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Molecular Biology and Genetics |
Departments of Pathology [T. U., K. M. W., R. H. H., M. G.], Oncology [M. T., R. H. H., M. G.], Surgery [C. J. Y.], Epidemiology [H. S.], and Medicine [M. G.], Johns Hopkins School of Medicine and the Johns Hopkins School of Public Health, Baltimore, Maryland 21205, and the Department of Leukemia of University of Texas at MD Anderson Cancer Center, Houston, Texas 77030 [J-P. J. I.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These data indicate that aberrant methylation of ppENK and its transcriptional repression is a common event in pancreatic carcinogenesis.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pancreatic cancer is the fourth leading cause of cancer death in men and in women, and each year 
28,000 Americans die of the disease (3)
. Frequent genetic changes such as mutational activation of the K-ras oncogene and inactivation of the p16, DPC4, p53, MKK4, STK11, TGFBR2, TGFBR1, and ALK-4 tumor suppressor genes have been described in pancreatic cancer (4, 5, 6)
. Although we have identified previously genes aberrantly methylated in pancreatic cancers (7)
, there almost certainly are others. Costello et al. (8)
have estimated that 400 genes are aberrantly methylated in cancers and have found evidence for tumor-specific pattern of methylation. A better knowledge of the pattern of DNA methylation abnormalities in cancer may improve our understanding of the role of DNA methylation in tumorigenesis. In addition, the identification of differentially methylated CpG islands in cancer may lead to the discovery of novel genes with tumor suppressor properties. Finally, identified genes or loci could be used as cancer-specific markers for the early detection of cancer (9)
. In this study we used MCA3
coupled with RDA to recover CpG islands differentially methylated in pancreatic adenocarcinoma (10)
. We chose MCA/RDA because the subtractive and kinetic enrichment of differentially methylated sequence by RDA (Fig. 1A)
has the potential to clone out sequences methylated only in cancer (8
, 11)
.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MCA/RDA.
MCA/RDA was performed as described by Toyota et al. (10)
with some modifications that may have improved the efficiency of the MCA/RDA technique.4
Briefly, 5 µg of DNA was digested with SmaI and XmaI (New England Biolabs). The restriction fragments were ligated to RMCA adapter and amplified by PCR in 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.5 M betaine, 2% DMSO, 200 µM each deoxynucleotide triphosphate, 100 pmol of RMCA 24mer primer and 15 units of Taq polymerase (Life Technologies, Inc.) in a final reaction volume of 100 µl. The reaction mixture was incubated at 72°C for 5 min and at 95°C for 3 min, and then was subject to 25 cycles of 1 min at 95°C and 3 min at 77°C followed by a final extension of 10 min at 77°C. We included betaine in the PCR reaction and amplified the methylated templates under a higher annealing temperature (77°C). The combination of betaine and DMSO can uniformly amplify a mixture of DNA with different GC content (12)
. These modifications might have enhanced the amplification of distinct MICPs instead of Alu repetitive sequences that accounted for 60% of the recovered clones using the original protocol (10)
. The MCA amplicon from either the pancreatic cancer cell line PL3 or PL8 was used as the tester for RDA, and a MCA amplicon generated from a mixture of DNA from the normal pancreata of six different patients was used as the driver. RDA was performed on these MCA amplicons using different adapters, JMCA and NMCA. Sequences of adapters used for MCA/RDA are available at our website.5
After the third round of competitive hybridization and selective amplification, the RDA difference products of second and third round amplifications were cloned into pBluescript II plasmid vector (Stratagene).
DNA Sequencing of Clones and Dot Blot Hybridization.
The clones recovered from each cell line after MCA/RDA were amplified with T3 and T7 primers and then sequenced using KS primer as recommended by the manufacturer (Sequitherm Excel; Epicentre Technologies). To determine the methylation status of MCA/RDA MICPs in pancreatic cancer and normal pancreas, we first screened MICPs by hybridizing them to a dot blot of MCA products of pancreatic cancers and normal pancreata. Plasmid DNA containing each independent clone was prepared and digested with SmaI. DNA fragments were recovered from agarose gel and used as a probe for dot blot hybridization. Aliquots (1 µl) of the mixture of 10XSSC and MCA products from the driver and from the tester (PL3 and PL8) both before and after each of the three rounds of RDA competitive hybridization/selective amplification were blotted onto nylon membranes in duplicate. Similarly, MCA products from six pancreatic cell lines (CAPAN1, CAPAN2, Panc1, Hs766T, MiaPaca2, and Colo357) and from eight other normal pancreata were also blotted onto the membranes. The membranes were hybridized with 32P-labeled probes overnight, washed, and exposed to a Kodak X-ray film.
