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Molecular Cancer Research 1:569-576 (2003)
© 2003 American Association for Cancer Research

Cancer Genes and Genomics

Epigenetic Regulation of Proprotein Convertase PACE4 Gene Expression in Human Ovarian Cancer Cells1

Yangxin Fu1, Elizabeth J. Campbell1, Trevor G. Shepherd1 and Mark W. Nachtigal1

Department of Pharmacology, Dalhousie University, Halifax, Canada

Requests for reprints: Mark W. Nachtigal, Department of Pharmacology, Tupper Medical Building, Room 5B1, 5850 College Street, Dalhousie University, Halifax, Nova Scotia, B3H 1X5 Canada. Phone: (902) 494-6348; Fax: (902) 494-1388. E-mail: Mark.Nachtigal{at}Dal.Ca


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Proprotein convertases (PC) are a family of serine endoproteases that play important roles in regulating cell function by converting proproteins to biologically active molecules. Several lines of evidence suggest that overexpression of PCs contributes to tumor formation and progression in various types of cancer. In this study, we examined PC expression in six normal ovarian surface epithelium (OSE) cultures, nine primary ovarian cancer (OC) cultures, and three established OC cell lines (Hey, HeyC2, and OCC-1). Our results show that furin and PC7 expression in OC cells was comparable to that in normal OSE. However, PACE4 expression was greatly reduced in all OC samples studied. PACE4 promoter activity was measured in HeyC2 and OCC-1 cells using transiently transfected luciferase reporter plasmids. Both cell lines supported PACE4 promoter activity, showing that the transcription factors critical for PACE4 expression are present in OC cells. The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, led to the hypothesis that reduced expression may be due to epigenetic modification of the PACE4 gene, such as DNA methylation and histone deacetylation. Methylation analysis of 79 CpG dinucleotides within the PACE4 promoter and exon I (-196/+340) revealed that the percentage of methylated cytosine nucleotides was 8–9% in normal OSE, but 58–93% in OC cells. Treatment with the demethylating agent 5-aza-2'-deoxycytidine and/or the histone deacetylase inhibitor trichostatin A greatly increased PACE4 expression in OC cells. These data suggest that the reduction of PACE4 expression in OC cells is caused, in part, by DNA hypermethylation and histone deacetylation.

Key Words: PACE4 • proprotein convertase • ovarian cancer • methylation • histone deacetylation


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The proprotein convertase (PC) family of calcium-dependent serine endoproteases plays a vital role in normal cellular physiology by converting proproteins to biologically active molecules. In mammals, eight PCs have been identified and cloned: furin; PC1/3; PC2; PC4; PACE4; PC5/6; PC7; and SKI-1/SIP (reviewed in Ref. 1). PCs are related to bacterial subtilisin, yeast kex2, and the Caenorhabditis elegans blisterase serine endoproteases that cleave precursor proteins at the general motif (K/R)-(X)n-(K/R), where K = lysine, R = arginine, X is any amino acid, and n = 0, 2, 4, or 6. The most recently cloned member, SKI-1/SIP, cleaves at non-basic residues (2). These enzymes work individually or coordinately to achieve the specific cleavage and bioactivation of precursor molecules. For example, PC1/3 and PC2 activities coordinately regulate processing of proopiomelanocortin (POMC) into its hormone (adrenocorticotropin) or neuropeptide (ß endorphin) cleavage products (3), whereas pro-transforming growth factor ß (TGF-ß) is cleaved by furin (4). In vitro PC molecules can display overlapping substrate specificity, but gene ablation studies clearly demonstrate that these molecules are not completely redundant functionally. Furin, PC1, and PACE4 null mice are embryonic lethal and display distinct lethality phenotypes (5–7), whereas PC2 null mice are diabetic (8), and male PC4 null mice are infertile (9). To date, there is only one confirmed case of a human with a germ line PC defect. This female patient has genetic mutations leading to loss of active PC1/3, resulting in diabetes, obesity, and hypocortisolism, most likely due to loss of bioactivation of proinsulin and POMC (10).

