[CANCER RESEARCH 51, 3702-3709, July 15, 1991]
Patterns of DNA Methylation in Selected Human Genes in Different Hodgkin's
Lymphoma and Leukemia Cell Lines and in Normal Human Lymphocytes1
Sabine Achten,2 Annett Behn-Krappa, Manfred Jücker,JörgSprengel, Irmgard Hölker,Birgit Schmitz, Hans Tesch,
Volker Diehl, and Walter Doerfler3
Institute of Genetics [S. A., A. B-K., J. S., I. H., B. S., W. D.J, and Department of Internal Medicine ¡M.]., H. T., V. D.J, University of Cologne, Cologne, Germany
ABSTRACT
The human genome, like many other genomes, harbors highly specific
patterns of DNA methylation which have not yet been systematically
studied. In a limited investigation on the genes for tumor necrosis
factors-a and -,1,a surprising interindividual concordance in the patterns
of DNA methylation at the nucleotide level has been demonstrated earlier
by using the genomic sequencing method on DNA from individuals of
very different ethnic origins. Patterns of DNA methylation could perhaps
serve as indicators for genetic activities. These activities would not have
to be restricted to gene transcription but could relate to other genetic
activities in the cell. DNA methylation patterns are known to be cell
type-specific. We have now initiated a study of these DNA patterns in
human lymphocytes and in human cell lines of different malignant origins.
Several of the proto-oncogenes, parts of the genes for tumor necrosis
factors-a and -,<, the insulin receptor and lamin C have been used as
hybridization probes. We have relied to some extent on the documented
observation that the methylation patterns at 5'-CCGG-3' (Hpall/Mspl)
sequences yield a reflection of patterns at all 5'-CG-3' sequences. Three
main types of patterns have been observed. Some of the probed segments
are completely unmethylated; others are fully methylated, most of the
areas are partly methylated exhibiting complex patterns at the S'-CCGG3' sites. In different tumor cell lines, different DNA methylation patterns
are apparent for the same DNA probes. Comparisons of the methylation
patterns in a given DNA segment between DNA from primary normal
human lymphocytes and DNA from different tumor cell lines reveal
changes in these patterns in several instances.
INTRODUCTION
One of
eukaryotic
long-term
However,
the biological functions of DNA methylation in
systems has been sought and documented in the
silencing of promoters (for reviews see Refs. 1-4).
5-mC4 residues in specific DNA sequences can be
more generally viewed as modulators of highly specific inter
actions between DNA sequence motifs and DNA-binding pro
teins. Since these interactions are at the core of many biological
functions, it can be expected that sequence-specific methylations will affect diverse cellular functions. Patterns of DNA
methylation in virus-transformed cell lines (5) and in specific
segments of the human genome (6, 7) can be very stable. In
human populations of diverse ethnic origins, a high degree of
interindividual concordance in patterns of DNA methylation
has been observed for parts of the human genome, at the
nucleotide (6, 7) and at the kilobase pair levels (60).
Levels of DNA methylation have been analyzed in virusinduced tumor cells (5, 8-11) and in human tumors (12, 13).
Received 12/26/90; accepted 5/1/91.
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.
' This research was supported by grants to H. T. and W. D. in the Forscher
gruppe "Immundysregulation und menschliche Lymphome" of the Deutsche
Forschungsgemeinschaft (Di 184/9-I).
2 Present address: Boehringer-Ingelheim Foundation, Stuttgart, Germany.
3 To whom requests for reprints should be addressed, at Institute of Genetics,
University of Cologne, Weyertal 121. D-5000 Cologne 41, Germany.
' The abbreviations used are: 5-mC, 5-methyldeoxycytidine; TNF, tumor ne
crosis factor; SDS, sodium dodecyl sulfate; 2 x SSC, 0.3 M NaCI, 0.03 M sodium
citrate; kbp, kilobase pair.
