Affleck Joslynn G, Walker Virginia K
Department of Biology, Biosciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Toxicol Sci. 2007 Oct;99(2):522-31. doi: 10.1093/toxsci/kfm123. Epub 2007 May 22.
The folic acid analog methotrexate (MTX), a competitive inhibitor of dihydrofolate reductase (DHFR), is used to treat a variety of cancers and autoimmune disorders. However, MTX also causes a wide range of toxic effects in healthy cells and is an established teratogen. Efforts to "rescue" the defects caused by MTX by administering a folate analog or by transgenic expression of a DHFR with an altered affinity for MTX have been attempted in a variety of mammals but limited protection was conferred. As a result, our understanding of the effect of MTX at the molecular genetic level remains incomplete and, in addition, continued mammalian sacrifice is not ideal. Due to the similarity of teratogenic effects produced by MTX in Drosophila melanogaster these insects were transformed with DHFR alleles to determine if rescue could be achieved. The resulting "MTX-resistant" flies were subsequently used to investigate changes in gene expression in response to MTX using semiquantitative reverse transcription PCR. The majority (12/14) of key transcripts that were affected in MTX-exposed females including transcripts involved in cell cycle, defense response, and transport were "rescued" in the "MTX-resistant" transgenic flies. These studies illustrate the utility of this invertebrate model for the investigation of molecular effects of MTX-induced teratogenicity, MTX-resistant DHFRs for gene therapy techniques, and teratogenic protection.
叶酸类似物甲氨蝶呤（MTX）是二氢叶酸还原酶（DHFR）的竞争性抑制剂，用于治疗多种癌症和自身免疫性疾病。然而，MTX也会在健康细胞中引发广泛的毒性作用，并且是一种已确定的致畸剂。人们尝试通过给予叶酸类似物或通过转基因表达对MTX具有改变亲和力的DHFR来“挽救”由MTX引起的缺陷，在多种哺乳动物中进行了尝试，但所提供的保护有限。因此，我们对MTX在分子遗传水平上的作用的理解仍然不完整，此外，持续进行哺乳动物牺牲并不理想。由于MTX在黑腹果蝇中产生的致畸作用具有相似性，因此对这些昆虫进行了DHFR等位基因转化，以确定是否可以实现挽救。随后，使用半定量逆转录PCR，将产生的“MTX抗性”果蝇用于研究基因表达对MTX的响应变化。在暴露于MTX的雌性果蝇中受影响的大多数（12/14）关键转录本，包括参与细胞周期、防御反应和转运的转录本，在“MTX抗性”转基因果蝇中得到了“挽救”。这些研究说明了这种无脊椎动物模型在研究MTX诱导的致畸性的分子效应、用于基因治疗技术的MTX抗性DHFR以及致畸保护方面的实用性。
