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首页> 外文学位 >The genomic response of MCF-7 breast cancer cells in the selection of resistance to taxane and anthracycline chemotherapeutics.
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The genomic response of MCF-7 breast cancer cells in the selection of resistance to taxane and anthracycline chemotherapeutics.

机译：MCF-7乳腺癌细胞在对紫杉烷和蒽环类药物的耐药性选择中的基因组反应。

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摘要

Multi-drug resistance is a well-studied phenomenon in cancer research both in vitro and in vivo. Often times, individuals studying chemotherapeutic resistance in vitro compare cells that have been developed to exhibit extreme resistance at pharmacologically insignificant doses, atypical of the clinical setting. In order to profile trends in the development of resistance, we have utilized a stepwise dose escalation protocol starting at 1000 fold below the measured IC50 with a 3-fold increase (one dose) in drug concentration every 14 days (for the majority of cell lines). Consistent with this protocol, a panel of isogenic MCF-7 resistant cell lines to paclitaxel (MCF-7TAX), docetaxel (MCF-7TXT), doxorubicin (MCF-7DOX), and epirubicin (MCF-7 EPI), in combination with an untreated co-cultured control (MCF-7 CC), were developed. Characterization of resistance and cross resistance profiles for the resistant MCF-7 cell panel was carried out through clonogenic assay. To identify significant gene candidates in the development of resistance, cDNA microarray profiling was used in conjunction with Q-PCR verification of identified gene transcripts. Genes were selected as potentially significant in the establishment of drug resistance through differential analysis of expression levels at known resistance thresholds. In addition, genes coinciding with the accumulation of resistance were identified through the calculation of Pearson product-moment correlations with increasing resistance levels. Additional Q-PCR profiling of commonly identified drug transporters (eg., P-gp) was also conducted in order to establish if genes varied concomitantly with increasing resistance.; Investigation of IC50 profiles for the resistant MCF-7 cell panel indicated that initial resistance occurs at the 9th 3-fold increase in drug concentration for paclitaxel (3.7 nM), docetaxel (3.3 nM), and epirubicin (31.5 nM), but at the 10th increase in drug concentration for doxorubicin (43.6 nM). In addition, the MCF-7DOX cells were not seen to develop significant cross resistance to the taxanes despite showing considerable resistance to anthracycline based compounds. All other resistant cell lines were seen to develop extreme cross resistance to both taxane, and anthracycline chemotherapeutics beginning at dose 9. Microarray profiling of gene expression patterns for the resistant MCF-7 cell lines has revealed a total of 49 genes with unique expression patterns for individual cell lines. Q-PCR verification of 9 microarray identified gene transcripts has shown an approximate 80% verification rate, whereas further Q-PCR analysis of the well known ABC transporter ABCB1/P-glycoprotein has revealed increased mRNA transcript levels for the MCF-7EPI (19,482x), MCF-7TXT (3,989x), and MCF-7TAX (7,811x) cell lines. Interestingly, ABCB1/P-gp transcript levels in MCF-7DOX cells is relatively low (∼2x) compared to other resistant cell lines, suggesting the presence of alternate mechanisms in the development of MCF-7DOX resistance.; The progressive resistance shown in the incrementally selected MCF-7 cell lines to taxane and anthracycline chemotherapeutics has demonstrated that significant, high level resistance occurs at sub-clinical dose range, and persists into clinically specified plasma-drug concentration parameters. Furthermore, the incremental escalation of drug concentration to promote the development of resistance in the MCF-7 cell lines has identified significant gene targets that correlate with resistance factors, suggesting a role for alternate and/or co-existing mechanisms in the development of xenobiotic resistance.; Keywords: multidrug resistance, pharmacogenomics, breast cancer, microarray, Q-PCR, taxanes, anthracyclines.
机译：在体外和体内癌症研究中，对多种药物的耐药性都是一个经过充分研究的现象。通常，在体外研究化学疗法抗药性的个体会比较那些已发展为在药理学上无关紧要的剂量下表现出极强抗药性的细胞，这是临床环境中的非典型现象。为了描述耐药性发展的趋势，我们采用了逐步剂量递增方案，从低于测得的IC50的1000倍开始，每14天药物浓度每3倍增加3倍（一次剂量）（对于大多数细胞系） ）。与该协议一致，一组对紫杉醇（MCF-7TAX），多西他赛（MCF-7TXT），阿霉素（MCF-7DOX）和表柔比星（MCF-7 EPI）的同基因MCF-7耐药细胞系与开发了未经处理的共培养对照（MCF-7 CC）。通过克隆形成试验对MCF-7耐药细胞组的耐药性和交叉耐药性进行了表征。为了在耐药性的发展中鉴定出重要的基因候选物，将cDNA微阵列分析与鉴定出的基因转录物的Q-PCR结合使用。通过在已知抗性阈值下对表达水平进行差异分析，选择在建立抗药性中潜在重要的基因。另外，通过计算随着抗性水平增加的皮尔逊产物-矩相关性，鉴定出与抗性积累相吻合的基因。为了确定基因是否随耐药性的增加而变化，还对常用的药物转运蛋白（如P-gp）进行了附加的Q-PCR分析。对耐药MCF-7细胞小组的IC50谱研究表明，最初的耐药发生在紫杉醇（3.7 nM），多西他赛（3.3 nM）和表柔比星（31.5 nM）的药物浓度增加9倍3倍时，但在阿霉素（43.6 nM）的药物浓度第十次增加。另外，尽管MCF-7DOX细胞对基于蒽环类的化合物显示出相当大的抗性，但未发现其对紫杉烷类具有显着的交叉抗性。从剂量9开始，观察到所有其他耐药细胞系对紫杉烷和蒽环类化学疗法均产生极强的交叉耐药性。针对耐药MCF-7细胞系的基因表达模式的微阵列分析揭示了总共49个具有独特表达模式的基因。单个细胞系。对9个微阵列鉴定的基因转录本进行Q-PCR验证显示出大约80％的验证率，而对著名ABC转运蛋白ABCB1 / P-糖蛋白的进一步Q-PCR分析显示MCF-7EPI的mRNA转录水平增加（19,482x ），MCF-7TXT（3,989x）和MCF-7TAX（7,811x）细胞系。有趣的是，与其他抗性细胞系相比，MCF-7DOX细胞中的ABCB1 / P-gp转录水平相对较低（约2倍），表明在MCF-7DOX抗性的形成中存在其他机制。在递增选择的MCF-7细胞系中对紫杉烷和蒽环类化学治疗药物显示出的逐步耐药性表明，在亚临床剂量范围内会出现明显的高水平耐药性，并持续存在临床指定的血浆药物浓度参数中。此外，药物浓度的逐步升高以促进MCF-7细胞系中耐药性的发展，已经发现了与耐药性因子相关的重要基因靶标，这表明异种和/或共存机制在异源生物耐药性发展中的作用。;关键词：多药耐药性，药物基因组学，乳腺癌，微阵列，Q-PCR，紫杉烷类，蒽环类。

著录项

  • 作者

    Veitch, Zachary William Neil.;

  • 作者单位

    Laurentian University (Canada).;

  • 授予单位 Laurentian University (Canada).;
  • 学科 Biology Molecular.; Biology Cell.; Health Sciences Pharmacology.
  • 学位 M.Sc.
  • 年度 2007
  • 页码 129 p.
  • 总页数 129
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;细胞生物学;药理学;
  • 关键词

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