DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas
Yan Nie1,
Guang-yu Yang1,
Yunlong Song1,
Xin Zhao1,2,
Chi So1,
Jie Liao1,
Li-Dong Wang2 and
Chung S. Yang1,3
1 Laboratory for Cancer Research, College of Pharmacy, RutgersThe State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA and
2 Henan Medical University, Zhengzhou, Henan 457500, China
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Abstract
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The three human leukocyte antigen (HLA) class I antigens, HLA-A, HLA-B and HLA-C, play important roles in the elimination of transformed cells by cytotoxic T cells. Frequent loss of expression of these antigens at the cell surface has been observed in many human cancers. Various mechanisms for post-transcriptional regulation have been proposed and tested but the molecular mechanisms for transcriptional regulation are not clear. We show by immunohistochemistry that the HLA class I antigens are absent in 26 of 29 (89%) samples of human esophageal squamous cell carcinomas (ESCC). Eleven of the 26 ESCC samples lost mRNA expression for at least one of the HLA genes, as shown by RTPCR. DNA from the 29 pairs of ESCC and neighboring normal epithelium were examined for CpG island hypermethylation, homozygous deletion, microsatellite instability (MSI) and loss of heterozygosity (LOH). DNA from normal epithelial tissues had no detectable methylation of the CpG islands of any of these gene loci. Thirteen of 29 ESCC samples (45%) exhibited methylation of one or more of the three HLA loci and six samples (21%) exhibited methylation of all three loci. The HLA-B gene locus was most frequently methylated (38%). HLA-B mRNA expression in an ESCC cell line, where HLA-B was hypermethylated and did not express mRNA, was activated after treatment with 5-aza-2'-deoxycytidine. Homozygous deletion of these three gene loci was not observed. Relatively low rates of LOH and MSI were observed for the microsatellite markers D6S306, D6S258, D6S273 and D6S1666, close to the HLA-A, -B and -C loci, although a high ratio of LOH was observed at a nearby locus (represented by the markers D6S1051 and D6S1560), where the tumor suppressor gene p21Waf1 resides. A strong correlation between genetic alterations and mRNA inactivation was observed in the ESCC samples. Our results indicate that HLA class I gene expression was frequently down-regulated in ESCC at both the protein and mRNA levels and that hypermethylation of the promoter regions of the HLA-A, -B and -C genes is a major mechanism of transcriptional inactivation.
Abbreviations: BCH, basal cell hyperplasia; ß2-M, ß2-microglobin; ESCC, esophageal squamous cell carcinoma; GAPDH, glyceraldehyde phosphate dehydrogenase gene; HD, homozygous deletion; HLA, human leukocyte antigen; IHC, immunohistochemistry; LOH, loss of heterozygosity; MSI, microsatellite instability.
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Introduction
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The human leukocyte antigen (HLA) class I antigens, HLA-A, HLA-B, and HLA-C, which form the class I major histocompatibility complex in humans, take part in the recognition of virally infected, grafted or transformed cells by cytotoxic T cells (1). The HLA genes are located on chromosome 6p21 and are expressed in most somatic tissues. Selective loss of expression of these loci has frequently been observed in human tumors and this event is thought to help tumor cells escape immune surveillance by T cells and natural killer cells (2,3). Expression of HLA class I antigens has been studied by immunohistochemistry (IHC) in various tumors in correlation with clinicopathological characteristics. Reduced expression has been observed in kidney, prostate, stomach, colon and germ cell testicular cancers and has been associated with tumor invasiveness and aggressiveness (2).
Loss of HLA class I antigen expression can occur at the genetic, transcriptional and post-transcriptional levels. Chromosomal loss in the short arm of chromosome 6 was observed in tumors of the colon (13.8%) and larynx (17.6%) and in melanomas (15.3%) (4). In another study, comparison of restriction enzyme digestion patterns of DNA from normal and tumor tissues indicated that the genes were generally intact (5). Recently, two mutations in the HLA-B gene were detected in cervical cancer, leading to loss of allelic expression (6). However, these genetic alterations may account for only a few cases of loss of HLA expression, considering the relatively high frequency of alterations detected in tumors. A direct association between lack of protein expression at the cell surface and a decrease in mRNA levels has been observed for HLA class I genes, suggesting transcriptional regulation.