In different human tumors, the patterns of DNA methylation
in selected human genome segments are frequently altered in
comparison to the DNA in nontumor cells (14, 15). Since
patterns of DNA methylation reflect in a complex way the
states of transcriptional, replicational, recombinatorial, and
other genetic activities in mammalian cells, it is not surprising
to find altered patterns in tumor cells which have a different
program of transcriptional and other genetic activities in com
parison to normal cells (16). In the present communication, we
have initiated an investigation of methylation patterns in several
human proto-oncogenes, as well as in parts of the TNF-a and
-ß,the insulin receptor, and the lamin C genes. The DNAs from
unstimulated or from cytokine-stimulated normal human lym
phocytes, from human B-cell and T-cell leukemia lines, from
myeloid leukemia cell lines, and from Hodgkin's lymphoma
cell lines as clonal derivatives of tumor cells (cf. Table 1) have
been subjected to Hpall and Mspl restriction analyses (17).
MATERIALS
AND METHODS
Cell Lines and Cellular DNA. Primary human lymphocytes were
prepared by the Ficoll-Paque technique from freshly drawn blood of
human volunteers (6). The human cell lines KB (human oral carci
noma), HeLa (human cervix carcinoma), or 293 (adenovirus type 5transformed human embryonic kidney cell line) were grown in culture
in Dulbecco's medium containing 10% fetal calf serum. A number of
established human lymphoma or leukemia cell lines from patients with
Hodgkin's disease (L428, L540, DEV, L591), with B-cell leukemia
(L660), with T-cell leukemias (Jur, CEM) or with myeloid leukemias
(HL60, U937) were grown in RPMI 1640 medium supplemented with
10% fetal calf serum (Table 1).
Isolation of DNA. A volume of 5 ml of human blood was diluted
with 45 ml of ice-cold dilution buffer (0.32 M sucrose, 5 IHMMgCI2,
1% Triton X-100, 0.01 M Tris-HCl, pH 7.5) and mixed for 10 min at
4°C.The nuclei liberated by the detergent treatment were sedimented
at 2900 x g for 15 min at 4°C,resuspended in 0.5 ml 0.01 M Tris-HCl,
pH 7.5, 1 mM EDTA, and 50 v\ each of 10% SDS and of proteinase K
(5 mg/ml) were added. The mixture was incubated overnight at 37°C.
Subsequently, the DNA was purified by repeated phenol:chloroform
(1:1) and chloroform extractions. The DNA from the cell lines (5) or
from primary cells (6) was extracted by standard procedures.
Restriction Analyses and Southern Blot Hybridization. DNAs were
cleaved with restriction endonucleases according to standard protocols.
For the restriction endonucleases Hpall and Mspl, 120 units of enzyme
were used to cleave 10 ^g of genomic DNA in a total volume of 100 to
300 n\. By using higher concentrations of restriction endonucleases, the
cleavage patterns for different DNAs, as determined by Southern blot
hybridization (33), did not change. In this way, it was ascertained that
DNA samples were completely cleaved. DNA fragments were separated
by electrophoresis on 1.0% agarose gels, and 10 ¿tg/slotof cleaved
cellular DNA was applied. After electrophoresis, the DNA was trans
ferred to Hybond N membranes (nylon; Amersham) or to nitrocellulose
filters (BA; Schleicher & Schuell). The overnight transfer of DNA to
the Hybond N membranes was effected in 0.25 N NaOH, 1.5 M NaCI,
and the membranes were then preincubated for 4 h or overnight at
68°Cin 2 x SSC, 0.5% low fat milk powder, 1% SDS, and 1 mg of
salmon sperm DNA/ml. Subsequently, the membranes were incubated
for 16-20 h at 68°Cin the same solution which then also contained
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DNA METHYLATION
PATTERNS IN HUMAN LYMPHOMA AND LEUKEMIA CELL LINES
Table l Human cell lines used in the analyses of DNA methylation patterns
Origin
Cell line
lymphocytes was either isolated directly or the lymphocytes were seeded
at a concentration of 0.5 x IO6to 10" cells/ml of RPMI 1640 medium.