Bisulfite Modification, Bisulfite-modified Genomic Sequencing, and MSP.
The bisulfite treatment was carried out for 16 h at 50°C using 1 µg of genomic DNA, as reported previously (7)
. Genomic sequencing was performed on bisulfite-treated DNA to examine the methylation status of 10–20 CpG dinucleotides located in and/or around SmaI sites of each clone in 22 pancreatic tissues (8 cancer cell lines, 6 primary adenocarcinomas, and 8 normal pancreata; Ref. 7
). Genomic sequencing of the coding sequence of cyclin G was also performed in PL8. We interpreted the level of methylation of each clone by quantifying the level of methylation of each CpG site by comparing the intensity of unconverted cytosine with that of cytosine plus thymidine. Generally, in pancreatic cancer cell lines, the level of methylation observed at each CpG dinucleotide was consistent throughout the CpG island. Therefore, we graded the average level of methylation of each sequence into 4 grades: 0–10%, 11–30%, 31–70%, and 71–100%.
MSP was performed as described previously (13) and to acquire optimal specificity, each primer pair contained four to six CpG sites, and high specific annealing temperatures were used. The primers and the specific annealing temperatures for each clone are available at our website.6 If validated MSP primers sets specific for methylated and unmethylated templates reveal that there is only amplification of methylated templates, we conclude that the sample is 100% methylated. Methylated and unmethylated templates were identified by bisulfite-modified sequencing. In describing MSP results performed on CpG islands that were normally unmethylated in non-neoplastic pancreas, we termed a pancreas cancer sample as "methylated" if MSP yielded any methylated templates.
RT-PCR and 5Aza-dC Treatment.
Five pancreatic cancer cell lines (PL3, PL8, CAPAN2, Panc1, and MiaPaca2) and four normal pancreata were used for RT-PCR analysis. The cell lines were treated with demethylating agent 5Aza-dC (Sigma Chemical Co.) at a final concentration of 1 µM for 5 days. Total RNA was prepared using TRIzol (Life Technologies, Inc.), reverse-transcribed and amplified. As a control for cDNA integrity, GAPDH was also amplified. Primer sequences for RT-PCR are available at our website.7
Statistics.
The primary outcome variable in this study was the observed number of 7 MICP loci found to be methylated in 64 pancreatic cancers. Wilcoxon’s rank-sum test compared the observed number of methylated loci by tumor differentiation (poorly versus well or moderately differentiated), lymph node status (0 or 1 versus >1 node positive), and prior CIMP classification (CIMP positive versus CIMP negative). Simple linear regression assessed the relationship between the observed number of methylated loci and these covariates: age, age squared, and tumor diameter (in cm). Multivariate linear regression assessed the simultaneous contribution of the clinicopathological and demographic variables to the observed number of methylated loci. All of the tests were two-sided. A P of < 0.05 signified statistical significance.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These 42 MICPs were also variably methylated in the other 6 pancreatic cancer cell lines examined (data not shown).8
All of the 42 MICPs had a GC content of >50%, and 40 (95%) had a sequence uniqueness sustaining the criteria of CpG island (14)
. The DNA homology search of each clone with the BLAST program (National Center for Biotechnology Information) demonstrated that 38 of the 42 (90%) MICPs had significant homologies to known human sequences including 11 MICPs matched to human gene sequences and 10 MICPs matched to human ESTs. Five MICPs were also matched or contained a part of CpG islands isolated previously (10
, 15)
, and 12 MICPs had significant homology to high-throughput genome sequences in the three international nucleotide sequence databases: DDBJ (DNA Databank of Japan), European Molecular Biology Laboratory, and GenBank. The remaining 4 had no significant homology to known sequences. Interestingly, 3 MICPs (MICP1, 14, and 23) matched to CpG islands originally recovered from colorectal cancer cell line using the same technique (named MINT 23, 20, and 32, respectively; Ref. 10
).
Characterization of the Methylation Status of Cloned CpG Islands by Bisulfite Sequencing.
