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Biology of Reproduction 62, 491-500 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Expression and Action of Hepatocyte Growth Factor in Human and Bovine Normal Ovarian Surface Epithelium and Ovarian Cancer1

Jeff A. Parrott3,a, and Michael K. Skinner2,a

a Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99164-4231


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
More than 95% of ovarian cancers originate from the epithelial cells on the surface of the ovary, which are termed ovarian surface epithelium (OSE). These OSE cells are modified peritoneal mesothelial cells separated from underlying ovarian surface stromal tissue by a basal lamina of dense collagenous connective tissue. Mesenchymal-epithelial cell interactions between stromal cells and OSE cells are postulated to be important for normal OSE biology and for the onset of ovarian cancer. Hepatocyte growth factor (HGF) is a mesenchymal-derived growth factor that mediates mesenchymal-epithelial cell interactions in a number of different tissues. The current study was an investigation of the expression and actions of HGF in normal OSE and ovarian cancer. Human epithelial cells from borderline and stage III ovarian cancer cases were found to express HGF protein in the epithelial cell component by immunocytochemistry analysis. The stromal cell component of human ovarian tumors contained little or no HGF immunostaining. Normal bovine ovaries have a similar physiology and endocrinology to human ovaries and are used as a model system to investigate normal OSE functions. HGF protein was detected in the OSE from both normal human and bovine ovaries. Adjacent ovarian stromal tissue contained light but positive HGF immunostaining. RNA was collected from normal bovine ovarian stromal cells to examine HGF gene expression. HGF transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. Using a quantitative reverse transcription-polymerase chain reaction procedure, HGF gene expression was found to be high in freshly isolated OSE but low in freshly isolated stroma. Levels of HGF gene expression after culture of stroma increased. Observations indicate that normal OSE express high levels of HGF in vivo and in vitro. Expression of HGF by normal epithelial cells versus stromal cells was unexpected and suggests that HGF may be important in an autocrine regulation of OSE. HGF actions on normal OSE cells and ovarian cancer cells were investigated. HGF was found to stimulate the growth of normal OSE cells in a manner similar to such growth stimulated by epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to grow in response to HGF. This observation suggests that HGF may be involved in sustaining growth of ovarian tumors. These results are the first to demonstrate the production and action of HGF in normal OSE cells and ovarian cancer cells. This appears to be an example of HGF production by an epithelial cell, such that a mesenchymal-epithelial mixed phenotype is present. The autocrine stimulation of OSE growth by the local production and action of HGF provides insight into how the OSE may develop abnormal growth characteristics involved in the onset and progression of ovarian cancer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The abnormal expression and action of growth factors such as hepatocyte growth factor (HGF) is speculated to be involved in the onset and progression of ovarian cancer. Growth factors such as HGF can stimulate abnormal cellular proliferation, changes in cellular morphology, altered cellular motility, and invasiveness, which are required to support tumor progression. HGF is a mesenchymal-derived growth factor that mediates stromal-epithelial cell interactions in a number of tissues including the ovary [1, 2]. Stromal cell-epithelial cell interactions are proposed to be important in many different cancers. The current study was designed to investigate the expression and action of HGF in ovarian surface epithelium (OSE) and ovarian cancer.

Although a small number of ovarian cancers originate from cells associated with the ovarian follicle, more than 95% of ovarian cancers originate in the epithelial cells on the surface of the ovary [3, 4]. These OSE cells are modified peritoneal mesothelial cells that undergo a mesenchymal to epithelial cell transition during development [5]. The OSE is a simple epithelium separated from underlying ovarian stromal tissue by a basal lamina of dense collagenous connective tissue [6]. Both the OSE and stroma appear to contribute to the formation of various extracellular matrix components that separate the two cell types [7]. During normal ovarian function, the OSE undergoes cyclic changes including the release of enzymes that contribute to the breakdown of the underlying stroma that overlies the preovulatory follicle [8, 9]. After ovulation, the OSE proliferates and covers the area affected by follicular rupture [10]. The alteration in OSE function and growth at ovulation implies that cellular association with the underlying stroma influences the OSE. The cellular associations between OSE and stroma also have been shown to influence the intermediate filaments in the OSE that may be compared with the early stages of neoplastic progression [11]. Tumorigenic tissue derived from the OSE also has close associations with stromal tissue. Tumor invasion often requires an association with host stromal tissue, and most ovarian tumors have a stromal-like component [12, 13]. Therefore, stromal-epithelial cell interactions appear to have a critical role in the function and growth of normal and tumorigenic OSE. This stromal-epithelial cell interaction may be similar to other classic mesenchymal-epithelial cell interactions and involve similar growth factors.