There is a growing body of literature to support a role for PC molecules in human cancer (11). Bassi et al. (12) found that furin overexpression in head and neck squamous cell carcinoma (HNSCC) correlated with tumor cell invasiveness. Treatment of HNSCC cells with the furin inhibitor {alpha}1-PDX altered the malignant phenotype and produced a decrease or complete block in tumorigenicity in vivo that correlated with a decrease in matrix metalloproteinase 2 processing and activity. Similarly, furin was overexpressed 10- to 25-fold in nonsmall cell lung carcinoma (NSCLC) compared to control lung tissue (13), and both furin and PC1 expression were elevated in human breast tumors (14). In their examination of an animal model of squamous cell carcinoma (SCC) progression to spindle cell carcinoma (SPCC), Hubbard et al. (15) found that PACE4 expression was increased in SPCC cells. Transfection and overexpression of PACE4 in SCC cell lines with low invasive ability promoted their in vivo invasiveness. The most likely cause for their increased invasiveness was through the observed gain of function to process prostromelysin-3, a matrix metalloproteinase expressed in tissues undergoing active remodeling, such as tumor invasion (16). In addition to these data, it has been demonstrated that PCs process several molecules implicated in human tumorigenesis, including stromelysin-3, membrane type 1 matrix metalloproteinases, and TGF-ß (3). Thus, there are several lines of evidence to suggest that altered PC biology plays a role in human tumor formation (reviewed in Ref. 11). This has led to intensive research attempting to discover peptide, protein, or small molecule inhibitors that may be used as anti-PC chemotherapeutic agents (17–19).

In the present study, we evaluated the potential for alterations in PC biology in ovarian cancer (OC) cells. We screened PC expression in human normal ovarian surface epithelial (OSE) and OC cells and determined that one member of the human PC family, PACE4, is expressed in normal OSE cells, and this expression is greatly reduced in primary OC cells and established OC cell lines. Recently Matei et al. (20) published a report on gene expression array analysis comparing normal ovaries with epithelial ovarian carcinoma, where they show that PACE4 expression was 4.5-fold lower in OC versus normal ovarian tissue. In this paper, we show that PACE4 gene hypermethylation and histone deacetylation play a role in the reduction of PACE4 expression in human OC cells.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
PC Messenger RNA Expression in Normal OSE and OC Cells
We screened primary cultures of normal OSE and OC cells as well as established OC cell lines (Hey, HeyC2, OCC-1) for PC expression. The expression of PC7 and furin in OC cells are comparable with that in normal OSE (Fig. 1A). As expected, none of these cells expressed PC1/3 or PC2, which are primarily produced by neuroendocrine cells (data not shown; reviewed in Ref. 3). The result that was most striking, however, was the reduced expression of PACE4 mRNA in all primary human OC cell cultures and established OC cell lines as compared with normal OSE (Fig. 1A). Fold differences in expression were analyzed by Southern analysis of PACE4 reverse transcription (RT)-PCR products (Fig. 1B).



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  		FIGURE 1. Proprotein convertase mRNA expression in normal OSE, primary OC cells, and established OC cell lines. A. Proprotein convertase PACE4, furin, and PC7 mRNA expression were assessed in normal OSE (OSE1–6), primary OC cells (OC#), and the human OC cell lines (Hey, HeyC2, OCC-1) by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a PCR and gel loading control. B. The PACE4 RT-PCR products depicted in A were transferred to nylon membrane for Southern blotting. The PACE4 PCR product was detected using full-length PACE4 cDNA. Fold changes in PACE4 expression are indicated; the signal for OSE4, which expresses the lowest amount of PACE4 in the normal OSE cells, was set as 1.

