Identification of HRK As a Target of Epigenetic Inactivation in Colorectal and Gastric Cancer

6410 Vol. 9, 6410–6418, December 15, 2003 Clinical Cancer Research

Identification of HRK as a Target of Epigenetic Inactivation in Colorectal and Gastric Cancer

Toshiro Obata,1 Minoru Toyota,1,2 Ayumi Satoh,1 primary colorectal cancers that showed methylation of multi- Yasushi Sasaki,2 Kazuhiro Ogi,2 ple genes, including p16INK4A and hMLH1, and was associ- Kimishige Akino,1,2 Hiromu Suzuki,1 ated with wild-type p53. 1 1,2 Conclusion: HRK methylation can be a useful molecular Masafumi Murai, Takefumi Kikuchi, target for cancer therapy in a subset of colorectal and gastric 2 1 3 Hiroaki Mita, Fumio Itoh, Jean-Pierre J. Issa, cancers. Takashi Tokino,2 and Kohzoh Imai1 1First Department of Internal Medicine and 2Department of Molecular INTRODUCTION Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan; and 3Department of Leukemia, The University of Dysregulation of proliferation, together with defective control Texas M. D. Anderson Cancer Center, Houston, Texas of apoptosis, are hallmarks of human tumors (1). Among the genes involved in the regulation of apoptosis, p53 is a major player whose downstream target genes participate in several apoptosis pathways ABSTRACT and are disrupted in cancer (2). BAX, one of the target genes of Purpose: Aberrant methylation of CpG islands can be a p53, has been shown to be mutated in a subset of colorectal and good molecular marker for identifying genes inactivated in gastric cancers (3). In addition to genetic changes, much evi- cancer. We found the proapoptotic gene HRK to be a target dence now suggests that by silencing transcription of certain for hypermethylation in human cancers and examined the genes, aberrant methylation of CpG islands (CGIs) also plays a role of such methylation in silencing the gene’s expression. key role in tumorigenesis (4, 5). Indeed, an increasing number of Experimental Design: Methylation of HRK was evaluated genes related to cell cycle control, DNA damage repair, the by bisulfite-PCR and bisulfite sequencing in a group of colo- invasiveness of tumors, and growth factor responses are now rectal and gastric cancer cell lines and primary cancers. Gene known to be inactivated by DNA methylation in gastrointestinal expression and histone acetylation were examined by reverse malignancies. Much less is known about epigenetic inactivation transcription-PCR and chromatin immunoprecipitation anal- of proapoptotic genes, however (6–8). yses, respectively. Apoptosis of cancer cells after treatment Aberrantly methylated CGIs can be good molecular markers with a DNA methyltransferase inhibitor and/or histone de- with which to identify genes inactivated in cancer. Several tech- acetylase inhibitor was examined with fluorescence-activated niques, including restriction landmark genome scanning and meth- cell-sorting analysis. ylation-sensitive arbitrary primer PCR, have been used to screen Results: The region around the HRK transcription start for aberrantly methylated DNA fragments as molecular markers of site was methylated in 36% of colorectal and 32% of gastric genes inactivated by DNA methylation (9–11). We have developed cancer cell lines and was closely associated with loss of expres- a novel methylation screening technique, methylated CGI amplifi- sion in those cell types. HRK expression was restored by treat- cation, which we used to identify DNA fragments hypermethylated ment with a methyltransferase inhibitor, 5-aza-deoxycytidine, in cancer (12). Moreover, coupling methylated CGI amplification and enhanced further by addition of histone deacetylase inhib- to representational difference analysis allowed us to identify genes itor trichostatin A or depsipeptide. Such restoration of HRK inactivated by hypermethylation (13, 14). One of the DNA frag- expression was well correlated with induction of apoptosis and ments identified with methylated CGI amplification, MINT17 (12, enhancement of Adriamycin-induced apoptosis. Expression of 15), was mapped to a part of the CGI of harakiri (HRK), a other proapoptotic genes, including BAX, BAD, BID, and proapoptotic gene belonging to the BH3-only subfamily of bcl-2. PUMA, was unaffected by treatment with 5-aza-deoxycytidine. HRK was originally isolated as a bcl-xL- and bcl-2-interacting Aberrant methylation of HRK was also frequently detected in protein (16) whose expression was induced by withdrawal of growth factors and which was involved in apoptosis of hematopoietic and neuronal cells (17, 18). Little is known about the function of HRK in human tumors. The aim of the present study, therefore, was to determine the extent to which DNA methylation affects Received 3/31/03; revised 9/15/03; accepted 9/15/03. Grant support: Grants-in-Aid for Scientific Research on Priority Areas expression of HRK and induction of apoptosis in a group of human from the Ministry of Education, Culture, Sports, Science and Technol- cancer cell lines and primary colorectal and gastric cancers. ogy (to M. T., F. I., T. T., and K. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked MATERIALS AND METHODS advertisement in accordance with 18 U.S.C. Section 1734 solely to Cell Lines and Specimens. We used 11 colorectal cancer indicate this fact. cell lines (Caco2, RKO, SW48, HCT116, DLD-1, LoVo, HT29, Requests for reprints: Minoru Toyota, Department of Molecular Bi- ology, Cancer Research Institute, Sapporo Medical University, South 1, Colo205, Colo201, SW480, and Colo320DM), 19 gastric cancer West 17, Chuo-ku, Sapporo 060-8556, Japan. Phone: 81-11-611-2111, cell lines (MKN7, MKN28, MKN45, MKN74, JRST, KatoIII, ext. 2387; Fax 81-11-618-3313; E-mail: [email protected]. AZ521, NUGC3, NUGC4, SNU1, SNU638, SH101, HSC39,

