|M.Sc Student||Chosha Tina|
|Subject||Molecular Factors that Inhibit Human Prostate Cancer|
|Department||Department of Medicine||Supervisor||Assistant Professor Fuad Fares|
|Full Thesis text - in Hebrew|
Background: NDRG1 is a member of the N-myc down-regulated gene family, which belongs to the α/β hydrolase superfamily. This gene was previously identified as an up- regulator of cellular differentiation, and was found to be down regulated during colon, breast and prostate tumor progression. NDRG1 is regulated by several factors including androgen, p53 and N-myc. The biological function of NDRG1 and the physiological relevance of its role in the cellular context remain elusive. To clarify the functional role of NDRG1 in prostate cancer cells, we over-expressed the NDRG1 in three prostate cancer cell lines with different differentiation levels.
Methods: Normal trophoblast cells were used in order to clone the NDRG1 cDNA. The coding sequence of NDRG1 was amplified by PCR and cloned into the eukaryotic expression vector, PCDNA 3.1. The plasmid was transfected into human prostate cancer cell lines; LNCaP (well differentiated), DU145 (moderately differentiated) and PC3 (poorly differentiated). Expression of NDRG1 was detected in the transcription and translation levels. The differentiation factors p21 and cytokeratin 8/18 were detected by western blotting.
Results: All the three cell lines expressed NDRG1. High levels were detected in poorly and moderately differentiated whereas low levels were found in the well differentiated cell line.
The cDNAs from all cell lines were used as templates for amplification of the NDRG1 by PCR. Sequencing results of NDRG1 showed that there are no mutations in the coding sequence of the gene.
To investigate the role of NDRG1 in the prostate cancer cell lines, the cells were transfected with molk vector and with NDRG1 expression plasmid. The expression of NDRG1 in the cloned cells was tested by RT-PCR and further confirmed by Western blot.
The NDRG1 transfected cells were examined for the rate of proliferation in comparison to molk transfections. The results revealed that there were no significant differences from the control cells. However, it was found that over-expression of NDRG1 up-regulated p21 and c8/18, therefore causing or progressing differentiation in cancer cell lines.