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Departments of Obstetrics and Gynecology (D.M.N., S.A.H., J.J.P.) and Anatomy (B.A.W.), University of Connecticut Health Center, Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: John J. Peluso, Ph.D., Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: peluso{at}nso2.uchc.edu
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Abstract |
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Introduction |
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To more completely elucidate the mechanisms associated with the oncogenic transformation of ovarian surface epithelial cells, Hoffman and associates have developed several different rat ovarian surface epithelial (ROSE) cell lines (7). Collectively, these cell lines seem to represent different stages of oncogenesis. For example, early-passage (EP)-ROSE-179 cells are similar to normal ROSE cells (7). Late passage (LP; >20 passages) ROSE-179 cells show an increase in their rate of proliferation, indicating that they have a reduced ability to negatively regulate cell cycle traverse (7). ROSE cells, transformed with sv-40, have even a shorter cell doubling time and are metastatic. Finally, ROSE cells transformed with both sv-40 and ras (T-ras ROSE cells) have the shortest cell doubling time, are metastatic, and form tumors when transplanted into host animals (7). This progression is thought to be caused by a progressive increase in endogenous ras expression and activity (7), which is a common alteration observed in human ovarian cancers (8, 9, 10).
Though ras activity facilitates entry into the cell cycle by increasing in the expression of the G1 regulatory protein, cyclin D1 (11), the loss of cell cycle control may also be caused by an inability to express cell cycle inhibitors. In ovarian granulosa cells, steroidogenic factor-1 (SF-1) induces the differentiated state (12, 13) and suppresses mitosis (14). Because the ovarian surface epithelial cells and granulosa cells share a common cellular lineage (15), it is possible that ovarian surface epithelial cells express SF-1 and that SF-1 plays an important role in regulating mitosis of both primary and transformed ovarian surface epithelial cells. The present studies were designed to test this hypothesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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60 days of age) and immature female Wistar rats (22
days of age) were obtained from Charles River Laboratory (Wilmington,
MA) and housed under controlled conditions of temperature, humidity,
and photoperiod (12 h light, 12 h dark; lights on at 07:00 h). On
the day of the experiment, immature animals were 23–25 days of age. In
some experiments, immature animals (23 days of age) were injected ip
with 20 IU of eCG and autopsied 48 h later. All rats were
cervically dislocated between 0930 and 1000 h. The ovaries were
removed and prepared for in situ hybridization. In total,
ovaries from six rats were analyzed for SF-1 expression (three mature;
one immature, and two eCG-primed immature rats). This protocol was
approved by the Animal Care Committee of the University of Connecticut
Health Center.
ROSE cell lines
The ROSE cell lines that were used in these studies
(EP-ROSE-179, LP-ROSE-179, T-sv-40 ROSE, and T-ras ROSE) were provided
by Dr. Robert Burghardt (Texas A & M University, College Station, TX).
These cells were cultured as previously described (7). Briefly, all the
ROSE cell lines were cultured in DMEM/F-12 medium (Sigma Chemical
Co., St. Louis, MO) that was supplemented with 5% FBS (7). The
cultures were maintained in a 5% CO2-95% air
atmosphere at 37 C. For cell proliferation studies, ROSE cell lines
were plated in 35-mm culture dishes at 5 x 104
cells/ml. Cells that were used for cell proliferation and/or in
situ localization of SF-1 messenger RNA (mRNA) were plated in
8-chamber glass Lab-Tek slides (Nunc Inc., Naperville, IL) at 6 x
104 cells/400 µl. Cells were also plated in
25-cm2 tissue culture flasks (Becton Dickinson and
Co. Labware, Franklin Lakes, NJ) at 4 x 105
cells/ml, transfected and/or assessed for stage of cell cycle/apoptosis
by fluorescence-activated cell sorting (FACS). Studies involving cell
sorting and subsequent RT-PCR analysis were plated in
75-cm2 tissue culture flasks at a density of 4 x
105 cells/ml.
Detection of SF-1 by Northern blot hybridization
RNA was isolated from ROSE cell lines by the RNAzol method
(Biotecx Laboratories Inc., Houston, TX), following manufacturer’s
instructions (16). The relative level of SF-1 mRNA was assessed by
Northern blot hybridization using 1% agarose/formaldehyde gels, as
described (16). The SF-1 complementary DNA (cDNA) clones have been
described (17). The SF-1 probe was labeled with
32P-deoxycycidine triphosphate using a Rad-Prime random
primer kit (Life Technologies, Gaithersburg, MD).
