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DNA Methylation in Ovarian Cancer
Gynecologic Oncology 82, 299 –304 (2001) doi:10.1006/gyno.2001.6284, available online at http://www.idealibrary.com on
DNA Methylation in Ovarian Cancer II. Expression of DNA Methyltransferases in Ovarian Cancer Cell Lines and Normal Ovarian Epithelial Cells 1 A. Ahluwalia,* J. A. Hurteau,† R. M. Bigsby,† and K. P. Nephew* ,† ,2 *Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405; and †Department of OB-GYN, Indiana University School of Medicine, Indianapolis, Indiana 46202 Received January 22, 2001
INTRODUCTION
Objective. The aim of this study was to investigate whether expression of the enzymes that catalyze cytosine CpG island methylation, DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b is altered in human ovarian cancer. Aberrations in DNA methylation are common in cancer and have important roles in tumor initiation and progression. Tumors that display frequent and concurrent inactivation of multiple genes by methylation are designated as having a CpG Island methylator phenotype, or CIMP. To date, colon, gastric, and most recently ovarian cancers meet the CIMP criteria for cancer. We hypothesized that altered expression of DNA methyltransferases can result in hypermethylation events seen in CIMP cancers. Methods. DNMT1, DNMT3a, and DNMT3b mRNA levels in eight ovarian cancer cells lines (Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5) were compared to DNMT expression in normal ovarian surface epithelial cells using semiquantitative reverse transcription-polymerase chain reaction. Results. In HeyA8 and HeyC2 ovarian cancer cells, DNMT1 expression levels were up to threefold higher (P < 0.05) than in normal ovarian surface epithelial cells. SK-OV-3 and PA-1 displayed increased DNMT3b expression (P < 0.05) compared to normal ovarian surface epithelial cells. Transcript levels for DNMT3a, however, were similar in cancer and normal ovarian cells. Conclusions. We observed differential expression of the DNMT genes in some ovarian cancer cell lines and conclude that alterations in DNMT expression might contribute to the CIMP phenotype in ovarian cancer. However, based on the lack of aberrant DNMT expression in some of the cancer cell lines examined, we further suggest that another mechanism(s), in addition to DNMT overexpression, accounts for methylation anomalies commonly observed in ovarian cancer. © 2001 Academic Press Key Words: ovarian cancer; DNA methyltransferase; DNA methylation; CpG islands. 1
The process of DNA methylation in normal adult cells of vertebrates largely involves maintaining the general patterns characteristic of all cells and selected patterns associated with specific cell types [1, 2]. In normal somatic cells, the enzyme DNA methyltransferase (DNMT) stably maintains the methylation pattern of DNA. This enzyme plays a key role in human carcinogenesis by methylating cytosines 5⬘ to guanosines on a DNA strand. In fact, changes in the status of DNA methylation are one of the most common molecular alterations in human neoplasia [3, 4]. Decrease in the overall content of 5-methyl cytosine [5], demethylation of specific loci [6], and de novo methylation of CpG islands [7] have all been observed in cancer. Inactivation of several genes, including tumor suppressor genes, by aberrant methylation of CpG islands has been documented in many cancers including ovarian cancer [8 –11]. The methylation machinery is composed of three known catalytically active DNMTs, DNMT1, DNMT3a, and DNMT3b. The predominant mammalian DNA methyltransferase enzyme is DNMT1 [12, 13] and it has a marked preference for hemimethylated rather than unmethylated DNA [14]. During DNA replication, DNMT1 can recognize the normally methylated CpG sites in the parent strand and catalyze cytosine methylation in the corresponding CpG site of the daughter strand. Active localization of the enzyme to the sites of DNA replication in dividing cells may facilitate this maintenance role of DNMT1 [15]. Recently, two additional mammalian DNA (cytotosine-5)-methyltransferase genes have been identified, DNMT3a and DNMT3b [16]. These are essential for mammalian development and are responsible for de novo methylation during embryogenesis [17]. All three DNMT genes are ubiquitously expressed in adult and fetal tissues, with fetal tissues expressing significantly higher levels than adult tissues [18]. In human cancer, increased DNMT enzyme activity is associated with cancer progression [19, 20], and upregulation of
This study was funded by the Gynecologic Oncology Group through NIH CA27469-18. 2 To whom correspondence should be addressed at Indiana University School of Medicine, Medical Sciences, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401. Fax: (812) 855-4436. E-mail: knephew@ indiana.edu. 299