HGF is an 87-kDa protein composed of a 69-kDa a subunit and a 34-kDa b subunit and is important for the organogenesis and morphogenesis of various tissues and organs [14–20]. HGF is primarily produced by mesenchymal-derived cells in many tissues and acts as an epithelial cell-specific mitogen. The receptor to HGF is the product of the c-met protooncogene (p190MET) that is primarily localized to epithelial cells [21–25] but can also be expressed by macrophages, neurons, endothelial cells, muscle cells, and cytotrophoblasts [26–29]. Two alternately spliced forms of HGF, known as NK1 and NK2, have been documented that may act as HGF agonists or antagonists [30–33]. HGF mediates mesenchymal-epithelial cell interactions in many tissues including the ovary [1, 2, 34]. Expression of HGF can be regulated in an endocrine manner in many tissues such as the kidney, spleen, lung, and prostate [15, 35–38]. In the ovary, HGF mediates cell-cell interactions between theca cells and granulosa cells, and expression can be regulated by estradiol and the LH-like factor hCG [1]. Theca cells and ovarian surface stromal cells are derived from the same ovarian interstitial cell population. Therefore, some similarities may exist between theca cell-granulosa cell interactions and ovarian surface stromal-OSE interactions. These observations suggest that HGF may be involved in stromal cell-OSE interactions in normal OSE biology and in ovarian cancer.

Overexpression of HGF or its receptor, c-Met, has been observed in tumors from a wide variety of organs [39–46]. A subset of ovarian cancers expresses high levels of c-Met, the HGF receptor [21, 47, 48]. HGF can stimulate motility, chemotaxis, and mitogenesis in ovarian carcinoma cells that overexpress c-Met [49] and may provide a selective growth advantage to these cells. Expression of c-Met has been studied in ovarian cancer, but expression of HGF in ovarian tumors has been limited. In addition, little information is available on the expression and action of HGF in normal OSE biology. In order to study the potential role of HGF in ovarian cancer, it is important to examine the expression and action of HGF on normal and abnormal OSE cells.

Normal OSE of the rat, rabbit, and human have been isolated and cultured [50–52], but the size and availability of these tissues often limits the use of these models. Bovine ovaries present a useful model for OSE and ovarian stromal interactions. The bovine ovary has essentially the same physiology and size as the human ovary. The cow is a mono-ovulator that ovulates regularly and has an ovarian cycle similar to that of the human. Bovine ovarian cancer has been reported [53], suggesting that bovine OSE have tumorigenic potential similar to that of human OSE. Therefore, the bovine ovary provides a useful model for examining the specific cell-cell interactions involving normal OSE. Once established, specific cellular interactions can be compared with those of human OSE and ovarian tumor cells.

The current study was designed to examine the local production and action of a specific stromal-epithelial cell factor, HGF, in human and bovine ovarian surface epithelium. The hypothesis tested is that HGF may have an important role in normal OSE as well as in ovarian cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissues

Fresh human adult ovarian tissues were obtained from Dr. Bethan Powell in the Department of Obstetrics and Gynecology at the University of California, San Francisco, CA. The normal human tissues were collected from salpingo-oophorectomy specimens removed for benign diseases from women of child-bearing age. Human cancer tissues were surgically collected from women with borderline and stage III ovarian cancer. Bovine ovaries were obtained from young nonpregnant cycling heifers less than 10 min after slaughter. When required, ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. Sections were stored at 4°C until immunocytochemistry (ICC) was performed.

Embedding, Histology, and ICC for HGF

Tissues were fixed in 4% paraformaldehyde and embedded in paraffin according to standard procedures. ICC for HGF was performed according to standard procedures. Briefly, 5-µm sections were deparaffinized and rehydrated, quenched in 20% methanol/3% hydrogen peroxide, and blocked in 5% serum for several hours at room temperature before incubation with primary antibody monoclonal anti-human HGF (R & D Systems, Minneapolis, MN) overnight at 4°C. Secondary antibody (biotinylated goat anti-mouse IgG from Vector Laboratories, Burlingame, CA) was detected by using the Vectastain kit (Vector) and diaminobenzadine. Slides were counterstained lightly with hematoxylin to visualize the tissue. HGF-positive cells are stained brown.