 
PACE4 Promoter Activity in HeyC2 and OCC-1 Cells
To study if the reduced PACE4 mRNA expression was due to loss of PACE4 promoter activity, the PACE4 promoter and part of exon 1 (-837 to +315) was PCR amplified and cloned into a luciferase reporter plasmid (pGL2-PACE4). The relative luciferase activities of pGL2-PACE4 were 125- and 53-fold higher than the promoterless luciferase reporter (pGL2-basic) in HeyC2 and OCC-1 cells, respectively, indicating that the PACE4 promoter can be activated in these cells (Fig. 2). This result suggests that transcription factors necessary for PACE4 expression are present and able to support PACE4 promoter activity in OC cells.



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  		FIGURE 2. PACE4 promoter activity in OC cell lines. PACE4 promoter activity was examined in the established OC cell lines HeyC2 and OCC-1. Cells were transiently transfected with pGL2-basic or pGL2-PACE4 plasmids, and co-transfected with pCMV.ßgal. Luciferase activity (RLUs, relative light units) was normalized to ß-galactosidase activity and is expressed as mean fold increase in activity compared with that from untreated pGL2-basic. Data were obtained from three independent experiments.

 
Methylation Status of PACE4 Promoter and Exon I in Normal OSE and OC Cells
The observation that established OC cell lines have reduced PACE4 expression, but maintained the ability to support PACE4 promoter activity, suggested that reduced expression may be due to epigenetic modification of the PACE4 gene, such as promoter DNA methylation or histone deacetylation. The DNA sequence of the PACE4 promoter region and first exon is highly GC rich (21), and contains a CpG island that provides a putative target for aberrant alteration of chromatin architecture leading to gene silencing. Our next goal was to determine the methylation status of the endogenous PACE4 promoter and exon I CpG-rich region (-196 to +340), which was assessed by bisulfite genomic sequencing. Bisulfite treatment converts cytosine to uracil and the latter is changed to thymidine during a subsequent PCR process. However, if cytosine nucleotides are methylated, they will not be converted to uracil (22). Genomic DNA from two normal OSE, the established HeyC2 cell line, and four primary OC samples was modified by bisulfite treatment. PACE4 genomic DNA (-233/+608) was then PCR amplified and subcloned. Due to the high CpG content of the PACE4 promoter/exon 1 sequence, these primers were chosen based on their lack of CpG dinucleotide sites and their ability to hybridize to modified DNA. Eight clones from each cell sample were selected for sequence analysis and 79 CpG dinucleotides in the PACE4 gene (-196 to -25 in 5'-flanking DNA; +9 to +340 in exon I) were examined. Fig. 3A is a representative sequencing gel showing that the majority of cytosines were converted to thymidine in bisulfite-treated DNA from normal OSE. In contrast, although non-CpG cytosines were changed to thymidine (Fig. 3A), the majority of cytosines in CpG dinucleotides in the PACE4 DNA isolated from HeyC2 cells remained unchanged (Fig. 3A, arrowhead), demonstrating that these cytosines were methylated. The methylation status of the PACE4 promoter/exon I from all cells is summarized in Fig. 3B. Normal OSE had 8–9% methylation in the 79 CpG dinucleotides examined, whereas the range of methylated CpG cytosine nucleotides in OC cells was 58–93%. These data show that the PACE4 gene is hypermethylated in primary OC cells and established cell lines compared with normal OSE, suggesting that DNA hypermethylation plays a role in the reduction of PACE4 expression in OC cells.



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  		FIGURE 3. Methylation status of the endogenous PACE4 promoter. Methylation status of 79 CpG dinucleotides in the PACE4 promoter (-196/-25) and exon I (+9/+340) was determined using bisulfite genomic sequencing. A. Representative bisulfite sequencing gel indicates complete conversion of non-CpG cytosines in both normal OSE and HeyC2 cells. Methylated cytosines in CpG dinucleotides remained unchanged (arrowhead). An example of the wild-type PACE4 DNA sequence is shown with arrowheads indicating methylated CpG nucleotides, while non-CpG cytosine nucleotides have been converted to thymidine. B. Summary of bisulfite genomic sequencing in normal OSE (OSE6 and OSE7), HeyC2, and primary OC (OC4, 7, 13, and 15) cells. Genomic DNA of eight individual clones (C1–8) from each cell sample was sequenced; the methylation status is summarized as the percentage of methylated cytosine in CpG dinucleotides. Note: Only seven clones of exon I were sequenced for OSE7. Each row is one sequenced allele. Each circle is a CpG dinucleotide; closed circle, methylated; open circle, un-methylated. The relative position of 79 CpG dinucleotides in the PACE4 gene (-200 to +350) is schematically represented at the top of the figure.

