Quantitation of estrogen receptor mRNA copy numbers in breast cancer cell lines and tumors.
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.
Poola
I
I
Department of Pharmacology, Howard University School of Medicine, Washington, DC 20059, USA.
Williams
D M
DM
Koduri
S
S
Ramprakash
J
J
Taylor
R E
RE
Hankins
W D
WD
D001943:Breast Neoplasms / Q000235:genetics*; D018076:DNA, Complementary; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases