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Molecular Cancer Research 3:261-269 (2005)
© 2005 American Association for Cancer Research

Cancer Genes and Genomics

Discovery of Epigenetically Masked Tumor Suppressor Genes in Endometrial Cancer

Noriyuki Takai1,5, Norihiko Kawamata1, Christine S. Walsh1,2, Sigal Gery1, Julian C. Desmond1, Sadie Whittaker1, Jonathan W. Said3, Laura M. Popoviciu3, Peter A. Jones4, Isao Miyakawa5 and H. Phillip Koeffler1

1 Division of Hematology/Oncology and 2 Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center and 3 Department of Pathology, Center of Health Science, University of California at Los Angeles School of Medicine; 4 Department of Biochemistry, School of Medicine, University of Southern California, Los Angeles, CA and 5 Department of Obstetrics and Gynecology, Oita University Faculty of Medicine, Oita, Japan

Requests for reprints: Norihiko Kawamata, Division of Hematology/Oncology, Cedars-Sinai Medical Center/University of California at Los Angeles School of Medicine, 8700 Beverly Boulevard, Los Angeles, CA 90048. Phone: 310-423-7736; Fax: 310-423-0443. E-mail: kawamatan{at}cshs.org


   Abstract
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 Notes
 Abstract
Introduction
 Results
 Materials and Methods
 References
 
Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated the discovery of novel tumor suppressor genes. We used a variety of research tools to search for genes that are epigenetically silenced in human endometrial cancers. Changes in global gene expression of the endometrial cancer cell line Ishikawa was analyzed after treatment with the demethylating agent 5-aza-2'-deoxycytidine combined with the histone deacetylase inhibitor suberoylanilide bishydroxamide. By screening over 22,000 genes, candidate tumor suppressor genes were identified. Additional microarray analysis and real-time reverse transcription-PCR of normal and cancerous endometrial samples and search for CpG islands further refined the list. Tazarotene-induced gene-1 (Tig1) and CCAAT/enhancer binding protein-{alpha} (C/ebp{alpha}) were chosen for further study. Expression of both genes was low in endometrial cancer cell lines and clinical samples but high in normal endometrial tissues. Bisulfite sequencing, restriction analysis, and/or methylation-specific PCR revealed aberrant methylation of the CpG island in the Tig1 gene of all 6 endometrial cancer cell lines examined and 4 of 18 clinical endometrial cancers, whereas the C/ebp{alpha} promoter remained unmethylated in endometrial cancers. Chromatin immunoprecipitation showed increased acetylated histone H3 bound to both Tig1 and C/ebp{alpha} genes after treatment with 5-aza-2'-deoxycytidine and/or suberoylanilide bishydroxamide. Forced expression of either TIG1 or C/EBP{alpha} led to significant growth reduction of Ishikawa cells. Our data suggest that C/ebp{alpha} and Tig1 function as tumor suppressor proteins in endometrial cancers and that their reexpression may be a therapeutic target.


   Introduction
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 Notes
 Abstract
 Introduction
Results
 Materials and Methods
 References
 
Endometrial cancer has been characterized by multiple genetic and epigenetic alterations (1, 2). In cancer cells, aberrant methylation of CpG islands has been found in the 5' end of the regulatory regions and/or the first exons of tumor suppressor genes and genes responsible for genomic stability (3, 4). Epigenetic changes can silence the transcription of these important genes, leading to clonal proliferation of tumor cells (3, 4). Epigenetic modifications of cytosine residues in DNA and the NH2 termini of histone proteins have emerged as key mechanisms in chromatin remodeling, affecting transcriptional regulation. Growing evidence exists that interplay occurs between cytosine methylation and histone modification. The methyl-CpG binding protein, MeCP2, has been found to be associated with histone deacetylase (HDAC) activity, providing a pathway by which histone modification can be induced by DNA methylation changes (4-6). The identification and characterization of genes whose CpG islands in the promoter are selectively hypermethylated in cancers not only improves our understanding of the role of epigenetic alterations in tumorigenesis but may also lead to the discovery of novel tumor suppressor genes.

Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in colorectal (7), esophageal (8), and pancreatic (9) cancers. In this study, a variety of techniques were implemented to identify genes silenced by DNA methylation and histone deacetylation in endometrial cancers. After screening >22,000 genes, we focused on two, tazarotene-induced gene-1 (Tig1) and CCAAT/enhancer binding protein-{alpha} (C/ebp{alpha}), and have begun to analyze their function.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Materials and Methods
 References
 
Pharmacologic Unmasking of Transcriptionally Repressed Genes
We used the cDNA microarray technique to identify genes significantly (P < 0.05) up-regulated in an endometrial cancer cell line, Ishikawa, after treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR; 3 days), which blocks DNA methylation, and suberoylanilide bishydroxamide (SAHA; 1 day) to inhibit HDAC. The gene expression profile was compared before and after treatment using Affymetrix (Santa Clara, CA) human genome U133A microarray chips containing 22,283 transcripts. Treatment with these agents resulted in up-regulation (defined as a ≥2.0-fold increase) of 676 genes. We focused on 101 genes that were either not expressed or expressed only at low levels (raw values <500) before treatment of the cell line. To narrow further the list of candidate genes, we queried their expression status in six endometrial cancer samples as determined by microarray analysis.6 The clinical samples were individually analyzed by microarray. Thirty-six of the 101 genes were expressed at very low levels in the clinical cancer samples (Table 1).


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  		Table 1. Epigenetically Silenced Genes in Ishikawa Cell

 
The 5' regulatory regions of these genes were analyzed in the BLAST database and the CpG Island Searcher (10) to determine whether they contained CpG islands. Thirty-two of the 36 (89%) genes harbored CpG sites in the promoter region (Table 1).

Expression of these 36 genes was examined in six endometrial cancer cell lines, including Ishikawa, with or without 5-Aza-CdR and SAHA using real-time quantitative reverse transcription-PCR (RT-PCR; data available on request; Ishikawa data in Table 1). The Tig1 gene was completely silenced in all six endometrial cancer cell lines, and its expression was induced in each cell line after exposure to 5-Aza-CdR and SAHA (Fig. 1A). Five genes (Sh3d5, Ngfr, Rpl3l, Rgc32, and C/ebp{alpha}) were up-regulated after a similar treatment in five of six cell lines (C/ebp{alpha} data in Fig. 1B; additional data available on request).



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  		FIGURE 1. Expression of the Tig1 and C/ebp{alpha} genes in cell lines treated with SAHA and/or 5-Aza-CdR (DAC). A and B. The two genes were silenced in endometrial carcinoma cell lines and reactivated by 5-Aza-CdR and/or SAHA. Expression patterns of the Tig1 (A) and C/ebp{alpha} (B) genes before and after exposed to SAHA (2.5 µmol/L, 1 day), 5-Aza-CdR (5 µmol/L, 3 days), or combination of both in six endometrial cancer cell lines. Expression of transcripts of each gene was measured by real-time RT-PCR relative to expression of 18S rRNA. Treatment with both agents showed more potent induction of gene expression than with either SAHA or 5-Aza-CdR alone. Real-time PCR was done thrice on each sample. Columns, mean; bars, SD.

 
Then, we analyzed the methylation status of the promoter regions and/or first exons of these six candidate genes in Ishikawa cells using bisulfite sequencing and/or combined bisulfite restriction analysis (COBRA). Methylation was only detected in the Tig1 gene. The transcription factor C/ebp{alpha} is important in the terminal differentiation of granulocytes, hepatocytes, and adipocytes; mutations of this gene are detected in acute myeloid leukemias (11, 12). C/EBP{alpha} seems to be down-regulated in a large proportion of lung cancers and its forced expression has growth-inhibitory properties in the epithelial cells of the airway (13). The Tig1 gene had been shown previously to be expressed in normal prostate but not in prostate cancers, where its overexpression is antiproliferative (14).

Therefore, we chose for further analysis the Tig1 gene, which was methylated in the cell line, and C/ebp{alpha}, which is associated with differentiation in a variety of tissues. To elucidate the expression levels of the Tig1 and C/ebp{alpha} genes in clinical samples, we did real-time PCR on seven endometrial cancers and seven normal endometrial samples. Expression of both genes was low in the clinical endometrial cancer samples and they were relatively high in normal endometrial tissues (Fig. 2A and B). Therefore, these two genes fulfilled our criteria of being putative tumor suppressor genes in endometrial tissue.