OSE and Stromal Cell Isolation and Cell Culture

OSE cells and ovarian stromal cells were isolated fresh (for quantitative reverse transcription [RT]-polymerase chain reaction [PCR] studies) or cultured (for Northern blot and growth studies). Similar procedures were used for both human and bovine ovaries. OSE cells were scraped from the surface of the ovary with a rubber policeman as previously described [54]. Sheets of epithelial cells were suspended in Hanks' buffered salt solution and then pelleted and washed before suspension for plating. After the removal of OSE cells, the ovarian surface stromal cells were microdissected from areas of the ovary devoid of follicles. A section of surface stromal cells 1–2 mm wide by 5–8 mm long and 1 mm deep was collected. The tissue piece was minced and digested with 1 mg/ml collagenase and 1 mg/ml hyaluronidase for 2 h at 37°C or 18 h at 4°C. Cells were plated with an initial density of approximately 106 cells/2 cm2 and were maintained at 37°C in a 5% CO2 atmosphere in Ham's F-12 (Gibco Labs., Grand Island, NY) supplemented with 10% calf serum. Medium was changed every 48–72 h. Once the cells had grown to confluence, the cells were trypsinized and split into appropriate plates. For isolation of RNA from cultured cells, OSE and stromal cells were plated in 100-mm large culture plates (Nunc, Naperville, IL) and maintained in Ham's F-12 supplemented with 10% calf serum. For growth assays, OSE cells were plated in 24-well plates in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% calf serum. When cells achieved 50–70% confluence, cells were washed in DMEM containing 0.1% calf serum for growth assays. The purity of OSE isolated by this procedure is greater than 98% by keratin staining, with no detectable stromal contamination [55]. Human ovarian cancer cell lines SKOV3 and OCC1 were obtained from the American Type Culture Collection (Rockville, MD).

RNA Preparation

Total RNA was prepared from freshly isolated or cultured cells using Trizol reagent (Gibco). Trizol was added directly to freshly isolated cells or to the culture plate to prevent RNA degradation. Total RNA was used to purify mRNA using the FastTrack 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, CA). RNA was stored at -70°C until use.

Northern Blot Analysis

Total RNA and mRNA from OSE cells and ovarian stromal cells were isolated as described above. Approximately 6 µg of total RNA and 6 µg mRNA were fractionated on a 1% formaldehyde-agarose gel. After fractionation, the RNA in the gel was transferred into nylon membrane (Hybond N+, Amersham) in single-strength 3-(n-morpholino) propanesulfonic acid (MOPS) buffer and UV cross-linked. The membranes were then prehybridized (500 mM phosphate buffer pH 7.2, 1 mM EDTA, 1% BSA, 7% SDS) for 2 h at 60°C. The hybridization was carried out at 60°C overnight with 32P-labeled HGF probe obtained by random primer extension (Prime-It II, Stratagene, La Jolla, CA) of a bovine HGF partial cDNA [1]. The membrane was washed in wash buffer (0.2-strength SSC [single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate], 0.1% SDS) at room temperature for 10 min and then 60°C for 20 min. Membranes were exposed to x-ray film (X-OMAT, Eastman Kodak, Rochester, NY) overnight at -70°C using an intensifying screen. The membrane was subsequently stripped and rehybridized with bovine cyclophilin using a similar procedure.

