Susceptibility of Nonpromoter CpG Islands to De Novo Methylation in Normal and Neoplastic Cells

Carvell Nguyen, Gangning Liang, TuDung T. Nguyen, Denice Tsao-Wei, Susan Groshen, Michael Lübbert, Jain-Hua Zhou, William F. Benedict, Peter A. Jones

Affiliations of authors: C. Nguyen, G. Liang, T. T. Nguyen, P. A. Jones (Department of Biochemistry and Molecular Biology), D. T.-W. Wei, S. Groshen (Department of Preventive Medicine), University of Southern California (USC)/Norris Comprehensive Cancer Center, Keck School of Medicine of the USC, Los Angeles; M. Lübbert, Department of Medicine, Division of Hematology/Oncology, University of Freiburg Medical Center, Germany; J.-H. Zhou, W. F. Benedict, Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston.

Correspondence to: Peter A. Jones, Ph.D., D.Sc., Department of Biochemistry and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, 1441 Eastlake Ave., Rm. 8302L, Los Angeles, CA (e-mail: jones_p{at}ccnt.hsc.usc.edu).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Many cancers display alterations in methylation patterns of CpG islands—stretches of DNA rich in CpG dinucleotides often associated with gene promoters that are involved in initiation of gene transcription. This methylation may perturb expression of genes critical to the regulation of cell proliferation. Aberrant methylation is not limited to a few genes or to promoter regions but has been found on a genome-wide scale in a variety of neoplasias, including colorectal cancer and acute myelogenous leukemia. Our goal was to characterize, in a quantitative manner, the profiles of abnormally methylated genes that may be specific for different cancers. Methods: Using a quantitative assay, methylation-sensitive single nucleotide primer extension (MS-SNuPE), we have analyzed the methylation levels of promoter and exonic (coding region) CpG islands of two cyclin-dependent kinase inhibitors [p15(INK4B) and p16(INK4A)] and the PAX6 gene, which encodes a transcriptional factor involved in neuronal proliferation, in DNA samples taken from patients with chronic myelogenous leukemia, acute myelogenous leukemia, myelodysplastic syndrome, and colorectal cancer. Results: De novo methylation of all three exonic loci in tumors—relative to baseline levels found in nontumor tissue or blood—was observed in hematologic neoplasias and in solid tumors as well as in normal colonic tissue. However, methylation of promoter regions was more limited. Moreover, two different patterns of promoter methylation distinguished the leukemias from colorectal cancer: p15 promoter hypermethylation was found only in the leukemias, and p16 promoter hypermethylation occurred only in colon tumors. However, we did not address this issue prospectively; therefore, such an observation is only hypothesis generating. Conclusions: The methylation patterns that we observed suggest that exonic CpG islands are more susceptible to de novo methylation than promoter islands and that methylation may be seeded in exonic regions, from which it can spread to other islands, including promoter regions. Subsequent selection of cells with a growth advantage conferred by spread of methylation into and inactivation of a particular promoter might then contribute to the genesis of a specific type of cancer.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alteration of methylation patterns is a common event in human neoplasia. These changes occur in regions of DNA, called CpG islands, that exhibit the expected distribution of CpG dinucleotides relative to the rest of the CpG-depleted genome. Hypermethylation of promoter CpG islands in critical cell cycle control genes occurs frequently in cancer and has been associated with decreased expression of these genes (1,2). Thus, methylation of CpG dinucleotides in the promoters of tumor suppressor genes has been implicated as a pathway by which these genes may be silenced, contributing to the loss of proliferation control in transformed cells. Methylation-induced inactivation of cell cycle control genes, such as those in the cyclin-dependent kinase inhibitor family (p15INK4B and p16INK4A), has been demonstrated in a variety of cancers, ranging from lung and colorectal cancers to leukemias and lymphomas (1,3,4).

Although methylation patterns of promoter regions in tumor suppressor genes have been studied extensively, hypermethylation of CpG islands that are not located in known promoters has been largely overlooked. Not all CpG islands are located in promoters (5), and it has been demonstrated that hypermethylation of exonic CpG islands can occur without concomitant methylation of the promoter or inactivation of the gene itself (6). For example, exon 5 of PAX6 has been found to be frequently methylated in bladder cancer; however, this exonic methylation does not block transcription of the gene (7). Although such data show that DNA hypermethylation is a common alteration in cancer, the genome-wide patterns of CpG island methylation and mechanisms by which these islands acquire aberrant methylation remain largely unknown.