0090-8258/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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DNMT gene expression has been demonstrated in lung [19], liver [21], leukemia [22], and breast [20] cancers. Cancers that display frequent and concurrent inactivation of multiple genes by hypermethylation have been designated CpG Island methylator phenotype (CIMP). These include colon [23], gastric [24], and most recently ovarian cancers [11]. The mechanism responsible for the increased methylation seen in colon cancer has been postulated to include alterations in the expression of DNMTs [25]. However, the mechanisms contributing to the increased methylation anomalies in ovarian cancer cells are unknown. To investigate a possible mechanism associated with aberrant methylation in ovarian cancer, we studied the expression of DNMT1, DNMT3a, and DNMT3b in ovarian cancer cell lines. MATERIALS AND METHODS Normal ovarian epithelial cells and ovarian cancer cell lines. Normal controls for this study consisted of normal ovarian surface epithelial (NOSE) and immortalized ovarian surface epithelial (IOSE). Because most ovarian tumors arise from the single layer of epithelium on the ovarian stromal surface, NOSE and IOSE cells represent the standard against which alterations in epithelial ovarian cancer are measured. NOSE cells were obtained from two patients at Indiana University School of Medicine (Department of Obstetrics and Gynecology) by scraping the ovarian surface epithelium. All protocols and procedures for human subjects were approved by Indiana University School of Medicine. Following cell culture conditions and procedures established by Dr. N. Auersperg [26], NOSE cells were placed in short-term culture and expanded (two to four passages). The purity of these ovarian cell cultures was confirmed by immunostaining for keratin and vimentin [26]. The IOSE cells routinely used in ovarian cancer studies [27, 28] were generously provided by Dr. N. Auersperg (University of British Columbia, Vancouver, Canada). Eight ovarian cancer cell lines, Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5, were utilized in this study. The Hey lines were obtained from Dr. G. Mills (MD Anderson Cancer Center, Houston, TX) and were derived from the peritoneal deposit of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary [29]. HeyA8 and HeyC2 cells were produced by passaging the parental Hey line in athymic mice. A2780 and A2780-P5 ovarian adenocarcinoma cell lines were obtained from Dr. J. Drummond (Biology Department, Indiana University, Bloomington, IN). The other ovarian cancer cell lines (OVCAR-3, from malignant ascites of a patient with ovarian adenocarcinoma; SK-OV-3, ovarian adenocarcinoma; PA-1, teratocarcinoma) were purchased from the American Type Culture Collection (Rockville, MD). Hey, HeyA8, HeyC2, and OVCAR-3 cells were grown in DMEM supplemented with 5% fetal bovine serum. SK-OV-3 cells were grown in McCoy’s medium supplemented with 10% fetal bovine serum. PA-1 cells were
grown in DMEM supplemented with 10% fetal bovine serum. A2780 and A2780-P5 cells were grown in DMEM supplemented with 10% fetal bovine serum and 2 U/ml of insulin. NOSE and IOSE cells were grown in Medium 199/105 supplemented with 15% fetal bovine serum. Cells were seeded at a density of 3– 6 ⫻ 10 5 cells/dish and harvested at 70% confluence. Reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was analyzed using semi-quantitative RTPCR using the RT-PCR kit from Applied Biosciences, as we have described previously [30]. Briefly, total cellular RNA was extracted from NOSE, IOSE, and the ovarian cancer cell lines by using TRI reagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s protocol and as we have described previously [31]. The total RNA preparation was treated with DNase prior to the RT reactions. Each RT reaction was carried out using 2.5 g of RNA in a volume of 20 l, as recommended by the manufacturer. The products of the RT reaction were divided half and used for subsequent analysis of DNMT1, DNMT3a, DNMT3b, and the control gene RPL-19, a constitutively expressed ribosomal gene [32]. Published sequences were used to design primers for the DNMTs [18]: DNMT1 sense, 5⬘-GAT CGA ATT CAT GCC GGC GCG TAC CGC CCC AG-3⬘; DNMT1 antisense, 5⬘-ATG GTG GTT TGC CTG GTG C-3⬘; DNMT3a sense, 5⬘-GGG GAC GTC CGC AGC GTC ACA A-3⬘; DNMT3a antisense, 5⬘-CAG GGT TGG ACT CGA GAA ATC GC-3⬘; DNMT3b sense, 5⬘-CCT GCT GAA TTA CTC ACG CCC C-3⬘; DNMT3b antisense, 5⬘-GTC TGT GTA GTG CAC AGG AAA GCC-3⬘; RPL-19 primer sequences have been described by us previously [32]. Amplification conditions were denaturation at 94°C for 2 min followed by n cycles at 94°C for 30 s, transcriptspecific annealing temperature for 1 min, and 72°C for 1 min. The number of cycles was optimized for each set of primers and RNA sample to keep amplification in the linear range (30 cycles for RPL-19 and 35 cycles for all DNMTs). Annealing temperatures were 58°C for DNMT1 and RPL-19 and 65°C for DNMT3a and DNMT3b. PCR products were electrophoresed on 2% agarose gels and quantified using a Molecular Dynamics densitometer. The sizes of the PCR products were 490 bp for DNMT1, 259 bp for DNMT3a, 237 bp for DNMT3b, and 486 bp for RPL-19. Each RT-PCR reaction was performed in triplicate. Fold increases in gene expression in the ovarian cancer cells were calculated with respect to the levels of the transcripts in the normal controls (the NOSE and IOSE cells). Levels of significance were calculated using the Statistical Analysis System and Student–Newman–Keuls test and Tukey’s honestly significant difference test. To confirm the specificity of the primers, the PCR products were sequenced using the Big Dye Terminator kit (Applied Biosystems, Foster City, CA) and the Applied Biosystems 3700 automated DNA sequencing system (Applied Biosystems). Sequence homology searches were performed using the Blast feature in GenBank.