-Interferon (7) and estrogen (8) increase surface expression of HLA class I antigens when introduced into cancer cell lines. Many experiments have been conducted to search for cis-transcriptional elements in the promoter regions of HLA class I genes. Several newly discovered elements, enhancer A and
/enhancer B, have been found to be capable of activating the transcription of HLA class I genes and an interferon-stimulated response element has been shown to significantly increase expression of HLA class I genes in response to interferon (9). In contrast, c-myc and the adenovirus E1A protein inhibit expression of HLA class I genes (2). In another report, down-regulation of the HLA class I genes was associated with DNA hypomethylation and N-myc expression in neuroectodermal tumor cell lines (10). Some experiments suggest that down-regulation of HLA class I antigens predominantly occurs at the post-transcription level. Expression of HLA class I antigens on the cell surface requires the association of their heavy chains with ß2-microglobulin (ß2-M). Absence of ß2-M abolishes transportation of HLA class I antigens. Palmisano et al. showed, by RTPCR, that in breast cancer the frequency of loss of expression of ß2-M gene expression was as high as 25% (11). It has also been reported that some mutations in ß2-M induced complete loss of expression of HLA class I antigens (12,13).
DNA methylation, especially 5'-CpG methylation, is an important mechanism in silencing the expression of genes (14,15). DNA methylation may directly interfere with the basal transcriptional machinery by altering the DNA secondary structure, especially the major groove conformation. DNA methylation can also induce chromosome remodeling through histone deacetylation, resulting in transcriptional repression. A group of methyl-CpG-binding proteins, which preferentially bind to methylated CpG dinucleotides, may interact with Sin3A. Sin3A in turn interacts with histone deacetylase (16) and is thought to be involved in this process (17,18). Aberrant DNA methylation has been found in various genes, including the putative tumor suppressor genes retinoblastoma and p16INK4a, leading to their down-regulation in tumors (19). Recently, Costello et al. conducted a global examination of CpG methylation in a large group of tumors and observed tumor type-specific patterns (20).
CpG islands are small regions of DNA ranging from 0.5 to 45 kb in length where the GC content is >60% and the CpG:GpC ratio is >0.6 (15). Using these criteria, we have conducted a search for well-preserved CpG islands in genes that have been reported to be inactivated in tumors. All three HLA class I genes showed exceptionally high CpG:GpC ratios (0.831.5), as well as a GC content >60% (Figure 1
). Hypermethylation of these genes has not been reported in either tumor or spermatogenic cells where HLA class I genes were not expressed (21). However, during a pilot study with four esophageal squamous cell carcinoma (ESCC) cell lines we detected hypermethylation of the HLA-B gene in two of them, suggesting a role for DNA hypermethylation in the down-regulation of HLA class I genes in ESCC.

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Fig. 1. CpG distribution of the HLA-A, -B and -C genes. The number of CpG dinucleotides was counted every 50 bp and is shown as a vertical bar. Exons of each gene are highlighted below. The dashed horizontal bar indicates the position of primers for the methylation- and non-methylation-specific PCR. The GC contents for the DNA fragments amplified by methylation-specific PCR were 60, 61 and 70% for HLA-A, -B and -C, respectively. The CpG:GpC ratios for the same fragments were 1.5, 1.2 and 0.83 for HLA-A, -B and -C, respectively.
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We have been studying the molecular alterations in ESCC samples from Linzhou (formally Linxian), a well-recognized high ESCC incidence region in northern China (22). A detailed alteration map of the 9p21 gene cluster (including p14ARF, p15INK4b and p16INK4a), especially the hypermethylation pattern, has been constructed. Many ESCC samples showed hypermethylation of the promoters of these genes, among which p16INK4a was the most frequently hypermethylated (40%) (23). Loss of expression of HLA class I antigens has previously been observed in esophageal cancer (24,25), but the molecular mechanisms are not clear.
In the present study we show, by IHC, that expression of the HLA class I antigens is frequently lost, accompanied by a high frequency of transcriptional inactivation. We examined the genetic alterations of the three HLA class I loci in terms of hypermethylation, loss of heterozygosity (LOH), microsatellite instability (MSI) and homozygous deletion (HD), as well as their gene expression in 29 pairs of surgically resected human ESCC tissues and their neighboring normal tissues. CpG island hypermethylation occurred at high frequency and was highly correlated with transcriptional inactivation. Inhibition of DNA methylation in a cell line that did not express HLA-B induced its mRNA expression. Our results suggest that DNA hypermethylation is a major mechanism for transcriptional inactivation of the HLA class I genes in ESCC.