In this study, we investigated the methylation patterns of three genes, p15(INK4B), p16(INK4A), and PAX6, each of which contain both a promoter and an exonic CpG island, in DNA taken from patients with chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), acute myelogenous leukemia (AML), and colorectal cancer. We used the quantitative methylation-sensitive single nucleotide primer extension (MS-SNuPE) assay to evaluate the methylation status of discrete CpG dinucleotide sites within each of the six CpG island loci. Average methylation values derived from this analysis were then analyzed for cancer- and locus-specific patterns.


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissue Collection and DNA Isolation

DNA from peripheral blood and bone marrow of patients with CML were obtained from the Los Angeles County/University of Southern California (USC) Hospital and the University of Freiburg Medical Center, Germany. A total of 22 CML samples were used: 10 from patients in chronic phase, six in accelerated phase, and six in blast crisis. In addition, DNAs from peripheral blood and bone marrow of six patients with MDS were provided by M. Lübbert, and the DNAs from peripheral blood and bone marrow of nine AML patients were obtained from W. F. Benedict. Control DNAs from peripheral blood and bone marrow were collected from seven healthy individuals. Fifteen matched sets of normal and tumor colon specimens were obtained from patients at the USC/Norris Comprehensive Cancer Center and the Los Angeles County/USC Medical Center. The DNA was extracted from the samples as described previously (8).

All subjects, with the exception of the patients with CML, gave written informed consent to the use of their tissue samples for research purposes. The studies for which the tissue samples were collected were reviewed and approved annually by the Institutional Review Board of the USC School of Medicine. Informed consent was not given by the patients with CML from whom some of the data in this study were derived. These specimens were collected, over the span from 1995 through 1998, as overage from complete blood cell count analyses that were to be discarded (samples came from leftover blood that had initially been drawn for CBC analysis), and informed consent was not required for such specimens at that period in time. However, procurement and analysis of these CML blood samples were done with the approval of the Institutional Review Board of the USC School of Medicine.

Analysis of Methylation by MS-SNuPE

Genomic DNA from patient samples (2 µg each) was digested with a restriction enzyme that did not cut within the DNA region to be amplified for 2 hours at 37 °C. After digestion, the DNA was denatured for 20 minutes at 95 °C and then treated with 3 M NaOH for 20 minutes at 45 °C. This solution was then treated with 3.6 M sodium bisulfite and 0.1 M hydroquinone for 16 hours at 55 °C in the dark. Treatment of DNA with bisulfite converts unmethylated cytosine residues to uracil, which is then converted to thymine after the primary bisulfite-specific polymerase chain reaction (PCR), leaving methylated cytosines unchanged. Bisulfite-converted DNA was purified with the Wizard System DNA Purification System (Promega Corp., Madison, WI), desulfanated by the addition of 3 M NaOH for 15 minutes at 40 °C, and ethanol precipitated.

The six CpG-rich target sequences were amplified by use of PCR primers specific for bisulfite-treated DNA (see Table 1Go) and run on 2% agarose gels for isolation. The DNA was purified by use of the Qiaquick gel purification kit (Qiagen, Valencia, CA).


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Table 1. Primer sequences for bisulfite polymerase chain reaction (PCR) and methylation-sensitive single nucleotide primer extension assay (MS-SNuPE)
 
Methylation levels—defined as the percent of DNA that is methylated—were measured by MS-SNuPE as described previously (9). Purified bisulfite-specific PCR products of each of the six CpG islands were used as templates for methylation analysis at three CpG sites per island analyzed by use of MS-SNuPE primers specific for each site (see Table 1Go) and reaction conditions specific for each island. These MS-SNuPE products were run out on a 15% polyacrylamide gel, and the resulting signals were quantitated by use of a PhosphorImager analysis system (Molecular Dynamics, Sunnyvale, CA). Methylation values were obtained by averaging the results of three CpG sites by use of the following equation: [methylated C/(methylated C + unmethylated T)] x 100.