Localization of SF-1 mRNA by in situ hybridization
Rat ovaries were fixed in 10% formalin, embedded in paraffin,
and sectioned at 10 µm. The sections were passed through a sequential
series of washes of histoclear, ethanol, and water to remove the
paraffin. The tissue sections were incubated with proteinase K (20
µg/ml) for 10 min at room temperature, washed in PBS three times for
5 min each, and fixed in 4% formaldehyde for 15 min at room
temperature. The cells from the four ROSE cell lines were plated in
duplicate wells in each of two 8-chamber Lab-Tek slides. One of these
slides was probed with antisense SF-1 and the other with the nonsense
probe. In this way, the hybridization conditions for all the cell lines
were identical. The ROSE cell lines were fixed with 10% formalin in
PBS for 5 min. Formalin was removed with two rinses of PBS. In
situ hybridization was carried out using 5' digoxigenin-labeled
oligonucleotides and analyzed using IP Lab Spectrum software (Signal
Analytics, Vienna, VA), as previously described (14).
To make semiquantitative measurements of SF-1 mRNA levels using in situ hybridization, it was necessary to determine that the amount of staining is proportional to the amount of mRNA detected. This was confirmed in a previous study in which R2C cells were stained for SF-1 mRNA using 0, 1, or 5 ng digoxigenin-labeled SF-1 oligonucleotide in 50 µl of buffer. At the lower concentrations of digoxigenin-labeled SF-1 probe, the amount of probe is limiting. Because the relative amount of SF-1 mRNA within each R2C cell is relatively constant, the amount of staining observed per cell at each concentration of digoxigenin-labeled SF-1 oligonucleotide is a direct reflection of the amount of mRNA detected. This study demonstrated that intensity of staining increased linearly with the amount of detected SF-1 mRNA (r2 = 0.9) (14). Therefore, these data indicate that semiquantitative measurements of SF-1 mRNA levels can be made using in situ hybridization.
The high reproducibility was assured, in part, by setting optical conditions to very precise standards. This was done by adjusting the optics and light levels so that a standard object gave the same OD reading before any cells were imaged.
Identification and isolation of quiescent, mitotically-active, and
apoptotic EP-ROSE-179 cells by FACS
Cells were fixed and stained for flow cytometry, as previously
described (18). Briefly, 5–10 x 106 cells were
harvested, washed in PBS, and fixed in 70% ethanol for 24 h. The
samples were stained with 50 µg/ml propidium iodide (PI) and 100 U/ml
ribonuclease 30 min before FACS analysis. The cells that were
sorted, and subsequently analyzed for SF-1 and hypoxanthine
phosphoribosyl transferase (HPRT) mRNAs, were not treated with
ribonuclease A (19).
Fluorescence from PI-stained cells was measured on either a FACScan flow cytometer (Becton Dickinson and Co.) using 488-nm laser excitation and a 585/42 BP filter in the FL-2 channel, or a FACSTAR Plus (Becton Dickinson and Co.) using a similar laser excitation with a 575/26 BP filter in the FL-2 channel. The FACSTAR Plus was used for the cell sorting experiments. At least 10,000 cells were collected in the G0/G1 phase of the cell cycle for each sample. The FL-2 Photo Multiplier Tube and amplifier gain were adjusted so that the G0/G1 peak fell approximately in the 400th channel, on a linear scale, to spread out subdiploid events and to electronically threshold out cellular debris and background artifacts below channel 40. The percentage of cells in different cell cycle phases was analyzed using CellQuest software (Becton Dickinson and Co.).
Detection of SF-1 mRNA by RT-PCR
RNA was isolated from FACS-sorted EP-ROSE-179 cells using the
Ultraspec RNA Isolation System (Biotecx Laboratories Inc.). The purity
and amount of RNA was assessed by measuring the
OD260/OD280 ratios. RT was performed using 50
ng of random primers (Gibco BRL, Gaithersburg, MD) per
0.88 µg RNA in a total vol of 12 µl. The mixture was incubated at
70 C for 10 min and then quickly chilled on ice. Seven microliters of a
mastermix containing 2 µl PCR buffer (10 x), 1 µl
MgCl2 (50 mM), 1 µl deoxynucleotide
triphosphate mix (10 mM each), 2 µl dithiothreitol (0.1
M), and 1 µl diethypyrocarbonate-treated
H2O was added and incubated for 25 min at 25 C. Two hundred
units of reverse transcriptase (Gibco BRL) per reaction
were added and the reaction was incubated at 25 C for 10 min and then
at 42 C for 50 min. The reverse transcriptase was inactivated at 70 C
for 15 min and the solution cooled on ice for 2 min.
