help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dunfield, L. D.
Right arrow Articles by Nachtigal, M. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dunfield, L. D.
Right arrow Articles by Nachtigal, M. W.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Ovarian Cancer
Endocrinology Vol. 143, No. 4 1174-1181
Copyright © 2002 by The Endocrine Society

CANCER

TGFß-Induced Smad Signaling Remains Intact in Primary Human Ovarian Cancer Cells

Lesley D. Dunfield, Elizabeth J. Campbell Dwyer and Mark W. Nachtigal

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Address all correspondence and requests for reprints to: Mark W. Nachtigal, Dalhousie University, Department of Pharmacology, Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: . mnachtig{at}is.dal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Disruptions in TGFß signaling have been implicated in various human cancers, including ovarian cancer. Our goal was to determine whether ovarian cancer cells isolated from patient ascites fluid were growth inhibited by TGFß1 treatment and further characterize the expression and activity profile of TGFß/Smad signaling components in human ovarian cancer cells. We found that 9 of 10 primary cultures of ovarian cancer cells (OC2–10) were growth inhibited by 16 pM TGFß1. One primary ovarian cancer sample (OC1) and the established ovarian cancer cell lines CaOV3 and SkOV3 continued to grow in the presence of TGFß1. All cells expressed components of the TGFß/Smad signaling pathway including TGFß1, TßRI, TßRII, Smad2, -3, -4, and Smad anchor for receptor activation. Although OC1, CaOV3, and SkOV3 are not growth inhibited by TGFß1, they can transmit the TGFß1 signal to turn on a transfected TGFß/Smad reporter gene, p3TP.lux. In addition, all cells up-regulate the endogenous TGFß target genes Smad7 and PAI-1. p15Ink4B mRNA is also up-regulated with TGFß1 treatment in OC2–9, whereas the p15Ink4B gene has been deleted in OC1, CaOV3, and SkOV3 cells. Homozygous deletion of p15Ink4B may account for TGFß resistance in some populations of ovarian cancer cells. Our data demonstrate that the TGFß/Smad signaling pathway remains functional in human ovarian cancer cells and suggest that if abnormalities exist in the cellular response of TGFß signals, they must lie downstream of the Smad proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN CANCER HAS the second highest incidence in gynecological cancers, ranks first in gynecologic cancer-related mortality (5-yr survival rate is ~20%), and is the fifth leading cause of cancer death among women (1, 2). If ovarian cancer is detected early, more than 93% of patients survive 5 yr; however, more than 75% of cases are diagnosed at advanced stages with a poor prognosis for survival (American Cancer Society). Most ovarian cancers are believed to arise from the ovarian surface epithelium (OSE), a modified peritoneal mesothelium derived embryologically from the coelomic epithelium (3). Characterizing the pathways that regulate the growth of OSE cells is a necessary first step toward understanding the events that lead to uncontrolled cellular proliferation and the development of ovarian cancer.

Human OSE cells express many of the receptors for hormones and growth factors produced by the ovarian surface epithelial, follicular, and stromal cells, leading to the suggestion that epithelial cell activity may be modified by autocrine and paracrine signals (4, 5, 6). OSE cells often form inclusion bodies within the ovarian cortex, and these are the predominant sites of epithelial dysplasia and cancer formation (3, 7, 8). It remains unclear what factors influence the transformation of the OSE in the inclusion cysts. Under circumstances in which women have reduced ovulation, such as with multiparity, lactation, and use of oral contraceptives, there is a decreased risk of developing ovarian cancer. The molecular basis for this phenomenon is unknown; however, recent research suggests that oral contraceptives may provide protection against development of ovarian cancer because they induce TGFß production in OSE cells, which initiates apoptosis and prevents OSE hyperplasia (9). Determining whether TGFß signaling activity is lost in ovarian cancer will give us insight into OSE cell transformation and may provide a novel basis for the prevention or treatment of ovarian cancer.

In humans, members of the TGFß superfamily can modulate growth of normal OSE cells in vitro (4, 10, 11, 12). Although mutations affecting TGFß receptors or their intracellular signaling molecules, the Smad proteins, are implicated in the development of human cancer (13, 14), the role that TGFß/Smad signaling plays in ovarian tumorigenesis is unclear. TGFß isoforms (TGFß1–3) and the type I (TßRI) and type II (TßRII) TGFß receptor subunits are expressed by normal human OSE, established human ovarian cancer cell lines, and primary ovarian cancer tissue and cells (4, 15, 16). Ito et al. (17) found that the downstream effectors for TGFß signaling Smad2 and Smad4 are expressed in the established human ovarian cancer cell line OVCAR 3. Because normal OSE produce and are growth inhibited by TGFß, it has been suggested that disruption to the TGFß signaling pathway may contribute to ovarian tumorigenesis. Recent reports (16, 18, 19, 20) suggest that deletion or inactivating mutations to components of the TGFß/Smad signaling pathway occur in human ovarian cancer. By contrast, Hurteau et al. (11) show that primary ovarian cancer cell cultures remain sensitive to the antiproliferative effect of TGFß, and Rodriguez et al. (21) demonstrate that TGFß treatment can enhance invasiveness of ovarian cancer cells through up-regulation of matrix metalloproteinases. Few studies have specifically determined whether primary human ovarian cancer cells maintain a functional TGFß signaling pathway (11, 16). In agreement with Hurteau et al. (11), we found that 9 of 10 primary cultures of ovarian cancer cells isolated from ascite fluid remained sensitive to TGFß growth inhibition and that these cells express all components of the TGFß/Smad signaling pathway. Further, we show that ovarian cancer cells treated with TGFß1 up-regulate endogenous TGFß/Smad target genes including p15Ink4B, Smad7, and plasminogen activator inhibitor 1 (PAI-1). Our data conclusively demonstrated that most primary human ovarian cancer cells remain sensitive to the antiproliferative effects of TGFß in vitro and that the TGFß/Smad signaling pathway remains functional.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary human ovarian cancer cells
Institutional approval for research with human materials was received before the initiation of these studies (QEII Health Sciences Center, Research Ethics Committee, no. QE-RS-99-016). Primary human ovarian cancer cells were isolated from ascite specimens by a technique modified from Hirte et al. (22). Briefly, ascitic fluid containing cells was mixed 1:1 with growth medium (MCDB105/M199 supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin/streptomycin). After 3 d, the ascitic fluid supernatant/medium was removed and attached cells were fed growth medium. A total of nine primary ovarian cancer (OC) cultures (OC1–9) were used for all experiments in the present study; however, only data from OC1–3 are shown. Expression of the epithelial marker E-cadherin was detected by RT-PCR analysis of the OC cultures, thus defining them as being derived from OSE and distinguishing them from reactive mesenchyme (23, 24, 25), a possible contaminant in cell preparations isolated form ascite fluid. Most experiments were performed at culture passages two through six, with the exception of those involving OC1 cells, which have been maintained in long-term culture and are currently at a higher passage number (>20).

