Abstract
Prostate-specific antigen (PSA) is expressed primarily by both normal prostate epithelium and the vast majority of prostate cancers. Increases in serum PSA during endocrine therapy are generally considered as evidence for prostate cancer recurrence or progression to androgen independence. The mechanisms by which PSA up-regulation occurs in androgen-refractory prostate cancer cells are unknown. In this study, by using LNCaP and its lineage-derived androgen-independent PSA-producing subline, C4-2, we identified two cis-elements within the 5.8-kilobase pair PSA promoter that are essential for the androgen-independent activity of PSA promoter in prostate cancer cells. First, a previously reported 440-bp androgen-responsive element enhancer core (AREc) was found to be important for the high basal PSA promoter activity in C4-2 cells. Both mutation analysis and supershift experiments demonstrated that androgen receptor (AR) binds to the AREs within the AREc and activate the basal PSA promoter activity in C4-2 cells under androgen-deprived conditions. Second, a 150-bp pN/H region was demonstrated to be a strong AR-independent positive-regulatory element of the PSA promoter in both LNCaP and C4-2 cells. Through DNase I footprinting and linker scan mutagenesis, a 17-bp RI site was identified as the key cis-element within the pN/H region. Data from electrophoretic mobility shift analysis and UV cross-linking experiments further indicated that a 45-kDa (p45) cell-specific transcription factor associates with RI in prostate cancer cells and may be responsible for driving the PSA promoter activity independent of androgen and AR. Furthermore, by juxtaposing AREc and pN/H, we produced a chimeric PSA promoter (supra-PSA) that exhibits 2-3-fold higher activity than the wild type PSA promoter in both LNCaP and C4-2 cells.
Highlights
Prostate-specific antigen (PSA) is a serum marker for prostate cancer
Through deletion analysis on PSA promoter, we provide evidence that androgen-responsive element enhancer core (AREc) and a pN/H element within the proximal promoter region are responsible for conferring the higher AI PSA promoter activity in C4-2 cells
Up-regulation of PSA Gene Expression in an Androgen-independent Prostate Cancer Cell Line, C4-2—C4-2, a lineage-derived LNCaP subline, was shown previously to be able to grow in castrated hosts and exhibit metastatic potential in vivo (30 –32)
Summary
PSA is a serum marker for prostate cancer. It has been shown that serum PSA is proportional to tumor volume and correlates positively with the clinical stage of the disease [11]. Due to the limited availability of AI yet PSA-producing prostate cancer cell lines, little is known about the androgenindependent regulation of PSA expression in hormone-refractory prostate cancer cells. The development of the androgenindependent PSA-producing C4-2 cell line from androgendependent parental LNCaP cells [29] has allowed us to study how PSA expression is regulated in an androgen- and growth factor-deprived environment. We have taken advantage of the remarkable difference in basal PSA expression between two lineage-related LNCaP cell lines, LNCaP and C4-2, for the analysis of androgen-independent regulation of PSA promoter in prostate cancer cells. An AR-dependent but androgen-independent pathway appears to be involved in AREc activation, whereas a 17-bp RI site within the pN/H was shown to associate with cell-specific transcription factor and regulates the PSA promoter activity independent of AR or androgen
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