Quantitative RT-PCR Assays

Steady-state levels of HGF and cyclophilin (i.e., 1B15) mRNAs were analyzed using a specific quantitative RT-PCR assay for each gene. These assays have previously been described in detail [1]. The primers used in this quantitative analysis of HGF and 1B15 were as follows: HGF, 5'-ACA GCT TTT TGC CTT CGA GCT ATC GGG GTA AAG ACC TAC AGG-3' (5' primer, 42-mer) and 5'-CAT CAA AGC CCT TGT CGG GAT A-3' (3' primer, 22-mer), which generated a specific 292-base pair (bp) HGF PCR product; and 1B15, 5'-ACA CGC CAT AAT GGC ACT GGT GGC AAG TCC ATC-3' (5' primer, 33-mer) and 5'-ATT TGC CAT GGA CAA GAT GCC AGG ACC TGT ATG-3' (3' primer, 33-mer), which generated a specific 105-bp product from all cell types, demonstrating the integrity of the RNA samples. Before RT, tubes containing total RNA and specific 3'-primers were heated to 65°C for 10 min to facilitate denaturing and cooled to room temperature to facilitate annealing. Total RNA (1 µg) was reverse-transcribed for 1 h at 37°C using the following conditions: 1 µg total RNA, 1 µM specific 3'-primers of interest (up to 4 different primers including 1B15), 0.1 mM dNTPs, 10 mM dithiothreitol, 40 units ribonuclease inhibitor (Promega, Madison, WI), and 200 Units M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) in 40 µl RT buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2). After 1 h, samples were heated to 95°C for 5 min to inactivate the reverse transcriptase enzyme. Samples were immediately diluted 2.5-fold, and carrier DNA (Bluescript plasmid, Stratagene) was added to a final concentration of 10 ng/µl. This concentration of Bluescript carrier DNA (10 ng/µl) was included in all subsequent dilutions of samples and standards. Immediately before amplification, each unknown sample was further diluted 1:10 in order to improve the fidelity of the PCR reaction. Plasmid DNAs (i.e., Bluescript) containing bovine HGF or 1B15 subclones were used to generate standard curves from 1 attogram/µl (10-15 g/µl) to 10 pg/µl (10 x 10-9 g/µl), each containing 10 ng/µl Bluescript carrier DNA. Identical 10-µl aliquots of each sample and standard were pipetted in duplicate into a 96-well reaction plate (Marsh Biomedical Products, Rochester, NY) and sealed with adhesive film (Marsh Biomedical Products) for PCR amplification. By this design, it was possible to simultaneously assay 5 known standard concentrations and 40 unknown samples for each gene. Amplification was performed in a Perkin Elmer (Foster City, CA) 9600 equipped with a heated lid using the following conditions: 0.4 µM each primer, 16 µM dNTPs, and 1.25 Units AmpliTaq polymerase in 50 µl GeneAmp PCR buffer (containing 1.5 mM MgCl2, Perkin Elmer). Each PCR amplification consisted of an initial denaturing reaction (5 min, 95°C); 25–31 cycles of denaturing (30 sec, 95°C), annealing (1 min, 60°C), and elongation (2 min, 72°C) reactions; and a final elongation reaction (10 min, 72°C). At least 0.25 µCi of 32P-labeled dCTP (Redivue, Amersham Life Sciences, Arlington Heights, IL) was included in each sample during amplification for detection purposes. Specific PCR products were quantitated by electrophoresing all samples on 4–5% polyacrylamide gels, simultaneously exposing the gels to a phosphor screen for 8–24 h, and then quantitating the specific bands on a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Each gene was assayed in separate PCR reactions from the same RT samples. Equivalent steady-state mRNA levels for each gene were determined by comparing each sample to the appropriate standard curve. All HGF data were normalized for 1B15.

Optimal cycle number for amplification was determined for each assay in order to achieve maximum sensitivity while maintaining linearity (i.e., logarithmic phase of PCR reactions). HGF quantitative PCR products were amplified for 33 cycles, and 1B15 PCR products were amplified for 25 cycles. The sensitivity of each quantitative PCR assay was below 1 fg, which corresponds to less than 125 fg target mRNA/µg total RNA. For each assay, all samples were simultaneously measured in duplicate, resulting in intraassay variabilities of 13.6% (HGF) and 6.5% (1B15).

Growth Assays

Cell growth was analyzed by quantifying [3H]thymidine incorporation into newly synthesized DNA. OSE cells were plated (approximately 1 million cells/cm2 providing 50% confluence) in 0.5 ml DMEM medium containing 0.1% calf serum. After 48 h, cells were treated with no growth factor (control), 50 ng/ml HGF, or 40 ng/ml epidermal growth factor (EGF). Cells were plated for 48 h and then treated for 20 h. After treatment, 0.5 ml DMEM containing 2.5 µCi [3H]thymidine was added to each well, and the cells were incubated for 4 h at 37°C and then sonicated. The quantity of [3H]thymidine incorporated into DNA was determined, as previously described [55]. Data were normalized to total DNA per well using an ethidium bromide procedure previously described.

