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

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 (P450_AROM) and the flavoprotein, NADPH-cytochrome P450 reductase. To evaluate P450_AROM mRNA levels in breast tissue, a specific competitive polymerase chain reaction amplification procedure was devised. In this method, a rat P450_AROM 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 primarily cells of stromal origin.