 
PACE4 Gene Expression Is Greatly Increased by Demethylation and/or Inhibition of Histone Deacetylation
Epigenetic alterations of chromatin structure, including DNA hypermethylation and histone deacetylation, can contribute to gene silencing. To study whether epigenetic alterations of the PACE4 gene contribute to the reduced PACE4 mRNA expression, we initially treated HeyC2 cells with the demethylating agent 5-aza-2'-deoxycitidine (Aza), the histone deacetylase inhibitor trichostatin A (TSA), or both. After treatment, the level of PACE4 expression was quantified by Southern analysis of RT-PCR products amplified using primers specific to the PACE4 cDNA (nts. 2896–3308). If promoter/exon I hypermethylation and histone deacetylation play a role in reduction of PACE4 expression, treatment of these cells with these inhibitors should increase or restore PACE4 mRNA expression. As shown in Fig. 4A, both Aza (1 or 10 µM) and TSA (0.1 or 1 µM) treatment result in increased PACE4 expression. In addition, a synergistic effect between Aza and TSA was observed in HeyC2 cells at all doses (Fig. 4A). The effect of each drug alone was maximal at the highest concentration tested; therefore, 10 mM Aza and 1 mM TSA were used to study the effect of these inhibitors on PACE4 expression in OCC-1 cells and three primary OC cell samples. To assess whether the treatments were specific for PACE4, PC7 expression was also analyzed. Aza treatment alone increased PACE4 expression in all cells (Fig. 4B). Although TSA alone failed to increase PACE4 expression in one of these cells (OC14), a synergistic effect between Aza and TSA was observed in all cells; for example, PACE4 expression was increased 22.6-fold in OCC-1 cells. Minimal increases were observed for PC7 expression, with a 1.6-fold increase as the greatest response. These data indicate that promoter hypermethylation and histone deacetylation can play a role in the reduced expression of PACE4 in OC cells.



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  		FIGURE 4. Increase in PACE4 gene expression by treatment with a demethylating agent and/or a histone deacetylase inhibitor. A. HeyC2 cells were treated with Aza (1 or 10 µM) or TSA (0.1 or 1 µM) or combination of various doses of Aza and TSA. Cells were cultured in the medium containing Aza for 3 days, with medium changes every day. TSA was applied for the last 18 h. PACE4 expression was examined using Southern analysis of RT-PCR products (isolated after 20 PCR cycles) using the full-length PACE4 cDNA as a probe. DMSO was used as a vehicle control. Expression in untreated normal OSE (OSE3) was used as a positive control. B. OCC-1 and three primary OC cell samples (OC13–15) were untreated (-) or treated (+) with 10 µM Aza, 1 µM TSA or both, and PACE4, PC7, or GAPDH expression was examined. Expression in untreated normal OSE (OSE3) was used as a positive control.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
This report shows PC expression by RT-PCR in normal OSE and OC cells, and demonstrates the striking reduction of PACE4 expression in OC cells. Overexpression of PCs has been demonstrated in various types of cancers, including lung, breast, and skin cancers, (11–16, 23), and is related to more aggressive behavior in some model systems. Indeed, transfection of a PACE4 expression vector into immortalized non-tumorigenic or papilloma-derived keratinocytes increased their invasiveness (24). In contrast to these previous reports, we found greatly reduced PACE4 expression levels in primary and established OC cells compared with normal human OSE. These findings are supported by a recent report that compared gene expression patterns in normal ovarian versus epithelial ovarian carcinoma using microarray analysis (20). Similar to our findings, they determined that PACE4 transcripts were expressed at a significantly higher level in normal ovaries than in epithelial ovarian carcinoma (4.5-fold, P = 0.04526). Collectively, these data demonstrate that alteration of PC expression in cancer cells varies in a tumor-specific manner, and raise the possibility that reduced PC expression may be involved in human tumor biology.