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  		FIGURE 2. Expression of the Tig1 and C/ebp{alpha} genes in cell lines and clinical samples. A and B. Real-time quantitative RT-PCR analysis of Tig1 and C/ebp{alpha} mRNA expression in endometrial cancer cell lines and samples from normal individuals and those with endometrial malignancy. Expression of the two genes, Tig1 (A) and C/ebp{alpha} (B), was examined by real-time RT-PCR. Results are arbitrarily expressed in units as the ratios of the transcripts/18S rRNA transcripts. The cell lines were cultured in DMEM containing 10% fetal bovine serum without SAHA and 5-Aza-CdR. Cell lines: (1) Ishikawa, (2) AN3CA, (3) HEC-1B, (4) HEC59, (5) RL95-2, and (6) KLE; (7-13) primary endometrial cancer samples; and (14-20) normal endometrial tissues. Real-time PCR was done thrice on each sample. Columns, mean; bars, SD.

 
Methylation Status Analysis of the CpG Islands in the Tig1 and C/ebp{alpha} Genes in Endometrial Cancer Cells and Normal Endometrium
To define the methylation status of the promoter region of Tig1 in Ishikawa cells and endometrial tissues, the bisulfite sequencing method was used (15). Genomic DNA extracted from each sample was treated with bisulfite and subjected to PCR amplification. The region analyzed contained 13 CpG sites and 6 independent clones (alleles) were sequenced in each sample. In Ishikawa cells, all of the sequenced alleles were hypermethylated (Fig. 3A), whereas we rarely detected methylated CpG sites in normal endometrial tissues (Fig. 3B). To investigate methylation status of the Tig1 gene in other endometrial cell lines, COBRA was employed (16). We used PCR primers covering exon1 of the Tig1 gene, in which CpG sequences were also methylated in Ishikawa cells but not in normal endometrial tissues as confirmed by bisulfite sequencing. Because this region contains seven recognition sites of the restriction enzyme HhaI (GCGC), using COBRA in this region with HhaI is a very sensitive means to detect the methylation of the Tig1 gene (Fig. 3C). The PCR products derived from methylated alleles can be cut into fragments with this enzyme, whereas the PCR products from unmethylated alleles cannot be cut because the restriction enzyme recognition sites disappear after bisulfite treatment. For all six endometrial cell lines, the PCR products were cut into small fragments (Fig. 3C), consistent with each of these cell lines being methylated in this region. As expected, the normal endometrial DNA treated with bisulfite and PCR amplified did not cut in the presence of HhaI, consistent with Tig1 gene being unmethylated as shown on Fig. 3C. In the C/ebp{alpha} gene, the region analyzed contained 31 CpG sites; in Ishikawa cells, only a few sites were methylated in a few alleles (data not shown).



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  		FIGURE 3. Methylation status of the Tig1 and C/ebp{alpha} genes. Methylation status of each CpG site in Ishikawa cells (A) and normal endometrial tissue (B) were determined by bisulfite sequencing. {blacksquare}, methylated CpG; {square}, unmethylated CpG. C. COBRA of the Tig1 gene. Exon 1 of the Tig1 gene was amplified from bisulfite-treated DNAs of six endometrial cell lines (Ishikawa, HEC59, HEC-1B, AN3CA, KLE, and RL95-2) and one normal endometrial sample. PCR products were digested with HhaI. Ishikawa cells and a normal endometrial sample were used as positive and negative controls, respectively. For all endometrial cancer lines, the PCR products were cut into fragments with HhaI, showing that these regions were methylated in these samples. The normal endometrial sample was not cut, consistent with Tig1 being unmethylated in these normal cells.

 
Further, we screened the methylation status of Tig1 gene in 18 clinical endometrial cancer samples by methylation-specific PCR (MSP). Four of the 18 cases showed methylation of the Tig1 gene (Fig. 4), and the positive cases were older (ages 69 versus 56 years; P = 0.01) and more frequently had metastatic disease (Table 2). Difference in the ages was statistically significant.



