛯䚹ィ 䛿400 ಸ䛾ಸ⋡䛷 10 ど㔝䛻䛴䛔䛶⾜䛔䠈ど㔝䛻䛚䛡
䜛⫢⣽⬊ᩘ䜒䛒䜟䛫䛶ィ 䛧䠈㝧ᛶ⣽⬊⋡䜢⟬ฟ䛧䛯䚹ᐇ㦂 4 䛻䛚䛔䛶䛿䠈GST-P–ኚ␗⫢
44 ⌧ᙉᗘ䛸ẚ㍑䛧䛯䚹ᐇ㦂5 䛿⏥≧⭢䠈⭤⬔䠈๓⫶䛚䜘䜃⭢⫶䛾ቑṪᛶኚ䠈ᐇ㦂 6 䛿⤖⭠䛚 䜘䜃༑ᣦ⭠䛾ቑṪᛶኚ䛻䛴䛔䛶䠈TMEM70䠈ATPB䠈GLUT1 䛚䜘䜃 G6PD 䛾Ⓨ⌧䜢䠈࿘ ᅖ䛾ᐇ㉁⣽⬊䛻䛚䛡䜛Ⓨ⌧ᙉᗘ䛸ẚ㍑䛧䛯䚹 Real-time RT-PCR ᐇ㦂 7 䛻䛚䛔䛶↓ฎ⨨ᑐ↷⩌䠈TAA ⩌䠈MP ⩌䛚䜘䜃 PMZ ⩌䛾⫢⮚䛛䜙䠈⥲ RNA 䜢
RNeasy Mini Kit (Qiagen) 䜢⏝䛔䛶ᢳฟ䛧 (n=6/⩌)䠈䛣䛾䛖䛱 2 μg 䜢⏝䛔䛶 SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific) 䛻䛶 1 ᮏ㙐 cDNA 䜢ྜᡂ䛧䛯䚹Real-time PCR 䛿䠈Power SYBR® Green PCR Master Mix 䛚䜘䜃 StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific) 䜢⏝䛔䠈〇㐀ඖ䛾䝥䝻䝖䝁䞊䝹䛻ᚑ䛳䛶ᐇ䛧䛯䚹PCR 䝥䝷䜲䝬䞊 䛿Primer Express V3.0 (Thermo Fisher Scientific) 䜢⏝䛔䛶タィ䛧䠈Table 2-3 䛻♧䛧䛯䚹ྛ㑇
ఏᏊ䛾mRNA Ⓨ⌧㔞䛿䠈↓ฎ⨨ᑐ↷⩌䛷䛾Ⓨ⌧್䛻ᑐ䛩䜛┦ᑐ್䛸䛧䛶ồ䜑䠈ෆᅉᛶ䝁䞁䝖 䝻䞊䝹䛸䛧䛶Actb 䛚䜘䜃 Hprt1 䛾᳨㔞⥺䜢ồ䜑 2-ΔΔC Tἲ [40] 䛻䛶⟬ฟ䛧䛯䚹 ⤫ ⤫ィゎᯒ 㻌 ᐃ㔞䝕䞊䝍䛻䛴䛔䛶ᖹᆒ್䛚䜘䜃ᶆ‽೫ᕪ䜢ồ䜑䛯䚹ከ⩌㛫ẚ㍑䛿䠈Bartlett ᳨ᐃ䛷➼ ศᩓ䜢☜ㄆ䛧䛯ᚋ䠈୍ඖ㓄⨨ศᩓศᯒ䜢⾜䛳䛯䚹᭷ពᕪ䛜ㄆ䜑䜙䜜䛯ሙྜ䛿Dunnett ᳨ᐃ䜢 ⾜䛳䛯䚹Bartlett ᳨ᐃ䛷➼ศᩓ䛷䛺䛛䛳䛯ሙྜ䠈Steel ᳨ᐃ䜢⾜䛳䛯䚹⩌㛫ẚ㍑䛿䠈F ᳨ᐃ䛻
