Thesis or Dissertation 腫瘍及び非腫瘍細胞由来アルカリ性ホスファターゼの阻害剤に対する反応性の相違

川村, 良

2015-03-25
Description
【目的】最近のiPS 細胞や腫瘍細胞に関する研究はALP が細胞の分化に関与することを示唆する. そこ で, 腫瘍細胞と非腫瘍細胞のALPを比較することを目的にALP阻害剤に対する反応性の違いを検討した. 【材料及び方法】市販のヒトの骨, 小腸, 胎盤及び肝臓由来のALP, 及びヒト腫瘍細胞である SaOs,Hela,P19 とP19 をレチノイン酸(RA)で処理して神経系細胞に分化させたRA-P19 のALP を使 用しALP 活性を測定した. また, ALP 阻害剤としてtetramisole,levamisole,L-homoarginine,etidronate 及びvanadate に対する反応性を調べた.【結果と考察】ヒト臓器由来のALP 活性の各阻害剤に対する反 応性はアイソザイムの型によって異なったが, ヒト腫瘍細胞由来のALP 活性の反応性には大きな相違は なく, 未分化な腫瘍細胞のALP は, 臓器の由来にかかわらず類似した性質を示す可能性が示唆された. P19 とRA-P19 のALP はetidronate 以外の阻害剤には類似した反応性を示したが, etidronate に対し ては異なった. EDTA でも同様の結果が得られたため, etidronate はキレート作用によりALP 活性を阻 害すると結論した. ALP 活性に必要なZn の両ALP 活性に対する作用を調べたところ, 低濃度ではALP 活性を促進したが, 高濃度では逆に阻害し, P19 には親和性の異なる2 種のZn 結合部位が存在すること が示唆された. さらにRA 処理によるZn の低親和性部位の変化が, P19 とRA-P19 のALP 活性の etidronate に対する反応性の相違を引き起こすことを示唆する結果を得た.【結論】腫瘍細胞のALP は臓 器特異的な阻害剤に対する反応性を喪失する可能性と活性発現に必須のZn 結合部位の変化も阻害剤に 対する反応性に影響を及ぼす可能性を示した.
[Objectives] Recent study on iPS and tumor cells suggests that ALP may be related to the differentiation of cells. Therefore we examined the difference in sensitivity to ALP inhibitors for the purpose of comparing the ALP of non-tumor cells with the tumor cells. [Materials and methods]We measured ALP activity using commercial ALP derived from human bone, small intestine, placenta and liver, and ALPs of human tumor cells, SaOs, Hela, P19 and RA-P19 which was differentiated into neurologic cells treated with retinoic acid (RA). Also, we examined sensitivity of ALP activity to tetramisole, levamisole, L-homoarginine, etidronate and vanadate as ALP inhibitor. [Results and Discussion]The sensitivity to each inhibitor of the ALP activity derived from a human organ varied depending of isozyme type. Sensitivities of the ALP activity derived from human tumor cells did not show the big difference, suggesting that ALP of undifferentiated tumor cells show similar characters regardless of the origin of the organ. The ALP of P19 and RA-P19 showed similar sensitivity to inhibitors except etidronate, but was different for etidronate. Because similar results were obtained with the EDTA, we concluded that etidronate inhibited ALP activity by chelate effects. We examined effects for P19 and RA-P19 ALP activity of Zn, which is necessary for ALP activity. Zn promoted ALP activity with the low concentration, but it inhibited the activity at the high concentration. It was suggested that two kinds of Zn binding sites with low and high affinity were present in P19. Furthermore, we obtained results to suggest that the change of the low affinity site of Zn by the RA treatment caused difference in the sensitivity for etidronate of the ALP activity in P19 and RA-P19. [Conclusion] The change of the Zn binding site which was essential to activity, changed the sensitivity to the inhibitor and that the ALP of tumor cells lost organ-specific sensitivity to inhibitors.
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Hokkaido University(北海道大学). 博士(歯学)
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http://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/60966/1/Ryo_Kawamura.pdf

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