PCR conditions for bisulfite-converted DNA: p15 promoter—95 °C for 3 minutes, 95 °C for 1 minute and 20 seconds, 55 °C for 1 minute, 72 °C for 1 minute and 20 seconds, cycled 38 times, then 72 °C for 4 minutes; p15 exon 2—95 °C for 2 minutes, 95 °C for 1 minute, 58 °C for 45 seconds, 72 °C for 1 minute and 15 seconds, cycled 40 times, then 72 °C for 4 minutes; p16 promoter—94 °C for 3 minutes, 94 °C for 1 minute, 67 °C for 30 seconds, 72 °C for 30 seconds, cycled 40 times, then 72 °C for 4 minutes; p16 exon 2—95 °C for 3 minutes, 95 °C for 1 minute, 60 °C for 45 seconds, 72 °C for 1 minute and 20 seconds, cycled 38 times, then 72 °C for 4 minutes; PAX6 promoter—95 °C for 2 minutes, 95 °C for 1 minute, 50 °C for 30 seconds, 72 °C for 1 minute, cycled 40 times, then 72 °C for 4 minutes; and PAX6 exon 5—95 °C for 2 minutes, 95 °C for 1 minute, 50 °C for 45 seconds, 72 °C for 1 minute, cycled 40 times, then 72 °C for 4 minutes.

MS-SNuPE conditions: p15 promoter—95 °C for 2 minutes, 46.5 °C for 2 minutes, and 72 °C for 1 minute; p15 exon 2—95 °C for 2 minutes, 50 °C for 2 minutes, and 72 °C for 1 minute; p16 promoter—95 °C for 2 minutes, 50 °C for 2 minutes, and 72 °C for 1 minute; p16 exon 2—95 °C for 2 minutes, 50 °C for 2 minutes, and 72 °C for 1 minute; PAX6 promoter—95 °C for 2 minutes, 49 °C for 2 minutes, and 72 °C for 1 minute; and PAX6 exon 5—95 °C for 2 minutes, 40 °C for 2 minutes, and 72 °C for 1 minute.

Statistical Methods

For each specimen, the quantitative MS-SNuPE assay was used to measure the methylation levels (read as percent of DNA that was methylated) for each of the six CpG islands. Before analysis, the percent of DNA methylated was transformed by use of the arcsin transformation to correct for the heteroscedasticity associated with binomial proportions (10). Means and 95% confidence intervals (CIs) for the means were calculated in the arcsin scale; values were transformed back to the original scale for purposes of presentation. To compare the actual methylation levels among groups of patients, we used F tests based on an analysis of variance to calculate P values; the least significant difference method of multiple comparisons was used for pairwise comparisons if the overall F test was statistically significant at the .05 level (10). The paired Student's t test was used to compare the levels of methylation between the colon tumor tissue and adjacent normal tissue for each of the six islands once the overall F test was statistically significant at the .05 level. The Spearman correlation coefficient was used to evaluate the association of the methylation levels between pairs of islands. Tolerance limits (95% CI for the 95th percentile), by use of the control white blood cell samples, were used to establish cutoffs for the upper limit of normal for each of the six islands (11). For the CML, AML, and MDS specimens, methylation levels above the cutoff were taken to represent de novo methylation. To evaluate the overall methylation pattern for each of the disease groups, we calculated the number of islands demonstrating de novo methylation. The Cochran–Mantel–Haenszel test was used to test for an association with disease progression (CML: chronic -> accelerated -> blast crisis; MDS -> AML) with the number of de novo methylated islands (12). Fisher's exact test was used to compare the frequency of de novo methylation among groups of specimens. All reported P values were two-sided.


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using the quantitative MS-SNuPE assay, we determined the extent of methylation in the promoter and exonic CpG islands of p15, p16, and PAX6 in samples from the four disease categories and from nondiseased control samples. Representative examples of the primary data obtained from MS-SNuPE analyses are displayed in Fig. 1Go. The six CpG islands that we analyzed are shown and characterized in Fig. 2Go. The MS-SNuPE data are quantitatively summarized in Table 2Go and are displayed qualitatively in subsequent figures. Hypermethylation of all three exonic loci was observed in the myelogenous neoplasias and solid tumors as well as in normal colonic tissue. In contrast, methylation of the three promoter islands was much more limited and seemed to be specific to the cancer type: Instances of p15 promoter hypermethylation were found only in the leukemias, p16 promoter hypermethylation occurred only in colon tumors, and the PAX6 promoter demonstrated no appreciable hypermethylation in any of the cancers.