During the first 48 h of lymphocyte cultivation, 1 to 2 pg of lipopolysaccharides, subsequently 70 to 80 units of interleukin 2 were added
per ml of growth medium (53). Upon lymphocyte replication, this
concentration was reduced to 20 to 30 units/ml. The DNA from
cultivated lymphocytes was purified as that from non-stimulated cells.
Computer Program. In some experiments, the results of Southern
blot hybridization experiments were graphically reproduced by using
the TEX program (54).
Reference
Human lymphoma or leu
kemia cell lines
L428
L540
18-23
DEV
L591
L660
Jur
CEM
HL60
U937
24
25
26
27,28
29
RESULTS
Other human cell lines
KB
HeLa
293
Human oral carcinoma line
30
Human cervix carcinoma line
31
Adenovirus type 5-trans32
formed human embryonic
kidney cells
* H, Hodgkin's disease; B, B-cell leukemia; T, T-cell leukemia; M, myeloid
leukemia.
10% dextran sulfate and the 32P-labeled DNA probe. After hybridiza
tion, the membranes were washed twice for 15 min at 68°Cin 2 x SSC,
0.5% SDS; twice for 15 min in 2 x SSC, 0.1% SDS, and once for 15
min in 0.1 x SSC. The Hybond N membranes could be used for
rehybridizations after a 15-min treatment with 0.2 N NaOH, followed
by two washes in 0.1 M sodium phosphate, pH 7.2. The transfer to
nitrocellulose filters was carried out as previously described (34).
Hybridization Probes. The different hybridization probes used in this
series of experiments and their derivations have been listed in Table 2.
These probes were excised from their vectors by appropriate restriction
endonucleases. Subsequently, the fragment probes were partly purified
by electrophoresis on 1.0% agarose gels and by electrocution (Biotrap,
Schleicher & Schuell). These probes still contained traces of the plasmici
vector sequences and thus also hybridized to the pBR322 marker DNA
fragments (cf. Figs. 1-5). The purified DNA fragments were then 32P
oligolabeled(Sl).
Lymphokine Stimulation of Human Lymphocytes. Human lympho
cytes were isolated from Thrombophob (80 ¿¿1/100
ml)-treated blood
by the Ficoll-Paque gradient technique (52). The DNA from these
Table 2 Hybridization probes used in the restriction analyses of human DNA
samples
GeneProtooncogenesc-mycregion5'
fragment2.4
Experimental Design, Selection of Probes. The patterns of
general genetic activities or inactivations, which could include
activities other than transcription, e.g., replication, repair, or
recombination, are reflected in patterns of DNA methylation.
The determination of these methylation patterns in specific
segments of the human genome in normal human lymphocytes
and in cell lines established from different human malignancies
would yield a composite, although complex, representation of
genetic activities in these different cell types. DNA was isolated
from human lymphocytes, or from several established cell lines
(Table 1) of Hodgkin's lymphomas, of a B-cell leukemia, of Tcell leukemias, or of myeloid leukemias. The DNAs were
cleaved with Hpall or Mspl, the fragments were separated by
electrophoresis on agarose gels, and blotted (33). As hybridi
zation probes, a series of different protooncogene segments or
segments from the TNF-a or -ßgene, the insulin receptor, or
the lamin C gene were used (Table 2). We will present the
results of a few typical Southern blot-hybridization experiments
(Figs. 1-5) and summarize the bulk of the data in tables (Tables
3 and 4). It had been ascertained in reconstitution experiments
that sufficient concentrations of restriction endonucleases had
been used. The standard amount (10 ng) of cellular DNA was
treated with 10,20, or 30 units of Hpall/ng of DNA and probed
with one or several DNA segments. Identical Hpall cleavage
patterns were observed.
Methylation Patterns in Segments of Different Human Genes.