To study the mechanism by which PACE4 expression is depressed in OC cells, we initially examined whether the PACE4 promoter was functional in transfected OC cell lines. Our results demonstrate that the PACE4 promoter can be activated in OC cells, suggesting that the transcriptional machinery that supports PACE4 expression remains unaltered. These data prompted us to examine whether the PACE4 expression was repressed due to epigenetic phenomena, such as DNA methylation or histone deacetylation. DNA hypermethylation and histone deacetylation are frequent epigenetic events in cancer that are associated with gene silencing (25, 26). GC-rich sequences, also known as CpG islands, are often found in promoter DNA and are targets of DNA methylation. Methyl CpG binding proteins (MBD1-1 and MeCP2) (27) can recruit histone deacetylases that act locally to produce a condensed chromatin structure that leads to transcriptional repression (25). In normal cells, CpG methylation plays an important role in regulating gene expression, whereas in cancer cells, aberrant promoter methylation or hypermethylation can lead to abnormal gene silencing, including repression of tumor suppressor genes. Studies to assess the patterns of CpG island methylation in human epithelial OCs (over 100 samples) using either methylation-specific PCR (28, 29) or differential methylation hybridization (30, 31) revealed frequently methylated CpG islands in many loci associated with human tumor formation, including the BRCA1 gene, the putative tumor suppressor gene HIC1, and MLH1. Moreover, expression of DNA methyltransferases (DNMT1 and DNMT3b) is elevated in established OC cell lines compared to normal OSE (32). These data strongly suggest that DNA methylation contribute to gene silencing in OC cells. Indeed, we found that the PACE4 promoter/exon I was hypermethylated in primary OC cells and cell lines compared to normal OSE. In addition, we found that treatment of human OC cells with demethylating agents and a histone deacetylase inhibitor greatly increased PACE4 gene expression. Treatment of cells with Aza alone enhanced PACE4 expression. However co-treatment with both Aza and TSA resulted in greatly enhanced levels of PACE4 mRNA production. Minimal effects were seen when PC7 expression was examined in the cells treated with these agents. These experiments further support the notion that both PACE4 DNA hypermethylation and histone deacetylation contribute to the reduction of PACE4 gene expression.

There are opposing views in the literature that argue the relevance of DNA methylation contributing to gene silencing when the analysis is conducted solely using cultured cells (33). Indeed, some investigations show that DNA methylation increases with increasing cell passage in vitro (34), whereas other studies indicate that gene methylation status of DNA from cultured cells closely reflects the methylation status of the original tumor material (35). Genomic DNA from all cells used for the current studies, whether primary OC or primary normal OSE cells, was harvested at early passage (1 to 2) and therefore, any alterations in DNA methylation status due to culture effects may apply equally to both normal and tumor cells. Our results indicate that there are dramatic differences between these two populations of cells grown in culture, normal OSE = 8–9% methylation compared to OC cells = 58–92% methylation. Although we have not analyzed the PACE4 gene methylation status in primary tumors, we believe that our data support the idea that DNA hypermethylation is involved in silencing PACE4 gene expression in human OC cells.