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  		FIGURE 4. MSP of Tig1 gene in clinical endometrial cancer samples. Top, MSP for methylated alleles of Tig1 gene. Ishikawa cells and normal endometrium were used as positive and negative controls, respectively. Samples from patients 1, 4, 6, and 8 show the PCR products for the methylated alleles of Tig1 gene. Bottom, MSP for unmethylated alleles of Tig1 gene. Ishikawa cells and normal endometrium were used as negative and positive controls, respectively. All cases, except Ishikawa cells, showed the PCR products for the unmethylated alleles of Tig1.

 

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  		Table 2. Clinical Data of Cases with Endometrial Cancer Examined

 
Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assays were done using Ishikawa cells treated with SAHA and/or 5-Aza-CdR to investigate the relative amounts of acetylated histone H3 and H4 bound to either Tig1 or C/ebp{alpha} gene (Fig. 5). The amount of acetylated histones (H3 and H4) binding to the Tig1 gene increased after exposure to either or both of the agents. In the C/ebp{alpha} gene, bound acetylated histone H3 increased after each treatment, whereas the amount of the acetylated histone H4 showed little change. Low number of cycles of the PCR reaction revealed identical results, which were indicative of an increase in the active transcription of these genes.



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  		FIGURE 5. SAHA and/or 5-Aza-CdR induced accumulation of acetylated histone H3 and H4 bound to the Tig1 and C/ebp{alpha} genes. DNA-chromatin complexes were immunoprecipitated with either anti-acetylated histone H3 (Ac-H3) or H4 (Ac-H4) antibodies or normal rabbit serum (cont) from Ishikawa cells cultured either with 5-Aza-CdR (5 µmol/L, 3 days) and/or SAHA (2.5 µmol/L, 1 day) or with no treatment (No Tx). The Tig1 and C/ebp{alpha} genes were amplified from the DNA isolated from the immunoprecipitated DNA-chromatin complexes.

 
Colony Formation Assay of the Tig1 and C/ebp{alpha} Genes
The effect of forced expression of either Tig1 or C/ebp{alpha} on the clonal growth of endometrial cancer cells was examined by transiently transfecting expression vectors into Ishikawa cells. Because anti-TIG1 antibody was not available, we inserted FLAG tag sequences into the NH2-terminal end of Tig1. Overexpression of each gene was confirmed at the protein level by Western blot analysis following transfection (Fig. 6A and D). The cells were plated and selected with G418; after 14 days, the resistant colonies were stained and counted. For colony transformation assay, results represent the mean of three experiments. Overexpression of both TIG1 and C/EBP{alpha} proteins strongly suppressed clonal growth of Ishikawa cells (Fig. 6B, C, E, and F).



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  		FIGURE 6. Enforced expression of either Tig1 or C/ebp{alpha} suppressed clonogenic growth of Ishikawa cells. Ishikawa endometrial cancer cells were transfected with either pcDNA3.1 (Empty vector), pcDNA3-FLAG-Tig1 (TIG), or pcDNA3-C/ebp{alpha} (C/EBP{alpha}). Protein expression levels at 24 hours after transfection were confirmed by Western blot using either anti-FLAG or anti-C/EBP{alpha} antibody (A and D). Transfected cells were selected with G418, and resistant colonies (after 14 days of culture in G418) were stained with crystal violet (B and E). The numbers of the colonies were counted (C and F). Columns, mean of experiments repeated thrice with triplicate dishes per experimental point; bars, SD.

 
Discussion
Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors (3, 17). Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. Aberrant hypermethylation of CpG islands in the promoter region of genes is a major mechanism by which tumor suppressor genes can become inactivated in cancer. Inhibitors of DNA methylation are becoming promising agents for cancer treatment. The nucleoside analogues 5-aza-cytidine and 5-Aza-CdR are potent inhibitors of DNA methylation (18) and are currently being tested as treatments for myelodysplastic syndromes and other diseases (19).

Recent investigations have shown the importance of the post-translational modification of histones as another mechanism of transcriptional control (20, 21). DNA methylation and histone modification interplay with each other to regulate the transcription of genes. Acetylation of Lys9 and methylation of Lys4 in histone H3 are associated with an open chromatin configuration, which is observed in transcriptionally active promoters. In contrast, methylation of Lys9 in histone H3 is a marker of condensed, inactive chromatin, which is most notably observed in the inactive X chromosome and at pericentromeric heterochromatin (22). In cancer therapy, therefore, the combination of a DNA methylation inhibitor and a HDAC inhibitor may be more efficient than either alone in leading to the reexpression of tumor suppressor genes previously silenced in cancers.