In previous work, we have demonstrated that the extent of
DNA methylation in the 5'-CCGG-3' sequences reflected the
overall levels of DNA methylation in all 5'-CG-3' sequences as
determined
by the genomic sequencing method (55, 56) in
parts of the TNF-a and
good correspondence in
a few segments of the mammalian genome and in integrated
foreign DNA did not prove that methylation patterns in test
Pst\2.0
kbp
sequences were always faithful indicators for levels of DNA
£coRIl.OkbpAamHI-fcoRI1.7kbpSau3A3.5
kbp
methylation in all 5'-CG-3' sequences.
c-myc, L-myc. The patterns of DNA methylation were pre
sented at the 5'-CCGG-3' sequences in a 2.4-kbp Hindlll-
integrated adenovirus genomes and in
1Exon
region + exon
Hindlll-EcoRll.SkbpSac!1.8kbpSmaI-£coRI2.3
kbp
(promoter)c-mycL-mycc-merc-mybN-mycc-irc2c-ro/1Ha-rosKi-rasN-rasp53
-ßgenes (6, 7, 57, 58). Of course, the
flankingsequencesExon
2+
3'-flank-ing
2+
sequencesExon
S'-flank-ing
2+
sequencesExons
+3'-flanking
11 + 12
se
quencesExons
11Entire8'and gene + 5
regionscDNA"5'
3'
(tumor sup
gene)NononcogenesTNF-a
pressor
15 region + exon
(promoter)TNF-/3
' region + exon
(promoter)TNF-0Insulin1(untranslated)Exons
3cDNAcDNACloned
2+
receptorLamin
CGene
°cDNA, complementary DNA
EcoRl fragment of the promoter (Fig. la) and in the exon 2
regions including the flanking sequences (1.5-kbp Sad frag
ment) of the human c-myc gene (Fig. 1Z>),as well as in parts of
exon 2 and intron 2 of the L-myc gene (Fig. le). Fragment sizes
£coRI1.8kbp£coRI-Jk7>aI1.8kbp£coRI0.8
kbp
were as expected and were included in Fig. 1 and all the
following figures, but were not listed separately in the text.
Hybridizations against the 1000- and 800-base pair fragments
of the c-myc promoter served as internal controls in all experi
EcoR\1
kbp
ments. The promoter region of the c-myc gene was nonmethylEcoRl-Pstl0.8
.2 kbp
ated in the 5'-CCGG-3' sequences in all cell lines investigated
Pst\3.6
kbp
(Fig. 1). The 5'-CCGG-3' sequences in the c-myc promoter of
£coRI1.9kbp£coRIReference3535363738,3940414243,4444454647,4847,4847,484950
kbp
cell lines L591, L660, CEM, and Jur, and of lymphokinestimulated or of unstimulated lymphocytes from several indi£coRI6.6
kbp
II3.0kbp ««mi
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DNA METHYLATION
PATTERNS IN HUMAN LYMPHOMA
AND LEUKEMIA CELL LINES
Lyml
Lym 2
unstsL591 L6M CEM Jur 'stim'stim
Lym1 Lym2
unst1L591 L660 CEM Jur 'stim'stim
(Sìbp3613M•«—
'H M"H M"HM"H M"H M"H M"H
Fig. l. DNA methylation patterns in parts
of the human c-myc and L-myc genes. DNAs
(10 jig) from human cell lines or primar) hu
man lymphocytes as indicated were cleaved
with //pall (//) or Msp\ (M). and the frag
ments were separated by electrophoresis on
1.0% agarose gels. Details of the DNA transfer
and hybridization experiments were described
under "Materials and Methods." The follow
ing c-myc or L-myc DNA segments were used
as 32P-labeled hybridization probes: (a) c-myc
promoter; (¿)c-myc promoter and exon 2 with
flanking sequences of the c-myc gene; (c) cmyc promoter and the exon 2 and intron 2
segments of the L-myc gene. As size marker.
pBR322 DNA was cut with Psl\ plus EcoRl
(3613- and 750-base pair fragments) or with
Bgll and fcoRl (2319- and 875-base pair frag
ments), and these fragments were coelectrophoresed. Since all hybridization probes used in
Figs. 1. 2. and 4 contained traces of vector
DNA, the pBR322 marker DNA fragments
were also visualized on the autoradiograms.
Fragment lengths were indicated in base pairs
(bp). Lym stim or Lym unsi referred to the
DNA from lymphokine-stimulated or unstimulaled primary human lymphocytes, respec
tively, from two different individuals (6, 57).