For 30 of the 42 MICPs, methylation was detected in 2 or more of 8 normal pancreata by dot blot analysis, suggesting that these MICPs could be frequently methylated in normal pancreas. These CpG islands including those of 8 known genes [CSX, FLJ00083, GAD1, ICAM5, HLH (helix loop helix DNA binding protein), MCT3, PAX5, and SMO (smoothened gene)] were isolated by MCA/RDA, because relatively fewer DNA templates were methylated in normal pancreas. Therefore, only the remaining 12 MICPs (and MICP3 = FLJ0083) were additionally analyzed by bisulfite sequencing. For 7 of the MICPs (Cyclin G, ppENK, ZBP, MICP25, 27, 36, and 38), methylation was restricted to pancreatic cancers. A map of the CpG island of ppENK and cyclin G is shown in Fig. 2
. In the case of 6 MICPs methylation bisulfite sequencing revealed methylation in DNA from pancreata samples as well as cancer DNA. A summary of the level of methylation of these 13 MICPs is shown in Fig. 3
. DNA from pancreatic cancer cell lines and from pancreatic cancer xenografts does not have DNA from contaminating stroma; in these samples we were able to identify whether aberrant methylation in the carcinoma was complete or partial. Bisulfite sequencing revealed that the level of methylation of ppENK, MICP27, and MICP38 was often 100% (see Fig. 3
).
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; Table 1
). Because methylation of these 7 MICPs was not found in non-neoplastic pancreata, we defined any amplification of methylated templates as indicating "methylation." Such aberrant methylation of the 7 was detected in 9–100% of the pancreatic cancer cell lines analyzed and in 2–92% of the 64 (Pxs and Pcas) pancreatic adenocarcinomas and pancreatic cancer xenografts analyzed. Because the primary pancreatic carcinomas harbor neoplastic and non-neoplastic tissue, MSP typically identified the presence of both methylated and unmethylated templates. DNA from one chronic pancreatitis tissue containing PanIN (16)
harbored methylated templates of Cyclin G, ppENK, and MICP25, and DNA from another chronic pancreatitis sample also had methylated templates of ppENK. A summary of the results of the methylation profiles of these 7 MICPs in each of the 75 pancreatic adenocarcinomas is shown in Table 2
.
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). Of the 75 pancreatic cancers analyzed, the methylation status of 29 cases had been characterized previously using a panel of candidate genes methylated in cancer (7)
. These 29 cancers included 7 CIMP+, 7 CIMP-intermediate, and 15 CIMP- cancers. In this subset of 29 cases, pancreatic carcinomas identified previously as CIMP+ harbored methylation of a greater number of the 7 MICPs than those cancers classified previously as CIMP- (3.4 versus 1.25 methylations; P = 0.002).
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; Table 1
). Amplification of methylated templates of these MICPs was always weaker in normal mucosae compared with the primary pancreatic adenocarcinomas (Fig. 4B)
, suggesting that there were fewer methylated DNA templates in these mucosae. None of these 7 CpG islands were methylated in peripheral blood lymphocytes from normal individuals. Of the CpG islands that harbored methylation in histologically normal tissues that were bisulfite sequenced, the number of methylated CpGs in the CpG island was similar in the normals compared with the cancers, but the relative percentage of methylated templates were often less than in the cancer samples as determined by comparing the density of C to T at each CpG.
Expression of Cyclin G and ppENK in Pancreatic Cancer and Effect of 5-Azacytidine.
PpENK causes growth suppression (17
, 18)
, and Cyclin G is a target for transcriptional activation by p53 and p73 (19
, 20) and may augment apoptosis, although growth promoting properties for cyclin G have also been reported (21
, 22)
. Therefore, we examined expression of ppENK and Cyclin G using RT-PCR in 4 and 5 pancreatic cell lines, respectively. Partial methylation (50%) of the 5' CpG island of Cyclin G in PL8 (Fig. 4C)
was associated with decreased expression of Cyclin G by RT-PCR. The 5' CpG island of Cyclin G was not methylated in a panel of normal pancreata, and Cyclin G was expressed in 4 normal pancreata by RT-PCR. Treatment with 5Aza-dC increased the expression of Cyclin G in PL8 (Fig. 4C)
. Because cyclin G was 50% methylated, we sequenced the coding region of cyclin G in the PL8 cell line. No mutations were found suggesting that cyclin G was not biallelically inactivated in this cell line.