In the present study, we set about to uncover novel tumor suppressor genes that are the subject of epigenetic silencing in endometrial cancers. Treatment with the combination of a DNA demethylating agent (5-Aza-CdR) and a HDAC inhibitor (SAHA) efficiently unmasked the expression of a large subset of genes. Using microarray analysis and real-time RT-PCR, we compared the expression of genes in the treated endometrial cell lines with those of the untreated cells. We focused on six genes (Tig1, Sh3d5, Ngfr, Rpl3l, Rgc32, and C/ebp{alpha}) whose expression was induced by 5-Aza-CdR and SAHA in the cell lines, not expressed in clinical cancer samples, and highly expressed in normal endometrial tissues. The fold change of expression of the tumor suppressor candidate genes detected by real-time RT-PCR was higher than detected by the microarray technique. Thus, the data emphasize the importance of verifying the microarray data using another quantitative technique.

We first analyzed the methylation status of the six candidate genes (Tig1, Sh3d5, Ngfr, Rpl3l, Rgc32, and C/ebp{alpha}) in Ishikawa cells because the tumor suppressor genes can be silenced by methylation of their promoters and/or first exons. Although we found methylation only in the Tig1 gene, we also did further analysis on C/ebp{alpha} because loss of function and mutations of C/ebp{alpha} have been reported in ~10% of acute myeloid leukemia samples (11, 12). In addition, other investigators have suggested that C/ebp{alpha} is an important factor for cellular differentiation; at least in leukemia, it is very often dysregulated in expression (13). Therefore, we focused on these two candidates, C/ebp{alpha} and Tig1.

Tig1, also known as retinoic acid receptor responder 1, is a retinoic acid receptor–responsive gene that was originally isolated from the skin and whose expression is increased by the synthetic retinoid tazarotene (23). The gene is located at 3p12-13, and loss of this region has been documented in endometrial cancers (24). Furthermore, expression of Tig1 is very low in prostate cancers. Thus far, the function of Tig1 remains to be determined (14). In our in vitro experiments, Tig1 had antiproliferative activity, suggesting that it could behave as a tumor suppressor gene in endometrial cancers. Although we tried to establish stable cell lines expressing Tig1 to analyze the antiproliferative activity of Tig1, we were unsuccessful. In our transient transfection assay, efficiency of transfection into Ishikawa cell is <10%. We were not able to distinguish the cells killed by transfection from those killed by the antibiotic selection. Therefore, we could not detect differentiated cells after transfection of these two genes; thus, we could not discern if the decreased growth of endometrial cancer cells after forced expression of either Tig1 or C/ebp{alpha} was associated with apoptosis or differentiation.

Moreover, our finding that the Tig1 gene is highly methylated in endometrial cancers but not in normal endometrial samples supports the notion that the gene may behave as a tumor suppressor gene in endometrial cells. We found methylation of the Tig1 gene in all 6 endometrial cancer cell lines analyzed by COBRA and 4 of 18 clinical endometrial cancer samples. The cases with methylation of Tig1 were older and three of them showed metastatic diseases. Methylation of Tig1 may be associated with aggressiveness of the endometrial cancer. However, these cases with methylation of Tig1 did not show a worse prognosis.

While preparing this article, Tokumaru et al. reported hypermethylation of the Tig1 gene in a variety of nonendometrial human cancer cell lines, with 17 of 25 (68%) of these lines having a methylated Tig1 (25), and Tig1 was noted to be methylated in clinical samples of prostate cancer (26) and a variety of human cancers (27-29). Taken together, Tig1 may function as a tumor suppressor gene and prevent carcinogenesis not only in endometrial cells but also in a variety of tissues.

C/ebp{alpha} is a member of the basic leucine zipper family of transcription factors. This intron-less gene is located on chromosome 19q13.1. Two isoforms of the C/EBP{alpha} protein (MW 42,000 and 30,000) are generated from a single transcript through the use of two in-frame start codons. C/EBP{alpha} plays an important role in the terminal differentiation of myeloid cells, hepatocytes, and adipocytes (30, 31). C/EBP{alpha} also has prominent antimitotic activity, the mechanism of which involves direct up-regulation of the p21 cyclin-dependent kinase inhibitor gene in hepatocytes and the interaction of C/EBP{alpha} with the retinoblastoma/E2F protein complex in adipocytes and myeloid cells (32-34).