Table 1 Sequences for primers used in this study Size Primer set Sequences PCR conditionsa (bp) Enzyme HRK-GM1 F:b 5Ј-AAAYGTATAATATAAGGAGAAATTTGG- 60 (3), 58 (4), 56 (5), 54 (26) 161 TaqI R: 5Ј-RATACAAAAAACACRAACACATAAC-3Ј HRK-GM2 F: 5Ј-GYGAGGTTAGYGGTTATGTGTT-3Ј 58 (3), 56 (4), 54 (5), 52 (26) 170 HhaI R: 5Ј-CRCCRCCACATAATACACTAATAC-3Ј HRK-GM6 F: 5Ј-ATTATAGATGGGTTTAGGGTYGGAAG-3Ј 64 (3), 62 (4), 60 (5), 58 (26) 181 BsPT104I R: 5Ј-CCCCCCACRCTCCRACTAC-3Ј MINT17 F: 5Ј-TTTTTTTTTTGTTGGAGAGGTTA-3Ј 58 (3), 56 (4), 54 (5), 52 (26) 184 NruI R: 5Ј-CTTCRCCCACTTTTTTTCTAC-3Ј HRK-GM7 F: 5Ј-TGTYGGATTGGAAAAGGGGGAA-3Ј 62 (3), 60 (4), 58 (5), 56 (26) 186 TaqI/TaiI R: 5Ј-CCCAAAAAACCCTCRCAAAAAACT-3Ј HRK-GM8 F: 5Ј-TTAATATATAGGTTAGGTAGGTTGGTT-3 62 (3), 60 (4), 58 (5), 56 (26) 179 TaiI R: 5Ј-CTAAAATTACAAATATACACCACCAC-3Ј HRK-RT F: 5Ј-TGCTCGGCAGGCGGAACTTGTAG-3Ј 60 (35) 237 R: 5Ј-GCTTCCCCAGTCCCATTCTGTGTTT-3Ј HRK-ChIP F: 5Ј-CCCGCCCCCCTCCACCATGTGAC-3Ј 60 (35) 129 F: 5Ј-CGCCTCCTCTCCCTCCGGCCTCTG-3Ј BAD-GM1 F: 5Ј-GTGATTGGGTTAGGTYGTTGATAG-3Ј 60 (3), 58 (4), 56 (5), 54 (26) 194 TaqI R: 5Ј-ACTCAAAACCTCAATCTCCCCTC-3Ј BAX-GM1 F: 5Ј-TTATTGTTGGTATTTATYGGGAGATG-3Ј 62 (3), 60 (4), 58 (5), 56 (26) 172 HinfI R: 5Ј-AATCACRTAAAAACCCCRCTAA-3Ј BID-GM1 F: 5Ј-AAGGGTGGAAAGTTTGGTTTTTATTG-3Ј 58 (3), 56 (4), 54 (5), 52 (26) 189 TaqI R: 5Ј-TCRACTACCCRCTTCCTCCTTAT-3Ј BIM-GM1 F: 5Ј-TTGGAYGTAGAGTTTTAATAAATTGT-3Ј 58 (3), 56 (4), 54 (5), 52 (26) 176 TaiI R: 5Ј-RAAAAAAACRAAACTTACCAACC-3Ј PUMA-GM1 F: 5-GGGYGGGYGGTTTTTTTTAG-3Ј 62 (3), 60 (4), 58 (5), 56 (26) 185 TaqI R: 5-ACRACACCAACRATCCCAACAA-3Ј RT-PCR BAD-RT F: 5Ј-CAGCCAACCAGCAGCAGCCATCA-3Ј 60 (35) 320 F: 5Ј-CCCAAGTTCCGATCCCACCAGGAC-3Ј BID-RT F: 5Ј-CCTAGAGACATGGAGAAGGAGAAGAC-3 60 (35) 464 F: 5Ј-GGAAATAAAGGCACCGTGTGTAGAT-3Ј BAX-RT F: 5Ј-CGTCCACCAAGAAGCTGAGCG-3Ј 60 (35) 344 R: 5Ј-AGCACTCCCGCCACAAAGATG-3Ј NOXA-RT F: 5Ј-CCAGTTGGAGGCTGAGGTTC-3Ј 60 (35) 362 R: 5Ј-CGTTTCCAAGGGCACCCATG-3Ј a Temperature in °C (number of cycles). b F, forward; R, reverse; RT-PCR, reverse-transcription-PCR.