Cell lines
Two ovarian cancer cell lines were used in this study, CaOV3 and SkOV3. Cells were grown in monolayer and maintained in DMEM (Canadian Life Technologies, Inc., Burlington, Ontario, Canada) supplemented with 0.1 mM nonessential amino acids, 10% heat-inactivated FBS (Canadian Life Technologies, Inc.) and penicillin/streptomycin (100 U/ml). Mv1Lu cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in MEM supplemented with 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10% heat-inactivated FBS, and penicillin/streptomycin (100 U/ml). Mv1Lu cells are known to stop growing in response to TGFß treatment and were used as control cultures for growth curve analyses.

RT-PCR amplification, cRNA probes, and Northern analysis
Total cytoplasmic RNA for RT-PCR was isolated from ovarian cancer cells, cell lines, and tumor tissue using the single-step guanidinium isothiocyanate method (26). RT-PCR was used to detect expression of TGFß signaling components and TGFß/Smad target genes (30 cycles: 35 sec at 94 C, 35 sec at 59.5 C, 35 sec at 72 C). Complementary DNA fragments of human TGFß1 (nts. 655-1054), TßRI (801–1197), TßRII (751–1123), Smad2 (240–440), Smad3 (501–878), Smad4 (601–1050), Smad7 (748–1198), Smad anchor for receptor activation (SARA) (1027–1411), p15INK4B (69–449), and PAI-1 (159–717) were subcloned into pCRII.TOPO (Invitrogen, Carlsbad, CA) and the identity of the amplified fragment was verified by manual sequencing. 32P-Labeled antisense cRNA probes were generated and purified by PAGE.

For Northern analysis primary ovarian cancer cells and ovarian cancer cell lines were treated with 16 pM (0.1 ng/ml) TGFß1 (Sigma-Aldrich Corp., Oakville, Ontario, Canada) for 4 h. RNA was isolated using the Genelute mammalian total RNA kit (Sigma-Aldrich Corp.). Ten micrograms total RNA was separated on a 1.5% formaldehyde gel and transferred to BrightStar Plus membrane (Ambion, Inc., Austin, TX). Blots were incubated with 1 x 106 cpm/ml cRNA probe overnight at 60 C in hybridization buffer (400 mM sodium phosphate, 1 mM EDTA, 0.5% SDS, 1 mg/ml BSA, 0.2 mg/ml yeast tRNA, 50% formamide) and washed at 60 C in wash solution (0.1% SDS, 0.1x standard saline citrate, 1 mM EDTA). Signals were visualized by autoradiography and loading controlled by densitometry and normalized to the signal from glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The data shown are representative of two independent experiments.

Western analysis
Total cellular protein was isolated from ovarian cancer cells grown to 70–80% confluence on 100-mm plates. Cells were washed two times in ice-cold PBS, dissolved in lysis buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF, protease inhibitor mix), clarified by centrifugation (10 min at 15,000 g), and quantitated by Bradford analysis. Ten micrograms protein extract per lane were separated by SDS-PAGE in the presence of 1% ß-mercaptoethanol using 10% gels (30 µg protein run on 12% gels for Fig. 2Go). Rabbit polyclonal Smad antibodies (Zymed Laboratories, Inc., South San Francisco, CA), rabbit antiphospho-Smad2 (Upstate Biotechnology, Inc., Lake Placid, NY), rabbit antiactin (Sigma, St. Louis, MO), and rabbit polyclonal p15Ink4B antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used to detect protein expression with enhanced chemiluminescence (Perkin-Elmer Corp., Markham, Ontario, Canada).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 2. Smad2 phosphorylation in response to exogenous TGFß1. A, Western blot indicating phosphorylated Smad2 in cells untreated (-) or treated (+) with 16 pM TGFGß1 for 30 min, 30 µg total cellular protein per lane. Total Smad2 and ß-actin protein levels are shown in the middle and bottom panel, respectively. B, The histogram indicates the mean fold increase in phosphorylated Smad2 in response to TGFß1, compared with phospo-Smad2 in untreated cells; signal has been normalized to total Smad2 and ß-actin levels. These results were confirmed in two independent experiments.

 
Growth curves
Primary human ovarian cancer cells (OC1–10) and established ovarian cancer cell lines (4 x 104 cells/well) were plated on d 0 in growth medium in 6-well plates. On d 1, and then every third day, cells were fed medium and treated with 16 pM TGFß1. Cells were counted using a hemacytometer over a 6- to 9-d period. The data are representative of at least three independent experiments.

Transient transfection and luciferase assays
Cells were plated in a 12-well plate at 4 x 104 cells/well on d 0. On d 1, cells in triplicate wells were transfected with 300 ng pGL2-mod or 300 ng p3TP-lux (27) using FuGENE6 transfection reagent (Roche Molecular Biochemicals, Laval, Quebec, Canada) in normal growth media. Additional controls (negative) included cells either untransfected or transfected with 300 ng pCMV5. Cells were serum starved (growth medium with 0.2% FBS) for 8 h on d 2 and then treated with 16 pM TGFß1 in growth medium containing 0.2% FBS. Twenty hours after addition of TGFß1, cells were harvested and luciferase activity was determined using the Enhanced luciferase assay kit (BD PharMingen, Mississauga, Ontario, Canada). The results shown are mean data from three independent experiments.