Current models suggest that PC molecules participate in the development of human cancers when they are overexpressed and cause increased production of biologically active substrates leading to tumor formation (11). In contrast, we and others (20) show that PACE4 expression is reduced in OC cells, and we hypothesize that decreased substrate activity will lead to dysregulation of normal cellular activities. Putative substrates include growth factors, hormones, receptors, matrix metalloproteinases, and adhesion molecules (11). Thus, future goals are to examine the effect of ectopic PACE4 expression in OC cells, identify PACE4 substrates in normal OSE and OC cells, and determine whether these PACE4 substrates play a role in the development of human OC.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Primary Human OSE and OC Cells
Primary human OC cells were isolated from ascites specimens as described previously (36). Briefly, ascitic fluid containing cells was mixed 1:1 with growth medium [MCDB105/M199 supplemented with 10% fetal bovine serum (Cansera, Rexdale, Ontario, Canada) and 100 units/ml penicillin G, 100µg/ml streptomycin]. After 3 days, the ascitic fluid supernatant/medium was removed and attached cells were fed fresh growth medium. The normal surface epithelial cells from solid ovarian specimens were isolated by a method combining two protocols (37, 38). The surface of the ovary was treated with Dispase II (Roche Molecular Biochemicals, Laval, Quebec, Canada) for 30 min at 37°C with occasional agitation. The OSE cells were gently scraped directly into the same growth medium as that used for primary OC cells and allowed to adhere and grow for 4 days. All experiments using normal primary OSE were performed at culture passages 2–4 and passages 1–4 for OC cells.

Cell Lines
Established human OC cell lines (Hey, HeyC2, and OCC-1) were grown in monolayer and maintained in RPMI (Invitrogen/Gibco, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum.

RT-PCR Analysis
Total cytoplasmic RNA for RT-PCR was isolated from normal OSE, primary OC cells, and cell lines using the Genelute Mammalian Total RNA kit (Sigma-Aldrich, Oakville, Ontario, Canada). RT-PCR was used to detect expression of PCs (35 s at 94°C, 35 s at 59.5°C, 35 s at 72°C, 25 cycles for PACE4, PC7, and GAPDH; 30 cycles for furin). Cycles were chosen so that the PCR reaction produced amplicons within the linear range; determined by comparison of PCR signal at 20–35 cycles. cDNA fragments of human furin (nts. 847–1171), PACE4 (nts. 2896–3308), and PC7 (nts. 943–1323) were subcloned into pCRII.TOPO (Invitrogen Canada Inc., Burlington, Ontario, Canada) and the identity of the amplified fragment was verified by manual sequencing using a T7 DNA polymerase sequencing kit (Amersham Biosciences Corp., Baie d'Urfe, Quebec, Canada).

PACE4 Promoter Constructs and Luciferase Assay
A 1150-bp PACE4 gene fragment (-837 to +315), encompassing the promoter and part of exon 1, was PCR amplified using the GC Rich Amplification system (Roche Molecular Biochemicals, Laval, Quebec, Canada) from a BAC clone containing part of human chromosome 15 (RPCI-11, Research Genetics, Invitrogen Corp., Huntsville, AL); PACE4 is localized to human chromosome 15q26. This DNA fragment was cloned into pCRII.TOPO (Invitrogen Canada) and sequence identity verified by automated sequencing (Cortec DNA Services Laboratories, Inc., Kingston, Ontario, Canada). The 1150-bp PACE4 DNA fragment was then subcloned into the pGL2-Basic luciferase reporter plasmid (Promega Corp./Fisher Scientific, Nepean, Ontario, Canada) at XhoI and HindIII sites to generate pGL2-PACE4. For luciferase assays, HeyC2 or OCC-1 cells were seeded in a 12-well plate at 4 x 104 cells/well in triplicate, cultured overnight, and transfected with 300 ng pGL2-PACE4 or pGL2-basic and co-transfected with 300 ng pCMV.ßGal using FuGENE transfection reagent (Roche Molecular Biochemicals). Cells were harvested after 48 h of transfection and luciferase activity was determined using the Enhanced luciferase assay kit (BD PharMingen, Mississauga, Ontario, Canada). Results are normalized with ß-galactosidase activity, and shown as means of three independent experiments.

