Paclitaxel resistance by random mutagenesis of α‐tubulin
Paclitaxel resistance by random mutagenesis of α‐tubulin
复制标题
DOI：
10.1002/cm.21154
复制
发表时间：
2013-12
期刊：
影响因子：
2.9
通讯作者：
Shanghua Yin;C. Zeng;Malathi Hari;F. Cabral
中科院分区：
文献类型：
--
作者：
Shanghua Yin;C. Zeng;Malathi Hari;F. Cabral
许多赋予紫杉醇耐药性的哺乳动物β-微管蛋白突变已被表征，但目前对α-微管蛋白突变在耐药性中的作用知之甚少。先前使用二维凝胶电泳的研究表明，在选择对紫杉醇耐药的 CHO 细胞中，α-微管蛋白突变的发生频率与 β-微管蛋白突变的频率相同，但这些突变的身份很大程度上未知。我们现在已经对几种具有基因组 DNA 损伤的紫杉醇抗性 CHO 细胞系中的主要 α-微管蛋白基因进行了测序，并确定了主要影响蛋白质氨基末端部分的 5 个突变。我们还使用随机诱变和 α-微管蛋白 cDNA 转染来选择更多的紫杉醇抗性突变体，以消除可能限制突变多样性的基因组限制。这种方法导致鉴定出分布在整个 α-微管蛋白序列中的 16 个额外突变。这些突变被证实足以通过定点诱变赋予抗性，并且它们通过涉及微管组装减少的机制发挥作用。一种突变阻止了 α-微管蛋白的乙酰化，但在其他方面产生了与其他突变相似的表型。对文献的扫描显示，大量耐药突变与脑部疾病患者中报告的病变重叠或接近，这表明微管组装的改变是细胞耐药和发育缺陷的基础。 © 2013 Wiley 期刊公司。
Many mammalian β‐tubulin mutations that confer paclitaxel resistance have been characterized, but little is currently known about the role of α‐tubulin mutations in drug resistance. Previous studies using two‐dimensional gel electrophoresis showed that α‐tubulin mutations occur with a frequency equal to β‐tubulin mutations among CHO cells selected for resistance to paclitaxel but the identities of those mutations are largely unknown. We have now sequenced the major α‐tubulin gene in several paclitaxel resistant CHO cell lines with lesions in genomic DNA and identified five mutations that predominately affect the amino terminal part of the protein. We also used random mutagenesis and transfection of α‐tubulin cDNA to select further paclitaxel resistant mutants in an effort to remove genomic constraints that may limit the diversity of mutations. This approach led to the identification of 16 additional mutations that were distributed throughout the α‐tubulin sequence. The mutations were confirmed as sufficient to confer resistance by site‐directed mutagenesis, and they acted by a mechanism that involved reductions in microtubule assembly. One mutation prevented the acetylation of α‐tubulin but otherwise produced a phenotype similar to the other mutations. A scan of the literature revealed that a significant number of drug resistance mutations overlap or lie close to lesions that have been reported in patients with brain disorders suggesting that alterations in microtubule assembly underlie both cellular resistance and developmental defects. © 2013 Wiley Periodicals, Inc.