Yeung C Y, Riser M E, Kellems R E, Siciliano M J
J Biol Chem. 1983 Jul 10;258(13):8330-7.
JEG-3 is a human choriocarcinoma cell line characterized by low levels of adenosine deaminase expression. For the purpose of studying adenosine deaminase gene regulation in the JEG-3 cells, we attempted to select variant cells having increased adenosine deaminase expression. This was accomplished by selecting cells for resistance to the cytotoxic adenosine analogs 9-beta-D-arabinofuranosyl adenine (ara-A) or 9-beta-D-xylofuranosyl adenine (xyl-A), both of which could presumably be detoxified by the action of adenosine deaminase. Single step high dose selection was ineffective in obtaining cells with increased adenosine deaminase. However, multistep selection using either ara-A or xyl-A resulted in cell populations with increased adenosine deaminase activity. Removal of selective pressure resulted in decreased adenosine deaminase levels. Subclones of xyl-A-resistant cells belonged to one of three phenotypic classes characterized by either elevated adenosine deaminase levels, decreased adenosine kinase levels, or both of these features. One subclone (A3-1A7) with unaltered adenosine kinase expression showed a 20-fold increase in adenosine deaminase expression. Further selection of this subclone for increasing xyl-A resistance resulted in an additional 2-fold increase in adenosine deaminase expression, followed by loss of adenosine kinase expression. These adenosine kinase-deficient cells showed no subsequent increase in adenosine deaminase expression in response to further xyl-A selection pressure. These results confirmed that xyl-A toxicity was mediated through its phosphorylated form and indicated that resistance may result from increased adenosine deaminase levels and/or adenosine kinase deficiency. The increased adenosine deaminase expression of the A3-1A7 subclone was exclusively in the ADA 2 allelic form. However, cell fusion experiments between A3-1A7 cells and mouse C1-1D cells established the existence of functional copies of both ADA 1 and ADA 2 allelic genes in the A3-1A7 cells. The increased expression of only one of the two functional ADA alleles, the requirement for a stepwise selection protocol to obtain cells with increased adenosine deaminase, and the instability of the adenosine deaminase phenotype in the absence of selective pressure suggest that the alteration of adenosine deaminase phenotype in the drug-resistant cells was the result of adenosine deaminase gene amplification.
JEG - 3是一种人绒毛膜癌细胞系，其特征在于腺苷脱氨酶表达水平较低。为了研究JEG - 3细胞中腺苷脱氨酶基因的调控，我们试图筛选出腺苷脱氨酶表达增加的变异细胞。这是通过选择对细胞毒性腺苷类似物9 - β - D - 阿拉伯呋喃糖基腺嘌呤（ara - A）或9 - β - D - 木糖呋喃糖基腺嘌呤（xyl - A）具有抗性的细胞来实现的，这两种类似物都可能通过腺苷脱氨酶的作用被解毒。单步高剂量筛选在获得腺苷脱氨酶增加的细胞方面无效。然而，使用ara - A或xyl - A进行多步筛选导致细胞群体的腺苷脱氨酶活性增加。去除选择压力导致腺苷脱氨酶水平降低。对xyl - A抗性细胞的亚克隆属于三种表型类别之一，其特征是腺苷脱氨酶水平升高、腺苷激酶水平降低或这两种特征都有。一个腺苷激酶表达未改变的亚克隆（A3 - 1A7）显示腺苷脱氨酶表达增加了20倍。对该亚克隆进一步选择以增加对xyl - A的抗性导致腺苷脱氨酶表达又增加了2倍，随后腺苷激酶表达丧失。这些腺苷激酶缺陷细胞在进一步的xyl - A选择压力下，腺苷脱氨酶表达没有随后的增加。这些结果证实xyl - A毒性是通过其磷酸化形式介导的，并表明抗性可能是由于腺苷脱氨酶水平增加和/或腺苷激酶缺陷所致。A3 - 1A7亚克隆中增加的腺苷脱氨酶表达仅为ADA 2等位基因形式。然而，A3 - 1A7细胞与小鼠C1 - 1D细胞之间的细胞融合实验确定了A3 - 1A7细胞中ADA 1和ADA 2等位基因的功能拷贝都存在。两个功能性ADA等位基因中只有一个表达增加、需要逐步选择方案来获得腺苷脱氨酶增加的细胞以及在没有选择压力时腺苷脱氨酶表型的不稳定性表明，耐药细胞中腺苷脱氨酶表型的改变是腺苷脱氨酶基因扩增的结果。