DNA Assays

DNA was measured fluorometrically with ethidium bromide as previously described [55]. An aliquot of the sonicated cell suspension was added to an equal volume of ethidium bromide solution (0.25 mM ethidium bromide, 100 U/ml heparin in ethidium bromide buffer [EBB; 20 mM sodium chloride, 5 mM ethylene diamine tetraacetate, 10 mM Tris, pH 7.8]; Sigma), diluted 1:2 with EBB, and allowed to incubate at room temperature for 30 min. Fluorescent emission at 585 nm with 350 nm excitation was then monitored. A standard curve with calf thymus DNA was used to quantify DNA levels in the culture wells. This assay has a sensitivity of approximately 0.1 µg DNA.

Statistical Analysis

All data were analyzed by a JMP 3.1 statistical analysis program (SAS Institute Inc., Cary, NC). Effects of growth factors on [3H]thymidine incorporation into DNA and differences among fresh versus cultured OSE and stromal cell HGF expression were analyzed by a one-way ANOVA. Observed significance probabilities of 0.05 (Prob > F) or less were considered evidence that an ANOVA model fit the data. Significant differences between treated cells and control (untreated) cells were determined using Dunnett's test, which guards against the high alpha-size (Type I) error rate across the hypothesis [56]. Significant differences among fresh versus cultured OSE and stromal cell HGF expression were determined using the Tukey-Kramer HSD (honestly significant difference) test, which protects the significance tests of all combinations of pairs [57–59]. These multiple comparison tests are recommended for multiple comparisons [60].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of HGF protein was examined at different stages of ovarian cancer by ICC. Tissues were examined from cases of borderline cancers and stage III ovarian cancers. At both stages, HGF protein was detected in the epithelial cell component of the disease (Fig. 1). Light staining could also be detected in the stromal cells that border the epithelial cells, but the most intense staining was in the epithelial cells. Control slides were analyzed using nonimmune IgG and showed no staining (Fig. 1E). Results support the hypothesis that expression of HGF protein may be important during different stages of ovarian cancer in humans. Experiments were next designed to characterize HGF expression in normal OSE and ovarian stromal cells.



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  		FIG. 1. HGF protein expression in human ovarian tumors. Sections were visualized and photographed at approximately x400 magnification (published at 92%). Similar results were obtained in four separate experiments using four different ovarian tumors. A, B) Borderline ovarian cancer. C, D) Stage III ovarian cancers. E) Control section stained with nonimmune IgG. The brown/gray staining in the outer layer of OSE (A and B) and epithelial colonies (C and D) represents positive staining

Expression of HGF protein was examined in normal human and bovine ovaries by ICC. The surface morphology of the bovine ovary is very similar to that of the human as previously described [55]. A single layer of OSE is present on the outer surface of the ovary adjacent to multiple layers of ovarian surface stromal cells. In both human and bovine ovaries, HGF protein was detected in the OSE (Fig. 2). Light staining could also be detected in the stromal cells that border the epithelial cells. No staining was detected in control slides using nonimmune IgG. Similar results were obtained from human and bovine ovaries, confirming the bovine ovary as a useful model of normal OSE biology. Observations suggest that HGF protein may be expressed at high levels by normal OSE and to a lesser extent by ovarian stromal cells.



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  		FIG. 2. HGF protein expression in normal human and bovine ovarian tissue. Sections were visualized and photographed at approximately x400 magnification. Similar results were obtained in four separate experiments using four different human ovaries. A) Human ovary stained with HGF antibody. B) Human ovary stained with nonimmune IgG. C) Bovine ovary stained with HGF antibody. D) Bovine ovary stained with nonimmune IgG