45 䜘䜚➼ศᩓᛶ䛾☜ㄆ䜢⾜䛳䛯䚹➼ศᩓ䛜ㄆ䜑䜙䜜䛯ሙྜ䛿Student 䛾 t ᳨ᐃ䜢⾜䛔䠈➼ศᩓ䛜 ㄆ䜑䜙䜜䛺䛛䛳䛯ሙྜ䛿Aspin-Welch 䛾 t ᳨ᐃ䜢⾜䛳䛯䚹䜎䛯䠈ᐇ㦂 3 䛾ච⤌⧊ᏛᰁⰍ 䛾⤖ᯝ䛻䛴䛔䛶䛿䠈Fisher 䛾ṇ☜☜⋡᳨ᐃ䜢⾜䛔䠈GST-P+ቑṪᛶኚ (ኚ␗⫢⣽⬊ᕢ䠈⫢ ⣽⬊⭢⭘䠈⫢⣽⬊⒴) 㛫䛷䛭䜜䛮䜜ẚ㍑䜢⾜䛳䛯䚹䛺䛚䠈䛩䜉䛶䛾᳨ᐃ䛻䛚䛔䛶᭷ពỈ‽ 5%௨ୗ䜢᭷ពᕪ䛒䜚䛸䛧䛯䚹䛩䜉䛶䛾ゎᯒ䛿䠈⤫ィゎᯒ䜰䝗䜲䞁䝋䝣䝖 䜶䜽䝉䝹⤫ィ 2010 䜢 ⏝䛔䛶⾜䛳䛯 (ᰴᘧ♫♫ሗ䝃䞊䝡䝇)䚹
46 ⤖ ⤖㻌 ᯝ ቑṪᛶኚ䛾ホ౯ ᐇ㦂1 䛚䜘䜃 2 䛻䛚䛔䛶䠈䛩䜉䛶䛾⫢Ⓨ䛜䜣≀㉁䛺䛔䛧⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊䛾 6 㐌㛫䛺 䛔䛧8 㐌㛫䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈GST-P+ኚ␗⫢⣽⬊ᕢ䛾ᩘ䛚䜘䜃㠃✚䛜 DEN-alone ⩌䛸ẚ ㍑䛧䛶᭷ព䛻ቑຍ䛧䛯 (Table 2-4)䚹ᐇ㦂 3 䛷䛿䠈30 㐌㛫䛾 TAA ᢞ䛻䜘䜚䠈ከ䛟䛾⫢⣽⬊⭢ ⭘䛚䜘䜃⭢⒴ᕢ䛾ᙧᡂ䛜䜏䜙䜜䛯䚹 ᐇ㦂4䛷䛿䠈CF䛚䜘䜃DEHP䛾13㐌㛫䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈GST-P–ኚ␗⫢⣽⬊ᕢ䛾ᩘ䛚䜘 䜃㠃✚䛜DEN-alone⩌䛸ẚ㍑䛧䛶᭷ព䛻ቑຍ䛧䛯 (Table 2-5)䚹୍᪉䛷䠈GST-P+ኚ␗⫢⣽⬊ ᕢ䛾ᩘ䛿䠈CF䛚䜘䜃DEHP䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚ῶᑡ䛧䛯䚹 ᐇ㦂5䛷䛿䠈䛩䛷䛻ඛ⾜◊✲䛷ሗ࿌䛧䛶䛔䜛⤖ᯝ䛸ྠᵝ䛷䠈SDM䛻䜘䜛Ⓨࡀࢇಁ㐍䛻䜘䜚䠈 ⏥≧⭢䛻㝈ᒁᛶℐ⬊ୖ⓶⣽⬊䛾㐣ᙧᡂ䛜ㄆ䜑䜙䜜䛯䚹ྠᵝ䛻䛧䛶䠈PEITC䛻䜘䜛Ⓨࡀࢇಁ 㐍䛻䜘䜚䠈⭤⬔䛾⛣⾜ୖ⓶䛻䛚䛔䛶䠈༢⣧ᛶ㐣ᙧᡂ䠈䛺䛔䛧ங㢌≧䛺䛔䛧⤖⠇ᛶ㐣ᙧᡂ䛜ㄆ 