| [Background & Objectives]Ovarian cancer is the most lethal of all gynecological cancers and arises mostcommonly from the surface epithelium. Successful clinical management ofpatients with epithelial ovarian cancer is limited by the lack of a reliable andspecific method for early detection, and the frequent recurrence of chemoresistantdisease. Experimental models are of crucial importance not only to understandthe biological and genetic factors that influence the phenotypic characteristics ofthe disease but also to utilize as a basis for developing rational interventionstrategies. Ovarian cancer cell lines derived from ascites or primary ovariantumors have been used extensively and can be very effective for studying theprocesses controlling growth regulation and chemosensitivity or evaluating noveltherapeutics, both in vitro and in xenograft models.The tumorigenesis always to be recognized as multiple steps and multipleprocesses, including some oncogenes inactivate and some tumor suppressor geneslost their function. k-Ras and c-Myc are two oncogenes frequently detected inepithelial ovarian cancer, in order to determine their roles in tumorigenesis ofovarian cancer, we introduced k-Ras or/and c-Myc to the mouse ovarian surfaceepithelium cells(MOSE) by retroviral gene delivery system, study the phenotypiccharacteristic of the transgenic MOSE and tumorigenesis in vivo.OPCML (Opioid-binding protein/cell adhesion molecule-like ), and also calledOBCAM (Opioid-binding protein/ cell adhesion molecule) is located at 11q25,LOH(loss-of-heterozygosity) frequently can be found in this region. Therelationship between OPCML and tumor have been known very limited, it maybea new tumor suppressor gene. In order to understand the function of OPCML, wecloned OPCML gene, then deliver the OPCML to ovarian cancer cell lines bylentiviral vector, study the function of OPCML in vitro and in vivo, and also checkthe reliability of lentiviral vector transgene system.[Materials and Methods]we collected and cultured the MOSE cell from the CD1 mice(expressing TsAg),subcloned the mutant k-Ras and normal mouse c-Myc to Moloney retroviralvector pLPCX and pLHCX, constructed the retroviral plasmid pLPC-mMyc andpLHC-kRas, producted recombinant retroviruses carring the c-Myc or k-Ras bytransfected the pLPC-mMyc or pLHC-kRas to ecotropic Phoenix cells. Wedelivered the oncogenes to the MOSE cells by the recombinant retroviruses, andconstructed the cell lines of Ras (MOSE-Ras), Myc (MOSE-Myc) and RM(MOSE-kRas/cMyc) that expressed the Ras, Myc or both Ras and Myc protein.We studied the phenotypic characteristics of transgenic MOSE by cellproliferation assay, soft-agarose cloning formation, Matrigel invasion assay andxenograft in nude mice.We cloned the normal OPCML gene from CD1 mouse according to the sequencein GenBank. At first, we subcloned OPCML to a commercial vector pcDNA3(pcDNA3 -OPCML), then sequence and transfected to 293T to check if it canexpressed OPCML protein; at last , subcloned the OPCML to a lentiviral vectorpWPI-GFP, that have GFP marker, constructed the lentivirus expressional plasmidpWPI-OPCML. Produced recombinant lentiviruses by co-transfectingpWPI-OPCML+pCMV-dR874 +pMDG to 293T, then infected ovarian cancer celllines (A2780, OCC1, RM et al) by recombinant lentiviruses. Observed thephenotypic characteristics by cell proliferation assay, cell Aggregation assay andxenograft assay in vivo.[Results](1)After infected MOSE by the recombinant viruses, mRNA of k-Ras and/or c-Myc can be detected by RT-PCR in Ras, Myc and RM cell lines, their proteincan be checked by Western blot, k-Ras protein was 21kDa, c-Myc protein was 62kDa; and also have the same expression after many generations culture;(2)Cell proliferation assay showed that Ras, and RM cell lines growed fast comparing parent cells (MOSE) and Myc cells(P<0.01); RM growed fast compared to Ras (P<0.05)(3)Cell cloning formation assay showed that Ras and RM cells can form colonies in soft-agarose culture, but not in Myc cells and the control(MOSE); the number of colonies w...
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