[CANCER RESEARCH 58, 5489-5494, December 1, 1998]
Aging and DNA Methylation in Colorectal Mucosa and Cancer1
Nita Ahuja, Qing Li, Avinash L. Mohan, Stephen B. Baylin, and Jean-Pierre J. Issa2
The Johns Hopkins Oncology Center ¡N.A.. Q. L, A. L. M.. S. B. B., J-P. J. /./ and Departments of Surgery ¡N.A./ and Medicine ¡S.B. B.], The Johns Hopkins University School
of Medicine, Baltimore, Maryland 21231
ABSTRACT
DNA methylation of promoter-associated CpG islands may function as
an alternate mechanism of silencing tumor suppressor genes in multiple
neoplasias including colorectal cancer. De novo methylation of genes
appears to be an early and frequent event in most neoplasias. For the ER
and IGF2 genes, we have previously shown that methylation actually
begins in the normal colon mucosa as an age-related event and progresses
to hypermethylation in cancer. In this study, we have determined the
frequency of age-related methylation in normal colonie mucosa among the
genes hypermethylated in colorectal cancer. We studied six genes, includ
ing N33, MYOD,p¡6, HIC-l, THBSI, and CALCA. The N33 gene showed
partial methylation in normal colon mucosa, which was age-related
(r = 0.7; P = 0.003 using regression analysis). Adenomas and cancers
showed further hypermethylation at this locus. Similarly, the MYOD gene
showed age-related methylation in normal colon mucosa (r = 0.7;
/' < 0.00001 using regression analysis) and hypermethylation in cancers.
Age-related methylation seems to be gene specific, because pI6, THBSI,
HIC-l, and CALCA were not affected. Furthermore, this process may also
be modulated by tissue-specific factors. Our study suggests that aging is a
major contributing factor to hypermethylation in cancer.
INTRODUCTION
CGIs3 are areas rich in CpG dinucleotides that are found within the
promoter region of most housekeeping genes ( 1). These islands are
normally unmethylated regardless of the transcriptional status of the
gene, except for genes on the inactivated X chromosome in females
(2) and certain imprinted genes (3). De novo methylation of promoter-
associated CGIs has been associated with the transcriptional inacti-
vation of the gene (4) and seems to be functionally equivalent to an
inactivating mutation for the silencing of multiple tumor suppressor
genes (5, 6) such as pI6, VHL, and RB. Abnormal CpG island
methylation seems to be a frequent event in most neoplasias, including
CRCs (5, 6).
Aging is one of the most important risk factors for the development
of neoplasia (7). The incidence of cancer rises sharply after the age of
60 years (8). For example, CRC, which is one of the common solid
human malignancies, appears at a median age of 62 years in both men
and women. This risk factor has been attributed partly to the cumu
lative exposure to carcinogens over time and the multiple hits required
for the onset of neoplasia (7). Epigenetic modifications of DNA, such
as altered DNA methylation patterns, have long been postulated to
play a role in aging and the associated increased incidence of neopla
sia (9). However, most of the focus has been on the overall genomic
hypomethylation, which has been proposed to function as a "counting
mechanism" for cellular senescence (10, 11), or on regional areas of
hypermethylation outside the promoter region of genes (12), such as
Received 6/15/98; accepted 10/5/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Colon Cancer Spore Grant CA62924 and National Cancer
Institute Grant CA433I8. N. A. is supported by NIH Training Grant 1-T32-DK07713.
J-P. J. I. is a Kimme] Foundation Scholar.
2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology
Center. 424 North Bond Street. Baltimore. MD 21231. Phone: (410) 955-8506; Fax: (410)
614-9884; E-mail: jpissa@>welchlink.welch.jhu.edu.
' The abbreviations used are: CGI, CpG islands; CRC. colorectal cancer; MI, micro-
satellite instability.
5489
Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.
those in c-MYC (13) orc-FOS(\4, 15). The functional significance of
these events is somewhat unclear at present (16).
However, hypermethylation of promoter-associated CGIs has been
more clearly associated with gene silencing (5, 6). We have recently
shown that the estrogen receptor (£/?)gene on 6q gets hypermethyl
ated at its promoter CGI in all colonie neoplasms (17). This process
is associated with decreased ER expression that appears to start in the
normal colonie epithelium as a function of age. No methylation is
apparent in young individuals, but partial methylation appears in older
individuals, and the ER gene is hypermethylated in all colonie ade
nomas and neoplasms. Thus, aging itself or age-dependent events
appear to be a significant risk factor for hypermethylation of this gene.
We have subsequently observed that the insulin-like growth factor II
(IGF2) gene also shows age-related methylation in the colon, which
eventually progresses to hypermethylation in cancers (18). These
studies illustrate that aging itself may be a significant risk factor for
the hypermethylation of these genes in cancers.
In the current study, we have tried to determine the frequency of
age-related methylation for multiple additional genes that are hyper
methylated in CRC. Our data indicate that aging-related methylation
is a common event in CRCs, being present in four of the eight genes
known to be hypermethylated in this tumor type, and suggest that
aging is a major contributor to hypermethylation in CRC.
