Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase
Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase
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DOI：
10.1016/s0925-4439(02)00079-0
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发表时间：
2002-07-18
影响因子：
6.2
通讯作者：
Bertino, JR
中科院分区：
文献类型：
--
作者：
Banerjee, D;Mayer-Kuckuk, P;Bertino, JR
耐药性通常是成功化疗的限制因素。我们的实验室一直有兴趣研究对靶向胸苷酸生物合成途径的药物，特别是靶向胸苷酸合成酶（TS）和二氢叶酸还原酶（DHFR）的药物的耐药性机制。我们已经使用白血病作为模型系统来研究对甲氨蝶呤（MTX）的耐药性，并且使用结直肠癌作为模型系统来研究5-氟尿嘧啶（5-FU）耐药性。在白血病中，我们和其他人已经表明，转运，外排，多聚谷氨酰化和水解酶活性是MTX耐药的主要决定因素。我们进一步报道了一些白血病细胞DHFR基因拷贝数的增加可能有助于耐药表型。最近，我们已经开始详细研究的分子机制，管理翻译调节DHFR响应MTX作为一个额外的耐药机制。到目前为止，涉及从患者获得的结直肠肿瘤的研究主要集中在TS表达水平和p53突变在确定对5-FU的反应中的预测价值上。尽管这两种测量的预测值似乎是显著的，但是考虑到在细胞系中观察到的对5-FU的耐药性的多样性，这些不太可能是预测对该药物的应答或导致获得性耐药性的唯一测量。尿苷-胞苷一磷酸激酶（UMPK）是5-FU活化过程中必不可少的限速酶，而二氢嘧啶脱氢酶（DPD）是使5-FU失活的分解代谢酶。因此，UMPK和DPD的改变可以解释在TS或p53没有改变的情况下5-FU反应的失败。调节TS的转录因子也可能影响药物敏感性。我们发现E2 F家族转录因子的mRNA水平与TS信息水平相关，并且在结直肠癌的肺转移中高于肝转移。此外，E2 F-1基因的基因拷贝数似乎在大量得自结直肠癌转移的样品中增加。我们还通过随机和定点诱变产生了DHFR和TS的突变体，这些突变体赋予对MTX和5-FU的抗性。这些突变体单独使用或作为突变体的融合cDNA使用已被证明在移植研究中是有用的，其中将这些突变cDNA转移到骨髓细胞中已显示赋予受体耐药性。DHFR的融合cDNA如DHFR-单纯疱疹病毒I型胸苷激酶（HSVTK）也可用于使用MTX作为小分子调节剂来调节体内基因表达，所述小分子调节剂可通过正电子发射断层扫描（PET）扫描或通过使用融合构建体如DHFR-EGFP的光学成像来监测。(C)2002 Elsevier Science B. V.保留所有权利。
Drug resistance is often a limiting factor in successful chemotherapy. Our laboratory has been interested in studying mechanisms of resistance to drugs that are targeted to the thymidylate biosynthesis pathway especially those that target thymidylate synthase (TS) and dihydrofolate reductase (DHFR). We have used leukemia as a model system to study resistance to methotrexate (MTX) and colorectal cancer as the model system to study 5-fluorouracil (5-FU) resistance. In leukemias, we and others have shown that transport, efflux, polyglutamylation and hydrolase activities are major determinants of MTX resistance. We have further reported that some leukemic cells have an increase in DHFR gene copy number possibly contributing to the resistant phenotype. Recently, we have begun to study in detail the molecular mechanisms that govern translational regulation of DHFR in response to MTX as an additional resistance mechanism. Studies thus far involving colorectal tumors obtained from patients have focused predominantly on the predictive value of levels of TS expression and p53 mutations in determining response to 5-FU. Although the predictive value of these two measures appears to be significant, given the variety of resistance to 5-FU observed in cell lines, it is not likely that these are the only measures predictive of response or responsible for acquired resistance to this drug. The enzyme uridine-cytidine monophosphate kinase (UMPK) is an essential and rate-limiting enzyme in 5-FU activation while dihydropyrimidine dehydrogenase (DPD) is a catabolic enzyme that inactivates 5-FU. Alterations in UMPK and DPD may therefore explain failure of 5-FU response in the absence of alterations in TS or p53. Transcription factors that regulate TS may also influence drug sensitivity. We have found that mRNA levels of the E2F family of transcription factors correlates with TS message levels and are higher in lung metastases than in liver metastases of colorectal cancers. Moreover, gene copy number of the E2F-1 gene appears to be increased in a significant number of samples obtained from metastases of colorectal cancer. We have also generated mutants of both DHFR and TS that confer resistance to MTX as well as 5-FU by random as well as site-directed mutagenesis. These mutants used alone or as fusion cDNAs of the mutants have proven to be useful in transplant studies where transfer of these mutant cDNAs to bone marrow cells have been shown to confer drug resistance to recipients. The fusion cDNAs of DHFR such as the DHFR-herpes simplex virus type I thymidine kinase (HSVTK) are also useful for regulation of gene expression in vivo using MTX as the small molecule regulator that can be monitored by positron emission tomography (PET) scanning or by optical imaging using a fusion construct such as DHFR-EGFP. (C) 2002 Elsevier Science B.V. All rights reserved.