In order to determine the sites of HGF gene expression, steady-state levels of HGF mRNA were examined in bovine OSE and ovarian stromal cells by Northern blot analysis. Total RNA and poly(A) RNA from cultured OSE and ovarian stromal cells were probed with a previously obtained bovine HGF probe [1]. It was necessary to isolate RNA from cultured cells because of the relatively large amount of RNA required for Northern blots. A specific HGF transcript was observed in both OSE and stromal cells (Fig. 3). However, the level of expression was apparently higher in cultured ovarian stromal cells than in cultured OSE cells, even after densitometric scanning and normalization with 1B15. It was necessary to examine poly(A) RNA from OSE cells and ovarian stroma in order to obtain an intense HGF band by Northern analysis. A bovine stromal cell line (EBTr) was used as a positive control, and two human ovarian cancer cell lines, SKOV3 and OCC1, were also included for comparison. Blots were reprobed with a cyclophilin probe (1B15) to demonstrate integrity of the RNA. These results suggest that both normal OSE and ovarian surface stromal cells express the HGF gene. Stromal cells may be the predominant site of HGF mRNA synthesis in cell culture. Experiments were further designed to quantitate HGF gene expression in freshly isolated OSE and ovarian stromal cells by a quantitative PCR procedure.



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  		FIG. 3. HGF mRNA expression in OSE and ovarian stromal cells. Bovine OSE cells and ovarian stromal cells were isolated and grown to confluence as described in Materials and Methods. Total RNA was isolated from cultured cells, and mRNA was purified from total RNA. RNA was separated on 1.5% agarose gels, blotted to nylon membranes, and probed for HGF mRNA expression. A) Probes were prepared using random primer extension of a previously isolated HGF subclone isolated from bovine ovarian cells [1]. B) Membranes were re-probed with constitutively expressed cyclophilin (1B15) to control for differences in loading and transfer. The multiple transcripts at and between the 28S and 18S bands are shown

HGF gene expression in normal OSE and ovarian stromal cells was examined using sensitive quantitative RT-PCR assays [1]. Total RNA was isolated from 8 to 12 different preparations of freshly isolated normal OSE and ovarian stromal cells. Samples were reverse-transcribed using the specific 3' primers of the HGF and cyclophilin (i.e., 1B15) genes. Samples were then simultaneously amplified by PCR with known HGF or 1B15 standard plasmids to quantitate gene expression. Steady-state levels of HGF mRNA expression were determined and normalized for the constitutively expressed gene cyclophilin, termed 1B15. Normalization for 1B15 expression corrected for changes in cell number, for the amount and integrity of initial mRNA, and for small differences in the efficiency of reverse transcription between samples. Consistent with the results from Northern analysis in Figure 3, HGF gene expression was observed in cultured OSE and ovarian stromal cells (Fig. 4). The level of HGF expression varied between individual samples, but in general HGF expression was higher in fresh OSE cells than in fresh stromal cells. Fresh stromal cells had low but detectable levels of HGF gene expression. Observations suggest that HGF gene expression may change in different populations of fresh and cultured OSE and ovarian stromal cells.



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  		FIG. 4. Quantitation of HGF mRNA expression in OSE and ovarian stromal cells. Bovine OSE cells and ovarian stromal cells were isolated and grown to confluence as described in Materials and Methods. Total RNA was extracted from freshly isolated cells or cultured cells. Steady-state levels of HGF mRNA were determined in replicate using quantitative RT-PCR as described in Materials and Methods. Levels of HGF mRNA were determined and normalized to levels of cyclophilin (1B15) mRNA (fg HGF mRNA/fg 1B15 mRNA). Data are presented as the mean ± SEM of duplicate determinations from 8–12 different sets of RNA from each cell type. An ANOVA was performed, and significant differences among cell types were determined using the Tukey-Kramer HSD test. Bars with different superscript letters differ from each other (P < 0.05)

The ability of HGF to influence the growth of bovine OSE cells was investigated. OSE were plated at 50% confluence and maintained in 0.1% calf serum for 48 h. Cells were then treated with 50 ng/ml HGF for 20 h and incubated for 4 h with [3H]thymidine. Cells treated with 40 ng/ml EGF or 10% bovine calf serum served as positive controls. HGF was found to stimulate DNA synthesis in bovine OSE (Fig. 5) to levels similar to those stimulated by EGF. The ability of HGF to promote the proliferation of OSE suggests that HGF may be involved in the normal growth functions of OSE. Similar growth experiments were performed using two human ovarian cancer cell lines, SKVO3 and OCC1 [61]. HGF was found to stimulate DNA synthesis in both SKOV3 and OCC1 cells (Fig. 6). EGF and 10% calf serum were used as positive controls. Observations demonstrate that HGF may also be involved in regulating the growth of human ovarian tumor cells.