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Materials and methods
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ESCC specimen preparation and DNA extraction
Twenty-nine primary ESCC specimens containing neighboring normal epithelial tissues were collected from patients in the Linzhou region (formerly known as Linxian) of northern China, a well-recognized high risk area for ESCC. The samples were frozen in liquid nitrogen within 1 h after surgical resection and were stored in liquid nitrogen, on dry ice or at 80°C until use. For each specimen, two pieces of tissue, one from the tumor mass and the other from normal esophageal mucosa remote from the primary tumor mass, were dissected and embedded with tissue freeze medium (OTC). Ten micrometer thick serial sections were cryosected and used for DNA extraction. The presence of tumor or normal tissue in the samples was confirmed histopathologically on slides stained with hematoxylin and eosin. Precancerous lesions were microdissected using a laser capture microdissection system (Arcturus, Moutain View, CA) for the genetic assay. About 500 cells were dissected for each lesion. DNA was extracted with a Qiagen Tissue Kit (Qiagen, Valencia, CA) following the manufacturer's procedure. The DNA was stored in aliquots at 20°C until use.
Immunohistochemical staining
A monoclonal antibody (W6/32) against HLA-A, -B and -C (Dako, Carpinteria, CA) was used to detect the expression of HLA proteins. After dewaxing, the tissue slides were treated with target unmasking fluid and microwaved, incubated in 0.3% H2O2 to quench endogenous peroxidase activity and incubated in 1% normal horse serum to minimize non-specific binding. The slides were sequentially incubated with primary antibody overnight at 4°C, biotinylated secondary antibody for 30 min and ABC reagent for 45 min. 3,3'-Diaminobenzidine (Sigma, St Louis, MO) was used as the chromagen. Negative controls were established by replacing the primary antibody with phosphate-buffered saline and normal mouse serum. Specific staining was recognized by comparing the slides with negative controls.
Measurement of mRNA expression of the HLA-A, -B and -C genes
cDNA was synthesized using the Advantage RT-for-PCR kit (Clontech, Palo Alto, CA) with oligo(dT) priming as recommended in the protocol provided. The glyceraldehyde phosphate dehydrogenase gene (GAPDH) was used as a control. RTPCR for the three HLA genes was run separately to avoid cross-reaction. PCR cycle numbers (typically 2530 for GAPDH and 3035 for HLA class I genes) were experimentally determined in pilot studies to run the reaction in the linear stage. PCR products were resolved on 2% agarose gels and signal intensities were quantified using a computer imaging system. The levels of gene transcripts were quantified as the ratio of the intensity of the HLA signal to the intensity of GAPDH. Inactivated expression was scored when expression of an HLA gene in the tumor sample was <25% of its expression in the corresponding normal sample. This threshold was chosen because of an estimated upper limit of 2025% non-cancerous cell contamination in our tumor samples.
Methylation-specific PCR and sequencing
The tumor and normal DNA were modified by bisulfite reaction using the procedure developed by Herman et al. (26). For microdissected DNA, 1 µg salmon sperm DNA was added as a carrier followed by denaturation with NaOH (final concentration 0.2 M). The DNA, in a volume of 50 µl, was incubated with 275 µl of modification buffer (10 mM hydroquinone, 3 M sodium disulfite, pH 5.0) for 16 h at 50°C. Modified DNA was purified with a Qiagen gel purification kit and then treated with 0.3 M NaOH for 10 min at room temperature. The DNA was then again purified with a Qiagen gel purification kit and eluted into 50 µl of water. Methylation-specific and non-methylation-specific primers (Table I
) were designed using Primer3, developed and maintained by the Whitehead Institute/MIT Center for Genome Research. Complete methylation (either homozygous methylation or single allele methylation when the other allele was deleted) was determined by the presence of the methylation-specific and absence of non-methylation-specific PCR products. Heterozygous methylation was scored when products showed up in both methylation- and non-methylation-specific PCR. High annealing temperatures were used to ensure the specificity of both methylation- and non-methylation-specific PCR. About 2050 ng DNA were used in each PCR amplification. After 13 min of heat activation, the reaction was incubated for four cycles of 2 min at 95°C, 2 min at 65°C and then 2 min at 72°C. The PCR reaction then underwent 3545 cycles of 10 s at 95°C, 45 s at 62°C and 30 s at 72°C. A 30 min incubation at 72°C was used to finalize the PCR amplification. To verify the methylation status observed by the methylation-specific PCR method, products of the methylation-specific PCR for HLA-B were cloned using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) and sequenced.