Bisulfite Modification of Genomic DNA
DNA bisulfite modification was performed according to the method described by Grunau et al. (22) with minor modifications. Genomic DNA was isolated from cells according to the method described in Ref. (39). Briefly, genomic DNA (5 µg) was denatured in 0.3 M NaOH for 20 min at 42°C in 10 µl volume, mixed with 120 µl freshly prepared bisulfite solution (Sigma-Aldrich; ~4 M sodium bisulfite, pH 5.0, containing 10 mM hydroquinone), and incubated at 55°C for 4 h in a thermocycler (Techne Incorp., Princeton, NJ). The bisulfite-treated DNA was then desalted using a Wizard DNA Clean-Up Kit (Promega) and eluted in 100 µl H2O. DNA was then desulfonated in the presence of 0.3 M NaOH at 37°C for 20 min, neutralized by addition of 0.1 volume of 3 M NaOAc (pH 5.2), and precipitated with 2.5 volumes of 100% ethanol overnight at -20°C. Precipitated DNA was then washed with 70% ethanol, air-dried, dissolved in H2O, and used for PCR amplification or stored at -20°C.

Bisulfite Sequencing
After bisulfite modification, the PACE4 promoter and exon I sequences from normal OSE and OC cells were amplified by PCR using the following primers (sense, 5'-AATGTGTTTTTTTAGTTAGGTTTGGTT-3', -233/-198, and antisense, 5'-CACTCACCTAACCCAAATTAAAATACC-3', +589/+608). Primers were chosen based on their lack of CpG dinucleotide sequences. PCR was performed with the GC Rich Amplification system (Roche Molecular Biochemicals) in a 25-µl volume according to the manufacturer's instructions. PCR conditions were as follows: 1 cycle, 5 min at 95°C; 35 cycles, 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C; and 1 cycle, 10 min at 72°C. PCR products were gel purified and cloned into pCRII-TOPO according to the manufacturer's instructions. Plasmid DNA from individual transformed bacterial clones was purified using a QIAprep Spin Miniprep kit (Qiagen Inc., Mississauga, Ontario, Canada), and manually sequenced to examine promoter/exon I methylation status. Methylation status is expressed as percentage of methylated cytosines per total CpG dinucleotides (methylation (%) = methylated cytosines/total cytosines in CpG dinucleotides x 100).

Aza and TSA Treatment
HeyC2 and OCC-1 cells were seeded at 3 x 105 cells in 10-cm dishes. After culturing overnight, cells were treated with 1 or 10 µM Aza for 3 days. Cells were fed fresh medium containing Aza every day. TSA at 0.1 or 1 µM was applied alone or in combination with Aza for the last 18 h of culture. Primary OC cells were treated with 10 µM Aza for 6 days (due to their slow rate of doubling compared to OC cell lines) and 1 µM TSA was included for the last 18 h of culture. DMSO was used as a vehicle control. Total RNA was isolated for RT-PCR analysis of PACE4 and PC7 mRNA expression. The PCR reaction was terminated after 20 cycles, and PCR products were separated on a 1.5% agarose gel and transferred to BrightStar Plus membrane (Ambion Inc., Austin, TX). Blots were incubated with a radiolabeled full-length PACE4 or PC7 (nts. 943–1323) cDNA probes (1 x 106 cpm/ml) overnight at 42°C in ULTRAhyb buffer (Ambion) and washed at 42°C in wash solution (0.1% SDS, 0.1x SSC). Signals were visualized by autoradiography, scanned, and quantified using NIH Image 1.62. GAPDH mRNA was used as a positive control for PCR and used to normalize PACE4 and PC7 expression.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
The authors thank Drs. J. Blay, C. Sinal, P. Murphy, D.C. Lagace, and L.D. Dunfield for critical reading of this manuscript, Drs. R. Grimshaw and J. Bentley (QEII Health Science Centre) for providing human ovarian tumor samples, and Dr. T.F. Baskett and the staff of the QEII Ob/Gyn Unit for providing normal human ovarian tissue.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Canadian Institutes of Health Research (MT-15438); Nova Scotia Health Research Foundation Fellowship (Y.F.); CaRE Fellowship with funding from the Canadian Cancer Society (T.G.S.). Note: M.W.N. is a Canadian Institutes of Health Research Scholar. Back

Received October 22, 2002; revised April 1, 2003; accepted April 8, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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