Five µl of the RT reaction was used for PCR amplification. PCR was performed in a vol of 100 µl containing 1.5 mM MgCl2 and 2.5 U Taq polymerase (Sigma Chemical Co.). Oligonucleotide primers were designed to anneal at different exons to distinguish template source, cDNA, and contamination with chromosomal DNA, respectively. The following primers for SF-1 were chosen, using the published rat cDNA sequence (20), spanning a length of 427 bp: antisense: 5'-GGTTGTTGCGGGGCATCTCGTTGC-3', sense: 5'-GACCACATCTACCGCCAGGTCCAG-3'. PCR was performed for 30 cycles, consisting of denaturing at 94 C for 1 min, annealing at 60 C for 1 min, and elongation at 72 C for 1 min. An initial denaturation step for 3 min and a final elongation step for 10 min were added to this protocol. In parallel, RT-PCR of HPRT transcripts was performed using the same amount of cDNA from each sample as a template. The following primers were designed according to a previous report (21), with the exception of a 1-bp exchange for rat HPRT mRNA: antisense 5'-GTCAAGGGCATATCCAACAACAAAC-3' and sense 5'-CCTGCTGGATTACATTAAAGCGCTG-3'. The PCR reaction resulted in a 352-bp amplification product. An aliquot of the PCR was separated on a 1.5% agarose gel containing ethidium bromide (0.5 µg/ml) and visualized under UV light. To estimate the amount of RT-PCR reaction product, the gel was photographed (scanned into a computer). The RT-PCR reaction product was analyzed using IPLab Gel software (Signal Analytics).
Transient transfection of SF-1
Before transfection, the cells were cultured for 24 h in
DMEM/F-12 medium that was supplemented with 5% FBS. The transfections
were performed with SuperFect Transfection Reagent (Qiagen, Santa
Clare, CA), according to the manufacturer’s instructions. The SF-1
expression vector was a plasmid containing SF-1 DNA in the sense
orientation, CMV-SF-1 (+), whereas the control vector was a plasmid
containing SF-1 DNA in the antisense orientation, CMV-SF-1 (-). Both
constructs were generously provided by Dr. Keith Parker (Duke
University, Durham, NC). Regardless of the vector, the DNA was diluted
to a final concentration of 2.5 ng/µl. Five microliters of SuperFect
Transfection Reagent/µg of DNA was incubated for 10 min at room
temperature in antibiotic- and serum-free DMEM/F-12. Then, the
DNA-SuperFect mixture was added to serum-supplemented DMEM/F-12. This
mixture was incubated for 2 h in a 5% CO2-95% air
atmosphere at 37 C. The ROSE cells were cultured in this DNA-SuperFect
medium for 2 h. The DNA-SuperFect medium was then changed to
serum-supplemented DMEM/F-12, and the cultures continued for an
additional 24 h.
Assessment of ROSE cell proliferation
After 24 h of culture, the number of ROSE cells was counted
within 8 different 160-µm2 grids within either the 35-mm
dish or lab-tek well (22, 23). After an additional 24 h of
culture, the number of cells present in these same areas was
determined. Cell proliferation was expressed as the fold increase in
cell number over 24-h control values.
Assessment of mitosis or apoptosis of individual transfected
cells
To confirm the studies on populations of transfected cells,
LP-ROSE-179 cells were plated in 8-chamber lab-tek slides and then
cotransfected with a green fluorescent protein (GFP) expression vector
(24). In this study, the GFP expression vector (pEGFP-C1,
CLONTECH, Palo Alto, CA) was diluted to a final
concentration of 1.25 ng/µl and then was transfected with either
control or SF-1 expression vector, as previously described. Twenty-four
hours after transfection, the cultures were then observed under
epifluorescent optics using the fluorescein isothiocyanate filter set
and the number of GFP-positive (green fluorescent) cells within
specific grids in each well counted. The cultures were continued for an
additional 24 h, and the number of GFP-positive cells in each grid
was counted again. The rate of cell proliferation was expressed as a
fold increase in GFP-positive cells over 24-h control values. For
studies in which apoptosis was assessed, the cells were stained with
hydroethidine (14 µg/ml PBS, Polyscience, Inc., Warrington, PA)
48 h after transfection (25). The cells were observed under
epifluorescent optics using the fluorescein isothiocyanate filters to
identity transfected cells. The filters were then changed to the
rhodamine filter set. Under these conditions, the hydroethidine-stained
nuclei fluoresced red. The nuclear morphology of the GFP-positive cells
were then classified as either normal or apoptotic, according to the
previously defined criteria (25, 26). The percentage of GFP-positive
apoptotic cells was calculated.
