Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays.
Transfection of a human cytochrome P-450 gene into the human lymphoblastoid cell line, AHH-1, and use of the recombinant cell line in gene mutation assays.
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将人细胞色素 P-450 基因转染至人类淋巴母细胞系 AHH-1 中，并在基因突变测定中使用重组细胞系。
DOI：
10.1093/carcin/10.2.295
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发表时间：
1989
期刊：
影响因子：
4.7
通讯作者：
Davies,RL
中科院分区：
文献类型：
--
作者：
Crespi,CL;Langenbach,R;Rudo,K;Chen,YT;Davies,RL
我们已经证明，使用 pHEBo 载体和潮霉素选择可以将人细胞色素 P1-450 基因转染到 AHH-1 人淋巴母细胞系中。当来自单纯疱疹病毒胸苷激酶基因的调节序列以适当的方向掺入时，转染的基因得以表达。使用7-乙氧基试卤灵脱乙基酶、7-乙氧基香豆素脱乙基酶和苯并(a)芘羟化酶活性测定在酶水平上监测基因表达。与对照细胞群相比，大量转化细胞群的这些酶活性高出 2 至 3 倍。还获得了表达更高水平的7-乙氧基试卤灵脱乙基酶活性的大量亚克隆。在潮霉素 B 存在的情况下，转染的细胞色素 P1-450 基因的表达稳定 20-30 天。发现转化的细胞群适合用于基因位点突变测定，并检查了黄曲霉毒素-B1 和 2-乙酰氨基芴 (AAF) 的致突变性。研究发现，与对照细胞相比，黄曲霉毒素-B1 对具有转染细胞色素 P1-450 活性的细胞的诱变性高 2-3 倍。相反，没有观察到 AAF 致突变性的差异。 AAF 代谢物谱分析表明，表达转染细胞色素 P1-450 基因的细胞产生的 N-和 7-羟基-AAF 比对照细胞多 8 倍。对照细胞和带有转染的细胞色素P1-450基因的细胞之间诱变反应的相似性可能是由于AHH-1细胞的低脱乙酰酶活性所致。这些观察结果表明该载体和表达系统适合将新的代谢活性引入AHH-1细胞系。
We have demonstrated that the human cytochrome P1-450 gene can be transfected into the AHH-1 human lymphoblastoid cell line using the pHEBo vector and hygromycin selection. The transfected gene was expressed when regulatory sequences derived from the herpes simplex virus thymidine kinase gene were incorporated in appropriate orientations. Gene expression was monitored at the enzyme level using assays for 7-ethoxyresorufin deethylase, 7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase activities. Bulk transformed cell populations had 2- to 3-fold more of these enzyme activities compared with control populations. Subclones of the bulk population expressing still higher levels of 7-ethoxyresorufin deethylase activity were also obtained. Expression of the transfected cytochrome P1-450 gene was stable for 20–30 days in the presence of hygromycin B. The transformed cell populations were found to be suitable for use in gene locus mutation assays and the mutagenicity of aflatoxin-B1and 2-acetylaminofluorene (AAF) were examined. Aflatoxin-B1was found to be 2–3 times more mutagenic to cells bearing the transfected cytochrome P1-450 activity as compared with control cells. In contrast, no difference in AAF mutagenicity was observed. Analysis of the AAF metabolite profile indicated that cells expressing the transfected cytochrome P1-450 gene produced 8-fold moreN- and 7-hydroxy-AAF than control cells. The similarity in mutagenic responses between control cells and cells bearing the transfected cytochrome P1-450 gene may be due to the low deacetylase activity of AHH-1 cells. These observations indicate that this vector and expression system are suitable for introducing novel metabolic activities into the AHH-1 cell line.