The technical details of lymphocyte stimula
tion were described under "Materials and
Methods." The characteristics and derivations
*«...
M.yw~
'H M"H M"HM"H M"H M"H M"H
bp3613
—
2319-•«1000750
2319-875-750^•i
t-- -_.---—
...•bp•«
-««-—*«.«-—
«<®bp
x•S
-«200
HL60 U937 OEV L540 L428 293
HeLa
KB
•£
'H M"H M"HM"HM"H M"HM"H M"H M'z
*
*
-
«Jl
.
-
t
-I
of the human cell lines were outlined in Table
1.
HL60 U937 L540 L428 293 HeLa
KB
Ì
Lym1
•£
L591 L660 CEM Jur stim
•£
'H M"HM"HM"HM"H M"HM"HM' 2
bp
rj-
^
|
I
'H M"HM"HM"HM"H M'
bp
bp
= --
4363
2100-
750»
- 2118
- 1631
N 1S65
-
680
-
510
-«2100
Fig. 2. DNA methylation patterns in parts of
the human c-mel gene. Experimental design and
symbols were explained in the legend to Fig. 1.
As hybridization probe, a cloned, 32P-labeled
Pstl fragment 2.3-kbp long was used. As marker,
a mixture of the EcoRl [4363 base pairs (/>/>)).
the K.\«
I (2118, 1565, 680 base pairs), and the
Hin n (1631, S10 base pairs) fragments of
pBR322 DNA was coelectrophoresed.
-«750
viduals were unmethylated (Fig. 1, a and b). The same finding
held true for the c-myc promoter in cell lines HL60, U937,
DEV, L540, L428, and in cell lines 293, HeLa, and KB (Fig.
le). These results conformed to the expected inverse correlation
between the lack of methylation in the c-myc promoter and the
activity of the c-myc gene in almost all of these cell lines (Ref.
23; our data, not shown). The examined exon 2 and the flanking
sequences of the c-myc gene in cell lines L591, CEM, and in
lymphokine-stimulated or unstimulated lymphocytes were sig
nificantly methylated in the 5'-CCGG-3' sequences and com
onstrated that the DNA preparations investigated were meth
ylated to various extents.
c-myb. The probing of the DNA from several human tumor
cell lines and from human blood cells or from human lympho
cytes of six different individuals with a 2-kbp c-myb DNA
segment from the coding region of the gene revealed methyla
tion of the 5'-CCGG-3' sequences in this genome segment,
LinesP1'MBlood
L660"M
pletely methylated in cell line Jur. The same sequences were
almost unmethylated in cell line L660 (Fig. 1A), as evidenced
by the appearance of fragments 2100, 400, and 200 base pairs
long. The L-myc gene segment investigated (Fig. \c) was meth
ylated to different extents in the 5'-CCGG-3' sequences.
c-met. The methylation patterns at the 5'-CCGG-3' se
quences were also determined with a 2.3-kbp c-met probe (Table
2) in the DNAs from human tumor cell lines and primary
human lymphocytes (Fig. 2). The results of the //pall and Mspl
(fragments of 2100 and 750 base pairs) cleavage patterns dem
P3
Lym
P4 ' ' PS
Cell
P6 ' 'DEV
CEM
LS91
Jur
H'1
HP2 H"M H"MH' 'M H"M H' 'MH"MH"M H"M H"M
••
•§
= a »*
»t
tfP-W
•g
S
•
Fig. 3. DNA methylation patterns in parts of the human c-myb gene. Details
and experimental design were similar to those described in the legend to Fig. 1
or in the text.