The pGL2-mod reporter plasmid used as a negative control for the transfection experiments is a promoterless luciferase construct and is a modified version of pGL2-Basic (Promega Corp./Fisher Scientific, Nepean, Ontario, Canada). This plasmid was generated by digestion of pGL2-Basic with KpnI and HindIII, blunting the cohesive ends with T4 DNA polymerase, and religating.

Statistical analysis
Growth curve statistical analysis was done using a repeated measures two-way ANOVA with Tukey’s post hoc test (P < 0.01). Statistical analysis of the transfection data was done using a t test (P < 0.01).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ovarian cancer cells express known components of the TGFß/Smad signaling pathway. To characterize the primary ovarian cancer cells and established cell lines used for the present study, RT-PCR was done to determine whether these cells expressed components of the TGFß/Smad signaling pathway. A total of nine primary ovarian cancer cell cultures were used for the present study (OC1–9). Data from OC1–3 are shown, and the data for OC2 and OC3 are representative of all responses to TGFß1 treatment for OC2–9 (see below). We found that primary ovarian cancer cells (OC1–9), CaOV3, and SkOV3 cells all express TGFß1, TßRI and TßRII, Smad2, Smad3, and Smad4 (representative results are shown in Fig. 1AGo). In addition, we determined that these cells also express SARA mRNA, a protein critical for regulating Smad2 and Smad3 signaling (28). Relative levels of Smad2 and Smad4 expression were analyzed by Northern analysis (Fig. 1BGo). Expression levels of Smad2 and Smad4 are elevated in the established ovarian cancer cells lines and high-passage OC1 in comparison with those in the low-passage primary ovarian cancer cells (OC2 and 3). The significance of this observation is unknown; however, it does not appear to affect Smad protein expression or signaling function (see below). To determine whether Smad transcript levels were altered by addition of TGFß1, cells were treated with 16 pM TGFß1 for 4 h before total RNA was harvested. After the signal was normalized vs. the GAPDH mRNA signal (graph, Fig. 1BGo), the data indicate that neither Smad2 nor Smad4 transcript levels were altered with TGFß1 treatment.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Ovarian cancer cells express all components of the TGFß/Smad signaling pathway. A, Representative RT-PCR data from cultured primary ovarian cancer cells and established cell lines (SkOV3 and CaOV3) indicating expression of TGFß (400 bp), TßRI (397 bp), TßRII (373 bp), Smad2 (327 bp), Smad3 (378 bp), Smad4 (450 bp) and SARA (423 bp). B, Representative Northern data indicating expression of human Smad2 (3.8- and 2.5-kb transcripts) and Smad4 (2.9-kb transcripts) in primary human ovarian cancer cells isolated from patient ascites (OC1–3) and established SkOV3 and CaOV3 cell lines. All primary cultures tested (OC1–9) expressed similar levels of Smad2 and Smad4. Cells were treated without (-) or with (+) 16 pM TGFß1 for 4 h before harvesting. Equivalent amounts of RNA (10 µg) were loaded in each lane and the signal was normalized by densitometry of GAPDH mRNA. The histogram indicates the ratio of Smad:GAPDH signal. C, Representative Western data indicating expression of Smad2 and Smad3 in ovarian cancer cells.

 
Expression of Smad2 and Smad3 protein was confirmed by Western analysis (Fig. 1CGo). We were unable to detect Smad4 expression using commercially available antibodies (Transduction Laboratories, Lexington, KY), either from ovarian cancer cell extracts or from positive control extracts sent with the antibody.

Smad2 phosphorylation in response to exogenous TGFß1
As an initial step to determine whether primary ovarian cancer cells have an active TGFß signaling pathway, we examined whether Smad2 was phosphorylated in response to TGFß1 treatment. Cells were incubated in 0.2% FBS containing medium for at least 8 h before 30-min stimulation with 16 pM TGFß1. In all cases the amount of phosphorylated Smad2 increased in response to TGFß1 treatment (Fig. 2Go; range of response 1.6- to 3.2-fold), suggesting that the TGFß signaling pathway is intact in primary ovarian cancer cells and established cell lines. These results were confirmed in two independent experiments, and the data in Fig. 2BGo are expressed as mean fold increase in Smad2 phosphorylation.

Up-regulation of endogenous genes in primary ovarian cancer cells and cell lines
Smad7 and PAI-1 are both TGFß/Smad target genes whose transcription is rapidly induced by TGFß treatment (29). Expression of Smad7 was increased in all primary ovarian cancer cells (OC1–9) and established CaOV3 and SkOV3 cell lines in response to exogenous TGFß1 (Fig. 3Go). PAI-1 was also increased in response to TGFß1 treatment; however, the signal for PAI-1 was much weaker in CaOV3, SkOV3, and OC1 cells. The data for CaOV3, SkOV3, and OC1 cells represents a 4-d exposure of the Northern, whereas the data for OC2 and OC3 are a 24-h exposure. Data shown are representative from at least two independent experiments. The mean fold increase in Smad7 was: SkOV3: 2.9; CaOV3: 1.9; OC1: 1.7; OC2: 1.4; OC3: 1.3, whereas for PAI-1, the mean fold increase was: SkOV3: 2.5; CaOV3: 1.5; OC1: 6.6; OC2: 2.2; OC3: 1.3. Pooling the data from all of our primary ovarian cancer cultures, excluding OC1, shows that the average fold response for Smad7 is 1.5 ± 0.11 (range: 1.2–1.9) and PAI-1 is 1.64 ± 0.15 (range: 1.2–2.8) 4 h after TGFß treatment. Work by Afrakhte et al. (30) demonstrated that up-regulation of Smad7 mRNA in Mv1Lu cells occurs within 30 min of TGFß stimulation and peaks at 90 min. Thereafter the Smad7 mRNA levels decay but are still measurable after 4 h. We have replicated these observations in our laboratory using CaOV3 cells treated with TGFß and see an average 5-fold response in Smad7 transcript levels 1 h after TGFß treatment (Fu, Y., and M. W. Nachtigal, unpublished observation). These observations demonstrate that the TGFß/Smad signaling pathway is functional and capable of directing expression of endogenous target genes in human ovarian cancer cells.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 3. Up-regulation of endogenous TGFß target genes in primary ovarian cancer cells and established cell lines. Representative Northern analysis of endogenous TGFß target gene expression in primary ovarian cancer cells (OC1–3) and cell lines (CaOV3 and SkOV3). Cells were treated without (-) or with (+) 16 pM TGFß for 4 h before harvesting total RNA. Blots were hybridized with cRNA probes for human Smad7 and PAI-1. Equivalent amounts of RNA (10 µg) were loaded in each lane and signal was normalized by densitometry of GAPDH mRNA. Data shown are representative data from at least two independent experiments.

