DNA METHYLATION PREVENTS THE AMPLIFICATION OF TROP1, A TUMOR-ASSOCIATED CELL-SURFACE ANTIGEN GENE
DNA METHYLATION PREVENTS THE AMPLIFICATION OF TROP1, A TUMOR-ASSOCIATED CELL-SURFACE ANTIGEN GENE
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DOI：
10.1073/pnas.91.13.5833
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发表时间：
1994-06-21
影响因子：
11.1
通讯作者：
HERZENBERG, LA
中科院分区：
文献类型：
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作者：
ALBERTI, S;NUTINI, M;HERZENBERG, LA
我们测试了这样的假设：在可比较的选择条件下，不同的基因在转染后可以具有不同的扩增能力。将来自人淋巴样或绒毛膜癌细胞系的DNA转染至L细胞中，通过荧光激活细胞分选来选择CD5、CD8A、TROP1和TROP2、在淋巴细胞或滋养层和癌上表达的基因的转染子。为了选择转染基因的扩增，我们通过荧光激活细胞分选具有最高表达的转染子来克隆两次。我们分析了源自原始转染子的总共 38 个家族（1768 个克隆）。然后，我们通过Southern印迹分析表面表达增加最高的克隆，并确定每个转染基因的拷贝数。 CD5、CD8A和TROP2被高频且渐进地扩增，而TROP1基本上根本不被扩增。我们通过用 5-氮杂胞苷处理 JAR 绒毛膜癌细胞来减少 DNA 甲基化，检验了 DNA 甲基化阻止 TROP1 基因扩增的假设。处理后不同时间提取的DNA用于转染。当使用显示TROP1基因去甲基化的DNA时，获得了16个Trop-1转染子，并且发现其中6个转染子的每个单倍体基因组含有多达40个TROP1基因拷贝，因此，我们表明从去甲基化的TROP1基因获得的转染子被有效且渐进地扩增。我们提出DNA甲基化通过改变甲基化DNA序列的识别或通过改变甲基化片段染色质的构象来影响DNA扩增。我们推测DNA甲基化是体内基因扩增的决定因素，例如在肿瘤细胞中。
We tested the hypothesis that different genes can have different abilities to be amplified after transfection under comparable selection conditions. DNA from human lymphoid or choriocarcinoma cell lines was transfected into L cells, Transfectants for CD5, CD8A, TROP1, and TROP2, genes expressed on lymphocytes or trophoblast and carcinomas, were selected by fluorescence-activated cell sorting. To select for amplification of the transfected gene we cloned twice by fluorescence-activated cell sorting the transfectants with the highest expression. We analyzed a total of 38 families (1768 clones) derived from the original transfectants. We then analyzed by Southern blotting the clones with the highest increase in surface expression and determined the copy number of each transfected gene. CD5, CD8A, and TROP2 were amplified with high frequency and progressively, whereas TROP1 essentially was not amplified at all. We examined the hypothesis that DNA methylation prevents the amplification of the TROP1 gene by treating JAR choriocarcinoma cells with 5-azacytidine to decrease DNA methylation. DNA extracted at different times after the treatment was used for transfection. When DNA that showed demethylation of the TROP1 gene was used, 16 Trop-1 transfectants were obtained and 6 of them were found to contain up to 40 copies of the TROP1 gene per haploid genome, Thus, we showed that transfectants obtained from a demethylated TROP1 gene were amplified efficiently and progressively. We propose that DNA methylation affects DNA amplification either by altering the recognition of methylated DNA sequences or by changing the conformation of the chromatin of methylated segments, We speculate that DNA methylation is a determinant of gene amplification in vivo, for example in tumor cells.