MOLECULAR MECHANISMS OF ACCELERATED ERYTHROID APOPTOSIS DUE TO A GLYCOLYTIC ENZYME DEFECT

丙酮酸激酶(PK)缺乏是糖酵解酶缺陷引起遗传性非球形细胞溶血性贫血的最常见原因之一。我们以前曾报道过PK-1^&lt；SLC&gt；小鼠的溶血性贫血是由于小鼠PKLR基因的自发突变所致。脾组织中凋亡的红系细胞显著增加，而PK-1；lt；SLC&gt；转基因挽救的突变小鼠脾中的红系细胞明显减少。SLC3是从PK-1SLC&gt；小鼠体内建立的一种友好的红白血病细胞。在常规传代过程中，该细胞表现出自发的凋亡，与对照组相比，Friend细胞更容易发生凋亡，这是在葡萄糖剥夺或葡萄糖类似物2-脱氧葡萄糖诱导下发生的。一名患有严重PK缺乏症的四岁日本女孩接受了脾切除术，以减少输血需求。脾和…进一步进行组织学检查和脾造血祖细胞的检测，以评估PK缺陷受试者的髓外造血。PK缺陷患者脾中CFU-GM、BFU-E和CFU-GEMM造血祖细胞数量明显高于正常对照组，提示骨髓外造血功能增强。原位末端标记法显示PK缺乏症患者的脾红髓中有凋亡细胞。在分离的脾细胞和体外培养的细胞中检测到7A6抗原的表达，但仅在血糖蛋白A阳性的细胞中检测到7A6抗原的表达。这些结果提供了证据，PK缺乏不仅影响红系祖细胞的存活，而且影响红系祖细胞的成熟，导致细胞过早死亡，即细胞凋亡。为了评估R-PK在SLC3细胞凋亡中的作用，我们建立了两个稳定的人R-PK基因转染细胞，SLC3-hRPK.hi和lo，并检测了这些细胞的基因表达谱。用Affymetrix基因芯片小鼠表达阵列430A进行基因芯片分析，共分析了22690个基因。大约7%的基因在SLC3-hRPK.hi和Lo中都显著下调，包括ε珠蛋白、促红细胞生成素受体、β光谱蛋白、血糖蛋白A或Rh相关A糖蛋白的基因。另一方面，组蛋白1的H1c和H2bc亚型、Kcne3钾电压门控通道和成体α-珠蛋白等基因的表达上调仅为0.2%。这些观察结果表明，强制表达RPK促进了珠蛋白基因切换和红系终末分化。进一步的研究需要阐明RPK缺乏引起的细胞凋亡的机制。较少

Pyruvate kinase (PK) deficiency is one of the most prevalent causes of hereditary non-spherocytic hemolytic anemia due to glycolytic enzyme defects. We previously reported that hemolytic anemia of the Pk-1^<slc> mouse was due to a spontaneous mutant of the murine PKLR gene. Apoptotic erythroid cells were notably increased in spleen, and the transgenic rescue of the Pk-1^<slc> decreased apoptotic erythroid cells in the mutant spleen. SLC3 is a Friend erythroleukemic cell, which has been established from the Pk-1^<slc> mouse. The cell shows spontaneous apoptosis during routine passage and is more susceptible to apoptosis compared to control Friend cells, which is inducible with either glucose deprivation or glucose analogue, 2-deoxyglucose.In this study, possible adverse effects of PK deficiency on the maturation of erythroid progenitors were investigated. A four-year-old Japanese girl with severe PK deficiency underwent splenectomy to reduce her need for blood transfusions. The spleen w … More as examined a histologically and the hematopoietic progenitors in the spleen were assayed to evaluate the extramedullary hematopoiesis of the PK-deficient subject. The number of hematopoietic progenitors including CFU-GM, BFU-E and CFU-GEMM in the spleen of the PK-deficient patient was much higher than those found in control spleens, indicating enhanced extramedullary hematopoiesis. The TUNEL assay demonstrated apoptotic cells in the splenic red pulp of the PK-deficient patient. The expression of 7A6 antigen was detected in cells isolated from spleen and in cells cultured in vitro, but only in those cells that were positive for glycophorin A. These results provide evidence that the metabolic disturbances in PK deficiency affect not only the survival of red cells but also the maturation of erythroid progenitors, which results in premature cell death, i.e., apoptosis.To evaluate an involvement of R-PK in apoptosis of SLC3, we established two stable-transfectants with wild-type human R-PK cDNA, SLC3-hRPK.hi and lo, and examined gene expression profiles of these cells. DNA microarray analysis were performed by using Affymetrix GeneChip Mouse Expression Array 430A, and totally 22690 genes were analyzed. Approximately 7% of genes were significantly down-regulated both in SLC3-hRPK.hi and lo, including genes for ε-globin, erythropoietin receptor, β-spectrin, glycophorin A, or Rh-associated A glycoprotein. On the other hand, up-regulated genes was only about 0.2%, such as subtypes H1c and H2bc of histone 1, kcne3 potassium voltage-gated channel or adult α-globin.We identified that gene expression of several pro-apoptotic genes such as Bnip31, Pdcd6ip, Casp8ap2, Faf1 and Blk was significantly down-regulated by introducing RPK. These observations suggest that forced expression of RPK facilitated globin gene switching and terminal erythroid differentiation. Further studies will require elucidating mechanisms for apoptosis due to RPK deficiency. Less