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FIG. 1. RT-PCR analysis with total cellular RNA obtained from NOSE, IOSE, Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5. A. Transcript-specific primers were used to amplify DNMT1, DNMT3a, DNMT3b, and RPL-19 (a constitutively expressed gene) used as the control for the reverse transcription step. PCR products were electrophoresed on standard 2% agarose gels, stained with ethidium bromide, and photographed. Representative gels are shown. B. Quantitation of the relative levels of DNMT1, DNMT3a, and DNMT3b in NOSE, IOSE, and eight cell lines relative to RPL-19. Results are expressed as arbitrary expression units.
RESULTS The expression levels of the DNMT1, DNMT3a, or DNMT3b transcripts were determined by semi-quantitative RT-PCR analysis (Fig. 1A). A constitutively expressed ribosomal gene, RPL-19, was used as a control for normalizing the RT reaction in each assay. PCR products corresponding to DNMT1 (490 bp), DNMT3 (259 bp), DNMT3b (237 bp), and RPL-19 (486 bp) were observed. Two additional bands of 330 and 400 bp were seen in the RT-PCR reaction for DNMT3b (described below). DNMT mRNA expression levels in the cell lines relative to RPL-19 mRNA levels were determined (Fig. 1B). DNMT1 expression was higher (P ⬍ 0.05) in HeyA8 and HeyC2 cells compared to the NOSE and IOSE cells (mean expression level ⫾ standard deviation (SD): NOSE, 1.1 ⫾ 0.2; IOSE, 1.3 ⫾ 0.4; HeyA8, 2.3 ⫾ 0.4; HeyC2, 2.8 ⫾ 0.2). Increased (P ⬍ 0.05) DNMT3b expression was observed in SK-OV-3 and PA-1 compared to NOSE (mean expression level ⫾ SD: NOSE, 1.1 ⫾ 0.1; IOSE, 1.3 ⫾ 0.3; SK-OV-3, 2.1 ⫾ 0.7; PA-1, 1.9 ⫾ 0.5). The expression levels of DNMT3a in the ovarian cancer cell lines were similar to those in the NOSE and IOSE cells. Expression of DNMT1, DNMT3a, and DNMT3b mRNAs was also calculated as a fold increase in the ratio of expression levels in the cancer cell lines compared to either NOSE or
IOSE. This analysis confirmed that expression of DNMT1 was increased (P ⬍ 0.05) in HeyA8 and HeyC2 cells (fold increase ⫾ SD with respect to NOSE was 2.1 ⫾ 0.3 for HeyA8 and 2.6 ⫾ 0.4 for HeyC2; fold increase with respect to IOSE was 1.8 ⫾ 0.2 for HeyA8 and 2.3 ⫾ 0.4 for HeyC2). Furthermore, DNMT3b expression was increased (P ⬍ 0.05) in SK-OV-3 and PA-1 compared to NOSE and IOSE (fold increase with respect to NOSE ⫾ SD: SK-OV-3, 2.0 ⫾ 0.6; PA-1, 1.7 ⫾ 0.5; fold increase with respect to IOSE ⫾ SD: SK-OV-3, 1.7 ⫾ 0.5; PA-1, 1.5 ⫾ 0.5). DNMT3a expression was not different (P ⬎ 0.05) in the ovarian cancer cell lines compared to NOSE and IOSE cells. Three RT-PCR products were observed after amplification using DNMT3b specific primers (Fig. 2). Sequence analysis identified these as DNMT3b1 , DNMT3b3 , and DNMT3b4 , three of the known splice variants of DNMT3b [18]. Observations on expression of these three splice variants in the cell lines are summarized in Table 1. Expression of DNMT3b3 mRNA, the major splice variant, was seen in all the cell lines. Expression of both minor splice variants, DNMT3b1 and DNMT3b4 , was seen in Hey, HeyA8, PA-1, A2780, and A2780-P5. However, OVCAR-3 expressed only the DNMT3b4 minor variant, while HeyC2 and SK-OV-3 cells expressed only the DNMT3b1. As for the normal ovarian epithelial cells,
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FIG. 2. RT-PCR analysis of DNMT3b splice variants. Total cellular RNA obtained from NOSE, IOSE, Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5 was analyzed using specific primers for DNMT3b. PCR products were electrophoresed on standard 2% agarose gels, ethidium bromide stained, and photographed. Representative gels are shown. DNMT3b1, 400 bp (top band); DNMT3b4, 330 bp (middle band); DNMT3b3, 259 bp (bottom band).