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Fig. 1. Quantitative methylation analysis of three CpG sites in p15 5' region and exon 2, p16 promoter and exon 2, and PAX6 promoter and exon 5, with the use of methylation-sensitive single nucleotide primer extension assay (MS-SNuPE). Representative results from autoradiographs of MS-SNuPE products run on 15% polyacrylamide gels are shown. Methylation is a measure of the signal in the C lane, which represents the amount of methylated DNA molecules as a result of [32P]deoxycytidine triphosphate incorporation. A band in the T lane is the product of [32P]deoxythymidine triphosphate incorporation and represents unmethylated cytosine molecules at a particular CpG site. The three bands present at each locus represent cytosines at three individual CpG sites examined by specific MS-SNuPE primers. Percent methylation represents the average of three sites by use of the following equation on PhosphorImager quantitation: [methylated C/(methylated C + unmethylated T)] x 100.

 


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Fig. 2. Partial maps of the six CpG islands analyzed. Tick marks below the line represent CpG sites. Bent arrows represent transcription start sites, while straight arrows below the line represent the specific CpG sites assayed by methylation-sensitive single nucleotide primer extension assay (MS-SNuPE). Boxes above the line denote coding regions of the genes. GC = percentage of total number of bases in sequence of interest that are guanine plus cytosine; Obs/Exp = observed/expected.

 

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Table 2. Summary of methylation levels for normal white blood cells, acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML), and colon tumor and adjacent normal colon tissue
 
Using cutoff values based on the tolerance intervals of control DNA as the threshold between normal and de novo methylation, we found that all disease types tested—preleukemias, leukemias, and colon cancer—demonstrated hypermethylation of the three exonic CpG islands when compared with control DNA from noncancerous tissue. Fig. 3Go shows the methylation patterns of DNA samples from various stages of progression of myeloid leukemias compared with control samples. The data demonstrate increasing degree and frequency of hypermethylation in each of the three exonic CpG islands through the three stages of CML progression (Fig. 3Go). The data also show that later stages of CML disease are associated with higher percentages of patients exhibiting methylation in multiple CpG islands as well as an increase in the absolute number of methylated islands across all patients (Fig. 4Go). This trend was found to be statistically significant (P = .014) by use of the Cochran–Mantel–Haenszel test. The methylation changes that occur with CML progression did not appear to be attributable to age differences among the patient samples (data not shown).



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Fig. 3. Quantitative methylation patterns of six CpG islands in preleukemic and leukemic samples. CML = chronic myelogenous leukemia; MDS = myelodysplastic syndrome (all cases classified as refractory anemias with excess blasts ± transformation); AML = acute myelogenous leukemia; ND = not determined. Values indicated by the colors are percentage methylation within a DNA sample from each patient.

 


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Fig. 4. Percentage of patients within each myeloid disease category exhibiting 0–6 methylated CpG islands. A statistically significant trend (P = .014, Cochran–Mantel–Haenszel test) was observed between increasing methylation frequency (higher percentage of patients showing hypermethylation and increased number of methylated CpG islands) and chronic myelogenous leukemia (CML) progression. There was no such trend between myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) (P = .97). All P values were two-sided.

 
Hypermethylation of the exons of p15, p16, and PAX6 was also demonstrated in DNA from patients with MDS and AML when compared with control samples (Fig. 3Go). We performed the same statistical analysis for methylation trends between MDS and AML that we did for the various stages of CML progression, since a substantial percentage of MDS cases (10%–40%) will progress to AML. No statistically significant trend was found between the methylation levels of MDS and AML (Fig. 4Go). However, the exonic methylation profiles of CML samples were significantly different from those of MDS–AML, based on pairwise tests comparing the methylation averages of the three exonic CpG islands (P<.001 for p15 exon 2, P = .009 for p16 exon 2, and P = .045 for PAX6 exon 5).

Fig. 5Go shows the methylation levels of DNA samples from matched sets of tumor and adjacent normal tissue taken from patients with colorectal cancer. As with the DNA from myeloid neoplasias, DNA from tumor tissue demonstrates significant hypermethylation of each of the three exonic CpG islands analyzed. However, unlike the situation seen with the myeloid neoplasias, exonic hypermethylation was also seen in normal colonic tissue, albeit at lower levels than in tumors. The difference in the levels of methylation seen in each of the three exons in adjacent normal tissue compared with tumor was found to be statistically significant (P = .021 for p15 exon 2, P<.001 for p16 exon 2, and P<.001 for PAX6 exon 5 by paired Student's t test).