MATERIALS AND METHODS
Tissue Samples. Samples of all primary cancers including CRCs, meta-
static liver lesions, and normal colon and liver tissues were obtained troni
surgical resections of patients operated on at the Johns Hopkins Hospital. The
samples were immediately fro/.en in liquid nitrogen after resection and stored
at —70°Cuntil processing. For the separation of epithelium from stroma.
freshly resected samples were lightly scraped with a razor blade to collect
surface epithelium and incubated for 2 h with gentle agitation in HBSS without
calcium and phosphate supplemented with 30 IDM EDTA (pH 7.0). After
incubation, epithelial cells were floating in the solution and collected by
centrifugation. The remaining tissue contained stromal cells exclusively. The
purity of each fraction was greater than 90% as determined by immunohisto-
chemistry. Specimen collection procedures were approved by the Joint Com
mittee on Clinical Investigation of the Johns Hopkins Hospital in accordance
with the policies of the Department of Health and Human Services. Cell lines
were obtained from the American Type Culture Collection (Rockville. MD).
Southern Blot Analysis. DNA was extracted using standard techniques.
Five pig of genomic DNA were digested with 100 units of the appropriate
restriction enzyme as specified by the manufacturer (New England Biolabs).
DNA was precipitated, and the DNA fragments were separated on a 1%
agarose gel. transferred to a nylon membrane (Zetaprobe; Bio-Rad), and
hybridized with the various probes described below. The blots were then
exposed on a phosphor screen for 2-5 days and developed using a Phospho-
rlmager (Molecular Dynamics). Using ImageQuant software, image analysis
was carried out, and the density of methylation was determined as the density
of the higher molecular weight band as a percentage of the total density for that
sample.
Hybridization Probes. Probes used for the analysis of promoter methyla
tion have been described previously and included a 0.3-kb 5' ER probe (17).
a 0.65-kb 5' c-ABL probe ( 18), a 0.35-kb 5' pió probe ( 19), a 0.6-kb 5' THBSI
probe (19), a 1.1-kb 5' N33 probe (20) containing exon 1, a 0.6-kb CALCA
probe (21), a 0.65-kb exon 1 MYOD probe (21), and the highly polymorphic
pYNZ22.1 probe near the HIC-l locus (22). Information on the IR, CNP,
HASH!, hMSH2, and c-MYC probes is available upon request. All probes were
 AGING AND DNA METHYLATION IN CRC

labeled by random priming (Boehringer Mannheim) using [a-'2P]dCTP
ersham).
Statistics. All statistical analyses were performed using the Microsoft
Excel software program.
RESULTS
Genes Studied. We have previously shown age-related methyla-
tion of the normal colorectal mucosa for the ER and the IGF2 genes
(17, 18). In the present study, we analyzed six additional genes that
have previously been shown to be hypermethylated in CRC: (a) N33,
a ubiquitously expressed gene on 8p22 that was recently cloned from
an area of frequent allelic loss in prostate carcinomas and has been
shown to display promoter hypermethylation in several CRC cell lines
associated with a lack of expression of the N33 transcript (20); (b)
MYOI), a muscle-specific transcription factor located on 1Ip that was
one of the first genes shown to be hypermethylated in immortalized
cell lines and has subsequently been shown to be hypermethylated in
several cancers, including bladder, breast, and CRCs (23); (e) piò, a
cyclin-dependent kinase inhibitor on 9p21 with tumor-suppressor
properties that has been shown to be hypermethylated in most com
mon human cancers, including CRCs (24); (d) THBS1, a p53- and
/?/>-regulaied angiogenesis inhibitor on 15q that we have previously
shown to be methylated in sporadic CRC with MI (19); (e) HIC-1, a
zinc-finger transcription factor on I7pl3.3 that was cloned on the
basis of the presence of a large CGI in this area that becomes
frequently methylated in various cancers, including CRC (25); and (/)
CALCA (calcitonin), on 1Ipl5 that shows CGI hypermethylation in
(Am- lung and CRCs as well as in leukemias (26). Control genes that were
not known to be hypermethylated in CRC included c-MYC, IR (insulin
receptor), CNP (C-type natriuretic peptide), c-ABL, HASH! (human
achaete schule homologue), and !iMSH2.
N33 Methylation in Normal Colon Mucosa and CRCs. The N33
gene has a typical CGI spanning its promoter and first exon with
multiple methylation-sensitive restriction sites, including an Eagl site,
a Bi\yHlI site, and a Sacll site (Fig. \A). We investigated the status of
N33 methylation in both normal colon mucosa and in CRCs.
We first examined the methylation of the N33 CGI-associated
promoter in 44 normal colorectal mucosa samples from subjects
ranging in age from 8 to 90 years. We used Southern blot analysis of
genomic DNA that was first restricted with methylation-sensitive
enzymes and then hybridized with a 5' region probe. The N33 gene
showed a partial methylation in the normal colorectal mucosa that was
age-related, being barely detectable in young individuals and progres
sively increasing in older individuals (see the examples in Fig. Iß).To
better study this relationship, we used densitometric analysis to meas
ure the relative ratio of the methylated ( 1.1-kb) band in each lane. The
intensity of methylation was plotted as a function of age (Fig. 1C) and
showed that N33 methylation increased as a function of age (r = 0.7;
P = 0.003 using regression analysis). The intensity of methylation
averaged 15.1% in patients less than 20 years of age (n = 4), 37.1%
in patients 20-39 years of age (n = 10), 48.0% in patients 40-59
years of age (n = 9), and 58.4% in patients more than 60 years of age
(n = 21 ; Fig. 1C). Next we did a subset analysis comparing the extent
of N33 methylation with various clinical and pathological parameters.

100-
R-0.7
90-
p=0.003
80-
70-

A. «H
I
30
20-
10'

Age
Age

S 15 20 29 37 46 56 61 72 82 F F TI T2 T3 T4 T5 T6 T7 T8 T9 MAC MC

B. D.

OJ

Fig. l. Methylation status of the N33 gene CGI in normal colorectal mucosa and CRCs. A, partial restriction map of the N33 gene showing the first exon (•)and flanking region.