NOSE cells expressed DNMT3b4 , but IOSE cells did not express either of the minor splice variants. DISCUSSION While global alterations in methylation have been reported in ovarian carcinomas [9, 10, 33, 34], and ovarian cancer has recently been shown to meet the CIMP criteria [11, Ahluwalia et al., in press], mechanisms contributing to the increased methylation anomalies in ovarian cancer cells are unknown. In the present study, we examined ovarian cancer cell lines for alterations in expression of the DNMTs. We found that while DNMT1 and DNMT3b transcript levels were increased in some of the ovarian cancer cell lines, the expression of DNMT3a was similar in all ovarian cancer cell lines compared to normal ovarian cells. Interestingly, all ovarian cancer cells expressed one or both of the DNMT3b splice variants. The increased level of DNMT1 mRNA seen in HeyA8 and the HeyC2 cell lines could contribute to methylation-induced silencing of key tumor suppressor genes and hence might play a role in the more
aggressive nature of these ovarian cancer cells observed in xenograft experiments in athymic mice (our unpublished observations). Increased DNMT3b expression was seen in SKOV-3 and PA-1 cell lines that represent ovarian adenocarcinoma and teratocarcinoma, respectively, suggesting that overexpression of DNMT3b is not unique to epithelial ovarian cancer and might be manifested in all ovarian carcinomas. This report suggests that overexpression of the DNMT genes may play a role in establishing or maintaining the CIMP in some ovarian tumors. Multiple CIMP phenotypes have recently been demonstrated for ovarian carcinomas [11], and the differential DNMT expression we observed might reflect the CIMP tumor subtype from which the ovarian cancer cells were derived. However, based on the lack of aberrant expression of DNMTs in 50% of the cell lines examined, we suggest that in addition to DNMT overexpression, an alternate mechanism(s) exists to account for the methylation abnormalities commonly observed in ovarian cancer. Altered expression of DNMTs does not appear to be associated with other CIMP cancers. In colon cancer, only marginal increases in DNMT1 expression were reported [35], and overexpression of DNMTs was not associated with CpG island hypermethylation in colorectal carcinomas [36]. Further studies are required to more clearly understand the mechanisms associated with the CIMP cancer in general and in multiple methylator phenotypes in ovarian cancer in particular. Despite the fact that several DNMTs have been identified, the preferred substrates and roles of each DNMT and their splice variants remain unclear. We observed differential expression of two of the splice variants of DNMT3b, the functions of which are not known, but may reflect the capacity of ovarian cells to methylate DNA. We suggest that the DNMT splice variants may preferentially bind CpG-rich sequences like CpG islands and could be responsible for initiating the aberrant CpG island hypermethylation of such sequences in ovarian cancer. Furthermore, the normal ovarian epithelial cells did not express DNMT3b1, and perhaps expression of this
TABLE 1 Expression of DNMT3b Splice Variants Sample NOSE IOSE Hey HeyA8 HeyC2 OVCAR-3 SK-OV-3 PA-1 A2780 A2780-P5
DNMT3b1
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
DNMT3b4
DNMT3b3
⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Note. Expression of the splice variants is indicated by a ⫹ sign.
DNA METHYLATION IN OVARIAN CANCER II
splice variant is an indicator of the transformation of ovarian epithelia. This is the first study to report differential expression of DNMT mRNA levels in ovarian cancer cells. We observed a significant increase in DNMT1 and DNMT3b gene expression in some cell lines but not in others, suggesting that alternative mechanisms that could give rise to global methylation errors in ovarian cancer must be considered. The possibility that changes in mRNA stability and alterations in enzyme activity might exist for these DNMTs in ovarian cancer cannot be ruled out. We also must consider the possibility that yet uncharacterized DNMTs may play a role in ovarian cancer. Another DNMT, DNMT2 (PuMet), has been identified, but does not appear to possess any methyltransferase activity in vivo [37– 39]. A key finding in our study is the differential expression of some of the DNMT genes, and although the enzymatic activity of DNMTs in cancer has been reported [19, 20, 40], it is not possible at this time to determine which DNMT gene products are contributing to the activity. To our knowledge, no antibodies exist today that are able to discriminate between the DNMTs. Current enzymatic assays used to determine the DNMT activity define the total DNA methyltransferase activity in the cell and do not discriminate between the various DNMTs including DNMT1, DNMT3a, and DNMT3b and their splice variants. Clearly, future studies on the DNMTs and the splice variants are needed to shed light on their role in ovarian cancer progression. DNMT levels appear to vary during the cell cycle. In G 0/G1, DNMT1 and DNMT3b levels were reported to be downregulated but DNMT3a levels remained stable; furthermore, DNMT3a levels appear to be insensitive to cell cycle alterations [40]. In cancer cells, inappropriate expression of one or more of the DNMTs during a particular phase of the cell cycle could result in aberrant DNA methylation and cancer progression. Furthermore, each DNMT activity might be targeted to a different subcellular location. For example, it was recently reported that DNMT1 is retained in the cytoplasm during embryonic development [41], but it remains to be determined whether the subcellular localization of DNMTs is altered in cancer cells. While the current data indicate that there is at least an association between altered expression of the DNMTs and increased methylation in ovarian cancer, more definitive studies are clearly warranted to examine the mechanisms for establishing the CIMP phenotype in ovarian cancer. ACKNOWLEDGMENTS We thank Bernadette Allison and Andrea Caperell-Grant for excellent technical assistance.