Detection of HD, LOH and MSI
HD of the HLA genes was examined using methods previously established in our laboratory, in which a ß-actin fragment was used as an internal standard (27). To ensure the specificity of the primers, multiple sequence alignments were performed and only those primer pairs that differed by at least five bases from the other two genes were used for HD study. Six microsatellite markers (D6S306, D6S258, D6S273, D6S1666, D6S1051 and D6S1560) were used for the LOH and MSI study of the 6p21 loci (Figure 2
). The PCR primers for these markers were obtained from Genome Database. About 100 ng DNA were used for duplex PCR amplification. Forward primers of both the target gene and control were radiolabeled at the 5'-end with [
-33P]dATP. To ensure linear amplification, PCR cycle number (typically 2630) for tumor and normal DNA reactions were experimentally determined based on the product intensity versus cycle number plots. The PCR products were resolved on a 6% denaturing polyacrylamide gel followed by autoradiography. A computer imaging system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD) was employed for density and area quantitation. HD was scored if the normalized signal intensity (target/ß-actin) in the tumor sample was <25% of that in the normal sample. LOH was counted if the density difference between the two major bands in the tumor sample was >5:1, provided that both bands had the same density in the corresponding normal sample. This threshold was chosen because of an estimated upper limit of 2025% normal cell contamination in our tumor samples. MSI was counted if shifted or additional bands were observed in the tumor sample.

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Fig. 2. Genomic positions of the HLA-A, -B and -C and p21Waf1 genes and six microsatellite markers. These genes span the chromosome region 6p21.36p21.2. Genes are represented by shaded boxes and markers by solid boxes. The diagram is not drawn to scale.
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5-Aza-2'-deoxycytidine treatment of an esophageal cell line
Esophageal cell lines were grown in Ham's F12 medium containing supplemented calf serum. An esophageal cell line with HLA-B hypermethylation was used in the methylation inhibition study. It was grown in culture medium with and without 5-aza-2'-deoxycytidine at a concentration of 2 µg/ml for 6 days, with medium changes on days 1, 3 and 5 (28). Cells were harvested at the end of day 6 for extraction of genomic DNA and total RNA and tested for DNA methylation and gene expression.
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Results
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Expression of HLA-A, -B and -C in tumor tissues and precancerous lesions
Twenty-nine pairs of ESCC and normal tissues were immunohistochemically analyzed with antibody W6/32, which recognizes the HLA-A, -B and -C antigens (Figure 3
). Intense immunostaining of the HLA-A, -B and -C antigens was observed in morphologically normal esophageal epithelia. HLA-A, -B and -C positive cells were found in the basal and parabasal cell layers of the squamous epithelium and positive staining was mainly confined to the cell membrane. Some cells in the superficial layer adjacent to the parabasal layers also showed HLA-A, -B and -C immunostaining. Luminal surface cells in the superficial layers were negative for immunostaining. All the samples were either intensely stained in the epithelia or contained no or very few specifically stained cells and were categorized as positive and negative, respectively. Interestingly, absence of HLA-A, -B and -C staining was observed in 26 (89%) of these 29 ESCC samples. Three ESCC samples (008, 149 and 2553) showed positive staining for the HLA-A, -B and -C antigens (Table II
).

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Fig. 3. IHC staining of normal, BCH and ESCC tumor tissues with monoclonal antibody W6/32 against HLA-A, -B and -C antigens. (a) Intense immunostaining for the HLA-A, -B and -C antigens was observed in morphologically normal esophageal epithelia and positive staining was mainly confined to the cell membrane. (b) Similar immunostaining was observed in samples with BCH. (c) No HLA-A, -B and -C staining was observed in samples with ESCC.
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Six samples with dysplasia and 14 samples with basal cell hyperplasia (BCH) adjacent to the ESCC were analyzed by IHC for expression of HLA-A, -B and -C. All of the 14 samples with BCH lesions and five samples with dysplasia showed positive immunostaining and the staining intensity was similar to that of normal epithelia. Only one sample with dysplasia displayed relatively weak staining for HLA-A, -B and -C.