The C/ebp{alpha} gene did not seem to be methylated in Ishikawa cells. However, our results suggest that C/ebp{alpha} may function as a tumor suppressor gene in endometrial cancers. Not only have the point mutations of C/ebp{alpha} been found in acute leukemias, but methylation of this gene has also been reported in this disease (11, 12, 35). However, the frequency of methylation of C/ebp{alpha} is very infrequent in these leukemias, although point mutations of this gene are frequently detected (11, 12, 35). Endometrial cancers should also be examined for point mutations of the C/ebp{alpha} gene.

Recently, reports have found that many genes having no CpG islands in their promoter regions are still stimulated transcriptionally after treatment with DNA methylation inhibitors (36). Interestingly, restoration of expression of Tig1, which was highly methylated, was induced by the HDAC inhibitor alone; expression of C/ebp{alpha} gene, which was not methylated, was induced by the DNA methylation inhibitor alone in our experiments (Fig. 1). These could be target genes of transcriptional factors whose genes are silenced by methylation. Reactivation of these silenced genes could sequentially induce downstream target genes. Alternatively, unknown effects of DNA methylation inhibitors may induce the transcription of a set of genes irrespective of their promoters' CpG status. HDAC inhibitors could affect methylated genes by modulating expression of genes associated with DNA methylation/demethylation.

In summary, we have discovered two putative tumor suppressor genes that are stimulated by demethylating agents and/or HDAC inhibitors in endometrial cancers. In additional studies, we have found that these two classes of drugs were able to inhibit effectively the growth of human endometrial cancer cells both in vitro and growing as xenografts in immunodeficient mice (37). This study provides us with valuable insight into not only the role of epigenetic changes in endometrial carcinomas but also the mechanisms by which the use of DNA-modifying drugs may prove to be important therapeutic tools in the treatment of these malignancies.


   Materials and Methods
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 Notes
 Abstract
 Introduction
 Results
 Materials and Methods
References
 
Cell Lines and Patient Samples
Six endometrial cancer cell lines (HEC-1B, RL95-2, KLE, AN3CA, Ishikawa, and HEC59) were used in this study. HEC-1B, RL95-2, KLE, and AN3CA cell lines were obtained from American Type Culture Collection (Rockville, MD) and maintained according to their recommendations. Ishikawa was kindly provided by Dr. Bruce A. Lessey (University of North Carolina, Chapel Hill, NC), and HEC59 was generously given to us by Dr. Timothy J. Kinsella (Case Western Reserve University, Cleveland, OH). These were maintained in DMEM with 5% fetal bovine serum. Samples of endometrial cancer (18 samples) and normal tissues (7 samples) were obtained from patients or normal volunteers after their informed consents. All normal endometrial tissues were obtained from postmenopausal women. All normal control cases showed atrophy of the endometrial tissues. All cases, including patients with cancers and normal controls, were not taking any hormone replacement treatment. Histologic type and grade of the endometrial cancers were classified based on the WHO classification (38). Staging of endometrial cancers used the staging system (1988) of the International Federation of Gynecology and Obstetrics (39): stage IA: tumor limited to endometrium, stage IB: invasion to ≤50% of myometrium, stage IC: invasion >50% of myometrium, IIA: endocervical glandular involvement only, IIB: cervical stroma invasion, IIIA: tumor invasion of serosa and/or adnexae and/or positive peritoneal cytology, and IV: tumor invasion of bladder and/or bowel mucosa or distant metastases, including intra-abdominal and/or inguinal lymph node.

Treatment with 5-Aza-CdR and/or SAHA
Six endometrial cancer cell lines were treated with 5-Aza-CdR (Sigma, St. Louis, MO) and SAHA (Alexis, Lausen, Switzerland) either alone or in combination. Cells were exposed continuously to 5-Aza-CdR (5 µmol/L) for 3 days or to SAHA (2.5 µmol/L) for 24 hours. For combined treatment, cells were cultured in the presence of 5-Aza-CdR (5 µmol/L) for 3 days and exposed on the third day (24 hours) in combination with SAHA (2.5 µmol/L). Mock-treated cells were cultured similarly.