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DNA METHYLATION
PATTERNS IN HUMAN LYMPHOMA AND LEUKEMIA CELL LINES
MVktr HL60
U937
OEV
L540
U28
293
HcU
KB
M»»herL591
L660
CEM
Lym Him Lym unit
bp
4363 —
2118 —
2118 —
1565 —
1565 —
bp
4363 —
3613 —
Fig. 4. (a) DNA methylation patterns in
exon 2 and flanking sequences of the human Nmyc gene. (A) DNA methylation patterns in exons 11. 12. and the flanking sequences in the
human c-srr2 gene, (c) DNA methylation pat
terns in the human Ha-rai gene, (d) DNA meth
ylation patterns in the promoter region (exon 1
and 5'-flanking sequences) of the human TNFfi gene. Details were described in the legends to
previous figures. The data adduced from South
ern transfer experiments were schematically pre
sented. The TEX computer program (54) was
used to produce the schematic representations
of this figure. As size markers. pBR322 DNA
was cut with EcoR\ [4361 base pairs (bp)\, EcoR\
and Pstl (3613 and 750 base pairs). £VoRIand
Bgn (2319 and 875 base pairs). //in/I (1631 and
510 base pairs), or Rsa\ (2118. 1565. and 680
base pairs), and the fragments were coelectrophoresed. Smaller marker DNA fragments were
not shown.
L540
Lym Him Lym up«
23H —
2118 —
680 —
510 —
M»'k*f L591
L660
CEM
Jur
Uymttim Lym unit
Mjrk,r
HL60
U937
DEV
L54G
L428
»3
H«L«
KB
bp
3613-
except in cell line DEV in which some of these sequences were
unmethylated (Fig. 3).
In Fig. 4 the results of Hpall/Mspl restriction analyses of
parts of the N-myc, c-src2, Ha-ras, and the TNF-/3 genes,
respectively, were schematically presented for the DNAs of a
number of cell lines, as well as from unstimulated or from
lymphokine-stimulated human lymphocytes. Computer reconstructions were prepared from the original autoradiograms.
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DNA METHYLATION
CE M DEV
PATTERNS [N HUMAN LYMPHOMA
AND LEUKEMIA CELL LINES
N-myc. The investigated segment of the human N-myc gene
was methylated to varying extents in the DNA of cell lines
U937, L540, L428, KB, and L660, but unmethylated or hardly
methylated in the DNA from all the other cell lines and in the
DNA from human lymphocytes (Fig. 4a).
c-src2. The 5'-CCGG-3' sequences in exons 11 and 12 and
the 3'-flanking sequences (Table 2) in the c-src2 gene were
Jur L591HL60 L660
'H M"HM"HM"HM"HM"HM'
methylated to varying extents in the DNA of the cell lines
investigated, as well as in stimulated or unstimulated lympho
cytes (Fig. 4Ä).In HeLa cells this genome segment was not
strongly methylated.
Ha-ras. A 6.6-kbp fragment comprising the entire Ha-ras
gene and its 5'- and 3'-flanking regions was used as hybridiza
tion probe for the analysis of 5'-CCGG-3' methylation pat
LS91 Jur L660 HL60DEV CEM
terns. Both in the DNA of normal human lymphocytes and in
the DNA from the tumor cell lines investigated, the 5'-CCGG3' sequences exhibited complex patterns of DNA methylation
'M H"MH"MH"MH"MH"MH'
Fig. S. (a) DNA methylation pattern in a segment of the human insulin
receptor gene, (h) DNA methylation pattern in a segment of the human lamÃ-nC
gene. As hybridi/ation probes complementary DNA clones were used.
patterns"GeneProtooncogcnesc-myc
(Fig. 4c). The methylation patterns in the DNA from unstim
ulated and in lymphokine-stimulated lymphocytes were identi
cal in the Ha-ras segment (Fig. 4c).