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DNA Methylation in Ovarian Cancer II. Expression of DNA Methyltransferases in Ovarian Cancer Cell Lines and Normal Ovarian Epithelial Cells 1 A. Ahluwalia,* J. A. Hurteau,† R. M. Bigsby,† and K. P. Nephew* ,† ,2 *Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405; and †Department of OB-GYN, Indiana University School of Medicine, Indianapolis, Indiana 46202 Received January 22, 2001
INTRODUCTION
Objective. The aim of this study was to investigate whether expression of the enzymes that catalyze cytosine CpG island methylation, DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b is altered in human ovarian cancer. Aberrations in DNA methylation are common in cancer and have important roles in tumor initiation and progression. Tumors that display frequent and concurrent inactivation of multiple genes by methylation are designated as having a CpG Island methylator phenotype, or CIMP. To date, colon, gastric, and most recently ovarian cancers meet the CIMP criteria for cancer. We hypothesized that altered expression of DNA methyltransferases can result in hypermethylation events seen in CIMP cancers. Methods. DNMT1, DNMT3a, and DNMT3b mRNA levels in eight ovarian cancer cells lines (Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5) were compared to DNMT expression in normal ovarian surface epithelial cells using semiquantitative reverse transcription-polymerase chain reaction. Results. In HeyA8 and HeyC2 ovarian cancer cells, DNMT1 expression levels were up to threefold higher (P < 0.05) than in normal ovarian surface epithelial cells. SK-OV-3 and PA-1 displayed increased DNMT3b expression (P < 0.05) compared to normal ovarian surface epithelial cells. Transcript levels for DNMT3a, however, were similar in cancer and normal ovarian cells. Conclusions. We observed differential expression of the DNMT genes in some ovarian cancer cell lines and conclude that alterations in DNMT expression might contribute to the CIMP phenotype in ovarian cancer. However, based on the lack of aberrant DNMT expression in some of the cancer cell lines examined, we further suggest that another mechanism(s), in addition to DNMT overexpression, accounts for methylation anomalies commonly observed in ovarian cancer. © 2001 Academic Press Key Words: ovarian cancer; DNA methyltransferase; DNA methylation; CpG islands. 1
The process of DNA methylation in normal adult cells of vertebrates largely involves maintaining the general patterns characteristic of all cells and selected patterns associated with specific cell types [1, 2]. In normal somatic cells, the enzyme DNA methyltransferase (DNMT) stably maintains the methylation pattern of DNA. This enzyme plays a key role in human carcinogenesis by methylating cytosines 5⬘ to guanosines on a DNA strand. In fact, changes in the status of DNA methylation are one of the most common molecular alterations in human neoplasia [3, 4]. Decrease in the overall content of 5-methyl cytosine [5], demethylation of specific loci [6], and de novo methylation of CpG islands [7] have all been observed in cancer. Inactivation of several genes, including tumor suppressor genes, by aberrant methylation of CpG islands has been documented in many cancers including ovarian cancer [8 –11]. The methylation machinery is composed of three known catalytically active DNMTs, DNMT1, DNMT3a, and DNMT3b. The predominant mammalian DNA methyltransferase enzyme is DNMT1 [12, 13] and it has a marked preference for hemimethylated rather than unmethylated DNA [14]. During DNA replication, DNMT1 can recognize the normally methylated CpG sites in the parent strand and catalyze cytosine methylation in the corresponding CpG site of the daughter strand. Active localization of the enzyme to the sites of DNA replication in dividing cells may facilitate this maintenance role of DNMT1 [15]. Recently, two additional mammalian DNA (cytotosine-5)-methyltransferase genes have been identified, DNMT3a and DNMT3b [16]. These are essential for mammalian development and are responsible for de novo methylation during embryogenesis [17]. All three DNMT genes are ubiquitously expressed in adult and fetal tissues, with fetal tissues expressing significantly higher levels than adult tissues [18]. In human cancer, increased DNMT enzyme activity is associated with cancer progression [19, 20], and upregulation of
This study was funded by the Gynecologic Oncology Group through NIH CA27469-18. 2 To whom correspondence should be addressed at Indiana University School of Medicine, Medical Sciences, 302 Jordan Hall, 1001 East 3rd Street, Bloomington, IN 47405-4401. Fax: (812) 855-4436. E-mail: knephew@ indiana.edu. 299