 
Reporter gene analysis of the TGFß signaling pathway in established ovarian cancer cells
The expression of all known TGFß/Smad signaling components in CaOV3, SkOV3, and OC1 cells and up-regulation of endogenous TGFß target genes in response to TGFß1 treatment shows that an intact pathway exists in these cells. However, we also tested the ability of ovarian cancer cells to transmit the TGFß signal by examining the response of a transiently transfected TGFß/Smad reporter gene, p3TP-lux, to exogenous TGFß1. Cells were transfected with p3TP-lux and the day after transfection treated with 16 pM TGFß1 for 20 h. Cells were then harvested and luciferase activity was determined (Fig. 4Go). Many of the primary ovarian cancer samples proved difficult to transfect, and thus we could not perform TGFß reporter gene experiments on these cells. All cells transfected with p3TP-lux demonstrated an 8- to 12-fold increase in luciferase activity above background in response to TGFß1 (Fig. 4Go). Negative controls (negative and pGL2-mod) gave no demonstrable activity (<0.1-fold activity relative to p3TP-lux) in the presence or absence of TGFß1. Thus CaOV3, SkOV3, and OC1 are capable of transmitting the TGFß/Smad signal to the nucleus to activate both endogenous and transfected target genes.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Reporter gene analysis of the TGFß signaling pathway in established ovarian cancer cells. Cells were transiently transfected with a TGFß-responsive reporter gene (p3TP-lux) and treated with 16 pM TGFß1 for 20 h before harvesting and measuring luciferase activity. All cells demonstrated a basal level of p3TP-lux activity in growth medium containing 10% FBS that was increased 8- to 12-fold in the presence of TGFß1. Luciferase activity from negative controls (negative and pGL2-mod) was less than 0.1-fold activity relative to p3TP-lux in the presence or absence of TGFß1. Luciferase activity was measured in relative light units and is reported as fold difference from basal p3TP-lux activity. Statistical analysis was done using a t test (*, P < 0.005). Bars indicate SEM.

 
Effect of TGFß on ovarian cancer cell proliferation
To further confirm TGFß signaling activity, we tested the effect of TGFß1 treatment on growth of ovarian cancer cell cultures. A total of 10 primary ovarian cancer cell cultures were used for the growth study (OC1–10). Data from OC1–3 are shown, and the data for OC2 and 3 are representative of all responses to TGFß1 treatment for OC2–10. Beginning 24 h after cells were seeded, cultures were treated with 16 pM TGFß1 and the effect on cell proliferation determined over the next 6–9 d (Fig. 5Go). OC2–9 cells were growth inhibited by TGFß1 treatment (Fig. 5Go); Mv1Lu cells are known to stop growing in response to TGFß treatment and were used as control cultures (data not shown; 31). We found that 16 pM TGFß1 was sufficient to inhibit growth of the primary cells. By contrast, OC1 and the established cell lines CaOV3 and SkOV3 (Fig. 5Go) continued to proliferate in the presence of TGFß1 (16 pM-1.6 nM). Cell viability was not affected by treatment with TGFß1 as determined by the trypan blue dye exclusion technique (data not shown). The graphs show representative data from three separate experiments. Statistical analysis was done using a repeated measures two-way ANOVA with Tukey’s post hoc test (*, P < 0.01). These data show that the majority of ovarian cancer cells (90%) stop growing in response to TGFß1 treatment, in contrast to OC1 and the established cell lines.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. Effect of TGFß on ovarian cancer cell proliferation. Primary human ovarian cancer cells from patient ascites (OC1–3) and established human ovarian cancer cell lines (SkOV3 and CaOV3) were grown for a period of 6 d to determine the effect of TGFß1 on cellular proliferation. A total of nine primary ovarian cancer samples were analyzed; growth curves for OC2 and OC3 are shown as representative samples of the responses for OC2–9. The majority of primary samples, OC2–9, were growth inhibited in media containing 10% FBS + 16 pM TGFß1 ({blacksquare}), by contrast to OC1 and the established cell lines, which grow equally well in media containing 10% FBS ({square}) or 10% FBS + 16 pM TGFß1 ({blacksquare}). The graphs show representative data from three separate experiments. Statistical analysis was done using a repeated-measures two-way ANOVA with Tukey’s post hoc test (*, P < 0.01). Bars indicate SEM.