Characterization of mRNA expression for the HLA class I genes
Levels of the HLA-A, -B and -C transcripts were determined in the 29 frozen ESCC samples by RTPCR analysis. A 197 bp fragment of the HLA-A gene transcript, a 130 bp fragment of the HLA-B transcript or a 151 bp fragment of the HLA-C transcript was generated, with a 456 bp fragment of the GAPDH transcript as a control. As summarized in Table II
, inactivated mRNA expression was observed in five (17%) tumor samples for HLA-A, in 10 (34%) tumor samples for HLA-B and in eight (28%) tumor samples for HLA-C (Table II
and Figure 4
). All samples that showed inactivated HLA-A mRNA expression also showed inactivated HLA-B and C mRNA expression and thus five (17%) tumors showed inactivated mRNA expression for all of the three HLA class I genes. Eleven (38%) tumor samples showed inactivated mRNA expression for at least one of the HLA class I genes.

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Fig. 4. mRNA expression pattern of the HLA-A, -B and -C genes. N, normal samples; T, tumor samples; arrows, band positions of the genes being examined. The RTPCR products were resolved on 2.5% agarose gels. M, 100 bp DNA marker ladder. (a) Sample 17 displays loss of HLA-A transcripts in tumor but not in normal tissue. (b) Samples 17 and 27 show loss of expression of HLA-B transcript in the tumor and sample 41 shows dramatically decreased expression of HLA-B transcript in the tumor (<5% of normal). (c) Sample 17 shows no detectable expression of HLA-C in the tumor and sample 41 displays very low (<5% of normal) expression of HLA-C compared with normal tissue in the same sample. (d) All samples display similar expression of the GAPDH gene in both normal and tumor tissues.
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Hypermethylation of 5' CpG islands in HLA-A, -B and -C gene loci
The methylation status of the HLA-A, -B and -C CpG islands was investigated in 29 ESCC samples together with their adjacent normal epithelial tissue, using methylation-specific PCR (Table II
and Figure 5
). We detected hypermethylation of the HLA-A gene in seven cases (24%), of HLA-B in 11 cases (38%) and of HLA-C in nine cases (31%). Among these, two cases of HLA-A, one case of HLA-B and one case of HLA-C hypermethylation occurred at only one allele with the other allele unmethylated, demonstrated by presence of products in both methylation- and non-methylated-specific PCR (Table II
and Figure 5b
). CpG islands in all three HLA class I genes were concurrently methylated in six (21%) of the 29 samples. Another two samples harbored methylation in the CpG islands of both the HLA-B and HLA-C genes. HLA-A alone was methylated in one tumor sample, HLA-B alone was methylated in three tumor samples and HLA-C alone was methylated in one sample. No methylation-specific and only non-methylation-specific PCR signal was detected for all of the 29 normal samples. To verify the specificity of the PCR reaction, all of the products from HLA-B methylation-specific PCR were cloned and sequenced. As shown in Figure 5d
, most of the CpG dinucleotides were methylated and only a few CpG sites were unmethylated, indicating the high specificity of methylation-specific PCR.

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Fig. 5. Determination of the methylation status of CpG islands. (a) Complete DNA methylation. (Left) Methylation-specific PCR. The presence of CpG island hypermethylation is shown in tumor (T) but not in normal (N) DNA (sample 17). The arrow indicates the expected size of the PCR products. (Right) Non-methylation-specific PCR. Non-methylation-specific PCR products were present in all normal DNAs (sample 17). The small amounts of PCR products in tumor DNA may arise from non-tumor cells in tumor tissues. (b) Incomplete DNA methylation. PCR products were observed for both methylation- (right) and non-methylation-specific (left) PCR from a tumor sample (sample 21). (c) Methylation status changes of HLA-B in cell line KYSE 510 with and without 5-aza-2'-deoxycytidine treatment. MS, methylation-specific PCR; US, non-methylation-specific PCR. Methylated HLA-B is present under both conditions but unmethylated product is only present in cells treated with 5-aza-2'-deoxycytidine. (d) Sequencing results of the methylation-specific PCR products of HLA-B from the 11 tumor samples. Solid dots indicate methylated CpG and empty circles indicate unmethylated CpG dinucleotides. Most of the CpG dinucleotides were methylated, confirming the specificity of methylation-specific PCR.