0090-8258/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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DNMT gene expression has been demonstrated in lung [19], liver [21], leukemia [22], and breast [20] cancers. Cancers that display frequent and concurrent inactivation of multiple genes by hypermethylation have been designated CpG Island methylator phenotype (CIMP). These include colon [23], gastric [24], and most recently ovarian cancers [11]. The mechanism responsible for the increased methylation seen in colon cancer has been postulated to include alterations in the expression of DNMTs [25]. However, the mechanisms contributing to the increased methylation anomalies in ovarian cancer cells are unknown. To investigate a possible mechanism associated with aberrant methylation in ovarian cancer, we studied the expression of DNMT1, DNMT3a, and DNMT3b in ovarian cancer cell lines. MATERIALS AND METHODS Normal ovarian epithelial cells and ovarian cancer cell lines. Normal controls for this study consisted of normal ovarian surface epithelial (NOSE) and immortalized ovarian surface epithelial (IOSE). Because most ovarian tumors arise from the single layer of epithelium on the ovarian stromal surface, NOSE and IOSE cells represent the standard against which alterations in epithelial ovarian cancer are measured. NOSE cells were obtained from two patients at Indiana University School of Medicine (Department of Obstetrics and Gynecology) by scraping the ovarian surface epithelium. All protocols and procedures for human subjects were approved by Indiana University School of Medicine. Following cell culture conditions and procedures established by Dr. N. Auersperg [26], NOSE cells were placed in short-term culture and expanded (two to four passages). The purity of these ovarian cell cultures was confirmed by immunostaining for keratin and vimentin [26]. The IOSE cells routinely used in ovarian cancer studies [27, 28] were generously provided by Dr. N. Auersperg (University of British Columbia, Vancouver, Canada). Eight ovarian cancer cell lines, Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5, were utilized in this study. The Hey lines were obtained from Dr. G. Mills (MD Anderson Cancer Center, Houston, TX) and were derived from the peritoneal deposit of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary [29]. HeyA8 and HeyC2 cells were produced by passaging the parental Hey line in athymic mice. A2780 and A2780-P5 ovarian adenocarcinoma cell lines were obtained from Dr. J. Drummond (Biology Department, Indiana University, Bloomington, IN). The other ovarian cancer cell lines (OVCAR-3, from malignant ascites of a patient with ovarian adenocarcinoma; SK-OV-3, ovarian adenocarcinoma; PA-1, teratocarcinoma) were purchased from the American Type Culture Collection (Rockville, MD). Hey, HeyA8, HeyC2, and OVCAR-3 cells were grown in DMEM supplemented with 5% fetal bovine serum. SK-OV-3 cells were grown in McCoy’s medium supplemented with 10% fetal bovine serum. PA-1 cells were
grown in DMEM supplemented with 10% fetal bovine serum. A2780 and A2780-P5 cells were grown in DMEM supplemented with 10% fetal bovine serum and 2 U/ml of insulin. NOSE and IOSE cells were grown in Medium 199/105 supplemented with 15% fetal bovine serum. Cells were seeded at a density of 3– 6 ⫻ 10 5 cells/dish and harvested at 70% confluence. Reverse transcription-polymerase chain reaction (RT-PCR). Gene expression was analyzed using semi-quantitative RTPCR using the RT-PCR kit from Applied Biosciences, as we have described previously [30]. Briefly, total cellular RNA was extracted from NOSE, IOSE, and the ovarian cancer cell lines by using TRI reagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s protocol and as we have described previously [31]. The total RNA preparation was treated with DNase prior to the RT reactions. Each RT reaction was carried out using 2.5 g of RNA in a volume of 20 l, as recommended by the manufacturer. The products of the RT reaction were divided half and used for subsequent analysis of DNMT1, DNMT3a, DNMT3b, and the control gene RPL-19, a constitutively expressed ribosomal gene [32]. Published sequences were used to design primers for the DNMTs [18]: DNMT1 sense, 5⬘-GAT CGA ATT CAT GCC GGC GCG TAC CGC CCC AG-3⬘; DNMT1 antisense, 5⬘-ATG GTG GTT TGC CTG GTG C-3⬘; DNMT3a sense, 5⬘-GGG GAC GTC CGC AGC GTC ACA A-3⬘; DNMT3a antisense, 5⬘-CAG GGT TGG ACT CGA GAA ATC GC-3⬘; DNMT3b sense, 5⬘-CCT GCT GAA TTA CTC ACG CCC C-3⬘; DNMT3b antisense, 5⬘-GTC TGT GTA GTG CAC AGG AAA GCC-3⬘; RPL-19 primer sequences have been described by us previously [32]. Amplification conditions were denaturation at 94°C for 2 min followed by n cycles at 94°C for 30 s, transcriptspecific annealing temperature for 1 min, and 72°C for 1 min. The number of cycles was optimized for each set of primers and RNA sample to keep amplification in the linear range (30 cycles for RPL-19 and 35 cycles for all DNMTs). Annealing temperatures were 58°C for DNMT1 and RPL-19 and 65°C for DNMT3a and DNMT3b. PCR products were electrophoresed on 2% agarose gels and quantified using a Molecular Dynamics densitometer. The sizes of the PCR products were 490 bp for DNMT1, 259 bp for DNMT3a, 237 bp for DNMT3b, and 486 bp for RPL-19. Each RT-PCR reaction was performed in triplicate. Fold increases in gene expression in the ovarian cancer cells were calculated with respect to the levels of the transcripts in the normal controls (the NOSE and IOSE cells). Levels of significance were calculated using the Statistical Analysis System and Student–Newman–Keuls test and Tukey’s honestly significant difference test. To confirm the specificity of the primers, the PCR products were sequenced using the Big Dye Terminator kit (Applied Biosystems, Foster City, CA) and the Applied Biosystems 3700 automated DNA sequencing system (Applied Biosystems). Sequence homology searches were performed using the Blast feature in GenBank.
