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. 2006 Apr;8(4):268–278. doi: 10.1593/neo.05502

Candidate Tumor-Suppressor Gene DLEC1 Is Frequently Downregulated by Promoter Hypermethylation and Histone Hypoacetylation in Human Epithelial Ovarian Cancer1

Joseph Kwong *, Ji-Young Lee *, Kwong-Kwok Wong †, Xiaofeng Zhou ‡, David T W Wong ‡, Kwok-Wai Lo §, William R Welch , Ross S Berkowitz *, Samuel C Mok *
PMCID: PMC1600675  PMID: 16756719

Abstract

Suppression of ovarian tumor growth by chromosome 3p was demonstrated in a previous study. Deleted in Lung and Esophageal Cancer 1 (DLEC1) on 3p22.3 is a candidate tumor suppressor in lung, esophageal, and renal cancers. The potential involvement of DLEC1 in epithelial ovarian cancer remains unknown. In the present study, DLEC1 downregulation was found in ovarian cancer cell lines and primary ovarian tumors. Focus-expressed DLEC1 in two ovarian cancer cell lines resulted in 41% to 52% inhibition of colony formation. No chromosomal loss of chromosome 3p22.3 in any ovarian cancer cell line or tissue was found. Promoter hypermethylation of DLEC1 was detected in ovarian cancer cell lines with reduced DLEC1 transcripts, whereas methylation was not detected in normal ovarian epithelium and DLEC1-expressing ovarian cancer cell lines. Treatment with demethylating agent enhanced DLEC1 expression in 90% (9 of 10) of ovarian cancer cell lines. DLEC1 promoter methylation was examined in 13 high-grade ovarian tumor tissues with DLEC1 downregulation, in which 54% of the tumors showed DLEC1 methylation. In addition, 80% of ovarian cancer cell lines significantly upregulated DLEC1 transcripts after histone deacetylase inhibitor treatment. Therefore, our results suggested that DLEC1 suppressed the growth of ovarian cancer cells and that its downregulation was closely associated with promoter hypermethylation and histone hypoacetylation.

Keywords: DLEC1, chromosome 3p22.3, promoter hypermethylation, histone hypoacetylation, epithelial ovarian cancer

Abbreviations: DLEC1, Deleted in Lung and Esophageal Cancer 1; HOSE, human ovarian surface epithelium; LOH, loss of heterozygosity; CNA, copy number abnormality; MSP, methylation-specific PCR; ChIP, chromatin immunoprecipitation; AZA, 5-aza-2′-deoxycytidine; TSA, Trichostatin A

Introduction

Ovarian cancer ranks as the fifth leading cause of cancer deaths in women and has the highest mortality rate among gynecologic malignancies in the United States [1]. Although ovarian cancer susceptibility genes (e.g., BRCA1, BRCA2, and MSH2) have been identified [2,3], most cases are sporadic and somatic mutations in familial ovarian cancer genes are not a major feature of sporadic cancers. Loss of heterozygosity (LOH) involving several chromosome 3p regions, accompanied by chromosome 3p deletions, is frequently detected in many common sporadic cancers, including lung, breast, cervical, kidney, and head and neck cancers [4]. These 3p genetic alterations lead to the assumption that the short arm of human chromosome 3 may harbor several tumor-suppressor genes. In ovarian cancer, LOH on chromosome 3p has also been reported to occur at a frequency ranging from 38% to 52% [5’8], and several common LOH regions on chromosome 3 have been mapped, including 3p12-p13, 3p14.2, 3p21.1-p22, 3p24-p25, and 3p25-p26 [6–8]. Functional study demonstrated that microcell-mediated chromosomal transfer of chromosome 3 into an ovarian cancer cell line induced senescence, growth arrest, and suppression of tumorigenicity [9]. Multiple lines of evidence suggested that chromosome 3p might harbor tumor-suppressor genes in ovarian cancer. However, FHIT and VHL, two well-known tumor-suppressor genes located on 3p, are not commonly inactivated in ovarian carcinogenesis [10,11]. RASSF1A, located on 3p21.3, has been suggested as a new candidate tumor-suppressor gene in human cancers. Epigenetic silencing of RASSF1A was detected in 40% of ovarian cancer cell lines and tumor tissues [12–14]. These data suggested that RASSF1A is one of 3p candidate tumor suppressors in ovarian cancer. Recently, a gene located on chromosome 3p22.3, named Deleted in Lung and Esophageal Cancer 1 (DLEC1), has been discovered as a candidate tumor-suppressor gene in lung, esophageal, and renal cancers [15]. The DLEC1 gene contains 37 exons and spans approximately 59 kb. The predicted DLEC1 protein contains 1755 amino acids. However, its exact biologic function is still unclear because the predicted amino acid sequence of DLEC1 has no significant homology to any of the known proteins or domains [15]. Here we speculate the possible involvement of DLEC1 in ovarian cancer pathogenesis by evaluating the tumor-suppressing activity of DLEC1 and by delineating the mechanisms that are related to DLEC1 downregulation in ovarian cancer.