Microarray Analysis
Total RNA (10 µg) extracted from treated and untreated Ishikawa cells or clinical endometrial samples was used as starting material for the cRNA preparation. Ishikawa cells were cultured in the presence of 5-Aza-CdR (5 µmol/L) for 2 days, and on the third day, SAHA (2.5 µmol/L) was also added to the culture. The viability of the Ishikawa cells after this treatment was >80%. All clinical samples were endometrioid carcinoma.

cDNA was synthesized using an oligo(dT) primer containing a T7 RNA polymerase promoter site with SuperScript Choice System (Invitrogen, Carlsbad, CA). Labeled RNA was prepared using the BioArray High-Yield RNA Transcript Labeling kit (Enzo, Farmingdale, NY).

Human genome U133A was used for microarray analysis. Array hybridization and scanning was done at the University of California at Los Angeles Microarray Core Facility (Los Angeles, CA). The hybridized signals were scanned at 560 nm using a confocal laser scanning microscope (GeneArray Scanner G2500A, Hewlett-Packard, Palo Alto, CA). The fluorescence intensity was measured for each microarray and normalized to the average fluorescence intensity for the entire microarray. GeneChip image analysis was done using the Microarray Analysis Suite 5.0 (Affymetrix). To assure that the gene expression measured by microarray assay was not affected by degradation of the RNA, we used the Bioanalyzer System (Agilent Technologies, Waldbronn, Germany) to evaluate the quality of the RNA. Furthermore, the expression level of glyceraldehyde-3-phosphate dehydrogenase, as determined by GeneChip assay, was required to be >5,000 (raw data) and measured as "present" (Affymetrix Call) in all of the samples. The experiments were done in triplicates. Data were analyzed by the GeneSpring software version 4.2 (Silicon Genetics, San Carlos, CA). The genes whose Ps between raw data of three independent microarray analyses were <0.05 were selected for further study to confirm the reproducibility of the microarray analysis. Ps of raw data of glyceraldehyde-3-phosphate dehydrogenase between three independent microarray analyses were <0.05. Fold changes in signal intensities obtained from oligonucleotide microarrays between treatment and control groups were calculated with Microarray Analysis Suite 5.0.

Real-time Quantitative PCR
Gene expression was quantified by real-time quantitative RT-PCR using iCycler (Bio-Rad, Hercules, CA). TaqMan or SYBR Green methods were employed according to the manufacturer's protocol. The sequences of the primer/probe sets used for this analysis will be provided on request. To determine the relative expression level of each sample, 18S rRNA expression levels were measured as internal controls. We did real-time PCR thrice on each samples and show the means and SDs by error bars in Figs. 1 and 2. Expression levels of each of the genes in the cell lines were measured separately in Figs. 1 and 2.

Bisulfite Sequencing, COBRA, and MSP
Bisulfite modification of DNA was done with the CpGenome DNA Modification kit according to the manufacturer's recommendations (Intergen Co., Purchase, NY). After bisulfate modification, the following primers were used for PCR: Tig1-S-1: 5'-AAAAACACCAAATCCCTAAACTAAACTA-3', Tig1-AS-1: 5'-GTAGGGTTGGGTGTTTTTGGTTTA-3', C/ebp{alpha}-S-2: 5'-CCACCCCACACCTACAATTCCAAAT-3', and C/ebp{alpha}-AS-2: 5'-GTTTTTGTTTATTGATTTTTTGGTTTTGTT-3'. PCR products were subcloned into pGEM-T Easy (Promega, Madison, WI) for sequence analysis. Six independent clones were sequenced in all samples analyzed. For COBRA (16) analysis of methylation of the for Tig1 gene, PCR primers Tig1-S-3: 5'-GTGTATTTAGGTGTTATTTTTTAG-3' and Tig1-AS-3: 5'-CTTCACTTCTTCAACTTCCAATCC-3' were used. The PCR products were digested with restriction enzyme HhaI (Invitrogen) and then electrophoresed in 2% agarose gel. Primers for Sh3d5, Ngfr, Rpl3l, and Rgc32 genes are provided on request.

