Roles of k-Ras, p53 and Pax Genes in the Origin of Ovarian and Fallopian Tube Cancer

【摘要】： Purpose: The overall aim of this thesis was to develop a novel fallopian tube cancer model and to investigate the roles of the paired homeobox 2 (pax2) and paired homeobox 8 (pax8) genes in ovarian cancer using cell lines derived from the mouse ovarian surface epithelium. Hopefully the study of these two models will allow a better understanding of the processes that contribute to the development of ovarian cancer. Method: 1. Primary oviductal cells were obtained from wild-type p53 mice and p53~(loxP/loxP) mice using a tissue fragment culture method and a novel, revised tissue fragment culture method. Clonal primary oviductal cell lines were derived from the primary oviductal cell cultures using 96-well plates, cloning rings and 2μl sterile pipette tips. Primary oviductal epithelial cells were identified using primary antibodies reactive with epithelial or mesenchymal markers. 2. cDNAs for the pax2 and pax8 genes were cloned from mouse oviductal tissue. The pax2 and pax8 ORF were then subcloned into lentiviral vectors generating the lentiviral vectors: of WPI-pax2, WPI-pax8, WPI-RFP-pax2 and WPI-RFP-pax8. 3. Optimization of the lentiviral vector (LvV) generation using polyethyleneimine (PEI)-based transient transfection. Different titration method of LvV stocks, culture medium, culture duration, cell density and DNA amounts were compared to obtained an optimized procedure for the production of LvV. 4. MOSE cell lines transduced with pax2, pax8 and/or k-Ras genes and mouse oviductal epithelial cell lines (OECLs) transduced with k-Ras were characterized using cells growth assay, growth of transgene cell lines in soft agar, invasion assays, quantitative polymerase chain reaction (Q-PCR), and tumorigenicity assays following intraperitoneal injection of cells into severe combined immunodeficiency (SCID) mice. Results: 1. 22 oviduct clonal cell lines were obtained from the primary oviduct cell cultures using the oviductal tissue fragment culture method. In addition, three oviduct clonal cell lines were obtained from the primary oviduct cell cultures that were generated using the revised oviductal tissue fragment culture method. Three primary oviductal epithelial cell lines were screen out from 24 oviduct clonal cell lines using the expression of E-cadherin and cytokeratin 19 as inclusion criteria for the selection of cells with an epithelial phenotype. 2. Optimization of the production method for lentivirus vectors (LvV) was achieved using polyethyleneimine (PEI)-based transient transfections. OPTI-MEM serum-free medium can be used directly to product LvV and the LvV can be harvested 48 hours after transfection. A cell density of 15×106 cells/10-cm plate and 1×DNA concentration was selected for the production of LvV. Methods for titering LvV carrying fluorescent reporters were investigated and a very simple direct titration method was developed which is faster than standard methods but equally sensitive. 3. After transduction of LvV expressing the pax2 into mouse ovarian surface epithelium (MOSE) cell lines, the growth rate of MOSE-k-Ras/c-Myc (MOSE-RM) and the p53~(△2-10) MOSE cell lines which both lack functional p53, was significantly increased. The expression of pax2 in the MOSE-RM cells not only led to a significant increase in the numbers of colonies formed in soft agar when compared to normal RM cells, vector transduced RM cells or pax8 expressing RM cells but also the size of the colonies formed was also larger than those formed by the other MOSE-RM cells. The expression of c-Myc gene was increased by the pax2 in MOSE-RM cells using Q-PCR assay. In animal experiment, the survival time of the MOSE-RM-WPI-pax2 group was significantly shorter than the MOSE-RM-WPI group (P 0.0001). The pathological features in these two groups were significantly different. The expression of pax8 led to increased proliferation only of the MOSE-RM cell line. In the two non-tumorigenic MOSE cell lines, pax8 led to inhibition of proliferation. The growth rate of the p53~(loxP/loxP) MOSE cells was significantly promoted by the expression of the cre recombinase gene in the presence or absence of k-Ras presumably due to the cre mediated loss of a functional p53 gene. The expression of cre or k-Ras did not increase the proliferation of the p53Δ2-10 MOSE cells. The p53~(loxP/loxP) MOSE cell or p53Δ2-10 MOSE cells could be transformed to malignant cells by the expression of the k-Ras and acquired the ability to invade through basement membranes following expression of the k-Ras gene. 4. After transduction of the k-Ras genes to OECLs, the growth rate of the OECL (4)-Kras-IRES-eGFP-Cre and OECL (22)-Kras-IRES-eGFP-Cre was significantly higher than the OECL (4) and OECL (22) which carry a wild-type p53 gene. The growth rate was not different between the OECL (B)-Kras-IRES-eGFP-Cre and OECL (B) which lack the function of p53. These transgenic OECs could be transformed to malignant cells by the expression of the k-Ras and acquired the ability to invade through basement membranes following expression of the k-Ras gene. Conclusion: 1. A new, simple revised tissue fragment culture method for collecting oviductal epithelial cells was established. Three OECLs from wild-type p53 mice and p53~(flox/flox) mice were developed and characterized. To our knowledge, this was the first reported use of a method such as our revised tissue fragment culture method to establish, immortalize and characterize oviductal epithelial cells from transgenic mice. A new oviductal cancer model involving the k-Ras, p53, pax2 and pax8 genes was initially established. 2. The development of a scalable process for production of LvV by PEI-mediated transfection was established and optimized. The PEI-based method was easy to use, more reliable with a high degree of reproducibility and consistency. Despite using less DNA, the PEI transfection method led to viral titers that were the same as those achieved using the CaPO4-based method. 3. To our knowledge, this is the first time reported that the pax2 gene could promote proliferation of a tumorigenic cell line-MOSE-RM possibly by increasing expression of c-Myc. Pax2 gene not only play an important role in the origin and development of ovarian cancer, but also acted as a prognostic factor in our model of ovarian cancer. The Pax8 gene plays parallel roles in the development of epithelial ovarian cancer and may be dependent on the expression of other oncogenes.