Materials and Methods

Cell Lines, Cultures, and Tumor Specimens

Five primary cultures of normal human ovarian surface epithelium (HOSE) cells (HOSE2177, HOSE0706, HOSE2105, HOSE2107, and HOSE2170) were used in this study. These ovarian epithelial cells are scraped from the surface of the ovary in patients with benign gynecologic diseases. Five HOSE samples (HOSE1912, HOSE2085, HOSE2225, HOSE2237, and HOSE2270) were collected directly from ovarian epithelial brushing. Twelve ovarian cancer cell lines (CaOV3, DOV13, MCAS, OVCA3, OVCA420, OVCA429, OVCA432, OVCA433, OVCA633, PEO4, SKOV3, and TOV112D) were used. All ovarian cancer cell lines were obtained either by recovery of material from ascites or by procurement of explanted tissue from solid tumors, as described previously [16]. They were either established in the Laboratory of Gynecologic Oncology, Brigham and Women's Hospital (Boston, MA), or purchased from the American Type Culture Collection (Rockville, MD) and the Japanese Collection of Research Bioresources (Tokyo, Japan). In addition, 91 cases of primary ovarian tumor tissues [25 serous tumors with low malignant potential (LMP), 6 low-grade (grade 1, late stage) serous carcinomas, and 60 high-grade (grades 2 and 3, late stage) serious carcinomas] were included in the study. All patient-derived specimens were collected and archived under protocols approved by the Institutional Review Board.

Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (PCR)

Two micrograms of total RNA obtained from 5 primary cultures of HOSE cell lines and 12 ovarian cancer cell lines was reverse-transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Fifteen nanograms of total RNA obtained from 91 microdissected ovarian tumor tissues and 5 HOSE-scraped samples was amplified and reverse-transcribed by the Ovation Aminoallyl RNA amplification and labeling system (NuGEN Technologies, Inc., San Carlos, CA). The PCR primers used were 5′-GAC GAA GTG AGC GCA AGC-3′ (forward) and 5′-ATC CAG CCG CTG CTT ATA GA-3′ (reverse) for DLEC1, and 5′-CGC GAG AAG ATG ACC CAG AT-3′ (forward) and 5′-GTA CGG CCA GAG GCG TAC AG-3′ (reverse) for β-actin. Using SYBP Green PCR master mix (Applied Biosystems), PCR products were analyzed by the 7300 real-time PCR system (Applied Biosystems). All reactions were performed in triplicate. The relative DLEC1 mRNA level of each sample was normalized with β-actin and calculated using 2(-ΔΔCT) [17] by comparing the amount of mRNA of each sample to a HOSE sample.

Anchorage-Dependent Colony Formation Assay

Anchorage-dependent colony formation assay was performed by pcDNA3.1+ transfection of either a plasmid containing full-length DLEC1 cDNA (a gift from Dr. Y Nakamura) [15] or an empty vector (pcDNA3.1+) in ovarian cancer cells OVCA429 and OVCA432 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After transfection, cells were then split and grown in medium containing 500 µg/ml Geneticin (Invitrogen) for 10 days. Colonies were stained with crystal violet and counted. The transfection experiment was performed in triplicate and repeated twice.