 
Growth inhibition by TGFß is partially mediated by modulating levels and activities of cell cycle regulatory proteins, such as p15Ink4B (CDKN2B) (32, 33). Indeed, the promoter for the p15Ink4B gene contains positive TGFß/Smad response elements, and p15Ink4B transcription is rapidly up-regulated in response to TGFß treatment (34, 35). Northern analysis reveals that in ovarian cancer cells, which are growth inhibited by TGFß1, p15Ink4B mRNA increases when cells are incubated with TGFß1 (OC2, OC3, Fig. 6AGo; OC4–9 not shown). The increase in mRNA correlates with increasing p15 protein levels (Fig. 6BGo). By contrast, p15Ink4B mRNA and protein was not detected in the OC1, CaOV3, and SkOV3 cells, which are insensitive to TGFß1. The gene for p15Ink4B is deleted in SkOV3 cells (36). Using genomic PCR and Southern analysis we did not detect p15Ink4B genomic DNA from OC1, CaOV3, and SkOV3 cells (data not shown); however, p15 Ink4B genomic DNA was detected in the TGFß1-sensitive OC samples (OC2–9; data not shown).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 6. Loss of TGFß growth inhibition is likely because of deletion of TGFß target genes. A, Representative Northern analysis of endogenous TGFß target gene expression in primary ovarian cancer cells (OC1–3) and SkOV3 cells. Cells were treated without (-) or with (+) 16 pM TGFß for 4 h before harvesting total RNA. Blots were hybridized with a cRNA probe for human p15INK4B. Equivalent amounts of RNA (10 µg) were loaded in each lane and signal was normalized by densitometry of GAPDH mRNA. B, Western analysis confirms that an increase in p15INK4B protein (15 kDa) correlates with up-regulation of p15INK4B mRNA. Data shown are representative data from two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our work is the first to show that all known components of the TGFß/Smad signaling pathway, including TGFß1, TßRI, TßRII, Smad2, -3, -4, and SARA, are expressed and remain functional in primary human ovarian cancer cell cultures and two established ovarian cancer cell lines, SkOV3 and CaOV3. Furthermore, our analysis revealed that 9 of 10 primary ovarian cancer cell cultures isolated from ascite fluid remain sensitive to the growth inhibitory effects of TGFß1. The ability of TGFß1 to block ovarian cancer cell proliferation is likely to be partially mediated by the observed increase in p15Ink4B production in response to TGFß1 treatment. The failure of CaOV3, SkOV3, and OC1 to respond to the growth-inhibitory effects of TGFß1 is not owing to defects in the TGFß/Smad signaling pathway because we showed that these cells can up-regulate a transfected TGFß/Smad-responsive reporter gene. In addition, all primary and established cells can increase mRNA levels of endogenous Smad7 and PAI-1, demonstrating that human ovarian cancer cells maintain a functional TGFß/Smad signaling pathway.

Previous reports examining the effect of TGFß on ovarian cancer cell proliferation have yielded contradictory results. Initial studies showed that most established ovarian cancer cell lines are resistant to the antiproliferative effects of TGFß (4, 33). Many established cell lines have accumulated significant genetic alterations, including loss of p53 and p15Ink4B and do not accurately represent primary ovarian cancer cells (36, 37). Loss of heterozygosity of p15Ink4B is shown to occur in approximately 10% of primary ovarian tumors and may contribute to the development of a proportion of human ovarian cancer (38). Indeed, the inability of CaOV3, SkOV3, and OC1 cells to respond to the antiproliferative effects of TGFß1 may be because of defects in TGFß/Smad targets important for cell cycle regulation, including p21Cip1, p27Kip1, and c-myc; however, we hypothesized that the observed effect is owing to deletion in p15Ink4B in these cells.

Hurteau et al. (11) demonstrated that primary ovarian cancer cells isolated from ascites remain sensitive to the growth-inhibitory effects of TGFß1, which agrees with the present study. By contrast, results from Yamada et al. (16) suggest that ovarian cancer cell cultures isolated from solid tumors, which express functional TGFß receptors, can be insensitive to the antiproliferative effects of TGFß1. Yamada et al. (16) showed that 5 of 23 cultures were growth inhibited, 7 of 23 were growth stimulated, and 11 of 23 showed no significant change in cell growth. In these studies, proliferation was measured over a 48-h time course using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide growth assay, whereas our cell proliferation studies were conducted over a 6- to 9-d time course. Indeed, we saw little change in cell number at 48 h, and significant differences occurred only at later time points, typically at d 4–5. In agreement with our data, Yamada et al. (16) did examine the growth-inhibitory effects of TGFß1 on one primary cell culture isolated from ascites and saw growth inhibition in response to TGFß1 treatment. It remains possible that contradictory results may reflect differences in the biology of ovarian cancer cells originating from different sites (ascites vs. solid tumor explants).

Recent literature suggests that TGFß receptor mutants may contribute to the development of some ovarian tumors. Lynch et al. (18) identified 5 of 24 sporadic ovarian tumors with missense mutations in the conserved serine/threonine kinase or transmembrane domain of TßRII; these results correlated with a decrease or loss of TßRII protein production. A similar study by Wang et al. (19) failed to detect any TßRII mutations in their patient population; however, they determined that 10 of 32 (31.3%) primary ovarian cancer samples failed to express TßRI. More recently, Chen et al. (20) reported that 33% of archival primary ovarian cancer samples harbor missense mutations in the coding region of TßRI. Groups that have examined the genes for Smad2, -3, and -4 discovered some sequence polymorphisms but no putative inactivating mutations in ovarian cancer tissues (19, 39). Although these studies indicate that missense mutations are frequently observed in coding and noncoding regions of the TßRI and TßRII genes, functional biochemical analysis was not done to determine whether these mutations affect TGFß/Smad signaling. We did not find any evidence of disrupted TGFß/Smad signaling in ovarian cancer cells isolated from patient ascites. These data suggest that mutations affecting the TGFß signaling pathway, although occurring in some human ovarian tumors, are not a universal hallmark of ovarian cancer.

