Quantitation of estrogen receptor mRNA copy numbers in breast cancer cell lines and tumors
Quantitation of estrogen receptor mRNA copy numbers in breast cancer cell lines and tumors
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DOI：
10.1006/abio.1998.2629
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发表时间：
1998-05-01
影响因子：
2.9
通讯作者：
Hankins, WD
中科院分区：
文献类型：
--
作者：
Poola, I;Williams, DM;Hankins, WD
一些临床研究表明，乳腺肿瘤中雌激素受体（ER）的含量影响生存率、肿瘤复发和抗雌激素治疗的反应，因此，准确定量肿瘤组织中ER含量的能力将对乳腺癌患者有显著的益处。虽然免疫组化和聚合酶链反应（PCR）方法已被描述为检测和半定量ER，他们都没有精确定量ER拷贝数在肿瘤样本。在本报告中，我们描述了一种分子方法，准确定量ER mRNA拷贝数使用逆转录PCR（RT-PCR）模板竞争法。通过将不相关的核酸序列插入ER cDNA克隆来设计竞争模板。然后进行模板竞争性RT-PCR分析以确定ER mRNA的拷贝数。作为来自各种样品的ER mRNA拷贝数的参考标准，还定量组成型表达基因甘油醛-3-磷酸脱氢酶（GAPDH）的mRNA拷贝数。在三种阳性细胞系MCF-7、T47 D和ZR-75中进行ER定量。两例阳性肿瘤组织。我们的研究结果表明，在所研究的细胞系中，T47 D表达的ER拷贝数最高。我们还在此提出，通过模板竞争方法可以在肿瘤样品中检测和定量低至每10（5）个GAPDH拷贝10（3）个拷贝的ER。此外，分子方法可以同时检测，区分和定量ER的外显子缺失变体拷贝数。本报告中描述的结果表明，肿瘤组织中外显子7缺失变体与野生型的比率显著高于所研究的细胞系。(C)北京：科学出版社.
Several clinical studies have suggested that the content of estrogen receptor (ER) in breast tumors influences the survival, tumor recurrence, and response to antiestrogen therapies, Therefore, the ability to precisely quantitate the ER content in tumor tissues will be of significant benefit to women with breast cancer. Although immunohistochemical and polymerase chain reaction (PCR) methods have been described for the detection and semiquantitation of ER, none of them precisely quantitate ER copy numbers in tumor samples. In the present report we describe a molecular approach to accurately quantitate ER mRNA copy numbers using a reverse-transcription PCR (RT-PCR) template competition method. A competitor template was devised by inserting unrelated nucleic acid sequences into an ER cDNA clone. A template competitive RT-PCR analysis was then performed to determine the number of copies of ER mRNA, As a standard of reference for the ER mRNA copy numbers from various samples, the mRNA copy numbers of a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were also quantitated, The ER quantitations were performed in three positive cell lines, MCF-7, T47D, and ZR-75, and two positive tumor tissues by this approach. Our results described here show that among the cell lines studied, T47D expresses the highest copy numbers of ER. We also present here that ER as low as 10(3) copies per 10(5) copies of GAPDH can be detected and quantitated in tumor samples by the template competition method. In addition, the molecular approach can simultaneously detect, distinguish, and quantitate exon deletion variant copy numbers of ER. The results described in this report indicate that the ratios of exon 7 deletion variant to wild type in the tumor tissues are significantly higher than in the cell lines studied. (C) 1998 Academic Press.