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Fig. 5. Quantitative methylation patterns of six CpG islands in matched sets of normal and tumor colon samples. Values indicated by the colors are percentage methylation within a DNA sample from each patient.

 
We also analyzed the methylation patterns across all three exonic islands to determine whether or not a methylator phenotype existed. In CML, concurrent methylation of at least two of the three exonic CpG islands was observed in three (14%) of 22 cases. In MDS, concurrent methylation of at least two of the three islands occurred in six (100%) of six cases, while concurrent methylation of all three islands occurred in two (33%) of six cases. In AML, concurrent methylation of at least two islands was seen in seven (78%) of nine cases, while methylation of all three islands was observed in three (50%) of six cases. In the cases of colorectal cancer, 15 (100%) of 15 showed concurrent hypermethylation of at least two exonic CpG islands, while 11 (73%) of 15 showed hypermethylation of all three exonic islands. Spearman correlation analyses were performed on all leukemia and colon cancer samples combined, and it was found that hypermethylation of any one exonic CpG island correlated positively with hypermethylation of the other two exonic islands. These correlations were statistically significant with all P values less than .001.

While all of the cancer types analyzed demonstrated hypermethylation of all three exonic CpG islands, the promoter methylation profile proved to be much more specific between the different neoplasias. In all of the myeloid neoplasias studied, hypermethylation of the PAX6 and p16 promoters was never observed. Only the promoter of p15 was ever hypermethylated in the myeloid neoplasias: five (23%) of 22 CML cases, five (83%) of six MDS cases, and eight (89%) of nine AML cases. The reverse was seen in colorectal cancer, in which only the promoter of p16 was ever hypermethylated (three [20%] of 15 cases), with p15 and PAX6 promoters remaining unmethylated in all cases.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study using the quantitative MS-SNuPE assay, we demonstrated a generalized deregulation of CpG island methylation within exons and promoters in cancer cells. Our data suggest that exons having the characteristics of CpG islands are more susceptible to methylation than promoters and may be where aberrant methylation is seeded. In each of the four neoplastic conditions studied, instances of hypermethylation of p15 exon 2, p16 exon 2, and PAX6 exon 5 were invariably observed, whereas promoter hypermethylation was specific for the cancer type. It is, therefore, reasonable to suggest that nonpromoter CpG islands within the body of a gene are somehow less protected against de novo methylation than those associated with the 5' region of the gene. The exact molecular mechanisms by which CpG islands protect themselves from de novo methylation and failures within those systems are poorly understood at this time. One possible explanation is that promoter-associated CpG islands are protected from DNA methyltransferases because of steric hindrance by transcription–initiation complexes and various transcription factors (13,14). Differential chromatin structure at exonic versus promoter CpG islands could also explain the difference in their susceptibilities to methylation. For example, it has been demonstrated that inhibition of poly(adenosine diphosphate [ADP]-ribosyl)ation of histone H1 introduces de novo methylation in transfected foreign DNA and can induce chromatin compaction and hypermethylation of endogenous islands (15,16). An intriguing avenue of research would be to characterize the differences, if any, in the levels of ADP-ribose polymers associated with promoter versus nonpromoter CpG islands.

The data also show that de novo methylation of exonic CpG islands occurs concurrently in multiple islands, suggesting global methylation changes in these transformed cells. Such concurrence of de novo methylation might imply that there exists some characteristic common to nonpromoter CpG islands within the body of a gene that render them susceptible to methylation and that such de novo methylation is not gene specific. We, therefore, propose a model of transformation of cells in which a random and global defect in methylation leads to multiple abnormally methylated CpG islands. Evidence exists to support such a hypothesis: It has been shown that promoter hypermethylation in cancers, such as AML or non-small-cell lung cancer, is not limited to one or two genes but is observed concurrently in a spectrum of loci in the same cell, many of which have critical roles in cell proliferation control (17,18). Also, a CpG island methylator phenotype has been demonstrated in colorectal, pancreatic, and gastric cancers, in which multiple islands, some of which are linked to known tumor suppressor genes, were simultaneously hypermethylated (1921).