Concurrent Analysis of LOH and Copy Number Abnormality (CNA) on Chromosome 3

LOH and CNA of chromosome 3 in ovarian cancer cell lines were analyzed by the Affmetrix 10K SNP mapping array [18]. Briefly, genomic DNA of 11 human ovarian cancer cell lines was isolated using a Qiagen DNeasy kit (Qiagen GmbH, Hilden, Germany). A total of 250 ng of purified DNA was digested with XbaI, ligated to an adaptor, PCR-amplified, fragmented, and biotin-labeled according to the Affmetrix Single Primer Assay protocol. Subsequently, biotin-labeled DNA was hybridized to the Affmetrix 10K SNP mapping array. Image acquisition and data analysis were performed as previously described [18].

Determination of DLEC1 Copy Number by Real-Time Quantitative PCR

Genomic DNA (100 ng/µl) of peripheral blood lymphocytes (PBL) from healthy subjects, HOSE, and ovarian cancer cell lines were used to determine DNA copy number by quantitative PCR. The primers for the DLEC1 locus (STS marker SHGC-170056) were 5′-AGT GGG GAT TCA GCA GTG AAT AA-3′ and 5′-ATG TGA GTC TGA AGT TCA AGG GC-3′. Using SYBP Green PCR master mix (Applied Biosystems), PCR products were analyzed by the 7300 real-time PCR system (Applied Biosystems). Relative gene copy numbers were derived using the formula: 2ΔCT, where ΔCT is the difference in amplification cycles required to detect amplification products from equal starting concentrations of normal genomic DNA compared to tumor genomic DNA [19]. All assays were performed in triplicate. Quantitative PCR results were normalized to the D2S385 locus, which was chosen because it was located in a region of the genome that did not show any alterations in human ovarian tumors [20]. The primers for D2S385 were 5′-AGC TGT CAG TAG AAA TAA GCA GAG A-3′ and 5′-TCA ATA ACA CGC CAA AAG AC-3′. Besides D2S385, Line-1 loci (M80343) on the genome also served as controls for normalization. The primers for the Line-1 gene were 5′-AAA GCC GCT CAA CTA CAT GG-3′ and 5′-TGC TTT GAA TGC GTCCCAGAG-3′ [21].

Methylation-Specific PCR (MSP)

CpG islands of the DLEC1 promoter were identified by CpG Island Searcher (http://ccnt.hsc.usc.edu/cpgislands/) using the following criteria: GC content >55%, ObsCpG/ExpCpG >0.65, and minimum length >500 bp. Genomic DNA were bisulfite-modified by CpGenome DNA Modification kit (Chemicon International, Temecula, CA). MSP was performed on bisulfite-modified DNA using primer sequences for methylated reactions [5′-GAT TAT AGC GAT GAC GGG ATT C-3′ (forward) and 5′-ACC CGA CTA ATA ACG AAA TTA ACG-3′ (reverse)] and unmethylated reactions [5′-TGA TTA TAG TGA TGA TGG GAT TTG A-3′ (forward) and 5′-CCC AAC TAA TAA CAA AAT TAA CAC C-3′ (reverse)]. The annealing temperature of both methylated and unmethylated reactions was 60 °C, and both PCR products were 197 bp in size.

Bisulfite Sequencing

The bisulfite-modified DNA of ovarian cancer cell lines was prepared for bisulfite sequencing. To obtain products for sequencing, PCR amplification was performed on 100 ng of bisulfite-modified genomic DNA. The PCR primers used were 5′-ATT TTT AAA AGG ATA ATG TTG AAG ATA TA-3′ (forward) and 5′-TAA ACT AAC TAA AAC TAC TAA ACC C-3′ (reverse). The annealing temperature was 58°C, and the PCR product size was 255 bp. Eighteen CpG sites were located within the PCR product. Amplification fragments were cloned. Eight to 10 clones of each sample were sequenced by the Biopolymers Facility at Harvard Medical School (Boston, MA).