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  		FIG. 5. HGF regulation of DNA synthesis in bovine OSE cells. Bovine OSE cells were isolated as described in Materials and Methods. Cells were deprived of serum when approximately 70% confluent, and [3H]thymidine incorporation into DNA was determined after 20 h of treatment with 50 ng/ml HGF, 40 ng/ml EGF, or 10 % bovine calf serum. Cells were incubated with [3H]thymidine for 4 h. Data are representative of four different experiments done in replicate. An ANOVA was performed, and significant differences from control were determined using Dunnett's test. Bars with asterisks differ from control (P < 0.05)



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  		FIG. 6. HGF regulation of DNA synthesis in human ovarian cancer cells. Human ovarian cancer cell lines SKOV3 and OCC1 were maintained in cell culture in 10% bovine calf serum. Cells were deprived of serum when approximately 70% confluent, and [3H]thymidine incorporation into DNA was determined after 20 h of treatment with 50 ng/ml HGF, 40 ng/ml EGF, or 10% bovine calf serum. Cells were incubated with [3H]thymidine for 4 h. Data are representative of four different experiments done in replicate. An ANOVA was performed, and significant differences from control were determined using Dunnett's test. Bars with asterisks differ from control (P < 0.05). A) Results using SKOV3 cells. B) Results using OCC1 cells


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This year some 30 000 new cases of ovarian cancer will be diagnosed in the United States, and it is estimated that there will be about 20 000 deaths from ovarian cancer [62]. Ovarian cancer accounts for 4% of all cancers in women and ranks fifth as a cause of cancer deaths among women. Although a considerable amount of research concerning established ovarian tumors has been conducted, little is known about the mechanisms involved in the onset and early progression of the disease. Mesenchymal-epithelial cell interactions between ovarian stromal cells and OSE are postulated to be critical in normal OSE biology and in ovarian cancer.

HGF protein was immunologically observed in the epithelial components of borderline and stage III ovarian cancers. The presence of high levels of HGF at these stages of ovarian cancer suggests that expression of HGF may be important for the development and/or progression of the disease. HGF was also found to be expressed in the OSE of both human and bovine ovaries. Results obtained from Northern analysis indicated that steady-state levels of HGF mRNA were comparable in ovarian stromal cells and OSE cells. However, these experiments were performed on cultured cells. When a more sensitive quantitative RT-PCR procedure was utilized, steady-state levels of HGF mRNA were found to be higher in freshly isolated OSE than in freshly isolated stroma. HGF expression was elevated in ovarian stromal cells after cell culture, suggesting that these cells have the ability to express HGF. It is possible the OSE provides a negative regulation of HGF expression by stromal cells in the intact tissue. The observation that HGF is expressed in normal OSE cells provides insight into the unusual nature of this epithelium. The biology of HGF has demonstrated that HGF expression is primarily limited to cells of mesenchymal or stromal origin.

OSE cells are modified peritoneal mesothelial cells that are derived from the coelomic epithelium that overlies the gonadal ridge in the embryo [5]. Therefore, the OSE is of mesodermal origin and developmentally is closely related to the underlying stromal fibroblasts. The OSE has classic epithelial cell characteristics such as keratin, mucin, desmosomes, and apical microvilli, and a basal lamina. However, OSE cells also coexpress vimentin, a mesenchymal intermediate filament protein. During postovulatory repair, OSE cells reversibly modulate to a more fibroblast-like form. In culture, these cells produce epithelial (e.g., laminin and collagen type IV) and mesenchymal (e.g., collagen types I and III) components of extracellular matrix [63]. A variety of environmental cues cause OSE cells to change from an epithelial to mesenchymal morphology [52, 64]. Thus these cells may be relatively immature, uncommitted cells that express a dual, epithelio-mesenchymal phenotype [64]. As a result, the expression of HGF by this "epithelial" cell may be a significant marker of this unusual phenotype. The uncommitted differentiated state of this cell may be a factor in its susceptibility to becoming transformed and developing tumors.