The CpG density of the area is plotted ahove the map, and the high density of CpG dinucleotides indicates a CGI. The predicted restriction fragments using ///ndlll/Si/rll are shown
al the bottom: H. Hináltt; S, Sai II; £.Eagl; Sm, Smal; B. BssHll. B, partial methylation of the N33 gene in normal colorectal tissues. Representative Southern blots of DNA from normal
coloreclal mucosa are shown. /.<•/;. approximate sizes of the hands (in kh). Tup. age of the patient at the time the mucosa sample was obtained. All lanes show patients' DNA restricted
with //('.'itili, a flanking enzyme, and .S'nrII. a melhylation-sensitive restriction enzyme, except for Lane F. which shows DNA restricted with //(milIf alone. The l.l-kb band reflects
Ihe methylalion of the Sudi site. C. methylation of N33 CGI increases with age. The percentage of N33 methylation of DNA from normal colorectal mucosa was plotted against the
age of the patient including the 10 patients shown in Fig. \H and 34 other control patients. A regression analysis of all samples gave a linear fit, as shown by the straight line, with
a correlation coefficient of 0.7. which is statistically significant (P - 0.003). D, methylation of the N33 CGI in colonie tumors. Left, the size of the various bands (in kb) is shown.
The Ume F shows DNA restricted with the ////idlll flanking cn/yme alone, whereas all other lanes show samples restricted with ///ndlll/Si/rll. iMiiex T1-T8 are cancers, whereas Lane
TV is an adenoma. Lanes T1-T3 and TV are hypermethylated. Limes T4-T6 appear to be equivalently methylated to normal colonie mucosa, and Lanes T7-T8 are cancers that appear
to be hypomethylated at N33. On the right are two colorectal tumor/adjacent mucosa pairs. M, uninvolved mucosa; A. adenoma; C, carcinoma. Note that the small 0.3-kb unmethylated
band is visible in only some samples, depending on the length of time the samples were electrophoresed.
5490

Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.
 AGINO AND DNA METHYLATION
There were no differences in the intensity of N33 methylation based
on the gender of the patient (means: females = 44% (n = 21) and
males = 50% (n = 23); P = 0.3 by / test) or the site of origin of the
colorectal mucosa [means: left side = 46% and right side = 48%
(n = 22 each); P = 0.7 by / test]. Of the 44 cases examined, 22 were
from patients without neoplasia. Age-related methylation was also
observed in this group (from 10% methylation in patients less than 20
years of age to 43% methylation in patients over 40 years of age).
However, control patients were significantly younger than those with
neoplasia, precluding an accurate comparison of the two groups.
Next we studied the pattern of N33 methylation in 44 primary
sporadic CRCs. The median age of these patients was 68 years. Based
on the above data for aging and N33, the normal surrounding colon
mucosa of these samples would be predicted to have a high degree of
N33 methylation, averaging greater than 50%. The majority of CRCs
appeared to either get more hypermethylated (20 of 43 samples) or
remain equivalently methylated (19 of 43 samples) when compared to
similar age-matched normal colon samples (see the examples in Fig.
ID). However, there are some tumors that are clearly hypomethylated,
ranging from 2-20% methylation (4 of 43 samples) at the N33
promoter, but the data on the normal colon mucosa from these
particular samples were unavailable (Fig. ID). To further understand
the extent of N33 methylation in CRCs, we studied the pattern of N33
methylation in 11 paired samples containing both cancer and normal
mucosa from the same patient. The average age of these 11 paired
samples was 65.9 years. As expected, the normal colon tissue from
these samples showed a high degree of N33 methylation, averaging
67%. However, the paired tumor tissue from these samples showed a
significantly higher amount of N33 methylation, averaging 83%
(P = 0.03 using / test; see the examples in Fig. ID). We also measured
N33 methylation in 10 adenomatous colonie polyps. These polyps also
showed a high degree of N33 methylation, averaging 80% (n = 10;
see the examples in Fig. ID), which was equivalent to that seen in
carcinomas. Finally, we had previously observed an association be
tween MI and CGI methylation in CRC for some (P16 and THBS1)
but not all (ER) genes (19). MI data were available for 24 of the CRCs
analyzed. N33 methylation was not affected by the MI status of the
tumors; N33 methylation averaged 68% in 14 MI+ cases and 75% in
10 MI- cases (P = 0.33). Thus, N33 hypermethylation seems to be
a very early event in CRC pathogenesis, starting as an age-related
event in the normal colorectal mucosa and evolving further in adeno
mas and carcinomas.
Age-related MYOD Methylation. The MYOD gene has a large
CGI of about 3.0 kb that spans its promoter as well as the first three
exons of the gene. The MYOD CGI contains five Sacll sites as well as
multiple Smal sites (Fig. 2A). We looked at the methylation status of
the MYOD gene CGI in normal colorectal mucosa and CRCs using
Southern blot hybridization. There were 37 samples of normal colo
rectal mucosa of various ages, ranging from 8-90 years. Throughout
its entire CGI, the MYOD gene had age-related partial methylation in
the normal colorectal mucosa samples as measured by two different
methylation-sensitive restriction enzyme sites (see the examples of
Southern blots in Fig. 2B). The intensity of MYOD methylation was
most dense within the heart of the CGI in exon 1 but also involved the
CpG dinucleotides near the transcription start site in an age-related
fashion, as indicated by the 1.7-kb band in Fig. 2B. To quantitate this
relationship, the intensity of methylation at the Sacll site closest to the
transcription start site, designated as SI on Fig. 2A, was measured by
densitometric analysis and plotted as a function of age. As shown in
Fig. 2C, the MYOD gene promoter showed increased methylation of
its CGI as a function of age (r = 0.7; P < 0.00001 using regression
analysis) at the SI site. The intensity of methylation averaged 3.5% in
patients less than 20 years of age (n = 4), 5.5% in patients 20-39
5491
Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.