5-Aza-2′ -Deoxycytidine (AZA) and Trichostatin A (TSA) Treatment

For AZA treatment, ovarian cancer cells, which showed DLEC1 downregulation, were incubated in 0 and 1 µM AZA for 4 days (Sigma-Aldrich, Inc., St. Louis, MO). The medium and the drug were replaced every 24 hours. For TSA treatment, the cells were incubated with 0 and 500 ng/ml TSA (Sigma-Aldrich, Inc.) for 24 hours. After AZA and TSA treatments, total RNA and genomic DNA were collected.

Chromatin Immunoprecipitation (ChIP)

ChIP assays were carried out using the ChIP assay kit (Upstate, Charlottesville, VA). Briefly, chromatin in TSA-treated or untreated ovarian cancer cells was crosslinked by adding formaldehyde directly to the culture medium to a final concentration of 1% and incubated for 10 minutes at 37°C. Cells were washed twice using ice-cold phosphate-buffered saline with protease inhibitors. The cells were scraped, collected, and pelleted for 4 minutes at 2000 rpm at 4°C. Cell pellets were resuspended to 1 x 106 cells/200 µl of sodium dodecyl sulfate lysis buffer with protease inhibitors and incubated on ice for 10 minutes. Lysates were sonicated to shear DNA to an average length between 200 and 1000 bp. Then the samples were centrifuged for 10 minutes at 13,000 rpm at 4°C, and the supernatant was transferred to a new tube. A sonicated cell supernatant was diluted 10-fold in ChIP dilution buffer with protease inhibitors. A diluted cell supernatant (2 ml) was precleared with 75 µl of salmon sperm DNA/protein A agarose 50% slurry for 30 minutes at 4°C with agitation. Agarose was pelleted by brief centrifugation, and the supernatant was transferred into a new tube. Primary antibodies (anti-acetyl-histones H3 and H4, catalog nos. 06-599 and 06-866; Upstate) were added to the precleared 2-ml supernatant and incubated overnight at 4°C. Antibody/histone complexes were collected by adding 60 µl of salmon sperm DNA/protein A agarose slurry for 1 hour at 4°C with rotation. The agarose was pelleted by gentle centrifugation.

The protein A agarose/antibody/chromatin complex was further washed by a series of washing buffers. Immunocomplexes were eluted and reverse-crosslinked. Immunoprecipitated DNA was recovered by phenol/chloroform extraction and analyzed by PCR. The primer pairs used for ChIP analysis of the DLEC1 promoter region were 5′-ACA ATG ACC ACA GCG ATG AC-3′ (forward) and 5′-GCT GTA GTG GAA GGC CTC AG-3′ (reverse). PCR was performed for 30 to 33 cycles, the annealing temperature was 65°C, and the PCR product size was 275 bp.

Statistical Analysis

Quantitative PCR data were compared using either nonparametric Mann-Whitney U test or Kruskal-Wallis test. The level of critical significance was set at P < .05. All analyses were performed using SPSS version 10.0 (SPSS, Inc., Chicago, IL).

Results

DLEC1 Downregulation in Epithelial Ovarian Cancers

DLEC1 has been implicated as a candidate tumor suppressor located on chromosome 3p22.3 and has been shown to be downregulated in lung, esophageal, and renal cancers [15]. We attempted to examine the expression level of DLEC1 mRNA in normal HOSE cell lines, ovarian cancer cell lines, and ovarian tumor tissues. Because there are at least six alternative transcripts of DLEC1 in normal and cancer cells [15], we designed a primer set that flanks exons 1 and 2 and detected all DLEC1 transcripts. DLEC1 expression levels in ovarian cancer cell lines are lower than those in normal HOSE cell lines (P = .027) (Figure 1A). DLEC1 downregulation was also found in serous tumors of LMP, low-grade serous cancers, and high-grade serous cancers (Figure 1B). DLEC1 expression levels were significantly different among normal HOSE tumors, LMP tumors, low-grade serous cancers, and high-grade serous cancers (P = .001) (Figure 1C); between HOSE and LMP tumors (P = .009); and between HOSE and high-grade serous cancers (P = .001). These results demonstrated that DLEC1 was downregulated in a majority of borderline and invasive epithelial ovarian cancers.