Statistical analysis
All experiments were repeated two to three times. When
appropriate, the data were evaluated by either an ANOVA followed by a
Student-Newman-Keuls test or a Student’s t test. Regardless
of the statistical test, a P value of 
0.05 was
considered to be significantly different.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In addition, SF-1 mRNA was
detected in ovarian surface epithelial cells (Fig. 1). Further,
Northern blot hybridization analysis was conducted on RNA isolated from
cells derived from ROSE cells. This analysis demonstrated that
EP-ROSE-179 cells expressed a 3-kb SF-1 transcript (Fig. 2). SF-1 mRNA transcripts were not
detected in either LP-, T-sv-, or T-ras ROSE-179 cells by Northern blot
hybridization (data not shown). Similarly, in situ
hybridization studies revealed that SF-1 mRNA was detected by a
blue-staining reaction product distributed throughout the cytoplasm of
EP-ROSE-179 cells (Fig. 3, A and B).
Quantitative in situ hybridization studies revealed that the
amount of SF-1 mRNA detected in EP-ROSE-179 cells was 4- to 5-fold
greater than that observed for the other ROSE cell lines
(P < 0.05; Fig. 4).
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). This fold
increase in the number of cells present after 48 h of culture was
less for EP-ROSE-179 cells than for the other ROSE cell lines (Fig. 5).
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), were
assessed for SF-1 and HPRT mRNAs by RT-PCR (Fig. 6B). This study showed
that the amount of RT-PCR product generated by using the SF-1 primer
pairs was 2.5- to 3.0-fold higher in quiescent cells than in
mitotically-active cells, whereas the amount of RT-PCR product for HPRT
was independent of the stage of the cell cycle (Fig. 6B).
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; C, E, and G), whereas
SF-1 mRNA was readily detected in approximately 30% of the cells that
were transfected with the SF-1 expression vector (Fig. 3; D, F, and H).
As judged by the amount of staining, high levels of SF-1 mRNA were
maintained within these cells, even in the presence of serum. However,
the morphology of the transfected cells was similar to that of the
nontransfected cells. Depending on the cell line, the number of cells
present, 48 h after transfection with the control vector,
increased by approximately 2-fold, compared with the 24-h value (Fig. 7). Compared with control vector,
transfection with the SF-1 expression vector significantly reduced the
number of cells present, after 48 h of culture, by 20–30%
(P < 0.05; Fig. 7).
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), as defined by Boersma et
al. (27). In the absence of serum, about 18% of the cells were
considered apoptotic, regardless of whether the cells were transfected
with control or SF-1 expression vector (Fig. 8, A and C). Serum did not
influence the distribution of cells transfected with control vector
(Fig. 8B), but it significantly increased the percentage of apoptotic
cells that were transfected with SF-1 expression vector, to 27 ±
2% (P < 0.05; Fig. 8, compare C with D).
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). Cells transfected with SF-1
expression vector failed to undergo mitosis in response to serum (Fig. 9A). As would be predicted from the data in Fig. 8, serum significantly
increased the percentage of apoptotic GFP/SF-1 transfected cells,
compared with no-serum control and serum-treated control vector groups
(P < 0.05). Serum decreased the percentage of
apoptotic GFP/control vector transfected cells (Fig. 9B). This finding
is in contrast to the effect of serum on the number of apoptotic cells
shown in Fig. 8B. The reason for this contradiction is unclear. It is
likely that cells that retain GFP have intact membranes. These cells
may be better able to respond to the antiapoptotic action of serum,
compared with non-GFP cells, which may have been irreversibly damaged
by the transfection process.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to maintaining the differentiated state of granulosa cells, SF-1 may be involved in regulating granulosa cell proliferation. Previous studies have shown that SF-1 levels are reduced when granulosa cells enter the S-phase of the cell cycle (16). Further, FSH induces SF-1 expression and prevents the granulosa cells from undergoing mitogen-dependent mitosis (14). Finally, SF-1 antisense treatment reduces FSH-induced SF-1 mRNA levels and, subsequently, attenuates FSH’s ability to prevent mitosis (14). These data demonstrate that SF-1 is a negative regulator of granulosa cell mitosis. SF-1 may play a similar role in controlling ovarian surface epithelial cell mitosis in vivo. Currently, this hypothesis cannot be directly tested.
Like primary ovarian surface epithelial cells, EP-ROSE-179 cells express SF-1 mRNA. The Northern blot analysis demonstrates that these cells express a 3-kb transcript that is the same size as that expressed by steroidogenic MA-10 cells and granulosa cells (16). In addition, the EP-ROSE-179 cells retain normal epithelial morphology and proliferate at a relatively slow rate (the present study and Ref. 7). These findings indicate that the EP-ROSE-179 cells are similar to primary ovarian surface epithelial cells and provide a good model system to study SF-1’s role in regulating ovarian surface epithelial cell mitosis.