IN CRC
H«IIIHII HIM
A. III
S2i"ÎS»JSJ B
7» O tt 15 » F
R=0.8
p<0.00001
0 20 40 60 80 100
Age
D.
T6-12
N1T1 N2T2N3T3 N4T4N5T5
«l
Fig. 2. Methylation status of the MYOD gene CGI in normal colorectal mucosa and
CRC. A. partial restriction map of the MYOD gene showing the first three exons (•)and
the flanking region. Vertical barf, restriction sites; B. BatnHl: H. //mdlll; SI-S5. Sacll
sites. Sml-Sm6, Smal sites. The CpG density of the area is plotted above the map. and the
high density of CpG dinucleotides indicates a CGI. B. partial methylation of the MYOD
gene in normal colorectal tissues. Representative Southern blots of normal colorectal
mucosa using two different methylation-sensitive restriction enzymes are shown. On the
left of each panel are the approximate sizes of the bands (in kbl. Urne F shows DNA cut
with the flanking enzyme alone. U-fl panel. DNA of 10 patients of various ages (indicated
on top of each lane) restricted with BamHl. a flanking enzyme, and Suri I. a methylation-
sensitive restriction enzyme. The 0.36- and 0.71-kb bands are present if there is no
methylation of any Sudi site, whereas the other bands reflect the methylation of either S2
(1.1 kb), S3 (0.53 kb). S2 and S3 (1.3 kb), orali of S1-S3 (1.7 kb). RiKhi panel. DNA of
the same 10 patients restricted with BamHl and Srnai. another methylation-sensitive
restriction enzyme. The 0.34- and 0.56-kb bands arc present if there is no methylation of
the Smal sites, whereas the other bands reflect the methylation of Sm2 (0.9 kb). Sm2 and
Sm.3 (I.I kb). Sml and Sm2 (1.5 kb). and Sml-Sm3 (1.7 kb). C methylation of the
MYOD CGI increases with age. The percentage of MYUD melhylation of DNA from
normal colorectal mucosa was plotted against the age of the patient. /,<'// panel, the
methylation of the 5' SacII site. SI, plotted as a function of age for the 10 patients shown
above and 27 other patients. A regression analysis of all samples gives a linear fit, as
shown by the straight line, with a correlation coefficient of 0.7. which is statistically
significant (P < 0.00001). Righi panel, the melhylation of the 5' Smal site. Sml. plotted
asa function of age for a total of 23 patients ranging in age from 10-90 years. Again, the
data result in a linear fit by regression analysis, with a correlation coefficient of 0.8
(P < 0.00001 ). D, methylation of the MYOD CGI in CRCs. On the left are shown the siz.es
of the various bands (¡nkb). In this analysis of DNA restricted with ///»dill and Saill. the
0.36- and 1.1-kb bands are seen if there is no methylation at the MYOD locus, whereas the
higher molecular weight bands represent increasing methylation. The left panel shows five
pairs of normal (Wtumor (D specimens. Tumors show hypermethylalion compared to the
surrounding normal tissue. In the middle, Lanes T6-TI2 represent additional tumors
showing almost complete methylation. and the panel on the extreme righi shows hyper
methylation in various CRC cell lines (indicated at the top of each lane). Note that CaCO2,
SW480, OLD 1. and HT29 show almost 100% melhylation. and none of the cell lines have
alÃ-elesdevoid of methylation (i.e.. no bands at 0.36 and 1.1 kb).
years of age (n = 6), 11.8% in patients 40-59 years of age (n = 10),
and 20.0% for patients more than 60 years of age (n = 17; Fig. 2C).
The methylation of the Sacll sites within exon 1, namely S2 and S3,
 AGING AND DNA METHYLATION
.--— * —
c-abl THBSI
Fig. 3. Melhylation stalus of other genes in normal colorectal tissues. Representative Southern blots of multiple genes that show no evidence of age-related methylation are shown.
Lane F represents DNA cut with the Hanking enzyme alone, whereas the other lanes are DNA cut with both the flanking enzyme and a methylation-sensitive restriction enzyme (£.
ktixl: lì./f.v.vHII;S. Siu'll: M. Mlul; Nr. Nrul; N. Not]). The ages of patients are shown on the lop of each panel for the various samples of normal colorectal mucosa. In each case,
Ihe arrowheads point to the size of the methylated band (which is absent in all cases). In the THBSI blot, a nonspecific band can be seen in all of the lanes, including the flank lane.
also showed an age-related increase, as demonstrated by multiple
higher molecular weight bands on Southern blot analysis (Fig. 2B). In
fact, when we considered the methylation of either SI, S2, or S3, the
MYOD CGI showed a striking age-related methylation (r = 0.9;
P < O.CKXXXMusing regression analysis): the intensity of methylation
averaged 28.2% in patients less than 20 years of age (n = 4), 39.8%
in patients 20-39 years of age (n = 6), 61.7% in patients 40-59 years
of age (n = 10), and 70.3% in patients more than 60 years of age
(;i = 17). On subset analysis, there was no difference in the intensity
of MYOD methylation at the SI site based on gender (means: 13.5%
for females (n = 18) and 13.7% for males (n = 19); P = 0.9 by / test).