HSC40, HSC41, HSC42, HSC43, HSC44, and HSC45), 7 pancre- amplify both methylated and unmethylated alleles. After amplifi- atic cancer cell lines (Panc-1, BxPC3, MIAPACA2, KP-1NL, KP3, cation, the PCR products were digested with restriction enzymes CF-PAC, and Capan2), 5 hepatocellular cancer cell lines (CHC4, that cleaved the regions containing the CpG sites retained after CHC20, CHC32, HLE, and HuH7), and 14 hematopoietic cancer bisulfite treatment as a result of cytosine methylation. The PCR cell lines (HO3238, KMS12PE, RPMI8226, K562, KR12, Daudi, primers and cycling parameters used to examine methylation of BALL1, Jurkat, Hs-sultan, RPMI1788, SCC3, Raji, IM9, and various regions of HRK and five other proapoptotic genes are ATL2) in this study. With the exception of eight gastric cancer cell shown in Table 1. To sequence the bisulfite-PCR products, frag- lines (HSC39, HSC40, HSC41, HSC42, HSC43, HSC44, HSC45, ments amplified with primers HRKGM1F/HRKGM1R and and SH-101) kindly provided by Dr. Yanagihara (National Cancer HRKGM2F/HRKGM2R were cloned into pCR4 vector by use of Center, Research Institute, Tokyo, Japan) (19, 20), all of the cell a TOPO-cloning kit (Invitrogen). Cycle sequencing was then car- lines were obtained from either the American Type Culture Col- ried out with a BigDye terminator kit (Applied Biosystems), after lection (Manassas, VA) or from the Japanese Collection of Re- which the DNA was sequenced on an ABI 3100 automated se- search Bioresources (Tokyo, Japan). The 58 primary colorectal quencer (Applied Biosystems). cancers, 50 colorectal adenomas, and 23 primary gastric cancer Reverse transcription-PCR (RT-PCR). Total RNA was specimens, as well as the 38 normal colorectal mucosa specimens prepared from normal stomach and colon tissues and from lym- and 23 stomach mucosa specimens have been described previously phocytes and cancer cell lines, after which 5-␮g samples were (21, 22). reverse-transcribed with Superscript II (Life Technologies) to pre- Bisulfite-PCR and Sequencing. Bisulfite modification of pare first-strand cDNA. The primer sequences and PCR parameters DNA was carried out essentially as described previously (23), after used are shown in Table 1. Controls consisted of RNA treated which the DNA was purified by a DNA purification system (Pro- identically but without the addition of reverse transcriptase and mega), precipitated with ethanol, resuspended in water, and stored are labeled as negative (Ϫ). The integrity of the cDNA was con- at Ϫ20°C until use. Bisulfite-PCR was carried out with primers that firmed by amplifying glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) as described previously (21). Samples (10 ␮l) of ampli- 24 h. Thereafter, the cells were harvested with trypsin, incubated fied product were then subjected to 2.5% agarose gel electrophore- in 75 mM KCl for 30 min at room temperature, and centrifuged. sis and stained with ethidium bromide. The resultant supernatant was discarded, and the cells were Chromatin Immunoprecipitation (ChIP) Analysis. fixed for 10 min in 5 ml of methanol at room temperature. After ChIP analysis was carried out as described previously (24). the cells were centrifuged again, the supernatant was discarded, Briefly, 1 ϫ 106 cells were incubated with 1.0% formaldehyde and the pellet was resuspended in 100 ␮l of methanol. Ten- for 10 min at 37°C to cross-link the DNA. The cells were then milliliter aliquots of the resultant cell suspension were dropped washed with ice-cold PBS containing protease inhibitors and onto glass slides and allowed to dry. After staining with pro- resuspended in lysis buffer [1% SDS, 10 mM EDTA, 50 mM pidium iodide (PI), the cells were washed with PBS, a coverslip Tris-HCl (pH 8.0), protease inhibitor]. Nucleoprotein complexes was placed over them, and the edges were sealed with clear nail were sonicated to reduce the size of the DNA fragments to polish. The shape of the nucleus of each cell was then examined 200-1000 bp, after which immunoprecipitation with antiacety- under a fluorescence microscope (ZEISS). lated histone H3 or antiacetylated histone H4 antibody (Upstate Fluorescence-Activated Cell-Sorting Analysis. Cells Biotechnologies, Lake Placid, NY) was carried out for 16 h at (3 ϫ 105 cells/dish) used for fluorescence-activated cell-sorting 4°C with rotation, and the resultant immune complexes were analysis were first treated with 5-aza-dC and/or a histone collected on protein A-agarose beads. The DNA was then pu- deacetylase (HDAC) inhibitor and Adriamycin (Sigma). They rified by phenol–chloroform extraction, precipitated with etha- were then trypsinized, fixed with methanol, rehydrated with nol, and resuspended in water, after which they were subjected PBS, treated with 2 mg/ml RNase at 37°C for 30 min, and to PCR amplification with primers HRK-ChIPF and HRK- stained with PI (50 ␮g/ml of solution). Fluorescence-activated ChIPR (Table 1). The amplified products were subjected to cell-sorting analysis was then carried out with a Becton Dick- agarose gel electrophoresis, and the intensities of resultant bands inson FACScan flow cytometer (Braintree, MA). were determined by a Lane and Spot Analyzer (Atto, Japan). Detection of Apoptotic cells by Fluorescence Micros- copy. Cells (5 ϫ 102) grown in 60-mm plates at 37°C under an RESULTS

