Molecular strategies for selective inhibition of androgen receptor gene expression by oligonucleotides

Androgen receptor (AR) is a ligand-activated transcription factor, belonging to the steroid/thyroid hormone receptor superfamily. Androgen receptor plays a critical role in the male-specific sexual phenotype and in the development of the male reproductive organs. It is also involved in the etiology of diseases such as prostate carcinoma, benign prostatic hyperplasia, androgen insensitivity syndrome and Kennedy's disease. I have explored the possibility for regulation of AR gene expression at the level of both promoter function and mRNA inactivation; the former approach involved initial characterization of the AR promoter. In a first series of experiments, I found that the proximal 5{dollar}spprime{dollar} flanking promoter region of the AR gene contains a long purine-pyrimidine-rich region in human, rat and mouse. Mutations within this region result in a 3-4 fold decrease in promoter activity in human HepG2 and Hela cells, respectively, indicating that it functions as an important regulatory element. Study of the conformational state of the region with S1 nuclease, a single-strand specific nuclease, shows that it is able to form a non-B-DNA conformation.; In a second approach, I engineered two hammerhead ribozymes, H1 and H2, to specifically target AR mRNA without affecting expression of other genes. The HR2 hammerhead ribozyme is more active than the H1 hammerhead ribozyme. A hammerhead ribozyme with mutations in the catalytic domain (mut-HR2) and antisense oligodeoxynucleotide (antisense HR2 oligo) to the target AR mRNA fail to catalyze the cleavage of the target AR mRNAs in vitro. In vivo, the hammerhead ribozymes, H1, HR2, mut-HR2, and antisense HR2 oligo, were cloned into a mammalian expression vector under the control of an RNA pol II promoter (pCMV). The full length AR cDNA was cloned into a mammalian expression vector as a target vector (pCMV-AR). The mouse mammary tumor virus long terminal repeat promoter containing the AR response element was ligated with the chloramphenical acetyl transferase gene as a reporter vector (pMMTV-CAT). The results show that the hammerhead ribozymes can inhibit CAT activity, and decrease AR mRNA as well as AR protein, but do not affect {dollar}beta{dollar}-actin mRNA. On the other hand, the mut-HR2 and antisense HR2 also decrease AR gene expression, but are less effective compared to wild type hammerhead ribozyme. This suggests that the hammerhead ribozyme has an enzymatic property beyond an antisense effect and also shows that the decrease in AR expression occurs in a dose-dependent manner.; In summary, these results showed that AR gene expression can be regulated at both transcription and posttranscription levels. These studies also show that the AR gene promoter contains the purine-pyrimidine rich region which acts as an enhancer element and is capable of forming triplex DNA. Although mutations of the region result in a decline of AR gene transcription, triplex-forming oligonucleotides directed to this region does not affect promoter function. However, I demonstrated that hammerhead ribozymes are able to catalyze the AR mRNA at specific sites in vitro and in vivo. This provides a potential tool to inactivate gene expression. (Abstract shortened by UMI.)...