|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1998 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1997 : ¥2,000,000 (Direct Cost : ¥2,000,000)
In this study, I have analyzed molecular mechanism for switching in the tissue-specific use of multiple exons 1 of human aromatase (estrogen synthetase) gene, whcih is often found in the age-related diseases, such as breast cancer, osteoporosis, arteriosclerosis, and Alzheimer disease, and causes aberrant expression of the human aromatase gene. I have establised a cultured cell line derived from adipose stromal tissues of human breasts. The cells caused elevated expression of aromatase gene and a switching of the alternative exons 1, which are often observed in breast cancer tissues, by removal of serum from the culture medium or addition of PKA and PKC activators such as forskolin and phorbol ester (TPA). Then, I analyzed the promoter region determining preferentia transcription from exon 1b, which is mostly used in healthy breast tissues, by using a newly developed vector consisting of the multiple exons 1/promoter-CAT fused gene. Consequently, I showed the presence of essential elements for transcription from exon 1b within -500 bp upstream from the transcriptional start site. Then, I perfonned gel shift assays using nuclear extracts from serum-depleted or forskolin/TPA-treated cells, and untreated control cells. I identified two DNA sites at around the -300 and -450 bp which were bound with control nuclear factors, but not with serum-depleted or forskolin/TPA-treated nuclear factors. I regarded the binding protein as a nuclear factor essential for transcription from exon 1b. I am now screening the cDNA of the nuclear factors binding to the specific sites by using an yeast one-hybrid system.