Experiments with the various ROSE-179 lines have generated the following data that support the hypothesis that SF-1 negatively regulates ovarian surface epithelial cell mitosis. First, ROSE cell lines that have a reduced ability to express SF-1 undergo mitosis more frequently than EP-ROSE-179 cells. Second, forced expression of SF-1 prevents ovarian surface epithelial cells from proliferating. This antimitotic action is extremely effective, in that exogenous SF-1 inhibits nearly all of the transfected (i.e. GFP-positive) cells from undergoing mitosis in response to serum. Based on these observations, it seems that SF-1 functions as a molecular switch, such that when it is present, ovarian surface epithelial cells do not proliferate. When SF-1 expression is turned off, the cells can undergo mitosis in response to a mitogen. Consistent with this hypothesis is the observation that SF-1 expression is apparently reduced in mitotically active EP-ROSE-179 cells, compared with quiescent EP-ROSE-179 cells. However, this finding must be confirmed using a more quantitative method to assess SF-1 mRNA.
This type of molecular switch has been observed in other cell
types. For example, MyoD is a transcription factor that actively
represses proliferation and induces the phenotypic gene expression
associated with muscle cell differentiation (33). Similarly, C/EBP
is a transcription factor that is required for adipocyte
differentiation (34). Like MyoD, C/EBP mRNA levels are suppressed in
response to mitotic stimuli (34). Thus, SF-1 seems to be one of a
select group of differentiation factors that negatively regulate
mitosis. The molecular mechanism through which the expression of these
differentiation factors suppress cell proliferation requires further
study. Initial studies indicate that MyoD stimulates the expression of
the cell cycle-dependent kinase inhibitor, p21, (35) and subsequently
accounts for the withdrawal of differentiating myocytes from the cell
cycle (35). A similar molecular target may account for SF-1’s
antimitotic action.
Interestingly, if SF-1 expression is not turned off in response to a mitogenic signal, the cells undergo apoptosis rather than mitosis. In the present study, SF-1 mRNA levels are maintained in the transfected cells, even in the presence of a mitogen (i.e. serum), because SF-1 expression is under the control of the CMV promoter. It is commonly believed that, if cells are exposed to mitogens under conditions that are not appropriate for mitosis, then the cells undergo apoptosis (36, 37). The inappropriate presence of SF-1 during the serum-induced mitosis could be perceived by the cells as a condition that is inappropriate for mitosis and, thus, could account for the cells undergoing apoptosis, as opposed to mitosis. This hypothesis must now be rigorously tested.
Finally, the present studies demonstrate that the malignant
transformation of ROSE cells seems to be associated with a reduced
ability to express SF-1. This inability to express SF-1 mRNA is
associated with an increased rate of cell cycle traverse, and it
precedes alterations in the cell morphology (Fig. 3, compare A with C).
Similarly, SF-1 levels are not detectable in human ovarian sex cord
tumors that are derived from granulosa cells (38). This lack of SF-1
expression correlates with the loss of steroidogenic enzymes and, thus,
a loss of the differentiated function (38). Taken together, these
observations are consistent with the hypothesis that the inability to
express SF-1 is an important event in the oncogenesis of both ovarian
surface epithelial cells and granulosa cells. The specific mechanisms
responsible for this alteration in gene expression are unknown.
In summary, these studies have shown that forced SF-1 expression inhibits cells from different ovarian epithelial cell lines from proliferating. As such, SF-1 may function as a molecular switch that must be turned off to allow cells to enter the cell cycle. Further, the ability to express SF-1 seems to be lost in the oncogenesis of these cells. As a result, an ovarian-specific mechanism, which controls proliferation, is subverted and could potentially lead to an increased rate of cellular proliferation and subsequent malignant transformation of the ovarian surface epithelial cells. Finally, SF-1 retains its antimitotic function, even in the tumorigenic cell line (T-ras-ROSE). Although enhanced ras activity seems to be responsible for the tumorigenic nature of ROSE cell lines used in the present study (7), it remains to be determined whether forced expression of SF-1 will attenuate mitosis in other ovarian cancer cells whose transformation is mediated by factors other than enhanced ras activation. If forced SF-1 expression is effective in preventing the growth of ovarian cancer cells with differing etiologies, then SF-1 has the potential to be incorporated into a gene therapy regime for the treatment of ovarian cancer.
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Acknowledgments |
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Footnotes |
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Received March 4, 1998.
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