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Immortalization of human ovarian surface epithelium with a temperature sensitive immortalization agent

University of British Columbia

Although ovarian cancer is the leading cause of death from gynecological malignancies among North American women, the etiology and early developments in the progression of this disease are still among the least understood and characterized of all human malignancies. Understanding early events in the development of ovarian carcinogenesis is important. Over 90% of ovarian carcinomas are thought to arise from the ovarian surface epithelium (OSE) (Young et al., 1989). Previous attempts to study hOSE were hampered by lack of specimen availability and the limited lifespan of OSE in vitro. Immortalization with wild type SV40 large T antigen (Tag) caused certain characteristics of hOSE to be lost: For example, CA125 is lost when hOSE are immortalized with wild type Tag. In the present study, immortalization of hOSE with a temperature sensitive Tag protein was attempted as a possible solution to the loss of these phenotypic characteristics. Four cases of hOSE were infected with a tsTag A 209 construct, and 28 monoclonal and 4 polyclonal lines were isolated. Two monoclonal and two polyclonal lines were observed for morphology and characterized by proliferation assays, indicators of immortalization (Tag and p53), differentiation markers (keratin, collagen and CA125) and senescence (SA j3-galactosidase) at the permissive (34°C) and nonpermissive (39°C) temperatures. All clones were composed of small, compact epithelial cells at the permissive temperature, and the cells became larger and flatter at the nonpermissive temperature. tsIOSE clones underwent 52-71 population doublings (pd) post infection at the permissive temperature and 2 -4 pd post infection at the nonpermissive temperature. Two of four cases increased expression of keratin and collagen when shifting from the permissive to the nonpermissive temperature of tsTag. The other two keratin and collagen values remained consistent through the temperature shifts. All cases expressed the epithelial marker at the nonpermissive temperature, as well as mamtaining a low level of CA125 that did not modulate with temperature in any case. No clones expressed SA P-galactosidase at the permissive temperature and all the clones expressed SA P-galactosidase at the nonpermissive temperature. The immortalization, differentiation, and senescence characteristics changed asynchronously after a temperature shift from the permissive to the nonpermissive temperature. Tag was lost by 24h, yet it took p53 at least 2 weeks to return to baseline levels. Senescence associated P-galactosidase began to return in 6 h, and was completely positive after 24h whereas senescence associated morphology did not return until after 24h. Keratin and collagen HI levels that were lost or affected by tsTag began to change after 24h. CA125 levels did not seem to be affected by temperature shifts. One of four cases survived crisis and continued to replicate up to 150 population doublings past the expected lifespan of hOSE. This post crisis line maintained keratin at both temperatures despite the fact that its parent line was one that modulated keratin levels according to temperature shifts. This line has not formed subcutaneous or intraperitoneal tumors after four months in scid mice. Immortalization of hOSE with tsTag is superior to immortalization with wild type Tag because the system is capable of producing a large number of cells that can be phenotypically similar to hOSE. tsIOSE with inactivated Tag maintain the differentiated characteristics of hOSE better than cells immortalized with wt Tag, and thus are more like normal hOSE than IOSE. These cells are a potential source of cells on which large scale studies can be performed. hOSE was induced to form a continuous line, which is now available for further study.

Thesis/Dissertation

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2009-03-12

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http://hdl.handle.net/2429/5956

Reproductive and Developmental Sciences

University of British Columbia

UBCV

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