Ovarian cancer cells receive a myriad of signals from molecules present in ascitic fluid or in the tumor microenvironment, and the interpretation of these signals ultimately results in the cellular response (e.g. grow, die, migrate, invade). With respect to TGFß/Smad signaling, target gene transcription is dictated by nuclear accumulation of the activated Smad complex and subsequent interaction with nuclear transcription factors and cofactors on the target gene promoter (40). In addition to signals from TGFß-like molecules, nuclear accumulation of Smad proteins can be modulated by Ras-activated Erk kinases (reviewed in Ref. 40). Hyperphosphorylation of Smad2 and -3 by Erk kinases, stimulated by epidermal growth factor (EGF), hepatocyte growth factor, or oncogenic Ras, blocks their nuclear translocation and inhibits TGFß signaling. In addition, EGF can up-regulate inhibitory Smad6 and -7 mRNA (30), thus decreasing expression of endogenous gene targets. EGF and hepatocyte growth factor are autocrine growth factors produced by ovarian cancer cells that stimulate their proliferation, migration, and invasiveness (41, 42). Indeed, EGF is typically elevated in serum from women with epithelial ovarian cancer, compared with healthy age-matched women (42A ) Although ovarian cancer cells isolated from ascites remain responsive to the antiproliferative effects of TGFß1, we hypothesized that this cytostatic activity is modulated by additional environmental cues affecting Smad signaling activity, such as EGF, allowing the cells to alter their normal cellular response to TGFß in vivo. Precedence for this idea exists with the discovery that oncogenic Ras in mammary epithelial cells blocks the antiproliferative effects of TGFß and reprograms the cells to become invasive and metastatic in response to TGFß (43).

The role that TGFß biology plays in ovarian tumorigenesis is unclear. We show that the TGFß/Smad signaling pathway is functional in ovarian cancer cells and that in vitro these cells stop proliferating in response to TGFß1 treatment. In marked contrast, in vivo ovarian cancer cells continue to proliferate despite the presence of high amounts of TGFß in ascite fluid [median concentration 5.4 ng/ml TGFß1 (44)]. Many human tumors, including ovarian cancers (15, 45), overexpress TGFß mRNA and protein in vivo, and increased expression correlates with advanced stages of the disease. Cells that escape the growth-inhibitory effects of TGFß and produce large amounts of the protein may have a selective advantage for tumor cell survival because of the positive angiogenic and immunosuppressive effects that allow TGFß to promote tumor growth (13, 14). In addition, TGFß enhances the invasive properties of ovarian cancer cells, most likely through up-regulation of cellular matrix metalloproteinases (21). Thus, TGFß may play a dual role in OSE biology. Under nonpathologic conditions, normal OSE cells are growth inhibited by TGFß through an autocrine loop. Once tumorigenesis is initiated, however, TGFß signaling may be altered to enhance the metastatic process.


   Acknowledgments
 
The authors acknowledge Drs. Y. Fu, D. Guernsey, and C. R. McMaster for critical reading of this manuscript; Drs. R. Grimshaw and J. Bentley (QEII Health Science Center) for providing human ovarian tumor samples; Dr. L. Attisano for p3TP-lux; and L. C. Coolen for assistance in establishing primary ovarian cancer cultures.


   Footnotes
 
This work was supported by grants from the Dalhousie Medical Research Foundation and the National Cancer Institute of Canada with funds from the Terry Fox Run (no. 10289). L.D.D. is supported by a studentship from Cancer Research and Education, Nova Scotia, with funding from the Faculty of Medicine, Dalhousie University. M.W.N. is a Canadian Institutes of Health Research Scholar.

Abbreviations: EGF, Epidermal growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mv1Lu, mink lung epithelial; OSE, ovarian surface epithelium; PAI, plasminogen activator inhibitor; SARA, Smad anchor for receptor activation.

Received October 13, 2001.