atmosphere of 5% CO2–95% air were treated first for 72 h with Identification of HRK as a Target for Aberrant Meth- 1 ␮M 5-aza-deoxycytidine (5-aza-dC) and then were mock- ylation in Cancer. We previously used the genome screening treated or treated with trichostatin A (TSA) or depsipeptide for technique, methylated CGI amplification, to identify DNA frag-

Fig. 1 Schematic diagram of the sequence around MINT17. A, genomic structure of HRK: CpG sites are indicated by vertical bars; the transcription start site is indicated by an arrow; and the positions of the PCR products analyzed by bisulfite-PCR are indicated by horizontal bars. B, bisulfite-PCR analysis of the 5Ј region of HRK in the indicated (top) cancer cell lines. The primer sets used are shown on the left. M, methylated alleles.

ments aberrantly methylated in colorectal cancer (12). One of those fragments, termed MINT17, matched a genomic DNA sequence on chromosome 12 previously identified by the Hu- man Genome Project. Subsequent Blast analysis revealed that MINT17 is situated within the 2-kb CGI in the 5Ј region of a proapoptotic gene, HRK (Fig. 1A). By comparing genomic sequences obtained from the Human Genome Project (AC025687) and cDNA sequences obtained with an expressed sequence tag (BE247231) from a 5Ј-end-enriched cDNA library constructed by the oligo-capping method (25), the HRK transcription start site was determined to be 120 bp upstream of the translation start site (Fig. 1A, arrow). Using the promoter prediction pro- gram TESS (BCM launcher), we identified three putative SP1 binding sites, an AP1 binding site, and a transcription factor ID (TFIID) binding site, indicating a role for the CGI in the transcriptional regulation of the gene’s expression. The HRK CGI is relatively large, and it is not clear whether it is coordinately regulated with respect to protection from methylation. To address that issue, we designed six primer sets that together spanned the entire CGI and assessed the methylation status in a panel of colorectal and gastric cancer cell lines (Fig. 1). Methylation of the 5Ј edge of the CGI (Fig. 1, GM7) was detected in all 30 cell lines tested, as was methylation of the 3Ј edge (Fig. 1, GM8). By contrast, methylation of the region around transcription start site (Fig. 1, GM1 and GM2) was observed in only 4 of 11 (36%) colorectal cancer cell lines and 6 of 19 (32%) gastric cancer cell lines. Such methylation around the transcription start site was specific to gastrointestinal cancer and was not detected in any of the 7 pancreatic, 14 hematopoi- Fig. 2 Bisulfite sequencing of the 5Ј region of HRK. PCR products etic, or 5 hepatocellular cancer cell lines tested (Table 2; data were cloned into pCR4-TOPO vectors by use of a TOPO DNA cloning not shown). kit, and at least seven clones were sequenced. The CpG sites analyzed Bisulfite sequencing was then carried out