Isolation and characterization of the promoter of the human prostate cancer-specific DD3 gene

Recently, we have described a novel gene, DD3, which is one of the most pro state cancer-specific genes described to date (Bussemakers, M. J. G., van B okhoven, A., Verhaegh, G. W,, Smit, F. P., Karthaus, H. F. M., Schalken, J. A, Debruyne, F. M. J., Ru, N., and Isaacs, W. B. (1999) Cancer Res. 59, 59 75-5979). The prostate cancer-specific expression of DD3 indicates that the DD3 gene promoter is a promising tool for the treatment of prostate cancer . To identify the promoter elements that are responsible for the prostate c ancer-specific expression of DD3, we have isolated and characterized the DD 3 promoter. Sequence analysis of the DD3 B'-flanking region was performed a nd several promoter-human growth hormone reporter constructs were prepared, which were transiently transfected in the DD3-positive cell line LNCaP and several DD3-negative cell lines. Using a 500-base pair DD3 promoter constr uct, we could detect promoter activity in LNCaP cells, which was not affect ed by increasing the size of the constructs. Truncated constructs, however, showed an increased transcriptional activity, suggesting the presence of a silencer that negatively regulates the expression of DD3. DNase-I footprin t analysis, using nuclear extracts from LNCaP cells, revealed the presence of three DNase-I-protected areas within the DD3 proximal promoter. We show that the high mobility group I(Y) protein binds to one of the DNase-I-prote cted areas and recruits another, yet unidentified, protein to the DD3 promo ter in LNCaP cells.