Accepted for publication December 13, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tortolero-Luna G, Mitchell MF, Rhodes-Morris HE 1994 Epidemiology and screening of ovarian cancer. In: Gershenson DM, ed. Update on epithelial ovarian cancer. Philadelphia: WB Saunders Co.; vol 21:1–23
  2. Daly M, Obrams GI 1998 Epidemiology and risk assessment for ovarian cancer. Sem Oncol 25:255–264[Medline]
  3. Scully RE 1995 Pathology of ovarian cancer precursors. J Cell Biochem Suppl 23:208–218[Medline]
  4. Berchuk A, Rodriguez G, Olt G, Whitaker R, Boente MP, Arrick BA, Clarke-Pearson DL, Bast RC 1992 Regulation of growth of normal ovarian epithelial cells and ovarian cancer cell lines by transforming growth factor-ß. Am J Obstet Gynecol 166:6776–6684
  5. Karlan BY, Jones J, Greenwald M, Lagasse LD 1995 Steroid hormone effects on the proliferation of human ovarian surface epithelium in vitro. Am J Obstet Gynecol 173:97–104[CrossRef][Medline]
  6. Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung M-C 1997 Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation. J Biol Chem 272:27913–27918[Abstract/Free Full Text]
  7. Radisavljevic S 1976 The pathogenesis of ovarian inclusion cysts and cystomas. Obstet Gynecol 49:424–429
  8. Hamilton T 1992 Ovarian cancer. Part 1: biology. Curr Probl Cancer 16:1–57[Medline]
  9. Rodriguez GC, Nagarsheth NP, Walmer D, Cline M, Bentley R, Whitaker R, Eisner P, Dodge R, Hughes C 2001 Progestin induction of apoptosis in the macaque ovarian epithelium is associated with up-regulation of transforming growth factor ß. Proc 32nd Meeting of the Society of Gynecologic Oncologists (Abstract 4)
  10. Zhou L, Leung BS 1992 Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factor {alpha} and ß1. Biochim Biophys Acta 1180:130–136[Medline]
  11. Hurteau JA, Rodriguez GC, Whitaker RS, Shah S, Mills G, Bast RC, Berchuk A 1994 Transforming growth factor-ß inhibits proliferation of human ovarian cancer cells obtained from ascites. Cancer 74:93–99[CrossRef][Medline]
  12. Di Simone N, Crowley WF, Wang Q-F, Sluss PM, Schneyer AL 1996 Characterization of inhibin/activin subunit, follistatin, and activin type II receptors in human ovarian cancer cell lines: a potential role in autocrine growth regulation. Endocrinology 137:486–494[Abstract]
  13. Gold LI 1999 The role for transforming growth factor-ß (TGF-ß) in human cancer. Crit Rev Oncog 10:303–360[Medline]
  14. Massague J, Blain SW, Lo RS 2000 TGFß signaling in growth control, cancer, and heritable disorders [In Process Citation]. Cell 103:295–309[CrossRef][Medline]
  15. Henriksen R, Gobl A, Wilander E, Öberg K, Miyazono K, Funa K 1995 Expression and prognostic significance of TGF-ß isotypes, latent TGF-ß1 binding protein, TGF-ß type I and type II receptors, and endoglin in normal ovary and ovarian neoplasms. Lab Invest 73:213–220[Medline]
  16. Yamada SD, Baldwin RL, Karlan BY 1999 Ovarian carcinoma cell cultures are resistant to TGF-ß1-mediated growth inhibition despite expression of functional receptors. Gynecol Oncol 75:72–77[CrossRef][Medline]
  17. Ito I, Minegishi T, Fukuda J, Shinozaki H, Auersperg N, Leung PCK 2000 Presence of activin signal transduction in normal ovarian cells and epithelial ovarian carcinoma. Br J Cancer 82:1415–1420[Medline]
  18. Lynch MA, Nakashima R, Song H, DeGroff VL, Wang D, Enomoto T, Weghorst CM 1998 Mutational analysis of the transforming growth factor ß receptor type II gene in human ovarian carcinoma. Cancer Res 58:4227–4232[Abstract/Free Full Text]
  19. Wang D, Kanuma T, Mizunuma H, Takama F, Ibuki Y, Wake N, Mogi A, Shitara Y, Takenoshita S 2000 Analysis of specific gene mutations in the transforming growth factor-ß signal transduction pathway in human ovarian cancer. Cancer Res 60:4507–4512[Abstract/Free Full Text]
  20. Chen T, Triplett J, Dehner B, Hurst B, Colligan B, Pemberton J, Graff JR, Carter JH 2001 Transforming growth factor-ß receptor type I gene is frequently mutated in ovarian carcinomas. Cancer Res 61:4679–4682[Abstract/Free Full Text]
  21. Rodriguez GC, Haisley C, Hurteau J, Moser TL, Whitaker R, Bast Jr RC, Stack MS 2001 Regulation of invasion of epithelial ovarian cancer by transforming growth factor-ß. Gynecol Oncol 80:245–253[CrossRef][Medline]
  22. Hirte HW, Clark DA, Mazurka J, O’Connell G, Rusthoven J 1992 A rapid and simple method for the purification of tumor cells from ascitic fluid of ovarian carcinoma. Gynecol Oncol 44:223–226[CrossRef][Medline]
  23. Schofield K, D’Aquila T, Rimm DL 1997 The cell adhesion molecule, E-cadherin, distinguishes mesothelial cells from carcinoma cells in fluids. Cancer 81:293–298[CrossRef][Medline]
  24. Auersperg N, Pan J, Grove BD, Peterson T, Fisher J, Maines-Bandiera S, Somasiri A, Roskelley CD 1999 E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium. Proc Natl Acad Sci USA 96:6249–6254[Abstract/Free Full Text]
  25. Kitazume H, Kitamura K, Mukai K, Inayama Y, Kawano N, Nakamura N, Sano J, Mitsui K, Yoshida S, Nakatani Y 2000 Cytologic differential diagnosis among reactive mesothelial cells, malignant mesothelioma, and adenocarcinoma: utility of combined E-cadherin and calretinin immunostaining. Cancer 90:55–60[CrossRef][Medline]
  26. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  27. Carcamo J, Zentella A, Massague J 1995 Disruption of transforming growth factor ß signaling by a mutation that prevents transphosphorylation within the receptor complex. Mol Cell Biol 15:1573–1581[Abstract]
  28. Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL 1998 SARA, a FYVE domain protein that recruits Smad2 to the TGFß receptor. Cell 95:779–791[CrossRef][Medline]
  29. Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R, Itoh S, Kawabata M, Heldin NE, Heldin CH, ten Dijke P 1997 Identification of Smad7, a TGFß-inducible antagonist of TGF-ß signalling. Nature 389:631–635[CrossRef][Medline]
  30. Afrakhte M, Moren A, Jossan S, Itoh S, Sampath K, Westermark B, Heldin CH, Heldin NE, ten Dijke P 1998 Induction of inhibitory Smad6 and Smad7 mRNA by TGF-ß family members. Biochem Biophys Res Commun 249:505–511[CrossRef][Medline]
