USE OF ALTERNATIVE PROMOTERS TO EXPRESS THE AROMATASE CYTOCHROME-P450(CYP19) GENE IN BREAST ADIPOSE TISSUES OF CANCER-FREE AND BREAST-CANCER PATIENTS

Estrogen biosynthesis in adipose tissue has assumed great significance in terms of a number of estrogen-related diseases. Recent evidence su ggests that estrogen synthesized locally in the breast is of singular significance in the development of breast cancer in elderly women. The biosynthesis of estrogen from C-19 steroids is catalyzed by a specifi c form of cytochrome P450, namely aromatase cytochrome P450 (P450arom; the product of the CYP19 gene). The human CYP19 gene comprises nine c oding exons, II-X, and its transcripts are expressed in the ovary, pla centa, testes, adipose tissue, and brain. Tissue-specific expression o f the CYP19 gene is determined, at least in part, by the use of tissue -specific promoters, which give rise to transcripts with unique 5'-non coding termini. Transcripts in adipose tissue contain 5'-termini deriv ed from specific untranslated exons, corresponding to expression deriv ed from the proximal promoter II and its splice valiant I.3, as well a s a distal promoter, I.4. The object of the present study was to deter mine the distribution of these various exon-specific transcripts in br east adipose tissues from cancer-free women undergoing reduction mammo plasty and from patients with breast cancer, because this would provid e important clues as to the nature of the factors regulating aromatase expression in these sites. To achieve this, we employed competitive R T-PCR, utilizing an internal standard for each exon-specific transcrip t of the CYP19 gene, as well as for the coding region, to evaluate tot al CYP19 gene transcripts. In cancer patients (n = 18), total CYP19 ge ne transcript levels were significantly higher in adipose tissue proxi mal to a tumor in comparison with adipose tissue distal to a tumor, in agreement with previous findings. Moreover, total transcript levels w ere higher in breast adipose tissue of cancer patients in comparison w ith those of cancer-free individuals (n = 9), even when the adipose ti ssue from the cancer patient was taken from a quadrant with no detecta ble tumor. We observed that exon I.4-specific transcripts were predomi nant in breast adipose obtained from cancer-free women. In this tissue , promoter-II-specific and exon I.3-specific transcripts were present in low copy number. On the other hand, in breast cancer patients, CYP1 9 gene transcripts from breast adipose tissue had primarily promoter-I I-specific and exon I.3-specific sequence, whereas comparatively few t ranscripts had exon I.4-specific sequence at the 5'-terminus. We concl ude that CYP19 gene transcription in breast adipose tissue of cancer-f ree individuals uses preferably promoter I.4, implicating a role of gl ucocorticoids and members of the IL-6 cytokine family in the regulatio n of this expression. On the other hand, the increased expression in b reast adipose tissue bearing a carcinoma results from expression from promoters II and I.3, which are regulated by unknown factors acting vi a increased cAMP formation, which are presumably secreted by the tumor or associated cells.