Poola I, Williams D M, Koduri S, Ramprakash J, Taylor R E, Hankins W D
Department of Pharmacology, Howard University School of Medicine, Washington, DC 20059, USA.
Anal Biochem. 1998 May 1;258(2):209-15. doi: 10.1006/abio.1998.2629.
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.
多项临床研究表明，乳腺肿瘤中雌激素受体（ER）的含量会影响患者的生存率、肿瘤复发情况以及对抗雌激素疗法的反应。因此，精确测定肿瘤组织中ER含量的能力将对乳腺癌女性患者大有裨益。尽管已有免疫组织化学和聚合酶链反应（PCR）方法用于检测和半定量ER，但这些方法均无法精确测定肿瘤样本中ER的拷贝数。在本报告中，我们描述了一种分子方法，即使用逆转录PCR（RT-PCR）模板竞争法精确测定ER mRNA的拷贝数。通过将无关核酸序列插入ER cDNA克隆中设计出一个竞争模板。然后进行模板竞争性RT-PCR分析以确定ER mRNA的拷贝数。作为各种样本中ER mRNA拷贝数的参考标准，还对组成型表达基因甘油醛-3-磷酸脱氢酶（GAPDH）的mRNA拷贝数进行了定量。通过这种方法对三种阳性细胞系MCF-7、T47D和ZR-75以及两个阳性肿瘤组织进行了ER定量。我们在此描述的结果表明，在所研究的细胞系中，T47D表达的ER拷贝数最高。我们还在此表明，通过模板竞争法可在肿瘤样本中检测和定量低至每10^5份GAPDH中有10^3份的ER。此外，这种分子方法可同时检测、区分和定量ER外显子缺失变异体的拷贝数。本报告中描述的结果表明，肿瘤组织中外显子7缺失变异体与野生型的比例明显高于所研究的细胞系。
