Ellenberger T E, Beverley S M
Department of Biological Chemistry and Molecular Pharmacolgy, Harvard Medical School, Boston, Masschusetts 02115.
J Biol Chem. 1989 Sep 5;264(25):15094-103.
Amplification of the H region has been previously observed in methotrexate (MTX)-resistant strains of Leishmania major and in unselected laboratory stocks of L. tarentolae. We now show that selection of L. major with the structurally unrelated drugs primaquine or terbinafine generated resistant lines exhibiting H region amplification and 23- and 12-fold cross-resistance to MTX, respectively. These and other drug-resistant lines bearing H region amplification also exhibited weak cross-resistance to primaquine and terbinafine, associating the amplified H region with pleiotropic resistance to MTX and other drugs. In contrast, lines selected for chloroquine or pentamidine resistance did not show H region amplification or this pattern of drug cross-resistance. The primaquine- and terbinafine-selected lines exhibited wild-type levels of dihydrofolate reductase-thymidylate synthase and normal uptake and accumulation of MTX, and the MTX resistance of these lines was not reversed by verapamil. These data suggest that the mechanism of MTX cross-resistance associated with H region amplification is novel and distinct from that mediated by overexpression of MDR genes in multidrug-resistant mammalian cells. Structural studies indicated that the amplified H region DNA in these L. major lines was largely (possibly exclusively) extra-chromosomal and consisted of circular inverted repeats joined at two DNA rearrangement junctions. Southern blot analyses showed that these rearrangement junctions were identical in four independent cell lines, suggesting that these sites are "hotspots" for DNA rearrangement. H region amplification in all of these lines was conservative, defined as retention of the chromosomal H region locus without structural alteration or reduction in copy number. This finding is consistent with an over-replication/recombination model for amplification of the H region.
先前在对甲氨蝶呤（MTX）耐药的硕大利什曼原虫菌株以及未经过选择的墨西哥利什曼原虫实验室菌株中观察到了H区域的扩增。我们现在表明，用结构不相关的药物伯氨喹或特比萘芬对硕大利什曼原虫进行选择，产生了具有H区域扩增的耐药株系，并且分别对MTX有23倍和12倍的交叉耐药性。这些以及其他带有H区域扩增的耐药株系对伯氨喹和特比萘芬也表现出较弱的交叉耐药性，将扩增的H区域与对MTX和其他药物的多效耐药性联系起来。相比之下，选择的对氯喹或喷他脒耐药的株系未显示H区域扩增或这种药物交叉耐药模式。经伯氨喹和特比萘芬选择的株系表现出野生型水平的二氢叶酸还原酶 - 胸苷酸合成酶以及MTX的正常摄取和积累，并且这些株系的MTX耐药性不会被维拉帕米逆转。这些数据表明，与H区域扩增相关的MTX交叉耐药机制是新颖的，并且不同于多药耐药哺乳动物细胞中由MDR基因过表达介导的机制。结构研究表明，这些硕大利什曼原虫株系中扩增的H区域DNA在很大程度上（可能完全）是染色体外的，由在两个DNA重排连接点处连接的环状反向重复序列组成。Southern印迹分析表明，这些重排连接点在四个独立的细胞系中是相同的，表明这些位点是DNA重排的“热点”。所有这些株系中的H区域扩增都是保守的，定义为染色体H区域位点的保留，没有结构改变或拷贝数减少。这一发现与H区域扩增的过复制/重组模型一致。