Figure 1.

Figure 1

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(A) Relative quantification of DLEC1 mRNA in normal HOSE cell cultures and ovarian cancer cell lines. Experiments were performed in triplicate. DLEC1 downregulation was observed in ovarian cancer cell lines compared with HOSE cell lines (P = .027). The referent was HOSE2177, which was considered to have a value of 1. (B) Relative quantification of DLEC1 mRNA in HOSE samples and primary ovarian epithelial tumor tissues. DLEC1 expression was reduced in a majority of primary ovarian tumors. The referent was HOSE1912, which was considered to have a value of 1. (C) Relative quantification of DLEC1 among HOSE tumors, serous tumors of LMP, low-grade serous tumors, and high-grade serous tumors. The box is bounded above and below by the 75th and 25th percentiles, and the median is indicated by the line in the box. Expression differences were obtained between 5 HOSE, 25 LMP, 6 low-grade, and 60 high-grade tumors by Kruskal-Wallis test (P = .001).

DLEC1 Reexpression Reduced Tumor Growth in Ovarian Cancer Cells

The growth-inhibitory effect of DLEC1 in ovarian cancer was examined by anchorage-dependent colony formation assay. Two ovarian cancer cell lines (OVCA429 and OVCA432) were tested. After transfection of the full-length DLEC1 cDNA or the vector alone into OVCA429 cells, DLEC1 mRNA was detected at a significantly higher level in DLEC1 transfectants compared with mock transfectants (Figure 2A). Colony formation assay showed that the number of colonies formed in DLEC1-expressing OVCA429 and OVCA432 cells was significantly reduced by 51.8% and 41.1%, respectively, when compared to that of mock transfectants (Figure 2, B and C). The results demonstrated that DLEC1 reexpression reduces the growth of ovarian cancer cells.

Figure 2.

Figure 2

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(A) Relative quantification of DLEC1 after full-length DLEC1 cDNA transfection in the OVCA429 cell line. The DLEC1 mRNA level was significantly higher in transfectant cells with pcDNA3.1+/DLEC1 when compared to empty vector control (pcDNA3.1+) and water control in the Lipofectamine transfection experiment (H2O control). The referent was HOSE2177, which was considered to have a value of 1. (B) Anchorage-dependent colony formation assay using the OVCA429 cell line. The photograph showed dishes containing cells transfected either with DLEC1 (pcDNA3.1+/DLEC1) or with an empty vector (pcDNA3.1+). After G418 selection for 10 days, cells were stained and counted. (C) The number of colonies formed between transfectant cells with DLEC1 (pcDNA3.1+/DLEC1) and an empty vector (pcDNA3.1+). Each experiment was repeated thrice. The fraction of colonies (in percent) in each disc compared with vector-transfected cells is indicated.

Concurrent Analysis of LOH and CNA of Chromosome 3

DLEC1 downregulation may be due to the loss of chromosome 3p22.3. To prove it, both LOH and CNA on chromosome 3 were analyzed in 11 human ovarian cancer cell lines using the Affymetrix 10K SNP mapping array. An LOH region spanning 3p22.2-3p23 was detected in 7 of 11 (63.6%) ovarian cancer cells. However, no copy number change on the region was found in any ovarian cancer cell line compared to that of normal HOSE cultures (Figure 3A). By real-time quantitative PCR, no copy change of the DLEC1 locus (SHGC-170056) was detected in any of the ovarian cancer cell lines (Figure 3B). We also performed LOH and CNA studies on three primary ovarian tumor tissues (OVCA tumors 633, 656, and 702). Although the LOH of chromosomes 3p22-3p23 was detected in an ovarian tumor tissue, no chromosomal loss on 3p22.3 was observed in our CNA data (Figure 3C). Therefore, our results demonstrated that DLEC1 loci maintained two copies in ovarian cancer cell lines and tissues. That will lead to our suggestion that DLEC1 downregulation in ovarian tumors is not due to chromosomal loss of 3p.

Figure 3.