However, there was a marginally significant higher intensity of
MYOD methylation at the SI site in samples originating from left-
sided colorectal mucosa (mean, 14.2%; n = 24) compared to right-
sided colorectal mucosa [mean, 8.6%; n = 11 (P = 0.05 by t test)]. Of
the 37 samples studied, 22 were from controls, and 15 were from
patients with CRC. Age-related methylation was equivalent in the two
groups.
We also studied the methylation of the Sma\ sites in the MYOD CGI
and found a similar age-related increase in methylation at all of the
SÃ-milsites around exon 1 (examples of Southern blot, Fig. 2B). The
intensity of methylation at the Snuil site closest to the transcription
start site, which is designated as Sml ¡nFig. 2A, was measured by
densitometric analysis and plotted as a function of age. As shown in
Fig. 2C, the MYOD gene promoter also showed increased methylation
at the Sml site as a function of age (r = 0.8; P < 0.00001; range,
10-90 years; sample size, 23). The intensity of methylation averaged
0.7% in patients less than 20 years of age (n = 2), 1.1% in patients
20-39 years of age (n = 4), 6.7% in patients 40-59 years of age
(n = 7), and 19.9% for patients more than 60 years of age (n = 10;
Fig. 2C).
To determine the impact of age-related methylation at the MYOD
locus on hypermethylation in cancer, we studied 11 cases of paired
normal/tumor DNA from the same patients. In all cases, methylation
was significantly more extensive in the tumors (including 4 adenomas
and 10 carcinomas), with most alÃ-elesshowing hypermethylation of
all of the methylation-sensitive restriction enzyme sites in the area
(Fig. 2D). Methylation seemed to be similar in adenomas and carci
nomas, suggesting that MYOD hypermethylation is a very early event
in CRC pathogenesis. We also studied an additional 20 carcinomas,
and all showed high degrees of methylation (>80%), suggesting that
MYOD hypermethylation is nearly universal in CRCs (see Fig. 2D).
There was no difference in MYOD methylation by MI status because
100% of the tumors were hypermethylated. Finally, six of eight CRC
cell lines (RKO, CaCO2, SW480, SW48, DLD1, and HT29) analyzed
had more than 95% of the alÃ-elesmethylated at all Sodi sites in the
region, whereas the remaining two (Lovo and HCT116) had more than
50% of the alÃ-elessimilarly methylated. None of the cell lines had
evidence of alÃ-elescompletely devoid of methylation (i.e., bands at
5492
Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.
IN CRC
90 85 82 82 Svii S5yn ÃŒMlyo
F N N N
( '¡licitomi!
IR
0.36 and 1.1 kb; Fig. ID). Overall, these data suggest that MYOD
methylation starts in the normal-appearing colorectal mucosa as a
function of age and becomes more prominent in cancers, suggesting
either that the cells with preexisting methylation are selected for in the
neoplastic process, or that tumors greatly extend methylation at this
locus.
Age-related Methylation Is Gene Specific. In contrast to N33 and
MYOD, none of the other genes examined showed evidence of age-
related methylation using similar Southern blot analysis. In particular,
THBSI was analyzed in 47 samples of normal colon mucosa ranging
in age from 20-98 years, and no methylation was observed (see the
examples in Fig. 3). Similarly, HIC-1 (n = 36; range, 20-98 years),
pió (n = 26; range, 20-98 years), and CALCA (n = 23; range, 10-90
years) had no evidence of methylation in any normal colonie mucosa
examined (Fig. 3; data not shown). Finally, we analyzed the normal
colonie mucosa at several gene loci that have shown no hypermethy
lation in CRC, including IR, c-ABL, c-MYC, CNP, and liMSH2 (Fig.
3; data not shown). These genes were similarly unmethylated in the
normal colon in 23 patients ranging in age from 10-90 years.
Age-related Methylation Is Modulated by Tissue-specific Fac
tors. Whereas age-related methylation seems to be a common event
within the normal colon mucosa, it is unknown whether this process
is present to a similar degree in all aging tissues, or whether it is
restricted to the colon alone. We therefore examined the methylation
D stroma
•
epithelium
Fig. 4. Age-related melhylation in various fractions of the colon mucosa. The mucosa
was separated into stroma and epithelial components, and the density of methylalion was
calculated for each fraction. The two colonie fractions for each of the genes are plotted on
the x-axis. and the average density of methylation for each fraction is plotted on the y-axis.
Slroma contains primarily stroma! components, whereas the epithelium contains epithelial
cells only. Both N33 and MYOD show methylation in both the epithelial and straniai
components, whereas the ER CGI primarily shows methylation in its epithelial compo
nent.
 AGINO AND DNA METHYLATION
N33
•MyoD
60 * ER«
504030•
20
10
100
Fig. 5. Concordancy of age-related methylation. Data on N33, MYOD, and ER CGI
methylation was available in 16 patients. The density of methylaüonwas plotted as a
function of age for each of these genes in all 16 patients. The data for each gene shows
a linear fit by regression analysis that seems broadly similar. The overall density of
methylation varies for each gene, being highest for the N33 gene and lower for ER and
MYOD. The MYOD data reflect the methylation of only the 5' SucII site, SI. The linear
fit curves for MYOD and ER were nearly superimposable, and only one (ER) is shown for
clarity.
status of the N33 and ER promoter CGIs in different normal tissues
from the same patients. In three patients for whom samples of normal
liver and colonie mucosa were available, N33 methylation was higher
in the colon than in the liver (74 versus 17%), which was statistically
significant (P = 0.01 by t test). By contrast, ER methylation was
much higher in the liver than it was in the normal colonie mucosa (81
versus 40%; P = 0.02 by i test). CRC that metastasized to the liver
showed high levels of methylation at all loci, as expected. Thus, the
age-related methylation of genes such as N33 and ER is modulated by
tissue-specific factors.
