Physiological significance of pyruvate kinase isozymes in erythrocyte.

红细胞中丙酮酸激酶同工酶的生理意义。

• 批准号: 11670153
• 负责人: KANNO Hitoshi
• 金额: $ 2.3万
• 依托单位: NIHON UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670153/
• 关键词: hemolytic anemia; apoptosis; glycolysis; reactive oxygen species; disease model animals; Friend cells; 遺伝子治療; トランスジェニックマウス

Pyruvate kinase (PK) is a rate-limiting enzyme of the glycolytic pathway and PK deficiency is one of the most prevalent causes of hereditary non-spherocytic hemolytic anemia. The Pk-1^<slc> mouse is found to be a spontaneous mutant of the murine LR-type PK gene, and manifests hemolytic anemia and splenomegaly. We showed that apoptotic cells were notably increased in spleen of the Pk-1^<slc> by the TUNEL method, and that the erythroid-specific expression of normal PK gene ameliorated apoptosis in the genetic rescue experiment. Flow cytometric analysis showed that Annexin V / Ter119-double positive cells were significantly increased in the Pk-1slc mouse compared to wild type CBA mice, confirming apoptotic cells were of erythroid lineage. In this study, we examined a physiological role of glycolysis upon apoptosis of the erythroid cells using two Friend erythroleukemic cells, SLC and CBA.These cell lines have been establushed from the Pk-1^<slc> and a wild-type CBA mouse, respectively. As previously reported, apoptosis spontaneously occurred in the Friend cells and was inducible in both cells by glucose deprivation or 0.1-20mM 2-deoxyglucose (2DG). SLC cells were more susceptible to apoptosis by either shorter exposure to glucose deprivation or lower 2DG concentration. The detailed mechanism are still unclear, but we have shown that reactive oxygen species (ROS) might be accountable for these apoptotic changes.

丙酮酸激酶(PK)是糖酵解途径的限速酶，PK缺乏是遗传性非球形细胞溶血性贫血最常见的原因之一。小鼠PK-1是小鼠LR型PK基因的自发突变，表现为溶血性贫血和脾肿大。用TUNEL法检测PK-1、LT、SLC、GT小鼠脾组织中的凋亡细胞数，发现正常PK基因的红系特异性表达改善了PK-1、LT、SLC、GT小鼠脾细胞的凋亡。流式细胞仪分析显示，与野生型CBA小鼠相比，PK-1slc小鼠Annexin V/Ter119-双阳性细胞明显增多，证实了凋亡细胞为红系细胞。在这项研究中，我们使用两个Friend红白血病细胞SLC和CBA，研究了糖酵解对红系细胞凋亡的生理作用。这两个细胞系分别来自PK-1、SLC和GT；以及野生型CBA小鼠。正如先前报道的那样，Friend细胞自发地发生了凋亡，并且在两种细胞中都可以被葡萄糖剥夺或0.1-20 mM 2-脱氧葡萄糖(2DG)诱导。无论是较短的葡萄糖缺乏暴露时间或较低的2DG浓度，SLC细胞都更容易发生凋亡。具体的机制尚不清楚，但我们已经证明，活性氧物种(ROS)可能是这些细胞凋亡变化的原因。

项目成果

Taguchi M: "Molecular cloning and expression profiles of rat myocilin."Molec Genet Metab. 70. 75-80 (2000)

田口 M：“大鼠肌纤蛋白的分子克隆和表达谱。”Molec Genet Metab。

Koko Murakami: "Human HK_R isozyme:the organization of the Hexokinase-I gene, the erythroid-specific promoter and transcription initiation site."Mol Genet Metab. 67. 118-130 (1999)

Koko Murakami：“人类 HK_R 同工酶：己糖激酶-I 基因的组织、红细胞特异性启动子和转录起始位点。”Mol Genet Metab。

Minoru Okubo: "Glycogen storage disease III subtypes and muscle weakness during childhood"Hum Genet. 104. 112 (1999)

Minoru Okubo：“糖原累积病 III 亚型和儿童时期的肌肉无力”Hum Genet。

Aisaki K, Kanno H, Oyaizu N, Hara Y, Miwa S, Ikawa Y: "Apoptotic changes precede mitochondrial dysfunction in red cell type-pyruvate kinase mutant murine erythroleukemia cell lines."Jpn J Cancer Res. 90. 171-179 (1999)

Aisaki K、Kanno H、Oyaizu N、Hara Y、Miwa S、Ikawa Y：“在红细胞型丙酮酸激酶突变型鼠红白血病细胞系中，细胞凋亡变化先于线粒体功能障碍。”Jpn J Cancer Res。

Ken-ichi Aisaki: "Apoptotic changes precede mitochondrial dysfunction in red cell type-pyru-vate kinase mutant murine ervthroleukemia cell lines."Jpn J Cancer Res. 90. 171-179 (1999)

Ken-ichi Aisaki：“在红细胞型丙酮酸激酶突变型小鼠红白血病细胞系中，细胞凋亡变化先于线粒体功能障碍。”Jpn J Cancer Res。