CONDITIONAL IMMORTALIZATION OF HUMAN ENDOMETRIAL STROMAL CELLS WITH ATEMPERATURE-SENSITIVE SIMIAN VIRUS-40

The study of immortalization and other alterations associated with neo plastic transformation of endometrial stromal cells is important to un derstanding the development of uterine sarcomas and mixed tumors. Beca use stromal cells are important regulators of associated epithelial ce lls, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of e ndometrial carcinomas. To study immortalization and associated phenoty pic and genetic alterations of human endometrial stromal cells, cultur es were transfected with a plasmid containing an ori-, temperature-sen sitive mutant SV40, A209 (tsSV40). Morphologically transformed colonie s were selected and propagated at the permissive temperature until the y entered 'crisis'. In contrast to human fibroblasts, every clone test ed was immortalization competent. The frequency of immortalization was approximately 1 X 10(-6). One uncloned and six cloned cell lines esca ped from crisis and appear to be immortal. Two clones, M4 and B10T1, w ere selected for further study. Immortalization is conditional; prolif erative arrest occurs at the restrictive temperature for large T antig en function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. C olony-forming efficiency at the restrictive temperature was undetectab le. Immortalization is also associated with several genetic alteration s. The DNA content of tsSV40 transfected cells was either diploid or t etraploid in the precrisis stage of proliferation, but became aneuploi d upon immortalization. Several structural rearrangements of chromosom es were detected in the immortalized stromal cells which differ from t hose found in SV40 immortalized fibroblasts. Although their capacity f or anchorage-independent proliferation (AIP) is variable, tsSV40-immor talized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferati on prior to 'crisis'.