0021-972x/94/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1994 by The Endocrine Society Vol. 79, No. 6 Printed in U.S.A. CYP19 (Aromatase Cytochrome P450) Gene Expression in Human Malignant Endometrial Tumors* SERDAR E. BULUN, KATHERINE ECONOMOS, DAVID MILLER, AND EVAN R. SIMPSON Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Division of Gynecologic Oncology, Departments of Obstetrics-Gynecology and Biochemistry (S.E.B., D.M., E.R.S.), University of Texas Southwestern Medical Center, Dallas, Texas 75235; and the Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, New York Hospital-Cornell Medical Center (K.E.), New York, New York 10021 ABSTRACT Cl9 steroids are converted to estrogens in a number of human tissues by the aromatase enzyme complex, which consists of aromatase cyto- chrome P450 (P450arom: nroduct of the CYPl9 gene) and NADPH- cytochrome Pi50 reductaie. Aromatase activity rhas been previously demonstrated in endometrial tumors. In the present study, we investi- gated CYP19 gene expression and its regulation in endometrial tumor samples (n = 9). Using a specific method of competitive polymerase chain reaction after reverse transcription, varying levels of P450arom transcripts were detected in all endometrial adenocarcinomas (n = 8) and one mixed Miillerian tumor studied. No correlations were observed between P450arom transcript levels and histological type of the tumor, grade, myometrial invasion, stage of the disease, or patient age. We have recently demonstrated that the tissue-specific regulation of CYP19 gene transcription is in part the consequence of alternative promoter use. The use of each promoter gives rise to a P450arom transcript with a unique untranslated 5’-end. We analyzed the untrans- lated first exons in 5’-terminals of P450arom transcripts in endometrial adenocarcinomas using a specific reverse transcription-polymerase chain reaction/Southern hybridization method we recently developed. Our findings indicated the gonadal-type (promoter II) and one of the adipose stromal cell-type (1.3) promoters were primarily used for P450arom expression in adenocarcinomas. On the other hand, distri- bution of transcripts specific for 1.3, I.4 (another adipose-type pro- moter), and promoter II in one mixed Miillerian tumor was uniform. Placental promoter (1.1)~specific P450arom transcripts were not de- tected in endometrial tumors. As P450arom transcripts were detected in all endometrial malignancies studied, whereas they were not demon- strable in the disease-free endometrium, activation or failure of inhi- bition of aromatase expression in these tumors may serve to promote neoplastic proliferation. (J Clin EndocrinoZMetab 79: 1831-1834,1994) T HE CONVERSION of C,, steroids to estrogensis cata- lyzed by an enzyme complex termed aromatase, which consists of a specific form of cytochrome P450 known as aromatase cytochrome P450 (P450arom, product of the CYP19 gene), as well as a flavoprotein, NADPH-cytochrome P450 reductase (l-3). Aromatase is expressedin a number of human cells and tissues,including ovarian granulosa and luteal cells (4), testicular Leydig cells (5), placental syncytio- trophoblast (6), adipose stromal cells of both males and females (7, 8), and the fetal brain (9). Aromatase activity in human endometrial cancer tissue was first demonstrated by Tseng and colleagues (10). An endometrial cancer cell line was also reported to contain aromatase activity (11). Using sensitive methods, such as polymerase chain reaction (PCR), we were unable to detect P450arom messenger ribonucleic acid (mRNA) or aromatase activity in disease-free human endometrial and myometrial tissuesor cells in culture (12, 13). On the other hand, we demonstrated aromataseactivity and CYP19 gene expression in uterine leiomyomas (13). The present study was conducted to understand the regulation Received June 15, 1994. Revision received August 19, 1994. Accepted August 30, 1994. Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. * This work was supported in part by LJSPHS Grant AG-08174 and the SGI-Mead Johnson 1993-94 Research Award. of CYP19 (P450arom) gene expressionin human endometrial malignant tumors. The entire CYP19 gene is at least 75 kilobases (kb) in size (14); the region encoding the P450arom protein, however, spansabout 35 kb of DNA and contains 9 exons (II-X). Exon II contains the translation start site. Recent findings from our laboratory indicate that the human CYP19 gene contains a number of tissue-specific promoters that direct aromatase expressionin human placenta, ovary, and adipose tissue via alternative splicing (14, 15). The majority of placental tran- scripts contain sequencesencoded in exon 1.1, which lies some40 kb up-stream of the translation start site. In ovarian corpus luteum tissue, CYP19 gene transcription is initiated 120 basepairsup-stream of the translation initiation site in exon II (promoter II). Some 20 kb down-stream of exon 1.1, another untranslated first exon, exon I.4 (14) was found to be expressedin human adipose tissue and adipose stromal cells in culture. Exon 1.3, located 306 basepairs up-stream of the common splice junction in exon II, was also identified in adipose tissue. All of these untranslated exons are spliced into the identical site up-stream of the translation initiation site in exon II. Consequently, the sequence encoding the open reading frame is identical in each case. Thus, the expressed protein is the same regardless of the splicing pattern. As a different promoter up-stream of each untrans- lated exon directs transcription in a tissue-specificmanner, a rapid way of analyzing promoter use for aromataseexpres- 1831 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 December 2016. at 10:47 For personal use only. No other uses without permission. . All rights reserved.