We found that ppENK was expressed in normal pancreata, but in 4 pancreatic cancer cell lines with aberrant methylation of the 5'region no expression was observed (Fig. 4D)
. 5Aza-dC treatment restored ppENK expression in all 4 of the cell lines. Thus, hypermethylation of the 5' CpG island of ppENK coincides with absent expression in pancreatic cancers.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Having identified by MCA/RDA 7 MICPs aberrantly methylated in pancreatic carcinoma, we examined a population of pancreatic cancers comparing the methylation patterns of these 7 MICPs to the distribution of methylated loci we observed in a previous study, which used a panel of candidate genes that undergo aberrant methylation (7) . Pancreatic cancers found previously to have a high prevalence of aberrant methylation of candidate genes also harbored methylation of more MICPs (P = 0.002). This data provides additional evidence that some pancreatic carcinomas harbor greater numbers of aberrantly methylated CpG islands. Classifying pancreatic carcinomas into subsets depending on their prevalence of aberrant methylation at CpG islands (the so-called "CIMP" classification) may shed light on the clinical and biological significance of the differences in global methylation patterns seen in pancreatic and other cancers.
We also found that cancers with a high number of aberrantly methylated MICPs were more likely to be larger in size and to have come from older patients than cancers with little or no aberrantly methylated MICPs. The correlations between methylated loci, tumor size, and patient age were not strong suggesting that other factors influence the development of aberrant methylation, but they suggest that aberrant methylation is more likely to be observed with increasing age of the neoplasm. Because pancreatic neoplasms are usually malignant once they reach 1–2 cm in size, these data raise the possibility that some aberrant methylation events may continue to occur after the transition to malignancy. Aberrant methylation of some genes is known to occur in early benign neoplasms (24, 25, 26)
. As shown in Fig. 3
, for some genes biallelic methylation was commonly observed (ppENK, MICP27, and MICP3 8) suggesting that it arose during carcinogenesis and may have been clonally selected; for others biallelic methylation was not found (such as MICP3 6, cyclin G, or ZBP). Comparison of methylation patterns in primary carcinomas, their local recurrences, distant metastases and neoplastic precursors (PanINs) should shed light on the timing of methylation in cancer development and evolution. Another possibility for the relationship between tumor size and methylation status is that cancers with high levels of aberrant methylation may be more likely to present when the tumor is larger. These clinicopathological correlations will require confirmation in other studies. Other investigators have attempted to identify the basis of cancer-related methylation by looking for associations between clinicopathological variables and methylation. Salem et al. 25
found a relationship between bladder cancer stage and the number of methylated genes among a panel (PAX6, exon 2 of p16, DBC, and TPEF). Using a panel of candidate genes, Zochbauer-Muller et al. (27)
observed an association between cigarette smoking and p16 methylation in lung cancers.
The absence of methylated templates of ppENK in normal pancreas and the absence of MICP36 and MICP38 in any normal tissue examined raises the possibility that MSP could be used to detect aberrant methylation of these MICPs in clinical samples such as pancreatic juice, duodenal fluid, stool, or blood for the early detection of pancreatic cancers. This would be valuable for individuals at high risk of developing pancreatic adenocarcinoma such as those with a strong family history of the disease (28) .
The identification of several genes that harbor low-level methylation in normal pancreas highlights the need for care when assigning significance to certain cancer-related methylation data. A gene that is methylated in only a small percentage of normal cells may appear to undergo selection during carcinogenesis if it is completely methylated in a cancer. Such a methylation pattern could arise in a cancer if a non-neoplastic cell harboring "normal" methylation undergoes selection and neoplastic transformation as a result of subsequent genetic or epigenetic events. This phenomenon makes it much more difficult to assign causality to methylation phenomena in cancers compared with genetic events such as homozygous deletion. For normally unmethylated genes of which the function is well characterized such as hMLH1, for genes that are methylated as a second hit for a tumor suppressor gene (e.g., VHL and E-cadherin; Ref. 29 ), or for tumor suppressor genes alternatively targeted by genetic and epigenetic inactivation (e.g., p16 and RB; Ref. 2 ), the biological significance of "aberrant methylation" is well accepted. Methylation of all of the templates in a neoplastic specimen is often interpreted as indicating that both alleles are methylated. In some cases this can also result from methylation of one allele combined with loss of the other allele by LOH (29) . As additional genes are identified that are methylated in pancreatic and other cancers, it will be important to identify low-level methylation in normal tissues using sensitive techniques such as MSP (13) to help determine whether such genes have truly undergone selection through de novo methylation.
