Journal of Clinical Endocrinology & Metabolism, Vol 74, 1247-1252, Copyright © 1992 by Endocrine Society

ARTICLES

Determination of aromatase cytochrome P450 messenger ribonucleic acid in human breast tissue by competitive polymerase chain reaction amplification

T Price, J Aitken, J Head, M Mahendroo, G Means and E Simpson
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9051.

Local production of estrogen in breast tissue may influence the growth of breast cancers. Peripheral conversion of C19 steroids to estrogens is catalyzed by the aromatase enzyme complex which is comprised of a specific form of cytochrome P450, aromatase cytochrome P450 (P450AROM) and the flavoprotein, NADPH-cytochrome P450 reductase. To evaluate P450AROM mRNA levels in breast tissue, a specific competitive polymerase chain reaction amplification procedure was devised. In this method, a rat P450AROM complementary RNA is coamplified as an internal standard in order to compare amplification reactions. The amplification products are recognized by hybridization with 32P-labeled oligonucleotides specific for each species. Densitometry is used to quantitate autoradiographs. Initial studies using RNA from whole breast tissue obtained from reduction mammoplasty revealed linearity of the relationship between the densitometer signal from the human amplification product and total RNA concentration. Breast tissue was then separated into a floating adipocyte fraction and a pelleted fraction containing the other cellular elements by collagenase digestion and centrifugation. Comparison of specific content of aromatase amplification product per unit weight of RNA extracted from adipocytes and pelleted cells revealed considerably higher levels in the RNA from the nonadipocyte fraction. Immunocytochemical characterization of this fraction revealed the presence of several cell types including macrophages, ductal epithelial cells, and endothelial cells, but primary cells of stromal origin.

This article has been cited by other articles:

				N. Tagawa, R. Yuda, S. Kubota, M. Wakabayashi, Y. Yamaguchi, D. Kiyonaga, N. Mori, E. Minamitani, H. Masuzaki, and Y. Kobayashi 17{beta}-Estradiol inhibits 11{beta}-hydroxysteroid dehydrogenase type 1 activity in rodent adipocytes J. Endocrinol., July 1, 2009; 202(1): 131 - 139. [Abstract] [Full Text] [PDF]

				R. T. Falk, E. Gentzschein, F. Z. Stanczyk, L. A. Brinton, M. Garcia-Closas, O. B. Ioffe, and M. E. Sherman Measurement of Sex Steroid Hormones in Breast Adipocytes: Methods and Implications Cancer Epidemiol. Biomarkers Prev., August 1, 2008; 17(8): 1891 - 1895. [Abstract] [Full Text] [PDF]

				S. Deb, J. Zhou, S. A. Amin, A. G. Imir, M. B. Yilmaz, Z. Lin, and S. E. Bulun A Novel Role of Sodium Butyrate in the Regulation of Cancer-associated Aromatase Promoters I.3 and II by Disrupting a Transcriptional Complex in Breast Adipose Fibroblasts J. Biol. Chem., February 3, 2006; 281(5): 2585 - 2597. [Abstract] [Full Text] [PDF]

				S. E. Bulun, Z. Lin, G. Imir, S. Amin, M. Demura, B. Yilmaz, R. Martin, H. Utsunomiya, S. Thung, B. Gurates, et al. Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment Pharmacol. Rev., September 1, 2005; 57(3): 359 - 383. [Abstract] [Full Text] [PDF]

				G. L. Rubin, J. H. Duong, C. D. Clyne, C. J. Speed, Y. Murata, C. Gong, and E. R. Simpson Ligands for the Peroxisomal Proliferator-Activated Receptor {gamma} and the Retinoid X Receptor Inhibit Aromatase Cytochrome P450 (CYP19) Expression Mediated by Promoter II in Human Breast Adipose Endocrinology, August 1, 2002; 143(8): 2863 - 2871. [Abstract] [Full Text] [PDF]

				L. Meng, J. Zhou, H. Sasano, T. Suzuki, K. M. Zeitoun, and S. E. Bulun Tumor Necrosis Factor {{alpha}} and Interleukin 11 Secreted by Malignant Breast Epithelial Cells Inhibit Adipocyte Differentiation by Selectively Down-Regulating CCAAT/Enhancer Binding Protein {{alpha}} and Peroxisome Proliferator-activated Receptor {{gamma}}: Mechanism of Desmoplastic Reaction Cancer Res., March 1, 2001; 61(5): 2250 - 2255. [Abstract] [Full Text]

				J. Zhou, B. Gurates, S. Yang, S. Sebastian, and S. E. Bulun Malignant Breast Epithelial Cells Stimulate Aromatase Expression via Promoter II in Human Adipose Fibroblasts: An Epithelial-Stromal Interaction in Breast Tumors Mediated by CCAAT/Enhancer Binding Protein {beta} Cancer Res., March 1, 2001; 61(5): 2328 - 2334. [Abstract] [Full Text]

				G. L. Rubin, Y. Zhao, A. M. Kalus, and E. R. Simpson Peroxisome Proliferator-activated Receptor {{gamma}} Ligands Inhibit Estrogen Biosynthesis in Human Breast Adipose Tissue: Possible Implications for Breast Cancer Therapy Cancer Res., March 1, 2000; 60(6): 1604 - 1608. [Abstract] [Full Text]

				R. J. Pauley, S. J. Santner, L. R. Tait, R. K. Bright, and R. J. Santen Regulated CYP19 Aromatase Transcription in Breast Stromal Fibroblasts J. Clin. Endocrinol. Metab., February 1, 2000; 85(2): 837 - 846. [Abstract] [Full Text]

				C. M. Halleux, P. J. Declerck, S. L. Tran, R. Detry, and S. M. Brichard Hormonal Control of Plasminogen Activator Inhibitor-1 Gene Expression and Production in Human Adipose Tissue: Stimulation by Glucocorticoids and Inhibition by Catecholamines J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 4097 - 4105. [Abstract] [Full Text]

				J. Zhou, T. R. Kumar, M. M. Matzuk, and C. Bondy Insulin-Like Growth Factor I Regulates Gonadotropin Responsiveness in the Murine Ovary Mol. Endocrinol., December 1, 1997; 11(13): 1924 - 1933. [Abstract] [Full Text] [PDF]

				S. J. Santner, R. J. Pauley, L. Tait, J. Kaseta, and R. J. Santen Aromatase Activity and Expression in Breast Cancer and Benign Breast Tissue Stromal Cells J. Clin. Endocrinol. Metab., January 1, 1997; 82(1): 200 - 208. [Abstract] [Full Text] [PDF]

				Y. Zhao, J. E. Nichols, S. E. Bulun, C. R. Mendelson, and E. R. Simpson Aromatase P450 Gene Expression in Human Adipose Tissue J. Biol. Chem., July 7, 1995; 270(27): 16449 - 16457. [Abstract] [Full Text] [PDF]