Figure 3

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(A) Concurrent analysis of LOH and CNA on chromosome 3 in 11 ovarian cancer cell lines. Left panel: SNP markers of chromosome 3 examined in this study. Middle panel: Results of the LOH study: each column represents one cell line, and each row represents an SNP marker. Color codes: blue for LOH, yellow for retained, grey for uninformative, and white for no call. Right panel: The CNA study: copy number changes were represented by different intensities of red. The arrow indicates the location of the DLEC1 gene. (B) Analysis of the copy number on DLEC1 loci by quantitative PCR. The STS marker SHGC-170056 on chromosomal 3p22.3 was analyzed and normalized by microsatellite marker D2S385 and Line-1 locus. PBL DNA from healthy subjects. No deletion on SHGC-170056 was found in HOSE and ovarian cancer cell lines. (C) Concurrent analysis of LOH and CNA on chromosome 3 in three primary ovarian tumor tissues.

Promoter Hypermethylation and Histone Hypoacetylation Contribute to DLEC1 Downregulation in Ovarian Cancer

Because CpG island was found in 5′ UTR and exon 1 of DLEC1 (Figure 4A), we hypothesized that promoter hypermethylation may be the mechanism used to repress DLEC1 expression in ovarian cancer. To test this hypothesis, the methylation status of the DLEC1 promoter among HOSE and ovarian cancer cell lines was evaluated by MSP. CpG methylation was detected in the DLEC1 promoter in ovarian cancer cell lines with reduced DLEC1 transcripts: complete methylation of the DLEC1 promoter (only methylated alleles) was detected in cell lines CaOV3, OVCA3, OVCA429, OVCA432, OVCA633, SKOV3, and TOV112D, and partial methylation (both methylated and unmethylated alleles) was detected in cell lines DOV13, OVCA420, and OVCA433. DLEC1 methylation was not detected in normal HOSE cultures (HOSE2105, HOSE2170, HOSE2177, HOSE2107, and HOSE0706) and DLEC1-expressing cancer cell lines (MCAS and PEO4) (Figures 1A and 4A). DLEC1 methylation patterns were further confirmed by bisulfite sequencing on several ovarian cancer cell lines. Dense DLEC1 methylation was observed in OVCA432, OVCA633, and OVCA433 cells; partial methylation was observed in OVCA420 cells; and rare methylation was detected in MCAS and PEO4 cells (Figure 4B).

Figure 4.

Figure 4

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(A) Promoter methylation of DLEC1 in HOSE and ovarian cancer cell lines. The schematic diagram illustrated a CpG island on the DLEC1 promoter. Grey box, exon 1 of the DLEC1 gene; vertical lines, CpG dinucleotides; (+1), transcription initiation site; solid black line, CpG island; arrows, primer locations for MSP. The PCR products in lane M indicate the presence of methylated alleles; the PCR products in lane U show the presence of unmethylated alleles. In vitro methylated DNA served as methylated control. (B) Genomic bisulfite sequencing of the DLEC1 5′ CpG island in six representative ovarian cancer cell lines. Eighteen CpG dinucleotides were analyzed in the 5′ promoter and exon 1 of the DLEC1 gene (represented as 18 columns of circles). Eight to 10 sequences were analyzed for each sample. Each row represents an individual sequence. Open circles, unmethylated CpG sites; closed circles, methylated CpG sites. mRNA expression levels of DLEC1 on those six ovarian cancer cell lines are indicated on the right panel.

In the treatment of ovarian cancer cell lines that showed DLEC1 downregulation with demethylating agent (AZA), 90% (9 of 10; except the cell line DOV13) showed significant upregulation of the DLEC1 transcript after AZA treatment (Figure 5A). Partial demethylation of DLEC1 promoters was also detected in representative ovarian cancer cell lines (CaOV3 and OVCA3) after AZA treatment (Figure 5B). These findings again confirmed that DLEC1 downregulation is mediated by promoter hypermethylation.

Figure 5.

