Cowan K H, Goldsmith M E, Levine R M, Aitken S C, Douglass E, Clendeninn N, Nienhuis A W, Lippman M E
J Biol Chem. 1982 Dec 25;257(24):15079-86.
Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5' flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.
已获得对甲氨蝶呤耐药（MTXR）的人乳腺癌细胞，其对该药物的敏感性比产生它们的野生型MCF - 7细胞低1000倍。耐药细胞中靶酶二氢叶酸（DHF）还原酶的活性大约增加了25倍。酶抑制研究和甲氨蝶呤亲和力研究未能揭示MTXR细胞中存在的DHF还原酶与野生型MCF - 7细胞中的DHF还原酶有任何差异。细胞遗传学分析表明，耐药细胞中存在拉长的标记染色体，而亲本细胞系中未发现这种染色体。使用Southern印迹杂交分析MTXR和野生型MCF - 7细胞的DNA表明，MTXR MCF - 7细胞比野生型MCF - 7细胞含有更多拷贝的DHF还原酶基因序列。这些实验还表明，MTXR细胞中扩增的DHF还原酶基因序列在扩增过程中可能经历了涉及5'侧翼序列的一致结构重排。MTXR MCF - 7细胞通过增加细胞生长对雌二醇作出反应，并且在给予雌二醇后，MTXR细胞中DHF还原酶的水平进一步诱导。放射性标记研究表明，雌激素刺激人乳腺癌细胞中DHF还原酶的实际合成。