in five cell lines are indicated by vertical bars and shown at the top. Methylated and F E to assess the methylation of HRK in more detail (Fig. 2). HSC44 unmethylated CpG sites are indicated by and , respectively. The CpG sites examined by bisulfite-PCR are indicated at the top. The and KatoIII cells, which bisulfite-PCR showed to be densely positions of TATA box and restriction sites used for bisulfite-PCR are methylated, were methylated at almost all CpG sites in the shown at the top. The translation start site was shown by a horizontal region analyzed. The majority of CpG sites were unmethylated arrow at the top. in HCT116 cells, as would be predicted from the bisulfite-PCR. With respect to RKO and HT29 cells, which bisulfite-PCR showed to be partially methylated, the former exhibited heterogeneous methylation, whereas the latter showed methylation in Absence of Methylation in Other Bcl-2 Family Pro- only a limited region. apoptotic Genes in Gastric and Colorectal Cancers. We next examined the methylation status of various other proapoptotic bcl-2 family genes to determine the extent to which they are also affected by epigenetic inactivation in colorectal and Table 2 Frequency of HRK methylation in various human tumors gastric cancers. Of six BH3-only family genes tested, four Tissue type Frequency, n (%) (BAD, BID, PUMA, and BIM) contained CpGs in their 5Ј Cell lines regions; BIK and BLK did not have CGIs. BAX, another bcl-2 Colon 4/11 (36) family proapoptotic gene, was also found to contain a CGI. Gastric 6/19 (32) When bisulfite-PCR was carried out with primers that covered Liver 0/5 (0) the regions around transcription start sites, no methylation was Pancreas 0/7 (0) hematopoietic 0/14 (0) detected in any of the five CGI-containing genes in 11 colorectal Primary tumors and 19 gastric cancer cell lines (Fig. 3). Apparently, epigenetic Colorectal cancer 14/58 (24) inactivation of bcl-2 family genes other than HRK is not com- Colorectal adenoma 1/50 (2) mon in gastrointestinal cancers. Gastric cancer 4/23 (17) Hepatocellular cancer 0/20 (0) DNA Methylation and Histone Deacetylation Are Asso- Normal tissue ciated with Loss of HRK Expression. Although initial re- Colon 0/38 (0) ports suggested that HRK is expressed mainly in hematopoietic Stomach 0/23 (0) cells (16), our RT-PCR analysis revealed that HRK mRNA is Bone marrow 0/6 (0) expressed in a wide variety of tissues, including stomach and

Fig. 3 Absence of methylation in the proapoptotic genes BAD, BID, BAX, PUMA, and BIM. A, CGIs of the indicated Bcl-2 family proapoptotic genes. B, methylation of BAD, BAX, BID, PUMA, and BIM in various colorectal cancer cell lines. Shown is representative bisulfite-PCR analysis of the indicated colorectal and gastric cancer cell lines. The results for bisulfite-PCR using in vitro SssI methylase-treated DNA is shown on the right. M, methylated alleles.