We next determined the specific patterns of methylation within the
colonie mucosa itself. The normal colon is composed of both epithe
lial and stromal components, and it was unclear which fractions are
involved in age-related methylation. Therefore, we separated the
epithelial and stromal components of colorectal mucosa to determine
the differences in methylation patterns between the components in
genes showing age-related methylation. The N33 gene showed similar
levels of methylation in both epithelia (mean, 60%) and stroma (mean,
75%) in four samples. MYOD also showed equivalent levels of meth
ylation in both the epithelial (mean, 20%) and stromal components
(mean, 19%; P = 0.8) in two samples. This was in marked contrast to
the ER gene, which showed four times higher methylation in the
colonie epithelium (mean, 31%) than in the stroma (mean, 8%) in five
cases, and this difference was statistically significant (P = 0.02; data
summarized in Fig. 4).
Concordancy of Age-related Methylation. ER, N33, and MYOD
all show age-related methylation in the normal colon. Therefore, we
looked at the concordancy of methylation of these three genes. Data
on the methylation status of all three genes was available for 16
samples of normal colonie mucosa. As shown in Fig. 5, whereas the
amount of methylation varied for each gene, the overall pattern of
age-related methylation was very similar for all three genes. Of the 16
cases, 4 appeared to have concordant deviations from the average; 1
4 Q. Li, N. Ahuja, P. C. Burger, and J-P. J. Issa, Methylation and
case (a 30-year-old man) had relatively high methylation levels for all
thrombospondin-1 promoter in human cancer, submitted for publication.
three genes, whereas 3 cases (all of whom were over 60 years of age) 5 J-P. J. Issa, unpublished observation.
had relatively low methylation levels for all three genes. 6 J. Herman and J-P. J. Issa, unpublished observation.
5493
Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.
IN CRC
DISCUSSION
In the current study, we have shown a strikingly similar pattern of
age-related methylation in normal colorectal mucosa and frequent
hypermethylation in CRC affecting the N33 gene on chromosome 8p
and the MYOD gene on chromosome 1Ip. In fact, of eight genes that
are known to be hypermethylated in CRC, four (N33, MYOD, ER, and
IGF2) display this remarkable pattern of age- and neoplasia-related
methylation. Interestingly, all four of these genes are very frequently
(>90%) hypermethylated in CRC, whereas the methylation frequency
of the other genes ranges from 5-38%. These data suggest that many
hypermethylation events observed in cancer are related to the fact that
neoplasia begins in aging cell populations.
Although we have limited our observations to CRC in this study,
our data should have important implications for all hypermethylation
events in cancer. In fact, in a recent study of hypermethylation of
multiple genes in glioblastoma multiforme, we found a striking con
cordance in the methylation status of ER and N33, which were both
much more frequently methylated in the tumors of older patients (27).
By contrast, just as seen in the colon, pi6 and THBSÌ methylation was
not related to the age of the patients studied.4 Furthermore, HlC-1,
which is frequently methylated in prostate cancer, is also partially
methylated in the normal prostate as well, and preliminary observa
tions suggest that this methylation is also age related.5 An important
point to note in these analyses is that the "normal" tissue adjacent to
cancer may not always be representative of the cell populations that
are predisposed to neoplasia. For example, normal breast tissue is
largely composed of supportive stromal cells and is unmethylated at
the HIC-1 locus, whereas purified breast epithelium is highly meth
ylated at this CGI.5 This factor may lead to an underestimation of the
contribution of age-related methylation in normal tissues to the ulti
mate frequency of methylation in neoplasia.
These observations raise the question of whether all hypermethy
lation events in cancer are related to initial methylation in normal
tissues, whether age related or stem cell related. Using the very
sensitive methylation-specific PCR (MSP) technique, which can de
tect a methylated cell diluted with 104-105 normal cells (28), we were
unable to find methylation of the piò gene in colorectal mucosa;6
similarly, we found no evidence of pi5 gene methylation in purified
hematopoietic progenitor cells (29). Thus, there seem to be two
distinct types of CGI methylation events in cancer: (a) methylation
that can be detected in both normal aging cells and neoplastic cells;
and (b) methylation that can only be detected in the neoplastic cells.
At present, it is unclear why only certain genes show age-related
methylation changes. This process seems to be tissue-specific and
involves both genes that are expressed in the colon (ER and N33) and
genes that are not expressed or are expressed at low levels (IGF2 and
MYOD). Age-related methylation of the ER gene may provide a
selective growth advantage for the normal colonie cells, but this does
not seem to hold true for MYOD or N33. MYOD is not expressed in
the normal colon, and N33, which seems to be an oligo-saccharyl-
transferase enzyme, has not been shown to affect the growth of cancer
cells in vitro. Thus, it is not readily apparent why the loss of N33 gene
expression would provide a selective advantage for tumor growth.
