Prostatic gene expression: probasin, human prostatic acid phosphatase and macrophage inhibitory cytokine-1 as model genes
1University of Oulu, Faculty of Medicine, Research Center for Molecular Endocrinology
2University of Oulu, Faculty of Medicine, WHO Collaborating Centre for Research on Reproductive Health
3University of Oulu, Biocenter Oulu
|Online Access:||PDF Full Text (PDF, 1 MB)|
|Persistent link:|| http://urn.fi/urn:isbn:9514272730
|Publish Date:|| 2004-02-16
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium 9 of Oulu University Hospital, on February 16th, 2004, at 12 noon.
Professor Teuvo Tammela
Professor Tapio Visakorpi
Gene products that are only expressed in one tissue or cell type are useful models for investigating the biochemical and molecular mechanisms of tissue/cell-specific gene regulation. The regulatory regions of such genes are also practical tools in gene therapy.
In this work, prostate-specific and androgen-dependent gene regulation was investigated by using human prostatic acid phosphatase (hPAP) and rat probasin (rPB) as models. In DNase I footprinting, a protected 12 bp region was found in the PB gene between the nucleotides -251 and -240 only with nuclear extracts of prostatic origin. The sequence of this area was GAAAATATGATA. Weak interaction could be detected between the DNA-binding domain of AR and the prostatic transcription factor. The results also suggested that the prostatic regulatory protein makes AR binding to its response element more effective and concomitantly magnifies the effect of androgen.
A hPAP construct containing the sequence between the nucleotides -734 and +467 in front of the CAT reporter gene was highly expressed in the prostate of transgenic mice. Five homologues (A-E) for our previously identified prostate-specific GAAAATATGATA DNA-binding site were found in the area where the sites C and E could bind the regulatory protein in EMSA.
The prostatic transcription factor complex bound to the GAAAATATGATA site was purified and characterized from a suspension-adapted mass culture of PC-3 prostate cancer cells by using sequence-specific DNA affinity chromatography, mass spectrometry and supershifts. Several potential transcription factors were identified, but only USF2 was confirmed to be part of the transcription factor complex.
Two PC-3 cell line variants (anchorage-dependent and suspension-adapted, anchorage-independent variants) were used as a model for advanced, androgen-independent prostate cancer. Genes that were overexpressed in a suspension-adapted PC-3 cell line were further investigated, since they can be considered as putative markers of metastatic activity. The macrophage inhibitory cytokine-1 (MIC-1) gene, which was overexpressed in the suspension-adapted PC-3 cell line, was further investigated in order to clarify the mechanism behind aggressive cell growth and androgen-independent gene regulation. In patient specimens, MIC-1 had no or low expression in benign prostatic hyperplasia and normal prostate but high in prostatic cancer and therefore it could be a useful marker for aggressive prostate cancer. Indomethacin increased the expression of MIC-1 in PC-3 cells, and apoptosis was also induced in this cell line but not in saPC-3 cell line suggesting a block in MIC-1 inducible apoptosis pathway.
Acta Universitatis Ouluensis. D, Medica
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