intestine (Fig. 4A). To determine whether aberrant methylation mean that RKO cells are a heterogeneous population in which of HRK correlates with the silencing of its expression, we some cells are unmethylated or that expression is derived from carried out RT-PCR with cDNA from 8 colorectal and 14 gastric unmethylated alleles in all cells. cancer cell lines (Fig. 4, B and C). We found that expression of It was recently shown that histone modification is also HRK was readily detectable in the 6 colorectal and 10 gastric involved in the gene silencing caused by DNA methylation cancer cell lines that did not show methylation of HRK. Expres- (26, 27). We therefore treated various cell lines with the HDAC sion of HRK was not detected in the two colorectal (SW48 and inhibitor TSA and found that, by itself, TSA did not restore HT29) and four gastric cancer cell lines (MKN28, KatoIII, HRK expression (Fig. 6A). It did, however, synergistically en- HSC44, and HSC45) that showed dense methylation of HRK, hance the response to 5-aza-dC, indicating a role for histone however, which indicates that methylation of the 5Ј region of deacetylation in gene silencing. Furthermore, ChIP analysis HRK is associated with loss of expression. using antiacetylated histone H3 and H4 antibodies showed The levels of methylation in each region of the CGI and that acetylation of histones H3 and H4 correlated directly expression of HRK are summarized in Fig. 5. The cell lines with gene expression and inversely with DNA methylation tested could generally be divided into two groups based on the (Fig. 6B). methylation densities determined by bisulfite-PCR. Although Re-expression of HRK Is Correlated with Induction of methylation of the 5Ј and 3Ј edges of the CGI occurred fre- Apoptosis. To determine whether the silencing of proapop- quently, it did not affect HRK expression, and all cell lines totic genes correlated with the escape of cancer cells from belonging to group 1 expressed HRK mRNA at readily detect- apoptosis, we treated three gastric cancer cell lines in which able levels. On the other hand, cell lines belonging to group 2, HRK expression was silent because of methylation (HSC44, which were more densely methylated around the transcription HSC45, and KatoIII) with 5-aza-dC and then examined expres- start site, did not express the gene, indicating that methylation of sion of HRK, BAX, BAD, BID, and NOXA. We found that that region plays a critical role in silencing HRK expression. 5-aza-dC induced expression of HRK in all three cell lines (Fig. Notably, RKO cells did not fall into either group 1 or 2. They 7). Expression of the other genes was unaffected by 5-aza-dC, were methylated on ϳ50% of the alleles throughout the entire which is consistent with the finding that DNA methylation in the region analyzed, but nonetheless expressed the gene. This may 5Ј region of these genes was absent in cancers (Fig. 3). The

CGI was detected in 14 (24%) colorectal and 4 (17%) gastric cancer cases but in only 2% (1 of 50) of colorectal adenomas and in none of the 38 samples of colorectal mucosa and 23 samples of gastric mucosa from areas adjacent to the tumors.

Fig. 5 Correlation between methylation of the HRK CGIs and gene expression. The percentages of methylated alleles were determined by bisulfite-PCR as shown in Fig. 1; their averages are indicated by the columns. All cell lines belonging to group 1 (blue columns; Caco2, HCT116, DLD-1, LoVo, SW480, MKN7, MKN45, MKN74, JRST, AZ521, NUGC3, SNU1, HSC42, and HSC43) expressed the gene. Cell lines belonging to group2 (red columns; SW48, HT29, MKN28, KatoIII, SNU638, HSC44, and HSC45) generally did not express the gene, although a small amount of the mRNA was detected in SNU638 cells. Fig. 4 Expression of HRK in normal tissues (A) and in colorectal (B) and gastric (C) cancer cell lines. Expression of HRK by the indicated (top) cell lines was examined by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was also amplified to confirm the integrity of the cDNA. Corresponding negative controls (amplification without reverse transcription) are shown as RT-negative (Ϫ).