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FIG. 1. RT-PCR analysis with total cellular RNA obtained from NOSE, IOSE, Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5. A. Transcript-specific primers were used to amplify DNMT1, DNMT3a, DNMT3b, and RPL-19 (a constitutively expressed gene) used as the control for the reverse transcription step. PCR products were electrophoresed on standard 2% agarose gels, stained with ethidium bromide, and photographed. Representative gels are shown. B. Quantitation of the relative levels of DNMT1, DNMT3a, and DNMT3b in NOSE, IOSE, and eight cell lines relative to RPL-19. Results are expressed as arbitrary expression units.
RESULTS The expression levels of the DNMT1, DNMT3a, or DNMT3b transcripts were determined by semi-quantitative RT-PCR analysis (Fig. 1A). A constitutively expressed ribosomal gene, RPL-19, was used as a control for normalizing the RT reaction in each assay. PCR products corresponding to DNMT1 (490 bp), DNMT3 (259 bp), DNMT3b (237 bp), and RPL-19 (486 bp) were observed. Two additional bands of 330 and 400 bp were seen in the RT-PCR reaction for DNMT3b (described below). DNMT mRNA expression levels in the cell lines relative to RPL-19 mRNA levels were determined (Fig. 1B). DNMT1 expression was higher (P ⬍ 0.05) in HeyA8 and HeyC2 cells compared to the NOSE and IOSE cells (mean expression level ⫾ standard deviation (SD): NOSE, 1.1 ⫾ 0.2; IOSE, 1.3 ⫾ 0.4; HeyA8, 2.3 ⫾ 0.4; HeyC2, 2.8 ⫾ 0.2). Increased (P ⬍ 0.05) DNMT3b expression was observed in SK-OV-3 and PA-1 compared to NOSE (mean expression level ⫾ SD: NOSE, 1.1 ⫾ 0.1; IOSE, 1.3 ⫾ 0.3; SK-OV-3, 2.1 ⫾ 0.7; PA-1, 1.9 ⫾ 0.5). The expression levels of DNMT3a in the ovarian cancer cell lines were similar to those in the NOSE and IOSE cells. Expression of DNMT1, DNMT3a, and DNMT3b mRNAs was also calculated as a fold increase in the ratio of expression levels in the cancer cell lines compared to either NOSE or
IOSE. This analysis confirmed that expression of DNMT1 was increased (P ⬍ 0.05) in HeyA8 and HeyC2 cells (fold increase ⫾ SD with respect to NOSE was 2.1 ⫾ 0.3 for HeyA8 and 2.6 ⫾ 0.4 for HeyC2; fold increase with respect to IOSE was 1.8 ⫾ 0.2 for HeyA8 and 2.3 ⫾ 0.4 for HeyC2). Furthermore, DNMT3b expression was increased (P ⬍ 0.05) in SK-OV-3 and PA-1 compared to NOSE and IOSE (fold increase with respect to NOSE ⫾ SD: SK-OV-3, 2.0 ⫾ 0.6; PA-1, 1.7 ⫾ 0.5; fold increase with respect to IOSE ⫾ SD: SK-OV-3, 1.7 ⫾ 0.5; PA-1, 1.5 ⫾ 0.5). DNMT3a expression was not different (P ⬎ 0.05) in the ovarian cancer cell lines compared to NOSE and IOSE cells. Three RT-PCR products were observed after amplification using DNMT3b specific primers (Fig. 2). Sequence analysis identified these as DNMT3b1 , DNMT3b3 , and DNMT3b4 , three of the known splice variants of DNMT3b [18]. Observations on expression of these three splice variants in the cell lines are summarized in Table 1. Expression of DNMT3b3 mRNA, the major splice variant, was seen in all the cell lines. Expression of both minor splice variants, DNMT3b1 and DNMT3b4 , was seen in Hey, HeyA8, PA-1, A2780, and A2780-P5. However, OVCAR-3 expressed only the DNMT3b4 minor variant, while HeyC2 and SK-OV-3 cells expressed only the DNMT3b1. As for the normal ovarian epithelial cells,
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FIG. 2. RT-PCR analysis of DNMT3b splice variants. Total cellular RNA obtained from NOSE, IOSE, Hey, HeyA8, HeyC2, OVCAR-3, SK-OV-3, PA-1, A2780, and A2780-P5 was analyzed using specific primers for DNMT3b. PCR products were electrophoresed on standard 2% agarose gels, ethidium bromide stained, and photographed. Representative gels are shown. DNMT3b1, 400 bp (top band); DNMT3b4, 330 bp (middle band); DNMT3b3, 259 bp (bottom band).
