Functional Characterization of Two Novel Human Prostate Cancer Metastasis Related Genes

摘要

We propose to identify the functional characterization of two novel cancer-specific, metastasis-related genes whose constitutive expression may be pivotal for prostate cancer progression. Due to hurricane Katrina, we were able to replace our DNA constructs, cell lines, frozen tissue sections, cell lines, research supplies. In addition, we were able to replace our laboratory personnel including a postdoctoral fellow in May of 2007. Since then we were able to re-generate Seq1 and Seq 2 DNA construct using various approaches. We have characterized the full-length cDNAs of the Seq1 and Seq2 genes using at least three 5 and 3 rapid amplification of cDNA ends (RACE) commercial kits (Invitrogen Carlsbad, CA, BD Bioscience (Clontech Inc), and Seegene, Rockville, MD). Specific expression of metastasis related genes were detected by PCR and southern blot analysis in mRNA-based cDNA libraries of androgen independent prostate cancer cells DU-14 and PC-3 cells. We have also subcloned cDNAs in bacterial expression plasmids and were sequence verified for orientation and recombinant protein expression, in vitro translation, and antibody production. Unfortunately, we were not able to obtain gene products as fusion proteins. This may be attributed to small size of our target gene mRNAs or that these genes may not be translatable. Alternatively, posttranslational modification may play a role the expression of the target genes. As such, we are planning to use an eukaryotic expression system to assure proper expression, folding and compartmentalization of our target proteins.