incidence of apoptosis roughly paralleled the expression of HRK, reaching a peak after 72 h of treatment with 5-aza-dC (Fig. 8). Moreover, the effect was synergistically enhanced by addition of TSA or depsipeptide, another HDAC. By contrast, cell lines in which HRK was unmethylated were significantly less susceptible to 5-aza-dC-induced cell death (Fig. 8B; Fig. 9A). We next examined the extent to which pretreating cells with 5-aza-dC increases the susceptibility of cancer cells to apoptosis stimulated by a chemotherapeutic drug (Fig. 9B). Cell lines were first treated with 1 ␮M 5-aza-dC or mock-treated for 72 h and then with Adriamycin for 24 h, after which the percentage of apoptotic cells was determined by flow cytometry. In the cell lines showing HRK methylation, the numbers of apoptotic cells were significantly higher in cells pretreated by 5-aza-dC than in mock-treated cells. On the other hand, no significant effect of 5-aza-dC was seen in the cell lines in which Fig. 6 Involvement of histone deacetylation in DNA methylation-in- HRK was unmethylated. duced silencing of HRK. A, representative RT-PCR analysis of the Aberrant Methylation of HRK in Primary Tumors. HSC45 gastric cancer cell line treated with 0.2 ␮M 5-aza-dC, 300 nM To examine the methylation status of HRK in primary tumors, TSA, or both. B, acetylation status of the histone around the HRK CGI. Shown are the results of a representative ChIP assay carried out with we performed bisulfite-PCR using samples of DNA from 58 antiacetylated histone H3 antibody followed by PCR to amplify the 5Ј colorectal and 23 gastric cancer cases and corresponding sam- region of HRK. The amount of DNA used was monitored by a PCR that ples of normal gastric mucosa (Fig. 10). Methylation of the HRK amplified DNA prepared from the input solution.

Fig. 7 Expression of BH3-only family genes after treatment with the DNA methyltransferase inhibitor 5-aza-dC. Three gastric cancer cell lines that do not express HRK were treated with 1 ␮M 5-aza-dC and then harvested at the indicated times after incubation. Expression of HRK, BAD, BID, BAX, and NOXA was then examined by semiquantitative RT-PCR. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was also amplified to confirm the integrity of the RNA.

Thus, aberrant methylation of HRK appears to be a cancer- cers. There was no correlation between HRK methylation and specific phenomenon. age (69.4 years versus 64.8 years; P ϭ 0.175), gender, or Finally, we evaluated the correlation between HRK meth- location of the tumor. On the other hand, HRK methylation was ylation and the clinicopathological features of colorectal can- detected exclusively in tumors that showed simultaneous methylation of multiple CGIs, i.e., tumors that exhibited the CGI methylator phenotype (22). Among the cases studied, 14 of 34 (41%) CGI methylator phenotype-positive cases showed methylation of HRK, whereas none of the 24 CGI methylator phenotype-negative cases did so (P Ͻ 0.001). In addition, there was a significant correlation between methylation of HRK and the absence of p53 mutations in colorectal cancers [1 of 21 (5%) in cases with mutant p53 versus 13 of 37 (35%) in cases with wild-type p53; P Ͻ 0.05].

