Validation and Localization of Restricted Gene Expression in the Developing Prostate -- with Emphasis on PAX Gene Expression Profiling and Functional Studies During Murine Prostate Development

Download or read book Validation and Localization of Restricted Gene Expression in the Developing Prostate -- with Emphasis on PAX Gene Expression Profiling and Functional Studies During Murine Prostate Development written by Qian Chen and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Prostate cancer (PCa), being the most commonly diagnosed cancer in American men and the second leading cause of cancer deaths, drew great attention from scientists since last decade. As developmental processes share several features in common with metastatic cancer, I hypothesized that a better understanding of genes involved in prostate morphogenesis may identify molecules that could be useful for better diagnosis and therapy for prostate cancer. The prostate is derived from the urogenital sinus (UGS), which can be separated into four subdomains based on cellular compartment and dorsal-ventral patterning. Essentially these are the epithelium/mesenchymal (UGE/UGM) compartments and the dorsal/ventral halves (UGD/UGV). I believe that the localization of a gene's expression should be related to its particular function in prostate organogenesis, and will help us to better understand the mechanism of PCa development and metastasis. In the first part of this research, cDNA microarray (MA) analysis was performed to identify differentially expressed genes associated with each of the UGS sub-compartments. Results identified 530 genes (3.5%) in UGE/UGM, and 35 (0.23%) in UGD/UGV that exhibit spatially restricted expression. To validate the MA results, 22 genes were chosen based on the magnitude of the expression difference, their potential function and whether they are expressed differentially in both comparisons of UGS/UGM or UGD/UGV. Quantitative real time reverse transcription polymerase chain reaction (Q-RT-PCR or Q-PCR) was performed to observe relative mRNA levels and more than 70% of the genes showed results consistent with MA analysis. Immunofluorescence (IF) then was used to localize five of those genes confirmed in the predicted subdomains by Q-PCR. PAX2 was one of five genes that showed dorsal-epithelial (DE) restricted expression in the UGS, and was studied further. The paired-box (PAX) gene family encodes a group of 9 transcription factors that regulate organogenesis both temporally and spatially. Results of earlier studies showed that PAX2 null male mice fail to develop a mature urogenital system properly. In addition, it was reported recently that PAX2 is overexpressed in the androgen independent human prostate cancer cell line, PC3. These studies indicated that PAX2 is implicated in both prostate organogenesis and prostate cancer. Despite this evidence, the pattern of PAX2 expression during prostate development is unknown. To discover PAX2's temporal expression pattern, UGS or prostate tissue samples were collected at 10 different developmental stages from C3H male mice. Q-PCR was performed to determine PAX2 mRNA levels in each sample group. PAX2 mRNA levels are higher in early developmental stage prostate tissue (urogenital sinus, through budding up to early puberty - about P14) as compared to its levels in post-pubertal (after P35) prostate tissue. Both RT-PCR and Western blotting were employed to detect the spatial expression pattern of PAX2 using samples of each UGS/prostate sub-compartment. Cytoplasmic proteins of prostate tissue were separated from nuclear proteins, followed by Western blotting, to verify the subcellular localization of the PAX2 protein. The results showed that PAX2 is a DE restricted and nuclear localized protein in developing prostate. The dorsal UGS derived prostate gland consists of the anterior lobes (AP) and dorsal-lateral lobes (DLP). Androgen levels were manipulated in vivo by castration, through a 21 day time course, in adult C3H male mice and in a human prostate cancer cell line, LNCaP, in vitro using serum starvation and androgen replacement (1 nM R1881, 48 hr timed course) to study the regulation of PAX2 expression by androgens. The Q-PCR and WB results showed that PAX2 expression levels in the adult mouse prostate are low and are induced by castration in a time-dependent manner. Also, androgen treated LNCaP cells in vitro display decreased PAX2 expression as compared to controls. Both studies indicated a similar regulatory pattern, which is, PAX2 mRNA expression is regulated negatively by androgen. The glandular morphology of PAX2 knockout mouse prostates were compared to those of wild type during a developmental series. PAX2 KO mice do not develop normal prostates and exhibit severe defects in both patterning of the prostate rudiment and elongation of glandular buds. Heterozygous mice had delayed prostate development. RT-PCR compared the expression of genes related to cancer development (WT1), developmental patterning (Wnt family members) and prostate organogenesis (shh and NKX3.1) and found their levels were reduced greatly. These results support a critical role for PAX2 in prostate development, and strongly suggests that PAX2 is upstream and required for expression of WT1, shh and NKX3.1. Thus, PAX2 is a candidate for mediating androgen signaling between the stromal and epithelial compartments during glandular bud formation.