CYP19 gene expression

The human CYP19 (P450 arom) gene is localized at chromosome 15q21.21. It belongs to the cytochrome P-450 superfamily compromising over 460 members in 74 families, of which cytochrome P450 arom is the sole member of the family 19 (29). CYP19 encodes aromatase that is the key enzyme for oestrogen biosynthesis (30, 31) which is achieved by sequential hydroxylation, oxidation, and removal of the C-19 carbon and aromatization of the A ring of the steroid.

It is well established that the highest levels of aromatase are present in the ovaries of premenopausal women, in the placenta of pregnant women, and in the peripheral adipose tissues of postmenopausal women and men (10, 32, 33).

The CYP19 gene is found between markers stSG12786 and stSG47530 with the 3'-end of the gene centromeric to the 5'-end of the gene, showing the direction of transcription as from telomere to centromere. It spans about 123 Kb. Only the 30 kb (exon II-exon X) 3'-region encodes aromatase, whereas the large 93 kb 5'-flanking region serves as the regulatory unit of the gene. The unusually large regulatory region contains 10 tissue-specific promoters that are alternatively used in various cell types.

Further upstream of exon II, there are a number of alternative first-exons which are differently spliced into distinct 5'-untranslated regions (34, 35, 36). In addition, up to nine different transcriptional start sides with individual promoters permitting tissue-specific regulation of expression have been described. However, even though each tissue expresses a unique first-exon 5'-untranslated region by splicing into a highly promiscuous splice acceptor site (AG-GACT) of the exon II, coding regions and translated products are identical in all tissue sites of expression (34, 36). This means that although transcripts in different tissues have different 5'-termini, the coding region is the same and therefore the proteins expressed in these tissues remain the same.

The recently published Human Genome Project Data allowed us for the first time to precisely locate all known promoters and elucidate the extraordinarily complex organization of the entire human CYP19 gene. Each promoter is regulated by a distinct set of regulatory sequences in DNA and transcription factors that bind to these specific sequences.

The promoter I.7 was cloned by analyzing P450arom mRNA in breast cancer tissue levels. P450arom mRNA with exon 1.7 expression was significantly increased in breast cancer tissues and adipose tissue adjacent to tumours (37). This TATA-less promoter accounts for the transcription of 29-54% of P450arom mRNAs in breast cancer tissues. The in vivo cellular distribution and physiologic roles of promoter I.7 in healthy tissues, however, are not known.

It is now known that the aromatase gene expression is regulated in a tissue-specific manner by the use of alternative promoters (37). Normal breast adipose tissue maintains low levels of aromatase expression primarily via promoter I.4 that lies 73 kb upstream of the common coding region. Promoters 1.3 and II are used only minimally in normal breast adipose tissue. By performing primer-specific RT-PCR analyses (38, 39, 40), it was revealed that the two major exons (I.3 and PII) are present in aromatase mRNAs isolated from breast tumours. These results suggest that promoters 1.3 and II are the major promoters directing aromatase expression in breast cancer and surrounding stromal cells and fibroblasts. It appears that the prototype oestrogen-dependent malignancy breast cancer takes advantage of four promoters (II, 1.3, 1.7, and I.4) for aromatase expression. The sum of P450arom mRNA species arising from these four promoters markedly increases the total P450arom mRNA levels in breast cancer compared with the normal breast that uses almost exclusively promoter I.4.

Many studies showed that a switch from an adipose-specific exon 1 (exon 1b or exon I.4) promoter used in non-tumour breast tissues to the ovary-specific exon 1 (exon 1c or exon I.2) occurred in breast cancer tissue (41, 42).

Immunohistochemical studies have provided evidence for both an epithelial and stromal location for the aromatase enzyme complex (17, 43). Biochemical studies, however, have revealed higher a aromatase activity in the stromal rather than the epithelial component of breast tumours (44). Furthermore, measurements of aromatase activity in fibroblasts derived from breast tumours or MCF-7 cells have demonstrated a much higher level of aromatase activity in fibroblasts (45).



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