The program of androgen-responsive genes in neoplastic prostate epithelium

Abstract

The human prostate gland is an important target organ of androgenic hormones. Testosterone and dihydrotestosterone interact with the androgen receptor to regulate vital aspects of prostate growth and function including cellular proliferation, differentiation, apoptosis, metabolism, and secretory activity. Our objective in this study was to characterize the temporal program of transcription that reflects the cellular response to androgens and to identify specific androgen-regulated genes (ARGs) or gene networks that participate in these responses. We used cDNA microarrays representing about 20,000 distinct human genes to profile androgen-responsive transcripts in the LNCaP adenocarcinoma cell line and identified 146 genes with transcript alterations more than 3-fold. Of these, 103 encode proteins with described functional roles, and 43 represent transcripts that have yet to be characterized. Temporal gene expression profiles grouped the ARGs into four distinct cohorts. Five uncharacterized ARGs demonstrated exclusive or high expression levels in the prostate relative to other tissues studied. A search of available DNA sequence upstream of 28 ARGs identified 25 with homology to the androgen response-element consensus-binding motif. These results identify previously uncharacterized and unsuspected genes whose expression levels are directly or indirectly regulated by androgens; further, they provide a comprehensive temporal view of the transcriptional program of human androgen-responsive cells.

Figure 1

Temporal expression profiles of ARGs. (A) Microarrays composed of 3,000 prostate-derived cDNAs were used to acquire serial measurements of androgen-induced transcripts in the LNCaP cell line. RNAs showing at least a 2-fold change in expression after androgen exposure were clustered. Groups of genes with similar patterns of expression are indicated by vertical bars (A–D). Tick marks on the x axis of clusters A–D temporal profiles indicate the same time intervals as depicted at Left. (B) The expanded cohort of characterized genes with a temporal profile of expression corresponding to PSA are shown and named according to the HUGO gene nomenclature (www.gene.ucl.ac.uk/nomenclature/).

Figure 2

Androgen-regulated expression of characterized and previously uncharacterized genes. (A) Northern analysis confirmation of ARG expression in LNCaP prostate cancer cells treated with the synthetic androgen R1881 (+) or vehicle control (−) for 24 h. For each gene, the corresponding microarray-derived fold change in expression is provided below the gene name. (B) Hierarchical cluster analysis of uncharacterized genes exhibiting a temporal expression profile corresponding to PSA. *, genes to the left of the vertical bar represent characterized ARGs with tissue-expression profiles enhanced or restricted to the prostate. (C) Northern analysis confirmation of uncharacterized ARGs in LNCaP cells treated with androgen (+) or vehicle control (−) for 24 h. Multiple tissue Northern blot of selected uncharacterized ARGs demonstrating prostate-restricted or prostate-enhanced expression relative to other normal human tissues. §, one alternative spliced form of Hs.288821 exhibits prostate-enhanced expression.

Figure 3

Identification of ARE motifs. (A) Functional human AREs verified through experimentation are shown with positions relative to the transcriptional start site and the approximate genome location. A CLUSTALW alignment identifies highly conserved residues in black. A consensus sequence generated by WEBLOGO displays the frequency of each base in the consensus proportional to the character height with the height of the entire stack adjusted to signify the information content of the sequences at that position. (B) Putative human AREs identified by searching the 5′ regulatory regions of androgen target genes for a motif corresponding to the Transfac ARE consensus sequence. A CLUSTALW alignment identifies highly conserved residues in black. A consensus sequence indicates the relative frequency and importance of nucleotides in the motif.