Figure 5

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(A) Relative quantification of DLEC1 in ovarian cancer cell lines after AZA treatment. Ninety percent (9 of 10) of ovarian cancer cell lines (except DOV13) showed a significant upregulation of the DLEC1 transcript after AZA treatment. The referent was HOSE2177, which was considered to have a value of 1. (B) MSP results of DLEC1 in representative ovarian cancer cell lines (CaOV3 and OVCA3) after AZA treatment. An unmethylated sequence was detected in AZA-treated cells (1 µM). (C) MSP results of the DLEC1 promoter in 14 high-grade ovarian tumor tissues. (D) The bar chart is combined with both relative quantification of DLEC1 expression and methylation status of the DLEC1 promoter in primary ovarian tumor tissues. M represents detected DLEC1 promoter methylation; U represents detected unmethylated DLEC1 promoter.

DLEC1 methylation was also investigated in 14 high-grade ovarian tumor tissues. For tumor tissues expressing DLEC1 (G3S3-393), no CpG methylation was found in the DLEC1 promoter. Among the 13 tumor tissues that showed DLEC1 downregulation, 54% (7 of 13) demonstrated DLEC1 promoter methylation. However, 46% (6 of 13) showed unmethylation on the DLEC1 promoter (Figure 5, C and D). These results suggested that other epigenetic alterations might contribute to DLEC1 downregulation in ovarian cancer.

Histone acetylation was suggested to be another possible epigenetic alteration contributing to DLEC1 downregulation [22]. To evaluate that, ovarian cancer cell lines were treated with the histone deactylase (HDAC) inhibitor Trichostain A (TSA). After TSA treatment, significant upregulation of DLEC1 was observed in 80% (8 of 10) of ovarian cancer cell lines (DOV13, OVCA420, OVCA429, OVCA432, OVCA433, OVCA633, SKOV3, and TOV112D) (Figure 6A). By ChIP analysis, an enhanced association between acetylated histone (H3 and H4) and the DLEC1 promoter was found in representative ovarian cancer cell lines (DOV13, OVCA420, and OVCA433) (Figure 6B). These data indicated that histone hypoacetylation is another epigenetic mechanism used to suppress DLEC1 expression in ovarian cancers.

Figure 6.

Figure 6

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(A) Reexpression of DLEC1 in ovarian cancer cell lines after TSA treatment. Eighty percent (8 of 10) of ovarian cancer cell lines (DOV13, OVCA420, OVCA 420, OVCA432, OVCA433, OVCA633, SKOV3, and TOV112D) showed a significant upregulation of DLEC1 expression after TSA treatment. The referent was HOSE2177, which was considered to have a value of 1. (B) ChIP analysis of the 5′ region of DLEC1. Genomic DNA associated with acetylated histones H3 and H4 was prepared by immunoprecipitation using antibodies specific to acetylated histones H3 and H4. The reaction without antibody immunoprecipitation (no antibody) served as negative control. Enhancement of association between acetylated histones (H3 and H4) and the DLEC1 promoter was observed in representative ovarian cancer cell lines (DOV13, OVCA420, and OVCA433) by treatment with 500 ng/ml TSA for 24 hours.

Discussion

The short arm of chromosome 3 was thought to harbor tumor-suppressor genes in human cancers. However, only a limited number was found to be related to epithelial ovarian cancers. Here, we investigated whether DLEC1 (located on 3p22.3) was one of the candidate tumor-suppressor genes in ovarian cancer.

The tumor-suppressing properties of DLEC1 were supported by studies showing aberrant expression of DLEC1 in several human cancers, including lung, esophageal, and renal tumors [15]. By sequence analysis, six alternatively spliced transcripts of DLEC1 were identified in normal lung, esophageal, and kidney tissues. However, all these six alterative spliced transcripts were likely to encode nonfunctional proteins. In cancer cell lines and tumor samples, DLEC1 expression patterns were varied. Some tumor samples did not express any DLEC1 transcripts, whereas some of them expressed aberrant, but not normal, transcripts [15]. In the present study, our primer sets were flanking exons 1 and 2 of DLEC1, which could detect all alterative transcripts of the gene. We found that the expression level of DLEC1 transcripts (including the normal DLEC1 transcript) in ovarian cancer cell lines and tumor tissues was lower than that in normal HOSE samples. That would lead to our hypothesis that DLEC1 may be the tumor suppressor in ovarian cancer.