NOSE cells expressed DNMT3b4 , but IOSE cells did not express either of the minor splice variants. DISCUSSION While global alterations in methylation have been reported in ovarian carcinomas [9, 10, 33, 34], and ovarian cancer has recently been shown to meet the CIMP criteria [11, Ahluwalia et al., in press], mechanisms contributing to the increased methylation anomalies in ovarian cancer cells are unknown. In the present study, we examined ovarian cancer cell lines for alterations in expression of the DNMTs. We found that while DNMT1 and DNMT3b transcript levels were increased in some of the ovarian cancer cell lines, the expression of DNMT3a was similar in all ovarian cancer cell lines compared to normal ovarian cells. Interestingly, all ovarian cancer cells expressed one or both of the DNMT3b splice variants. The increased level of DNMT1 mRNA seen in HeyA8 and the HeyC2 cell lines could contribute to methylation-induced silencing of key tumor suppressor genes and hence might play a role in the more
aggressive nature of these ovarian cancer cells observed in xenograft experiments in athymic mice (our unpublished observations). Increased DNMT3b expression was seen in SKOV-3 and PA-1 cell lines that represent ovarian adenocarcinoma and teratocarcinoma, respectively, suggesting that overexpression of DNMT3b is not unique to epithelial ovarian cancer and might be manifested in all ovarian carcinomas. This report suggests that overexpression of the DNMT genes may play a role in establishing or maintaining the CIMP in some ovarian tumors. Multiple CIMP phenotypes have recently been demonstrated for ovarian carcinomas [11], and the differential DNMT expression we observed might reflect the CIMP tumor subtype from which the ovarian cancer cells were derived. However, based on the lack of aberrant expression of DNMTs in 50% of the cell lines examined, we suggest that in addition to DNMT overexpression, an alternate mechanism(s) exists to account for the methylation abnormalities commonly observed in ovarian cancer. Altered expression of DNMTs does not appear to be associated with other CIMP cancers. In colon cancer, only marginal increases in DNMT1 expression were reported [35], and overexpression of DNMTs was not associated with CpG island hypermethylation in colorectal carcinomas [36]. Further studies are required to more clearly understand the mechanisms associated with the CIMP cancer in general and in multiple methylator phenotypes in ovarian cancer in particular. Despite the fact that several DNMTs have been identified, the preferred substrates and roles of each DNMT and their splice variants remain unclear. We observed differential expression of two of the splice variants of DNMT3b, the functions of which are not known, but may reflect the capacity of ovarian cells to methylate DNA. We suggest that the DNMT splice variants may preferentially bind CpG-rich sequences like CpG islands and could be responsible for initiating the aberrant CpG island hypermethylation of such sequences in ovarian cancer. Furthermore, the normal ovarian epithelial cells did not express DNMT3b1, and perhaps expression of this
TABLE 1 Expression of DNMT3b Splice Variants Sample NOSE IOSE Hey HeyA8 HeyC2 OVCAR-3 SK-OV-3 PA-1 A2780 A2780-P5
DNMT3b1
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
DNMT3b4
DNMT3b3
⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫹ ⫹ ⫹ ⫹ ⫹ ⫹
Note. Expression of the splice variants is indicated by a ⫹ sign.
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splice variant is an indicator of the transformation of ovarian epithelia. This is the first study to report differential expression of DNMT mRNA levels in ovarian cancer cells. We observed a significant increase in DNMT1 and DNMT3b gene expression in some cell lines but not in others, suggesting that alternative mechanisms that could give rise to global methylation errors in ovarian cancer must be considered. The possibility that changes in mRNA stability and alterations in enzyme activity might exist for these DNMTs in ovarian cancer cannot be ruled out. We also must consider the possibility that yet uncharacterized DNMTs may play a role in ovarian cancer. Another DNMT, DNMT2 (PuMet), has been identified, but does not appear to possess any methyltransferase activity in vivo [37– 39]. A key finding in our study is the differential expression of some of the DNMT genes, and although the enzymatic activity of DNMTs in cancer has been reported [19, 20, 40], it is not possible at this time to determine which DNMT gene products are contributing to the activity. To our knowledge, no antibodies exist today that are able to discriminate between the DNMTs. Current enzymatic assays used to determine the DNMT activity define the total DNA methyltransferase activity in the cell and do not discriminate between the various DNMTs including DNMT1, DNMT3a, and DNMT3b and their splice variants. Clearly, future studies on the DNMTs and the splice variants are needed to shed light on their role in ovarian cancer progression. DNMT levels appear to vary during the cell cycle. In G 0/G1, DNMT1 and DNMT3b levels were reported to be downregulated but DNMT3a levels remained stable; furthermore, DNMT3a levels appear to be insensitive to cell cycle alterations [40]. In cancer cells, inappropriate expression of one or more of the DNMTs during a particular phase of the cell cycle could result in aberrant DNA methylation and cancer progression. Furthermore, each DNMT activity might be targeted to a different subcellular location. For example, it was recently reported that DNMT1 is retained in the cytoplasm during embryonic development [41], but it remains to be determined whether the subcellular localization of DNMTs is altered in cancer cells. While the current data indicate that there is at least an association between altered expression of the DNMTs and increased methylation in ovarian cancer, more definitive studies are clearly warranted to examine the mechanisms for establishing the CIMP phenotype in ovarian cancer. ACKNOWLEDGMENTS We thank Bernadette Allison and Andrea Caperell-Grant for excellent technical assistance.
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