Methylation of several CpG islands has also been observed in non-neoplastic colorectal (30 , 31) , bladder, and prostate tissues (11) . Methylation in normal tissues that is not the result of imprinting is frequently "age-related." This has been best shown in the colonic mucosa for genes such as ER (30 , 31) . We did not observe a pattern of age-related methylation in 15 histologically normal pancreata (mean age of 62) though we did observe some variability in the methylation of CpG islands in histologically normal pancreas. Our normal tissue population was not large enough to rule out trends in methylation with age. Methylation of ppENK, MICP25, and MICP27 in a significant percentage of the DNA templates within normal gastric, duodenal, and colonic mucosae, and its absence in normal pancreas and peripheral blood mononuclear cells highlights the fact that some CpG islands are methylated in a tissue-specific fashion (11) . Other genes have been shown to be methylated in histologically normal gastrointestinal tissue. For example, methylation of an APC gene promoter occurs in both normal and cancerous gastric and esophageal epithelia (32 , 33) . This low-level methylation of different normal tissues might also explain some of the tumor type-specific methylation patterns observed by other investigators (8) .
We also observed methylation of several MICPs in pancreata affected by chronic pancreatitis. Two of the 5 pancreata with chronic pancreatitis harbored aberrant methylation, 1 of these 2 pancreata contained a PanIN lesion, and this latter sample displayed methylation of 3 MICPs. Previous studies demonstrated that chronic pancreatitis is a significant risk factor for the development of pancreatic cancer (34) , and duct lesions (PanIN) often found in chronic pancreatitis are considered precursors to infiltrating pancreatic carcinoma (16) . The presence of aberrant methylation in DNA from chronic pancreatitis suggests that de novo methylation of CpG islands may be an early event in pancreatic cancer development in this setting.
Several approaches have been used to identify differentially methylated genes in cancer. The subtractive and kinetic enrichment of differentially methylated sequences by RDA may have advantages over other techniques to isolate differentially methylated sequences between normal tissue and cancer (8 , 11) . RLGS has been used successfully to profile methylation alterations in multiple tissues (8) , but it has limitations, because it cannot discriminate between methylation and deletion events, the latter of which is common in pancreatic carcinoma (35) . Furthermore, unlike MCA/RDA, RLGS and AP-PCR require the isolation and the subsequent cloning of identified spots or bands. Finally, MCA/RDA, like RLGS and AP-PCR, not only identifies absolute differences in methylation between cancer and normal DNA, it also will identify methylated sequences present both in cancer and normal DNA if there is low-level methylation in normal DNA as was observed for many CpG islands cloned in this study.
In conclusion, our results indicate that ppENK is commonly transcriptionally silenced by aberrant methylation in pancreatic adenocarcinomas. We have found that aberrant methylation of CpG islands in pancreatic adenocarcinomas is more common among older patients and those with larger tumors. The biological basis for these clinicopathological correlations and their clinical utility remain to be determined. Detection of methylated ppENK and other methylated genes may be a valuable biomarker for the early detection of pancreatic adenocarcinoma among patients at high risk of developing this disease.
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FOOTNOTES |
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1 Supported by the Lustgarten Foundation for Pancreatic Cancer Research, the Pancreatic Cancer Action Network (PANCAN), the Michael Rolfe Foundation, and the NIH SPORE Award (5P50CA62924-07). 
2 To whom requests for reprints should be addressed, at Departments of Pathology, Medicine, and Oncology, 632 Ross Building, The Johns Hopkins Hospital, 720 Rutland Avenue, Baltimore, MD 21205; Phone: (410) 614-3314; Fax (410) 614-0671; Email: mgoggins{at}jhmi.edu
3 The abbreviations used are: MCA, methylated CpG island amplification; MSP, methylation-specific PCR; RDA, representational difference analysis; 5Aza-dC, 5-aza-2'-deoxycytidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase gene; RT-PCR, reverse transcription-PCR; MICP, methylated in carcinoma of the pancreas; PanIN, pancreatic intraepithelial neoplasia; RLGS, restriction landmark genome scanning; CIMP, CpG island methylator phenotype.
4 Internet address: http://mdanderson.org/leukemia/methylation.
5 Internet address: http://pathology2.jhu.edu/pancreas/prim0425.htm.
6 Internet address: http://pathology2.jhu.edu/pancreas/prim0425.htm.
7 Internet address: http://pathology2.jhu.edu/pancreas/prim0425.htm.
8 Internet address: www.pathology.jhu.edu/pancreas/gogginslab for a table describing these 42 differentially methylated MICPs.
9 Internet address: http://www.psu.edu/ur/archives/intercom_1999/Nov11/research.html.
Received 3/19/01. Accepted 10/ 1/01.
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REFERENCES |
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, exons.