Primers for MSP of Tig1 gene are as follows: for methylated alleles, M-TIG-MSP-S: 5'-AGCGTCGTGCGCGGATAGGTA-3' and M-TIG-MSP-AS: 5'-TCGGATCGGTTCGTTTAGCGCGTTA-3'; for unmethylated alleles, U-TIG-MSP-S: 5'-TTGGGTTAGGGATGTGGTATG-3' and U-TIG-MSP-AS: 5'-AATACTAAAATACAACATCACCTCCA-3'. MSP was done on bisulfite-treated DNA according to the manufacturer's protocol (Intergen).

ChIP Assay
Cells were plated at a density of 1 x 106 cells per 100 mm dish and incubated overnight. The cells were cultured with or without SAHA, 5-Aza-CdR, or both as described above. ChIP assay was done using ChIP assay kit (Upstate, Inc., Chicago, IL) according to the manufacturer's protocol. In brief, the treated cells were chemically cross-linked in 1% formaldehyde. Cells (5 x 105) from each sample were pelleted and resuspended in 0.5 mL SDS lysis buffer and then sonicated with US sonicator. Aliquots were diluted 5-fold in immunoprecipitation buffer. Anti–acetylated histone H3 antibody (5 µg, Upstate), anti–acetylated histone H4 antibody (Upstate), or normal rabbit serum were added and incubated overnight at 4°C. Immune complexes were precipitated with protein A-Sepharose beads (Upstate) and eluted into elution buffer. The protein-DNA cross-linking was reversed under high-salt condition; precipitated DNA was purified by standard proteinase K phenol/chloroform extraction method and then suspended in 50 µL H2O. DNA isolated from ChIP experiments were used as templates for the PCR of Tig1 and C/ebp{alpha} genes. PCR products were electrophoresed in 2% agarose gels. The primer pairs used for Tig1 ChIP analysis were 5'-GAACTTTGCAACCCGTTGTT-3' and 5'-GATTCTTTGAGGCCGTGTGT-3'. The primers used for C/ebp{alpha} ChIP analysis were 5'-TGGACAAGAACAGCAACGAG-3' and 5'-TTGTCACTGGTCAGCTCCAG-3'.

Transfection and Colony Formation Assay
The Tig1 cDNA plasmid was kindly provided by Dr. Youqiang Ke (Royal Liverpool University, Liverpool, United Kingdom). The FLAG tag sequence (Sigma) was inserted into the 5' end of the coding region of the Tig1 cDNA by PCR. The FLAG-Tig1 cDNA was cloned into the pcDNA3.1(+) expression vector (Invitrogen). The entire coding region (nucleotides 132-1,230) of the wild-type C/ebp{alpha} was amplified as described previously (11). The fragments were gel purified and ligated into the pcDNA3.1(+) expression vector.

Colony formation assays were done in monolayer culture as described previously (40). Ishikawa cells were plated at 2 x 104 per well into six-well plates and transfected with either pcDNA-FLAG-Tig1, pcDNA-C/ebp{alpha}, or pcDNA3.1(+) empty vector (0.4 µg/well) using Effectene Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer's protocol. At 24 hours after the transfection, these cells were harvested and one third of each sample was plated onto 100 mm tissue culture dishes and two thirds of each sample were subjected to Western blot analysis to confirm the expression of the proteins. Cells were selected in medium containing G418 (800 µg/mL). The medium was replaced every 3 days. After 14 days, colonies were stained with 1% crystal violet and photographed. Colonies that were larger than 3 mm in diameter were counted as positive. For colony transformation assay, results represent the mean of three experiments.

Statistical Analysis
Statistical differences between means were analyzed by the t test. Ps < 0.05 were considered to indicate statistical significance.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Materials and Methods
 References
 
Grant support: NIH grant CA042710-20 (H.P. Koeffler), A. and S. Schwartz Fund, Women's Cancer Research Fund, and Parker Hughes Trust.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: N. Takai and N. Kawamata contributed equally to this work. H.P. Koeffler holds the Mark Goodson Endowed Chair of Oncology Research and is a member of the Jonsson Cancer Center and the Molecular Biology Institute of University of California at Los Angeles.

6 J.C. Desmond et al., unpublished data. Back

Received 6/12/04; revised 3/ 1/05; accepted 3/16/05.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Materials and Methods
 References
 

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