To prove this hypothesis, DLEC1 overexpression and anchorage-dependent colony formation assay were tested on two ovarian cancer cell lines. The number of colonies formed in DLEC1 transfectants was significantly lower than that of mock transfectants, which showed that DLEC1 suppressed the growth of ovarian cancer cells. Although we noted that the overexpression of any gene in the cells could cause growth suppression, we suggest that DLEC1 exhibits a certain feature as a tumor suppressor in ovarian cancer because a similar tumor-suppressing effect of DLEC1 was also reported in lung, esophageal, renal, and nasopharyngeal cancers [15] (K. W. Lo, personal communication). To further confirm the tumor-suppressor role of DLEC1, DLEC1 knockdown experiments by siRNA in normal HOSE cell lines are necessary.

Loss of chromosome 3p22.3 was found in several tumors, including cervical, renal, lung, and breast tumors [23,24]. Surprisingly, we have not found any loss of chromosome 3p22.3 in ovarian cancer cell lines and tumor tissues, although LOH was detected in some samples. Our previous study also demonstrated that no chromosome 3p22.3 copy number change was found in microdissected serous ovarian cancer tissues using a cDNA microarray platform [25]. Thus, our CNA data showed that two copies of the DLEC1 gene are maintained in ovarian cancer. It is possible to speculate that DLEC1 downregulation is not due to chromosomal loss of 3p22.3.

Epigenetic modifications on the promoter are related to transcriptional regulation of the gene. Promoter hypermethylation is associated with transcriptional repression of tumor-suppressor and cancer-related genes [26]. Aberrant methylation of multiple cancer-related genes (such as BRCA, RASSF1A, and OPCML) is a frequent event in epithelial cancer compared with normal ovarian surface epithelium [27–29]. Among those methylated genes, promoter methylation of the hMLH1 is critical to the loss of hMLH1 expression and drug resistance in ovarian cancer [30]. In this study, we have clearly demonstrated that the loss of DLEC1 expression in ovarian cancer is closely associated with promoter hypermethylation. Because hMLH1 is also located on chromosome 3p22.3, it is possible to speculate that promoter hypermethylation is a critical mechanism used to silence tumor-suppressor genes, which are located on 3p22.3 in ovarian cancer.

Histone hypoacetylation is another mechanism that cooperates with DNA methylation in gene silencing [22]. Two cancer-related genes TMS1 and PACE4 are also silenced in ovarian cancer by DNA methylation and histone hypoacetylation [31,32]. In this study, restoration of DLEC1 expression after histone deacetylase inhibitor treatment and enhanced association between acetylated histones (H3 and H4) and the DLEC1 promoter indicate that histone hypoacetylation is also involved in DLEC1 downregulation in ovarian cancer cells. Because several histone deacetylase inhibitors were found to exhibit antiproliferative activity and to induce apoptosis in human ovarian cancer cells [33], we suggest that the restoration of tumor-suppressor genes, such as DLEC1 and others, by HDAC inhibitors might contribute to antitumor effects.

In conclusion, we have demonstrated the loss of DLEC1 expression in ovarian cancers and the suppression of ovarian cancer cell growth by DLEC1 reexpression. The loss of DLEC1 expression in ovarian cancers is related to promoter hypermethylation and histone hypoacetylation, but not to loss of chromosome 3p22.3.

Acknowledgements

We thank Yataro Daigo and Yusuke Nakamura for providing us with pcDNA3.1+ DLEC1SS. We also thank Kwok-Wai Lo for sharing the results before publication.

Footnotes

1

This study was supported, in part, by the Dana Farber Harvard Ovarian Cancer SPORE grants P50CA165009, R33CA103595, and K22DE014847 from the National Institutes of Health, Department of Health and Human Services, Gillette Center For Women's Cancer, Adler Foundation, Inc., Edgar Astrove Fund, Morro Family Fund, Natalie Pohl Fund, Ovarian Cancer Research Fund, and Ruth W. White Gynecologic Oncology Research Fellowship.

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