䜑䜙䜜䠈catechol䛻䜘䜛Ⓨࡀࢇಁ㐍䛷䛿๓⫶䛻䛚䛔䛶䛿㐣ゅ䛺䛔䛧㘒ゅ䜢క䛳䛯ᡥᖹୖ ⓶㐣ᙧᡂ䛜䠈⭢⫶䛷䛿㝈ᒁᛶ㐣ᙧᡂ䛜ㄆ䜑䜙䜜䛯䚹 㻌 ᐇ㦂6䛻䛚䛡䜛AOMㄏⓎⓎ䛜䜣䝰䝕䝹䛷䛿䠈⤖⭠䛚䜘䜃༑ᣦ⭠䛻䠈」ᩘ䛾⭢⭘䛚䜘䜃 ⭢⒴䛜ㄆ䜑䜙䜜䛯䚹 ᐇ㦂7 䛻䛚䛔䛶䛿䠈TAA 䛾 28 ᪥䛚䜘䜃 90 ᪥㛫ᢞ䠈䛺䛔䛧 MP 䛾 90 ᪥㛫ᢞ䛻䜘䜚䠈 GST-P+ኚ␗⫢⣽⬊ᕢ䛾ᙧᡂ䛜䜏䜙䜜䠈ᩘ䛚䜘䜃㠃✚䛜ቑຍ䛧䛯 (Table 2-6)䚹
47 ᐇ ᐇ㦂1-3 䛾 GST-P+ቑṪᛶኚ䛻䛚䛡䜛TMEM70 䛾ච⤌⧊Ꮫⓗゎᯒ ᐇ㦂1 䛚䜘䜃 2 䛚䛔䛶䠈TMEM70 䛿 GST-P+ኚ␗⫢⣽⬊ᕢ䛾࿘ᅖ䛷䠈⫢⣽⬊䛾⣽⬊㉁䛻 㝧ᛶ䜢♧䛧䛯䚹㒊ศ䛾GST-P+ኚ␗⫢⣽⬊ᕢ䛷䛿䠈⫢⣽⬊⣽⬊㉁䛻䛚䛔䛶TMEM70 䛾Ⓨ ⌧䛜ㄆ䜑䜙䜜䛯䛜䠈୍㒊䛾 GST-P+ኚ␗⫢⣽⬊ᕢ䛷䛿䠈ኚ␗⫢⣽⬊ᕢ䛻୍⮴䛧䛶 TMEM70 䛜㝜ᛶ䜢♧䛧䛯 (Fig. 2-1)䚹ゎᯒ䛻⏝䛔䛯䛶䛾⫢Ⓨ䛜䜣≀㉁䛚䜘䜃⫢Ⓨ䛜䜣䝥䝻䝰䞊䝍䞊 䛾6 㐌䛺䛔䛧 8 㐌㛫䛾Ⓨ䛜䜣ಁ㐍䛻䜘䜚䠈TMEM70–/GST-P+ᕢ䛾ྜ䛜DEN-alone ⩌䛻ẚ㍑ 䛧䛶ቑຍ䛧䛯䚹 ᐇ㦂3 䛻䛚䛔䛶䛿䠈TAA 䛾 30 㐌㛫䛾Ⓨ䛜䜣ಁ㐍䛻䜘䛳䛶ㄏⓎ䛥䜜䛯⫢⮚䛾ኚ␗⫢⣽⬊ᕢ䠈 ⭢⭘䛚䜘䜃⭢⒴䛿䠈GST-P 䛻㝧ᛶ䛷䛒䛳䛯䚹➨ 1 ❶䛷ᚓ䜙䜜䛯⤖ᯝ䛸ྠᵝ䛻䠈GST-P 䛻㝧ᛶ 䜢♧䛩ቑṪᛶኚ࿘ᅖ䛾⫢⣽⬊䛻䛚䛔䛶 TMEM70 䛿䠈⣽⬊㉁䛻㝧ᛶ䜢♧䛧䛯䛜䠈GST-P+ ቑṪᛶኚ䛻䛚䛔䛶䛿䠈TMEM70 䛿䛔䛪䜜䜒Ⓨ⌧ῶᑡ䛧䠈⫢⣽⬊⭢⭘䛚䜘䜃⫢⣽⬊⒴䛷䛿 ኚ␗⫢⣽⬊ᕢ䛻ẚ䜉䛶㧗䛔㢖ᗘ䛷ቑṪᛶኚ䛻୍⮴䛧䛯 TMEM70 䛾Ⓨ⌧ῶᑡ䛜䜏䜙䜜 䛯䚹 ᐇ㦂1 䛚䜘䜃 2 䛾 GST-P+ኚ␗⫢⣽⬊ᕢ䛻䛚䛡䜛ATPB䠈HSP60䠈GLUT1 䛚䜘䜃 G6PD 䛾 ච⤌⧊Ꮫⓗゎᯒ ATPB 䛿䠈DEN-alone 䛷䛿 GST-P+ኚ␗⫢⣽⬊ᕢ䛾࿘ᅖ䛷䠈⫢⣽⬊䛾⣽⬊㉁䛻㝧ᛶ䜢♧䛧