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Hypermethylation of HLA-B in precancerous lesions
The HLA-B gene locus, where hypermethylation occurred at the highest frequency, was used to determine whether HLA class I locus methylation occurred in precancerous lesions. Thirteen samples that showed either dysplasia or BCH were used for this purpose. Two of these 13 samples showed hypermethylation of the p16INK4a gene using the procedure described (23), indicating that the methylation detection procedure was effective (data not shown). However, none of these samples showed hypermethylation of the HLA-B locus.
Homozygous deletion of HLA-A, -B and -C gene loci
Gene deletion was examined by differential PCR analysis of the genomic DNA. Multiplex PCR amplification was performed to generate a 142 bp fragment of the HLA-A gene, a 162 bp fragment of the HLA-B gene and a 175 bp fragment of the HLA-C gene, together with a 187 bp fragment of the ß-actin internal control (Figure 6
). Since all of these genes are extremely polymorphic, the conditions for duplex PCR were carefully optimized to ensure the highest specificity. None of the samples contained deletion of any of the genes, which is consistent with reports from other laboratories (12).

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Fig. 6. Homozygous deletion of HLA-A, -B and -C was not observed in tumor samples. PCR products of matched tumor (T) and normal (N) samples were loaded next to each other for comparison. Each sample had four products corresponding to HLA-A, -B and -C and ß-actin, indicated by the arrows. Five samples (17, 19, 20, 21 and 22) of tumor/normal pairs and placental DNA showed similar band density patterns for each gene product. No HD was present.
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LOH of HLA-A, -B and -C gene loci
Two microsatellite markers (D6S306 and D6S258) that flank the HLA-A gene locus and two (D6S273 and D6S1666) that are adjacent to the HLA-B and -C gene loci were selected for HLA class I gene locus LOH analysis. Another two markers (D6S1051 and D6S1560) in 6p21 that are adjacent to the tumor suppressor gene p21Waf1 were used as references (Figure 2
). All six microsatellite markers showed high rates of heterozygosity, with 18/29 (0.62) for D6S306, 24/29 (0.83) for D6S258, 24/29 (0.83) for D6S273, 16/29 (0.55) for D6S1666, 17/29 (0.59) for D6S1051 and 21/29 (0.72) for D6S1560. The LOH ratios of the HLA-A, -B and -C loci microsatellite markers were relatively low: 3/18 (17%) for D6S306, 2/24 (8%) for D6S258, 6/24 (25%) for D6S273 and 3/16 (19%) for D6S1666. A relatively high LOH ratio, however, was observed for the p21Waf1 locus, indicated by the markers D6S1051 and D6S1560 (Figure 7
): 8/17 (47%) for D6S1051 and 8/21 (38%) for D6S1560. Due to the physical proximity of D6S306 and D6S258, D6S273 and D6S1666 and D6S1051 and D6S1560, the LOH of a specific locus can be better estimated by combining the data for both the markers in a pair. The combination of D6S306 and D6S258 gave a LOH ratio of 3/29 (10%) at the HLA-A locus. The combination of D6S273 and D6S1666 gave a LOH ratio of 7/26 (27%) at the HLA-B and -C loci. The combination of D6S1051 and D6S1560 gave a LOH ratio of 11/25 (44%) at the p21Waf1 gene locus. MSI was observed in three cases, one each at D6S273, D6S1560 and D6S1051.

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Fig. 7. LOH and MSI at HLA class I loci in primary ESCC. N, normal samples; T, tumor samples. (a) LOH at microsatellite marker D6S306. Case 13 shows no LOH, indicated by the identical densities of the two major bands. Cases 6, 10 and 17 are non-informative. (b) LOH at D6S273. Cases 30 and 40 are non-informative. Case 28 contained LOH at this locus as in tumors the upper major band was much weaker than the lower major band while in normal tissues they were the same. Case 36 did not show LOH. (c) LOH and MSI at D6S1051. Case 2553 was non-informative. Case 2451 was informative but did not show LOH. Case 150 shows LOH at D6S1051 with a missing upper band in the tumor samples. Case 144 shows MSI, with the lower band in the tumor shifting to a shorter position.