  31. Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang XF, Massague J 1992 TGF ß signals through a heteromeric protein kinase receptor complex. Cell 71:1003–1014[CrossRef][Medline]
  32. Hannon GJ, Beach D 1994 p15INK4B is a potential effector of TGF-ß-induced cell cycle arrest. Nature 371:257–261[CrossRef][Medline]
  33. Elbendary A, Berchuck A, Davis P, Havrilesky L, Bast Jr RC, Iglehart JD, Marks JR 1994 Transforming growth factor ß 1 can induce CIP1/WAF1 expression independent of the p53 pathway in ovarian cancer cells. Cell Growth Differ 5:1301–1307[Abstract]
  34. Datto MB, Yu Y, Wang XF 1995 Functional analysis of the transforming growth factor ß responsive elements in the WAF1/Cip1/p21 promoter. J Biol Chem 270:28623–28628[Abstract/Free Full Text]
  35. Li JM, Nichols MA, Chandrasekharan S, Xiong Y, Wang XF 1995 Transforming growth factor ß activates the promoter of cyclin-dependent kinase inhibitor p15INK4B through an Sp1 consensus site. J Biol Chem 270:26750–26753[Abstract/Free Full Text]
  36. Watson JE, Gabra H, Taylor KJ, Rabiasz GJ, Morrison H, Perry P, Smyth JF, Porteous DJ 1999 Identification and characterization of a homozygous deletion found in ovarian ascites by representational difference analysis. Genome Res 9:226–233[Abstract/Free Full Text]
  37. Elbendary AA, Cirisano FD, Evans Jr AC, Davis PL, Iglehart JD, Marks JR, Berchuck A 1996 Relationship between p21 expression and mutation of the p53 tumor suppressor gene in normal and malignant ovarian epithelial cells. Clin Cancer Res 2:1571–1575[Abstract]
  38. Ichikawa Y, Yoshida S, Koyama Y, Hirai M, Ishikawa T, Nishida M, Tsunoda H, Kubo T, Miwa M, Uchida K 1996 Inactivation of p16/CDKN2 and p15/MTS2 genes in different histological types and clinical stages of primary ovarian tumors. Int J Cancer 69:466–470[CrossRef][Medline]
  39. Wang D, Kanuma T, Takama F, Mizumuma H, Ibuki Y, Wake N, Mogi A, Shitara Y, Hagiwara K, Takenoshita S 1999 Mutation analysis of the Smad3 gene in human ovarian cancers. Int J Oncol 15:949–953[Medline]
  40. Attisano L, Wrana JL 2000 Smads as transcriptional co-modulators. Curr Opin Cell Biol 12:235–243[CrossRef][Medline]
  41. Alper O, De Santis ML, Stromberg K, Hacker NF, Cho-Chung YS, Salomon DS 2000 Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells. Int J Cancer 88:566–574[CrossRef][Medline]
  42. Sowter HM, Corps AN, Smith SK 1999 Hepatocyte growth factor (HGF) in ovarian epithelial tumour fluids stimulates the migration of ovarian carcinoma cells. Int J Cancer 83:476–480[CrossRef][Medline]
  43. Baron AT, Lafky JM, Boardman CH, Balasubramaniam S, Suman VJ, Podratz KC, Maihle NJ 1999 Serum sErbB1 and epidermal growth factor levels as tumor biomarkers in women with stage III or IV epithelial ovarian cancer. Cancer Epidemiol Biomarkers Prev 8:129–137[Abstract/Free Full Text]
  44. Massague J, Chen YG 2000 Controlling TGF-ß signaling. Genes Dev 14:627–644[Free Full Text]
  45. Abendstein B, Stadlmann S, Knabbe C, Buck M, Muller-Holzner E, Zeimet AG, Marth C, Obrist P, Krugmann J, Offner FA 2000 Regulation of transforming growth factor-ß secretion by human peritoneal mesothelial and ovarian carcinoma cells. Cytokine 12:1115–1119[CrossRef][Medline]
  46. Bristow RE, Baldwin RL, Yamada SD, Korc M, Karlan BY 1999 Altered expression of transforming growth factor-ß ligands and receptors in primary and recurrent ovarian carcinoma. Cancer 85:658–668[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
J. S. Sunde, H. Donninger, K. Wu, M. E. Johnson, R. G. Pestell, G. S. Rose, S. C. Mok, J. Brady, T. Bonome, and M. J. Birrer
Expression Profiling Identifies Altered Expression of Genes That Contribute to the Inhibition of Transforming Growth Factor-{beta} Signaling in Ovarian Cancer.
Cancer Res., September 1, 2006; 66(17): 8404 - 8412.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
G. Xu, H. Zhou, Q. Wang, N. Auersperg, and C. Peng
Activin Receptor-Like Kinase 7 Induces Apoptosis through Up-Regulation of Bax and Down-Regulation of Xiap in Normal and Malignant Ovarian Epithelial Cell Lines
Mol. Cancer Res., April 1, 2006; 4(4): 235 - 246.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
J. N. Kloth, G. J. Fleuren, J. Oosting, R. X. de Menezes, P. H.C. Eilers, G. G. Kenter, and A. Gorter
Substantial changes in gene expression of Wnt, MAPK and TNF{alpha} pathways induced by TGF-{beta}1 in cervical cancer cell lines
Carcinogenesis, September 1, 2005; 26(9): 1493 - 1502.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
J.-S. Kim, S. J. Baek, T. Sali, and T. E. Eling
The conventional nonsteroidal anti-inflammatory drug sulindac sulfide arrests ovarian cancer cell growth via the expression of NAG-1/MIC-1/GDF-15
Mol. Cancer Ther., March 1, 2005; 4(3): 487 - 493.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
G. D'Andrilli, C. Kumar, G. Scambia, and A. Giordano
Cell Cycle Genes in Ovarian Cancer: Steps Toward Earlier Diagnosis and Novel Therapies
Clin. Cancer Res., December 15, 2004; 10(24): 8132 - 8141.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
G. Xu, Y. Zhong, S. Munir, B. B. Yang, B. K. Tsang, and C. Peng
Nodal Induces Apoptosis and Inhibits Proliferation in Human Epithelial Ovarian Cancer Cells via Activin Receptor-Like Kinase 7
J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5523 - 5534.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. G. Shepherd and M. W. Nachtigal
Identification of a Putative Autocrine Bone Morphogenetic Protein-Signaling Pathway in Human Ovarian Surface Epithelium and Ovarian Cancer Cells
Endocrinology, August 1, 2003; 144(8): 3306 - 3314.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
Y. Fu, E. J. Campbell, T. G. Shepherd, and M. W. Nachtigal
Epigenetic Regulation of Proprotein Convertase PACE4 Gene Expression in Human Ovarian Cancer Cells
Mol. Cancer Res., June 1, 2003; 1(8): 569 - 576.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dunfield, L. D.
Right arrow Articles by Nachtigal, M. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dunfield, L. D.
Right arrow Articles by Nachtigal, M. W.
Right arrowPubmed/NCBI databases
*Substance via MeSH
Medline Plus Health Information
*Ovarian Cancer

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. <