Of interest and in contrast to the situation observed in control white blood cell DNA, we observed hypermethylation of the three exonic CpG islands in adjacent normal colonic tissue, although methylation levels in normal colonic tissues were substantially lower than those measured in colon tumor. Therefore, even nonpromoter CpG islands in normal cells are vulnerable to de novo methylation. The de novo methylation present in normal colonic tissue could be a preneoplastic change in cells that are already primed to become cancerous, or such exonic methylation could be related to the natural progression of age. Such a phenomenon is not unprecedented; the presence of age-linked hypermethylation in histologically normal colonic tissue has been demonstrated previously for CpG islands in ER, PAX6, EGFR, and other genes but, in those cases, was found in promoter islands (22,23). Although the average age of our sample of patients (x = 63 years old) seems to support the idea of age-related de novo methylation, we cannot make a solid conclusion because of lack of control subjects. However, it is important to note that, although de novo methylation is observed in histologically normal colonic tissue, there still exists a statistically significant increase in exonic methylation levels from adjacent normal tissue to tumor tissue.

Comparison of the exonic methylation levels observed in colon with those measured in blood revealed another intriguing pattern. As has been emphasized throughout this article, exonic CpG islands, when compared with promoters, display increased susceptibility to de novo methylation. However, even the methylation patterns among exons are not homogeneous. For example, in normal and tumor colon, p15 exon 2 is equally, if not more, prone to becoming methylated compared with the exons of p16 and PAX6, whereas p15 exon 2 is methylated to a lesser extent than p16 exon 2 or PAX6 exon 5 in the leukemic samples. Just as the context of a CpG island (i.e., whether it is located within a promoter or an exon) seems to modulate its susceptibility to methylation, the cell type-specific environment of a CpG island (i.e., the complement of active genes specific to a cell type) may also play a role in the regulation of its methylation. For example, in the myeloid neoplasias, it was observed that, on average, the methylation level of the 5' region of p15 was higher than that of the exon, whereas the inverse is seen in colon tissue. It is possible that the methylation and, therefore, transcriptional status of the promoter of p15 influences the methylation of the downstream island at exon 2. In fact, hypermethylation of nonpromoter CpG islands within the body of a gene is sometimes correlated with increased expression (6,24,25). It has been suggested that transcription through downstream, nonpromoter islands may facilitate their de novo methylation, perhaps by perturbation of the chromatin structure at the island or by the creation of single-stranded DNA intermediates that are proven substrates for de novo methylation (26). Our data provide additional evidence for such a hypothesis.

A generalized defect in the control of methylation can lead to global aberrant methylation patterns, as supported by the concurrent hypermethylation of multiple exonic CpG islands in the neoplasias that we have studied. The cancer-specific promoter methylation that we have observed in this study might then be a result of spreading from exonic foci and selection of cells whose growth is deregulated by the inactivation of a tumor-suppressor gene (Fig. 5Go). The hypermethylation of the p15 5' region that we observed corroborates previous work showing that p15 is targeted by aberrant methylation in CML, MDS, and AML (1,2729). Presumably, the progenitor cells of the leukemic disorders were selected by the methylation-induced silencing of p15, while those of certain of the colon tumors were selected by inactivation of p16 or other critical loci not analyzed in our study.

We have demonstrated that the methylator phenotype observed in some cancers can be applied to nonpromoter as well as promoter CpG islands. The nonspecific and global presence of de novo methylation in exonic islands seems to correlate well with progression of the neoplastic state and could conceivably be used as a tumor marker in the tracking and prognosis of certain cancers. For example, the efficacy of demethylating agents, such as decitabine, in the treatment of some cancers may be tracked by measuring the changes in methylation levels at these de novo-methylated CpG islands. Moreover, although the presence of exonic CpG island methylation appears to be a global phenomenon across different cancers, the degree of methylation at those exonic islands may help in distinguishing certain neoplasias from others, as exemplified by the relative difference in the susceptibility of p15 exon 2 to methylation between colon and blood in our study.


    NOTES
 
Supported by Public Health Service grants R35CA49758 and P01CA55164 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received March 6, 2001; revised July 17, 2001; accepted August 3, 2001.


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