Furthermore, age-related methylation does not seem to be a simple
random event with selection of affected cells (based on a growth
advantage), because hypermethylation is not seen for the pl6 locus in
normal colon. It seems possible, therefore, that different CGIs have
different intrinsic rates of de novo methylation and protection against
silencing of the
 AGING AND DNA METHYLATION
methylation, and that the weakly protected islands are those that
display age-related methylation. Thus, age-related methylation of
genes may initially arise from local triggering factors (chromatin
structure or the proximity of Alu sequences) and be modulated by
rra/is-acting factors such as described in the APRT gene promoter,
where the CGI is protected from methylation by a cluster of binding
sites for the Spl transcription factor (30). It is also possible that for
some genes, this protection against de novo methylation may be lost
during aging. Additionally, several factors have been shown to mod
ulate CGI methylation in cancers such as defects in DNA repair
systems (19) or different types of carcinogen exposure (31). There
may also be genetic differences in the rate of age-related methylation
between different individuals, as suggested perhaps by the fact that a
few patients in our study appeared to have concordant differences in
methylation for all three genes when compared with the age-adjusted
average. Age-related methylation of promoter-associated CGIs may
then be related to both endogenous triggering factors and exogenous
modulating factors, such as carcinogen exposure.
Overall, our data suggest that many genes become methylated as a
function of age in the normal colon. Whereas some of the genes
affected are not expressed in the normal colon and may be of little
physiological relevance, others, such as the ER gene, seem to modu
late growth and differentiation in the normal colon (17). Thus, meth
ylation and the loss of expression of these latter genes may impart a
growth-selective advantage to the affected cells, which then become
more susceptible to acquiring further genetic defects that ultimately
lead to neoplasia. Therefore, we propose that age-related methylation
constitutes a type of field-defect in the colon, and that it partially
explains the dramatic age-related increase in CRC incidence. This
hypothesis predicts that patients with high levels of methylation in
their colorectal mucosa may be at higher risk for developing colorec-
tal adenomas and cancer, and that normal mucosa from CRC patients
would have higher levels of methylation than mucosa from patients
without cancer. In the current study, the limited number of patients
studied precludes such an analysis, which will require a carefully
designed epidemiological investigation. This hypothesis also provides
a potential explanation for the important finding that the reduction of
DNA-methyltransferase in a mouse model of CRC results in a marked
reduction in polyp incidence (32). Thus, reducing DNA-methyltrans
ferase levels may inhibit or slow the development of age-related
methylation, thus limiting the extent of the field defect and ultimately
decreasing the formation of tumors.
In summary, our study shows that age-related methylation is in fact
a frequent event among genes that are hypermethylated in cancer.
Age-related methylation may be one of the key events, accounting for
the fact that aging is the most important risk factor for most of the
common types of cancers in humans.
ACKNOWLEDGMENTS
We thank Dr. R. Bookstein (Canji Inc., San Diego, CA) for kindly providing
the N33 probe. Dr. S. Pearson-White (University of Virginia, Charlottesville,
VA) for kindly providing the MYOD probe, and Dr. S. Hamilton (Johns
Hopkins University, Baltimore, MD) for providing samples of normal and
neoplastic colon.
REFERENCES
1. Cross. S. H.. and Bird, A. P. CpG islands and genes. Curr. Opin. Genet. Dev., 5:
309-314, 1995.
2. Singer-Sam, J.. and Riggs. A. D. X chromosome inaciivation and DNA methylation.
EXS, 64: 358-384, 1993.
3. Barlow, D. P. Camelie imprinting in mammals. Science (Washington DC), 270;
1610-1613. 1995.
5494
Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.
IN CRC
4. Eden. S., and Cedar, H. Role of DNA methylation in the regulation of transcription.
Curr. Opin. Genet. Dev.. 4: 255-259. 1994.
5. Baylin. S. B.. Herman, J. G., Graff, J. R., Venino, P. M., and Issa, J. P. J. Alterations
in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res., 72:
141-196, 1997.
6. Jones. P. A. DNA methylation errors and cancer. Cancer Res., 56: 2463-2467, 1996.
7. Ershler, W. B.. and Longo, D. L. Aging and cancer: issues of basic and clinical
science. J. Nati. Cancer Inst., 89: 1489-1497. 1997.
8. tandis. S. H.. Murray, T., Bolden, S.. and Wingo. P. A. Cancer statistics, 1998. CA.
Cancer J. Clin., 48: 6-29, 1998.
9. Holliday, R. The inheritance of epigenelic defects. Science (Washington DC), 238:
163-170, 1987.
10. Mazin, A. L. Life span prediction from the rate of age-related DNA demethylation in
normal and cancer cell lines. Exp. Gerontol.. 30: 475-484, 1995.
11. Cooney. C. A. Are somatic cells inherently deficient in methylation metabolism? A
proposed mechanism for DNA methylation loss, senescence and aging. Growth Dev.
Aging. 57: 261-273, 1993.
12. Ghazi. H., Gonzales, F. A., and Jones, P. A. Methylation of CpG-island-containing
genes in human sperm, fetal and adult tissues. Gene (Amst.), 114: 203-210, 1992.
13. Halle, J. P., Schmidt, C.. and Adam, G. Changes of the methylation pattern of the
c-myc gene during in vitro aging of imr90 human embryonic fibroblasts. Mutât.Res..
316: 157-171, 1995.
14. Uehara. Y.. Ono. T., Kurishita, A., Kokuryu, H., and Okada, S. Age-dependent and
tissue-specific changes of DNA methylation within and around the c-fos gene in mice.
Oncogene. 4: 1023-1028, 1989.
15. Choi. E. K.. Uyeno. S.. Nishida, N.. Okumoto, T.. Fujimura. S., Aoki, Y.. Nata, M..
Sagisaka, K., Fukuda, Y.. Nakao. K., Yoshimoto. T., Kim, Y. S., and Ono, T.
