Permalink : http://hdl.handle.net/2115/82749

KEYWORDS : アルカリ性ホスファターゼ;阻害剤;レチノイン酸;キレート作用;alkaline phosphatase;ALP inhibitor;retinoic acid;chelate effect

【目的】最近のi P S 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，etidronat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] In a recent study on iPS and tumor cells, it was suggested that different types of cells have different levels of alkaline phosphatase (ALP) activity. Therefore, we examined differences in sensitivity to ALP inhibitors in both non-tumor cells and tumor cells.[Materials and methods] We measured ALP activity using commercial ALP derived from the small intestine, humanbone, placenta, and liver. ALP activity in human tumor cells: SaOs, Hela, P19, and RA-P19, which was differentiated into neurologic cells with retinoic acid (RA) treatment. Additionally, we examined the sensitivity of ALP activity to inhibitors using tetramisole, levamisole, L-homoarginine, etidronate, and vanadate. [Results and Discussion] The sensitivity to each inhibitor of ALP activity varied depending on the isozyme type and its human organ origin. The sensitivity of ALP activity derived from human tumor cells did not show large differences, suggesting that ALP activity of undifferentiated tumor cells has similar characteristics regardless of the organ origin. ALP activity in P19 and RA-P19 showed similar sensitivity to inhibitors, except for etidronate, which produced different sensitivity. Similar results were obtained with EDTA, thus etidronate inhibited ALP activity by chelation effects was concluded. We examined the effects of Zn, which is necessary for ALP activity, on ALP activity in P19 and RA-P19. Zn promoted ALP activity at a low concentration, but it inhibited ALP activity at a high concentration. It has been proposed that two types of Zn binding sites with either low- or high-affinity are present in P19 cells. Furthermore, our results suggested that changes in the low-affinity site of Zn with RA treatment caused differences in sensitivity of ALP activity to etidronate in P19 and RA-P19.[Conclusion] The changes in the Zn binding site, which are essential for inhibitory activity, changed sensitivity to the inhibitor along with ALP activity in tumor cells that lost organ-specific sensitivity to inhibitors.