| Summary: | 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.
|
|---|