Alterations of c-fos gene methylation in the processes of aging and tumorigenesis in
human liver. Mutai. Res., 354: 123-128, 1996.
16. Mays-Hoopes, L. L. DNA methylation in aging and cancer. J. Gerontol., 44: 35-36,
1989.
17. Issa. J. P.. Ottaviano, Y. L., Celano, P., Hamilton, S. R., Davidson, N. E., and Baylin,
S. B. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in
human colon. Nat. Genet.. 7: 536-540, 1994.
18. Issa. J. P. J., Vertino. P. M.. Boehm, C. D., Newsham, 1. F., and Baylin, S. B. Switch
from mono-allelic to bi-allelic human IGF2 promoter methylation during aging and
carcinogenesis. Proc. Nati. Acad. Sci. USA, 93: 11757-11762. 1996.
19. Ahuja, N., Mohan. A. L.. Li. Q.. Stoiker, J. M., Herman. J. G.. Hamilton. S. R..
Baylin, S. B.. and Issa, J. P. Association between CpG island methylation and
microsatellite instability in colorectal cancer. Cancer Res., 57: 3370-3374, 1997.
20. MacGrogan, D.. Levy, A., Bova. G. S., Isaacs. W. A., and Bookstein, R. Structure and
methylation-associated silencing of a gene within a homozygously deleted region of
human chromosome band 8p22. Genomics, 35: 55-65, 1996.
21. Venino, P. M., Issa, J. P., Pereira-Smith, O. M.. and Baylin, S. B. Stabilization of
DNA methyltransferase levels and CpG island hypermethylation precede SV40-
induced immortalization of human fibroblasts. Cell Growth Differ., 5: 1395-1402,
1994.
22. Issa, J. P. J., Zehnbauer, B. A.. Kaufmann, S. H.. Biel. M. A., and Baylin. S. B. HIC1
hypermethylation is a late event in hematopoietic neoplasms. Cancer Res., 57:
1678-1681. 1997.
23. Rideout, W. M., Ill, Eversole-Ciré.P., Spruck. C. H.. Ill, Hustad. C. M.. Coetzee.
G. A., Gonzales. F. A., and Jones, P. A. Progressive increases in the methylation
status and heterochromatinization of the myoD CpG island during oncogenic trans
formation. Mol. Cell. Biol.. 14: 6143-6152, 1994.
24. Herman. J. G., Merlo. A.. Mao. L., Lapidus, R. G.. Issa. J. P.. Davidson. N. E..
Sidransky. D.. and Baylin. S. B. Inactivalion of the CDKN2/pl6/MTSI gene is
frequently associated with aberrant DNA methylation in all common human cancers.
Cancer Res.. 55: 4525-4530. 1995.
25. Wales, M. M., Biel. M. A., el Deiry. W.. Nelkin, B. D.. Issa, J. P., Cavenee, W. K.,
Kuerbitz. S. J.. and Baylin, S. B. p53 activates expression of HIC-l. a new candidate
tumour suppressor gene on I7pl3.3. Nat. Med., /: 570-577. 1995.
26. Baylin, S. B.. Fearon. E. R.. Vogelstein. B., de Bustros. A.. Sharkis, S. J., Burke, P. J..
Staal. S. P.. and Nelkin. B. D. Hypermethylation of the 5' region of the calcitonin
gene is a property of human lymphoid and acute myeloid malignancies. Blood, 70:
412-417, 1987.
27. Li. Q., Jedlicka. A.. Ahuja. N.. Gibbons, M. C., Baylin, S. B., Burger, P. C.. and Issa.
J. P. J. Concordant methylation of the ER and N33 genes in glioblastoma multiforme.
Oncogene. 16: 3197-3202, 1998.
28. Herman, J. G.. Graff, J. R., Myohanen, S., Nelkin, B. D.. and Baylin, S. B. Methy-
lation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc.
Nati. Acad. Sci. USA, 93: 9821-9826. 1996.
29. Herman, J. G., Civin, C. I., Issa. J. P. J., Collector. M. !.. Sharkis, S. J., and Baylin,
S. B. Distinct patterns of /)/5""""1 and pl6INK4* characterize the major types of
hématologiemalignancies. Cancer Res., 57: 837-841, 1997.
30. Turker, M. S.. and Bestor, T. H. Formation of methylation patterns in the mammalian
genome. Mutât.Res.. 386: 119-130, 1997.
31. Issa. J. P. J.. Baylin. S. B.. and Belinsky. S. A. Methylation of the estrogen receptor
CpG island in lung tumors is related to the specific type of carcinogen exposure.
Cancer Res., 56: 3655-3658, 1996.
32. Laird, P. W., Jackson-Grusby, L., Fazeli, A., Dickinson. S. L., Jung, W. E., Li, E.,
Weinberg. R. A., and Jaenisch. R. Suppression of intestinal neoplasia by DNA
hypomethylation. Cell, 81: 197-205. 1995.
Aging and DNA Methylation in Colorectal Mucosa and Cancer
Nita Ahuja, Qing Li, Avinash L. Mohan, et al.

Cancer Res 1998;58:5489-5494.

Updated version Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/58/23/5489

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.

Permissions To request permission to re-use all or part of this article, use this link
http://cancerres.aacrjournals.org/content/58/23/5489.
Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
Rightslink site.

Downloaded from cancerres.aacrjournals.org on April 21, 2020. © 1998 American Association for Cancer Research.