DISCUSSION HRK, which was originally identified as a proapoptotic gene mediating apoptosis induced by diminished levels of cy- tokine in hematopoietic cells (16), belongs to the BH3-only subfamily of Bcl-2 genes, whose products act at points upstream in the apoptotic signal cascade (28). Alteration of these proteins may be a critical feature of cancer cells, enabling them to escape apoptosis resulting, for example, from the DNA damage caused by chemotherapeutic drugs (29, 30). In this report we have shown that expression of a proapoptotic gene, HRK, is silenced in colorectal and gastric cancer by DNA methylation and histone deacetylation. Interestingly, HRK is located in chromosome 12q13, where loss of heterozygosity has frequently been de- Fig. 8 Treatment with 5-aza-dC and HDAC inhibitors induces apoptosis tected in human tumors (31–33), which means that HRK may be in HSC45 gastric cancer cells. A, representative images showing apoptotic inactivated not only by DNA methylation but also by gene cells stained with PI. HSC45 cells were treated with the indicated drugs, deletion. However, it was aberrant methylation that we found to harvested, and stained with PI. B, numbers of apoptotic cells induced by be a specific feature of HRK in the various tumor cells studied treatment with 5-aza-dC and HDAC inhibitors. Columns show the percent- and that was not seen in any of the other BH3-only family genes ages of sub-G1 cells calculated by flow cytometry. Columns: 1, mock- treated; 2, 0.2 ␮M 5-aza-dC for 24 h; 3, 0.2 ␮M 5-aza-dC for 48 h; 4, 0.2 ␮M studied (BAD, BID, and PUMA). The reason for this specificity 5-aza-dC for 72 h; 5, 0.2 ␮M 5-aza-dC for 96 h; 6, 0.2 ␮M 5-aza-dC for 72 h remains unclear but may be related to the fact that expression of ϩ 10 nM depsipeptide for 24 h; 7, 0.2 ␮M 5-aza-dC for 72 h ϩ 300 nM TSA HRK, like that of NOXA, is regulated at the transcriptional level, for 24 h; 8, 2.0 ␮M 5-aza-dC for 24 h; 9, 2.0 ␮M 5-aza-dC for 48 h; 10, 2.0 whereas the others are regulated by post-transcriptional mech- ␮M 5-aza-dC for 72 h; 11, 2.0 ␮M 5-aza-dC for 96 h; 12, 2.0 ␮M 5-aza-dC for 72 h ϩ 10 nM depsipeptide for 24 h; 13, 2.0 ␮M 5-aza-dC for 72 h ϩ anisms. For example, whereas NOXA is induced by p53 and 300 nM TSA for 24 h. HRK by cellular stress caused by absence of growth factors (18,

34), the proapoptotic activity of BAD is controlled by the level of its phosphorylation (35). Thus, inactivation of BAD in cancer, if it occurs, is likely caused by altered protein modification (e.g., phosphorylation) rather than by transcriptional silencing. The role of HRK in apoptosis has mainly been described in hematopoietic and neuronal cells: loss of the growth signal in these cells induces transcription of HRK, leading to cell death (18, 36). Our findings, however, suggest that HRK is much more widely expressed, and significant increases in the numbers of apoptotic cells were observed when HSC44 and HSC45 gastric cancer cells were treated with 5-aza-dC plus Fig. 10 Aberrant methylation of HRK in primary colorectal and gastric TSA or depsipeptide. Indeed, the fact that significant induc- tumors. Bisulfite-PCR analysis of HRK in primary colorectal and gastric tion of apoptosis was observed when cancer cells re-express- cancers and in colorectal adenomas. M, methylated allele; N, normal; T, ing HRK were treated with 5-aza-dC suggests that the gene tumor. may be a good molecular target for chemotherapy. In that regard, recent studies have shown that the combined use of inhibitors of DNA methyltransferase and HDAC synergistically enhances expression of otherwise methylated genes (27, tended to be less sensitive to 5-aza-dC (Fig. 8B; Fig. 9). 37). Notably, cell lines in which HRK was unmethylated Further study will be necessary to determine whether cell lines that express HRK have defects in chemotherapeutic drug-activated pathways leading to apoptosis. Aberrant methylation of HRK was frequently detected in colorectal cancers that showed methylation of multiple CGIs, as well as in colorectal cancers that did not have p53 mutations. Epigenetic inactivation of HRK may thus lead to loss of apoptosis, even in cancer cells expressing wild-type p53.Itwas previously demonstrated that cancers expressing wild-type p53 tend to be resistant to gene therapy using Ad-p53 (38). Epige- netic therapy using 5-aza-dC may thus represent a useful alter- native approach for treating such cases. It is also noteworthy that a majority of genes methylated in tumors tend to also be methylated in premalignant regions, such as colorectal adenomas (13, 22, 39). This was not the case with HRK, however, suggesting that inactivation of HRK may involve a critical step in the progression from adenomas to cancers. In summary, we have shown that a proapoptotic gene, HRK, is inactivated by aberrant methylation of its CGI, which sheds new light on the mechanism by which cancers expressing wild-type p53 escape apoptosis. Furthermore, the fact that proapoptotic genes other than HRK appear to be unaffected by 5-aza-dC suggests that HRK might be selectively targetable for antimethylation and antihistone deacetylation therapy aimed at inducing apoptosis in cancer cells.

ACKNOWLEDGMENTS We thank Dr. William F. Goldman for editing the manuscript.

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Toshiro Obata, Minoru Toyota, Ayumi Satoh, et al.

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