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Restoration of HLA-B expression after 5-aza-2'-deoxycytidine treatment
Two of four cell lines examined showed HLA-B hypermethylation. Cell line KYSE 510 harbored a homozygous DNA hypermethylation and KYSE 150 a heterozygous hypermethylation at the CpG island of the HLA-B gene. RTPCR showed that KYSE 510 cells did not have detectable HLA-B expression. KYSE 150 cells showed mRNA levels comparable with the other two cell lines, suggesting that heterozygous hypermethylation did not inactivate expression of HLA-B. After treatment of KYSE 510 cells with 5-aza-2'-deoxycytidine, the HLA-B gene was unmethylated, as shown by the non-methylation-specific PCR product (Figure 5c
). RTPCR showed that expression of HLA-B was induced by 5-aza-2'-deoxycytidine treatment, confirming the role of DNA hypermethylation in inactivation of HLA-B expression (data not shown). The methylation-specific PCR product was still observable (Figure 5c
) after treatment because 5-aza-2'-deoxycytidine inhibits methylation of newly synthesized DNA and some of the hypermethylated DNA strands remained in the cells.
Correlation between molecular alterations, inactivation of mRNA expression and loss of HLA class I gene antigens at the cell surface
All HLA class I genes that showed complete hypermethylation (homozygous hypermethylation or one allele hypermethylated with the other allele deleted) showed transcriptional inactivation. In contrast, transcriptionally inactivated genes were always hypermethylated at CpG islands in their 5'-region (Table II
). LOH or MSI alone did not seem to affect mRNA expression of neighboring genes. Heterozygous deletion of a gene, as suggested by LOH of the neighboring microsatellite markers, may also play a role, as shown in sample 19, where the other allele was methylated. All three samples that showed positive IHC staining for the HLA class I antigens did not show transcriptional inactivation or DNA hypermethylation. The only molecular alteration observed in these samples was LOH at D6S1666. Samples with transcriptional inactivation of at least one of the HLA class I genes accounted for only 42% (11 of 26) of the samples with negative HLA staining, suggesting that other mechanisms, such as post-transcriptional modification and protein transportation, are also important in the regulation of HLA expression.
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Discussion
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The HLA-A, -B and -C antigens are expressed in most somatic cells, but their expression on the cell surface is not detectable in many tumors. Monoclonal antibody W6/32 is a specific antibody against the HLA-A, -B and -C antigens. Recent IHC work using this antibody showed that loss of HLA-A, -B and -C expression was a frequent event in human cancers (2931). Rockett et al. reported that in esophageal carcinomas as many as 54% of the cases had weak or no HLA class I expression (24). In our present study 26 of 29 (89%) tumor tissues displayed loss of HLA-A, -B and -C expression, whereas all of the 29 normal epithelial tissues stained intensely with antibody W6/32. Loss of immunosurveillance is one of the most important mechanisms for the development of cancer. The frequent loss of expression of HLA class I genes in ESCC suggests a significant role of this event in the development of ESCC.
Controversial results have been reported regarding whether down-regulation of the HLA class I genes occurs at the transcriptional level (11,32). In the present study we found that 11 of 26 ESCC samples which stained negatively with antibody W6/32 showed HLA mRNA expression inactivation. Fifteen of these 26 ESCC samples did not show mRNA inactivation, suggesting that post-transcriptional mechanisms also play a role.
CpG island hypermethylation has been suggested to be one of the major mechanisms for inactivation of genes that inhibit tumor progression and infiltration (15). Each of the three HLA class I genes contains a well-preserved CpG island in its 5'-region. Methylation-specific primers were designed to amplify the regions within or close to the promoters where methylation has the greatest impact on transcription. Primer and template multiple alignments were performed to ensure the specificity of PCR, since these molecules are highly homologous. Hypermethylation of each of the three loci was observed in some ESCC cases and methylation of HLA-B was confirmed by sequencing. Demethylation and re-expression of the HLA-B gene was obtained by treatment with 5-aza-2'-deoxycytidine in one of the esophageal cell lines, suggesting that DNA methylation was responsible for mRNA inactivation in this cell line. The HLA-B locus was most frequently hypermethylated, which is consistent with the observation that in human tumors down-regulation of HLA class I expression was B locus-specific (33). There was a high concurrence among all three HLA genes, which is similar to the case of the 9p21 gene cluster, where the p14ARF, p15INK4b and p16INK4a genes are located and frequently hypermethylated concurrently in ESCC (23). However, no correlation was observed between the methylation pattern of 9p21 genes and that of HLA class I genes in a subset of 18 samples in which the methylation status of 9p21 genes is available (data not shown), suggesting that methylation of these two gene clusters are independent events in ESCC development.
