STUDY OF BIOLOGICAL SIGNIFICANCE OF ANTIZYMES IN KNOCKOUT MICE

1)抗酶是多胺诱导的蛋白质，它负向调节细胞中的多胺。我们之前报道过混合遗传背景的纯合子AZ1敲除小鼠部分胚胎致死。C57BL/6J背景的纯合子胚胎在胚胎日(E) 13.5 ~ 16.5时死亡率较高。肝发育不全是最早发现的异常。随后的分析显示，E12.5-13.5的肝造血细胞凋亡减少，外周血中红细胞增多的贫血，表明AZ1基因敲除小鼠的肝脏中继发性胚胎造血是受影响最大的过程。我们的遗传分析表明，AZ1基因敲除小鼠的致死表型存在修饰基因。为了鉴定修饰基因，我们在MSKR小鼠（一种源自日本野生molossinus小鼠的菌株）中制备了一个同源系。2)对来自AZ1敲除小鼠的原代和永活培养细胞的研究表明，多胺在这些细胞中延缓了ODC的抑制，AZ2部分补偿了多胺的调节。因此，AZ2具有抑制ODC的活性，并以多胺依赖的方式抑制多胺摄取。3)我们还制备了AZ2敲除小鼠。纯合子AZ2敲除小鼠无明显表型。AZ1-AZ2双纯合子敲除小鼠E14.5完全胚胎致死。在肝脏形成之前，E10.5开始出现系统性发育迟缓，这表明双敲除胚胎的死亡原因与AZ1敲除胚胎不同。4)幸存者的长期观察未显示AZ1纯合子的肿瘤发生增加。然而，AZ1敲除小鼠与apc缺陷的mm小鼠杂交显示，在AZ1杂合突变存在的情况下，min小鼠肠腺瘤的数量和大小增加。

1) Antizymes are polyamine-induced proteins that negatively regulate cellular polyamines. We previously reported that homozygous AZ1 knockout mice with a mixed genetic background are partially embryonic lethal. Homozygous embryos with the C57BL/6J background showed a high mortality rate at embryonic day (E) 13.5-16.5. Hepatic hypoplasia was the earliest abnormality ever detected. Subsequent analysis revealed a reduction of hepatic hemopoietic cells with apoptotic morphology at E12.5-13.5 and anemia with increased erythroblasts in the peripheral circulation, indicating that the secondary embryonic hematopojesis in the liver was a most affected process in the AZ1 knockout mice. Presence of modifier gene(s) for the lethal phenotype of AZ1 knockout mice was indicated in our genetic analysis. To identify the modifier gene(s) using positional assignment, we have prepared a congenic line in MSKR mice, a strain originated from wild molossinus mice in Japan.2) Studies using primary and immortalized cultured cells derived from AZ1 knockout mice demonstrated that ODC repression by polyamines was retarded in these cells and that AZ2 partially compensated the polyamine regulation. Thus AZ2 has activities to repress ODC and inhibit polyamine uptake both in a polyamine-dependent manner.3) We also prepared AZ2 knockout mice. Homozygous AZ2 knockout mice showed no apparent phenotype. AZ1-AZ2 double homozygous knockout mice were complete embryonic lethal by E14.5. The systemic developmental retardation started by E10.5 before the formation of liver, suggesting that the cause of death in the double knockout embryos differs from AZ1 knockouts.4) Long term observation of survivors failed to show the increase in the tumorigenesis in AZ1 homozygotes. However, crossing of AZ1 knockout mice and APC-deficient mm mice demonstrated increases in the number and size of intestinal adenomas in the presence of the heterozygous AZ1 mutation in min mice.