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

Abstract

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.

17β-Estradiol inhibits 11β-hydroxysteroid dehydrogenase type 1 activity in rodent adipocytes

“…Adipose tissue is composed of mature adipocytes and stromal-vascular cells, with estrogen production occurring predominantly in stromal-vascular cells (Ackerman et al 1981, Price et al 1992, and 11b-HSD1 mRNA expression and enzyme activity occurring predominantly in adipocytes (Berger et al 2001, Atanasov et al 2004, Dieudonne et al 2006. Glucocorticoids activate aromatase activity in cultured stromal-vascular cells of adipose tissue (Simpson et al 1981, Folkerd & James 1983, Perel et al 1986, Dieudonne et al 2006.…”

Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment

“…This latter group of cells in the breast adipose tissue was characterized using immunohistochemical methods (Price et al, 1992). Ninety percent of these resident cells of adipose tissue are fibroblasts, i.e., the potential precursors of mature adipocytes, and another 7% represented endothelial cells.…”

Exploring Breast Cancer Estrogen Disposition: The Basis for Endocrine Manipulation

“…However, due to conflicting evidence, the relative contribution from circulating versus locally synthesized estrogens to intratumor E 2 levels in postmenopausal women has been controversial for 2 decades. Although intratumor aromatase expression has been detected at mRNA (16) and protein (17) levels, and aromatase activity has been detected in tumor specimens in vitro (18), the quantitative importance of local estrogen production within tumors and adjacent benign tissues versus circulatory uptake has not been defined.…”