References

Notes: [Summary] = Primary culture and immortalization of mouse ovarian survace epithelial cells.= The incidence of ovarian malignancies has significantly increased in the past decades in many countries, however, an appropriate animal model system enabling the study of ovarian cancer such as stable mouse ovarian epithelial cell lines has not yet been developed. Here we report the establishment of cell lines derived from mouse ovarian surface epithelium (MOSE) by two procedures - one, through the introduction of SV40 large T antigen dnA into C3H/He MOSE (T-Ag-MOSE) and another through spontaneous immortalization of cells from p53-deficient MOSE (p53-def-MOSE). p53-def-MOSE cell line did not show any transformed phenotype either in vitro culture system nor in vivo tumorigenicity assay. Tumors formed by the injection of T-Ag-MOSE were undifferentiated malignancies associated with heterologous mesothelial tissues such as those of the osteoid phenotype. The established MOSE cell lines are usefull in the molecular analysis of the multistep carcinogenesis of ovarian tissues in humans. [Materials and Methods] Ovaries were removed from adult female C3H/He mice (8 weeks of age) under septic conditions, and transferred to a culture dish containing the high-glucose version of Dulbecco's modified Eagle Medium (GIBCO laboratories) supplemented with 10% fetal bovine serum (FBS), (penicillin 100U/ml) and streptomycin (100ug/ml) (complete growth medium). After fallopian tubes and connective tissue surrounding the ovary were carefully removed, ovaries were washed three times by phosphate-buffered saline (PBS) and treated as a whole with 0.025% trypsin for 60 minutes. Then the ovaries were removed and the above mentioned complete growth medium was added to the reaction solution to dilute the trypsin. The cell suspension was then placed in tissue culture dish (LUX) and cultured under conditions of 5% CO2 in 37 C humidified air. The cultured cells did not trow well after several passages, and to obtain immortalized cells, the primary culture cells in the dish were transfected with elongation factor promoter driven SV40 large T antigen DNA containing plasmid pEF321-T by the polybrene DMSO method. Colonies of the OSE showing cobblestone appearance were isolated using cloning cylinders. Then the epithelial cell suspension was diluted and placed in 96-well cell culture plates. The wells containing only one cell were marked and a monoclonal cell line (T-Ag-MOSE) were obtained from these wells. T-Ag-MOSE) were obtained from these wells. T-Ag-MOSE cells have been passaged up to 50 times (1:10 splits).
Reference ID: 4980

Notes: Normal somatic cells are endowed with limited doubling potential in culture, and the process of immortalization is an inevitable step in neoplastic transformation of the cells. To examine the roles of p53 in this process, the cells of p53-deficient mice were examined for doubling potential. Fibroblast-like cells from a variety of tissues of these mice proliferated continuously without showing aging or crisis. The aneuploid cells overcome the population with passage, but cloning experiment indicated that chromosomal changes were not essential to this process. The enhanced proliferative potential in culture of cells from the p53-deficient mice was also observed in epithelial cells of lens, mammary glands and seminal vesicles and in neural precursor cells. Proliferation of bone marrow cells in response to stem cell factor was enhanced in long term culture, but not in in vitro colony assay; no permanent cell lines could be obtained. No effects of p53-deficiency were found in proliferation of cardiac muscle cells or hepatocytes.
Reference ID: 4981