A significant observation in the current study is the ability of OSE cells not only to express HGF but to respond to it in an autocrine manner. In cell culture, HGF stimulated the growth of normal OSE cells, SKOV3 cells, and OCC1 cells. All the cells had a doubling time of less than 48 h. Both the SKOV3 and OCC1 were found to express high levels of HGF (data not shown). This suggests that OSE cells have the ability to stimulate their own growth in an autocrine manner. Perhaps this unusual autocrine stimulation by HGF can lead to the inability to regulate the normal functional differentiation of the OSE. The inability to control functional differentiation and the ability to promote abnormal proliferation is proposed to be involved in the onset of ovarian cancer. It is possible that ovarian cancers generally derive from the OSE cell population since normal OSE cells display characteristics of a mesenchymal and epithelial cells (i.e., HGF expression and action). Abnormal proliferation of ovarian cancer cells may be sustained in a similar autocrine manner involving HGF.

HGF and its receptor, c-Met, have been implicated in other human tumors [47]. In a study of breast cancer, HGF concentrations correlated with disease relapse and reduced overall survival, suggesting that HGF may promote tumor progression [65]. HGF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer [39]. The c-Met/HGF receptor is overexpressed in a renal cell carcinoma cell line whose motility is triggered by HGF. Expression of the c-Met/HGF receptor may be involved in the onset and progression of renal cell carcinomas [45]. The c-Met/HGF receptor appears to be involved in the growth and behavior of pancreatic cancer and may contribute to the ductal phenotype of these tumors [44]. The c-met gene is expressed at late stages of melanoma progression, and the presence of c-Met/HGF receptor may contribute to the acquisition of an invasive phenotype [41]. The current study suggests that HGF may have a similar role in ovarian cancer.

Growth control of both normal and tumorigenic OSE is a critical cellular parameter to consider in understanding ovarian cancer. The majority of information available on ovarian growth factors relates to the developing ovarian follicle [66]. Several growth factors, however, have been shown to influence OSE. Normal OSE cells express the EGF receptor, and a large number of tumorigenic OSE cells also express the EGF receptor [67–69]. EGF can stimulate the proliferation of normal human [69] OSE and bovine [55] OSE cells. Transforming growth factor (TGF) alpha has been associated with ovarian cancer [70, 71] and may act as an autocrine growth factor to induce cell proliferation in both normal and tumorigenic OSE [72, 73]. Basic fibroblast growth factor (bFGF) and its receptor are expressed by human ovarian epithelial neoplasms [74, 75], suggesting that bFGF may also regulate ovarian cancer proliferation through an autocrine mechanism. Several ovarian cancer cell lines proliferate in response to bFGF [76]. TGFß is a multifunctional protein that has a major role in inhibiting the actions of growth stimulators such as EGF/TGF{alpha}, bFGF, and HGF. TGFß has been shown to be produced by OSE [77], and TGFß can inhibit the growth of normal OSE cells and some tumorigenic OSE cells [76–78]. It is likely that the combined actions of a number of different growth factors including HGF are involved in the onset and progression of ovarian cancer.

In the current study, the role that HGF may have in mediating cell-cell interactions involving OSE was investigated. Human and bovine OSE cells were shown to express HGF mRNA and protein. OSE cells from normal ovaries and ovarian cancers expressed HGF. Ovarian surface stromal cells were also shown to express HGF. These observations are the first to establish the potential role of HGF in normal OSE biology and ovarian cancer. Both OSE cells and ovarian surface stromal cells had the capacity to express high levels of HGF in vitro, but fresh OSE cells appeared to express higher levels of HGF than fresh stromal cells. HGF stimulated the growth of OSE cells, supporting the role of HGF in OSE biology. Established ovarian cancer lines, SKOV3 and OCC1, were also stimulated to grow in response to HGF. Further analysis of the actions of HGF is needed to elucidate the function of HGF in ovarian cancer. Of particular interest will be the combined effects with other growth factors. HGF is primarily a stromal cell-derived growth factor in other tissues. Expression of HGF by OSE cells indicates that this epithelial cell is normally in an altered differentiation state that is susceptible to transformation.


   ACKNOWLEDGMENTS
 
We thank Gene Herrington and Grace Kim for technical assistance. We also thank Susan Cobb for assistance in preparation of the manuscript.


   FOOTNOTES
 
First decision: 16 August 1999.

1 This work was supported by an Ovarian Cancer Program Project grant from the National Institutes of Health (NIH). Back

2 Correspondence: FAX: 509-335-2176; skinner{at}mail.wsu.edu Back

3 Current address: Atairgin Technologies Inc., 4 Jenner, Suite 180, Irvine CA 92618. Back

Accepted: October 15, 1999.

Received: June 17, 1999.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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