Mechanisms involved in loss of PI-anchored proteins (DAF and CD59) in human leukemia cell lines.

我们研究了糖基磷脂酰肌醇（GPI）锚定补体调节蛋白DAF和/或CD59在缺乏这些蛋白表面表达的一组人类白血病细胞系中的缺陷机制：U937 (DAF^+/CD59^-), CEM (DAF^<- e1s1 +>), TALL （DAF^-/CD59^-）和Ramos [Ramos (-)] （DAF^-/CD59^-）的一个亚株，Northe blot和逆转录聚合酶链反应（RT-PCR）显示，DAF和/或CD59缺乏的主要原因是除了Ramos（-）外，大多数细胞系的mRNA表达失败，从而使DAF和CD59 U937产生足够的mRNA。通过检测其他GPI锚定蛋白，CEM和TALL细胞在GPI锚定形成方面没有缺陷。Southern blot未检测到与DAF或CD59相关的基因异常。因此，除了Ramos（-）外，这些白血病细胞系中DAF和/CD59缺陷的原因实际上是无法检测到相关mRNA的稳态水平，很可能是由于这些细胞系中转录步骤的异常。另一方面，Ramos（-）细胞不能产生GPI锚，而它通常表达DAF和CD59转录本。将PIGA cDNA转染到Ramos（-）细胞后，DAF和CD59的表达得以恢复，这表明gpi锚蛋白形成缺陷的机制与缺乏PIGA基因产物的阵发性夜间血红蛋白尿（PNH）细胞相似。因此，人类白血病细胞系中DAF和/或CD59缺陷的机制并不统一，与引起PNH的机制大多不同。

We investigated the mechanisms of defects of glycosyl-phosphatidylinositol (GPI) -anchored complement regulatory proteins, DAF and/or CD59, in a panel of human leukemia cell lines that lack surface expression of these proteins : U937 (DAF^+/CD59^-), CEM (DAF^<-E1S1+>), TALL (DAF^-/CD59^-), and a substrains of Ramos [Ramos (-)] (DAF^-/CD59^-), Northe blot and reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that the main cause of the DAF and/or CD59 deficiency is the failure of mRNA expression in most of the cell lines except for Ramos (-) which allowed the generation of the sufficient mRNA of DAF and CD59 U937, CEM,and TALL cells were not defective in GPI anchor formation as assesse by the detection of other GPI-anchored proteins. No gene abnormality corresponding to DAF or CD59 was detected by Southern blotting. Thus, the cause of the defects of DAF and/CD59 in these leukemia cell lines except for Ramos (-) is virtually undetectable steady state levels of the relevant mRNA,most likely, is attributable to aberrance at the transcription steps in these cell lines. On the other hand, Ramos (-) cells failed to generate a GPI anchor, whereas it normally expressed DAF and CD59 transcripts. The transfection of PIGA cDNA to Ramos (-) cells restored the DAF and CD59 expression, indicating that the mechanism defective in GPI-anchor formation is similar to that of paroxysmal nocturnal hemoglobinuria (PNH) cells, a deficiency of the PIG-A gene product. Thus, the mechanisms of the defects of DAF and/or CD59 in human leukemia cell lines are not uniform and are mostly different from that proposed to cause PNH.