Hypermethylation of HLA-B was frequent in ESCC, but no detectable hypermethylation of HLA-B was observed in precancerous lesions. All of these precancerous lesions showing either BCH or dysplasia displayed strong staining for the HLA-A, -B and -C antigens, except for one sample with dysplasia, which showed relatively weak staining. This result suggests that loss of expression of the HLA class I genes may occur after the dysplasia stage. Activation of HLA class I antigen expression has been proposed as a therapeutic approach for cancer (34). Reactivation of tumor suppressor genes by inhibition of DNA methylation has been tested so as to inhibit cancer development (35). These approaches need to be studied further.
Multiple mechanisms may be involved in the inactivation of HLA class I molecules, including genomic alteration, transcriptional regulation and protein transportation. Previous results indicate that genomic alterations of these loci are not frequent (46). In this study we have conducted detailed analyses of these genomic alterations, including HD and LOH. The LOH ratio at HLA class I loci (825%) was within the base level range in tumors. Interestingly, at ~3.3 Mb away from these loci (6p21), where the tumor suppressor gene p21Waf1 resides, the LOH ratio was markedly higher (3847%). Down-regulation of p21Waf1 has been observed in various tumors but the molecular mechanisms are not clear. The LOH found at the p21Waf1 locus may result in down-regulation of this gene and provide one such molecular mechanism. This locus was used as a reference for the LOH analysis and the results suggest that the HLA class I genes are not a primary target within the 6p21 chromosome region for disruption by LOH in ESCC development. The absence of HD of the HLA class I loci, as evidenced by the multiplex PCR results, further strengthens the idea that genetic alteration of these loci is rare and thus does not play a major role in silencing of these loci.
By looking at the LOH of each single gene, we found that when LOH occurred in the HLA-A region, it also occurred in the HLA-B and -C and p21Waf1 regions and samples showing LOH in the HLA-B or -C regions also contained LOH in the p21Waf1 region. The LOH frequency was highest in the p21Waf1 region (44%) and lowest in the HLA-A region (10%), with an intermediate frequency of 27% in the HLA-B and -C regions. This may suggest that either p21Waf1 or a region further downstream is the target of the LOH events. On the other hand, this may also suggest that all these regions are targets of the LOH events but that methylation of the HLA genes helps to reduce LOH in the regions around them. It was reported that DNA hypomethylation elevates genomic instability but that DNA methylation reduces it (32,36). The negative correlation between DNA methylation and LOH in the HLA-A, -B and -C regions and the p21Waf1 region in our samples strengthens this idea.
In 29 ESCC samples we have found that 10 (34%) showed either hypermethylation or LOH at the HLA-A locus, 15 (52%) showed either hypermethylation, MSI or LOH at the HLA-B locus and 14 (48%) showed hypermethylation, MSI or LOH at the HLA-C locus. Nine (31%) of these 29 cases had genetic alterations at all three loci and 18 (62%) had genetic alterations at one or more of these loci. Genes that showed hypermethylation at both alleles or hypermethylation at one allele with the other allele deleted did not have active expression, suggesting that these genetic events affected expression of these genes at the transcription level. There is also a strong correlation between DNA methylation and mRNA transcriptional inactivation. It was reported that c-myc and the adenovirus E1A protein inhibit expression of HLA class I genes (2). We have previously observed that in ESCC activation of oncogenes such as ras and myc is rare (data not shown), so that they may not play an important role in down-regulation of the HLA class I genes.
In summary, our results show that down-regulation of the HLA class I antigens, which in turn results in the escape from immunosurveillance, is common in ESCC samples from Linzhou. Transcriptional inactivation of HLA class I genes, primarily caused by DNA hypermethylation, plays an important role in this process. Hypermethylation of these gene loci, particularly at the HLA-B locus, is frequent in these samples and may be a key mechanism for down-regulation of the HLA class I genes.
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Notes
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3 To whom correspondence should be addressed 
*The first two authors contributed equally to this study.
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Acknowledgments
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We thank Mr Darren N.Seril for helpful discussion and critical reading of the manuscript. We are also grateful to Ms Dongxuan Jia for her assistance in preparing the frozen tissue samples for our analysis. This work was supported by NIH Grant CA65781 and facilities from NIEHS Center Grant ES 05022 and NCI Cancer Center Supporting Grant CA 72030.
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Received September 13, 2000;
revised June 18, 2001;
accepted July 3, 2001.