In the DNAs from most cell lines investigated and in the
DNA from human lymphocytes, Mspl cleavage yielded frag
ments of about 1000, 580, 350, and 250 base pairs in the Ha
ras segment studied. In the DNAs from some cell lines (Jur,
L540, L428, HL60. HeLa, and KB) and from human lympho
cytes higher molecular mass Mspl fragments were also gener
ated, either in addition to the ~1000-base pair fragment or
instead of it. The Mspl fragments 580, 350, and 250 base pairs
long were uniformly present in all DNA preparations. Differ
ences between individuals in Mspl cleavage patterns in the
human Ha-ras gene were described previously (15, 59) and may
Table 3 Methylation
lines"N;nrin;il"lymphocytes
293—
L428
cell
L540
DEV
L591
L660
Jur
CEM
____________++
promoterc-myc
2)L-mycc-metc-mybN-mycc-src2c-rafHa-ra/v*Ki-rasN-rosp53NononcogenesTNF-ii-promoterTNF-ii-promoterTNF-0
(Exon
++±
+
±
++
++
±
+++
++
±+
++
±
+
±
±
±
ND
+++
+
+
±±±±
+
++
ND±±++
+
++
++
++
++
±±±
+
+
±ND
±ND±ND±+
±±±±
+
+±
+
+
+
+
+
+
+
±±±±±±ND±ND±±±+
++
++ND
+
+
+
+
++
+
+
+
+
+
+
+
+++
+±
+
+
+
+
+
++±
++±
±+
ND+
2/3)Insulin
(Exons
receptorLamin
CTumor
±
+±
NDf
ND
±
ND
ND
++
+
+
+
++
±
±±
±
++
+
±
+
++
++
++
+
+
±
±
±
±
++
±
HL60
U937
KB
HeLa
++
++
+
++
++
+
±
++
++
+
ND
±
+
ND
+
+
+
+
+
+
ND+
+
+
+
+
+
+
+
+
++
+
+
+
++
+
+
+
ND
ND
++
±
±
ND
ND
±
+
ND
ND
ND
" Methylation patterns could vary considerably and the chosen qualifications have to be considered approximations. The following designations were chosen for
Hpa\\ (//) and Msp\ (A/) cleavage patterns.
MH
MH
MH
MH
MH
* All cleavage patterns in the Ha-ras region were different, although they were tabulated ±.
' ND, not determined.
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DNA METHYLATION
PATTERNS IN HUMAN LYMPHOMA AND LEUKEMIA CELL LINES
ment patterns were reprobed with the -"P-labeled c-myc pro
moter fragment in which the 5'-CCGG-3' sequences proved
Table 4 Changes of methylation patterns in tumor cell lines in comparison to
normal human lymphocytes
Genec-myc
promoterc-myc
patterns"All
in patterns of
methylation*No
unmethylated in all DNA preparations analyzed (Table 3). In
this way, it could be proven that complex cleavage patterns
were due to methylated DNA sequences in specific DNA
segments.
The analyzed 5'-CCGG-3' sequences in the c-myc promoter
changeDecrease
notmethylatedComplex
identical:
2)L-mycc-metc-mybN-mycc-src2c-rafH-rasKi-raiN-rasp53TNF-«
(exon
methylationIncrease
in
patternsComplex
inmethylationDecrease
or decrease
patternsComplex
patternsAll
1:methylatedComplex
except
methylationNo
in
changeIncrease
patternsComplex
patternsAll
methylationIncrease
in
inmethylationNo
or decrease
identical:methylatedComplex
changeNo
changeNo
patternsAll
identical:methylatedAll changeNo
identical:methylatedAll changeNo
1:methylatedComplex
except
changeIncrease
promoterTNF-/J
methylationIncrease
in
patternsComplex
promoterTNF-/3
methylationIncrease
in
patternsComplex
2/3)Insulin
(exons
methylationDecrease
in
patternsComplex
methylationDecrease
in
patternsComplex
receptorLamÃ-n
in methylation
patternsChanges
CMethylation
°This summary is based on a comparison of the levels of DNA methylation
between normal human lymphocytes and human and leukemia cell lines as shown
in Table 3.
* These changes can be observed only in some of the leukemia and lymphoma
cell lines.
be due to a true polymorphism or, perhaps less likely, to
methylation of 5'-CC-3' sequences.
TNF-0 Gene. Hybridization of the //pall- or A/spI-cut DNAs
from the different cell lines or from human lymphocytes with
a "P-labeled probe, which contained exon 1 and the 5'-untrans
lated region of the TNF-0 gene, revealed complex patterns and
marked methylation in the 5'-CCGG-3' sequences (Fig. 4d).
Insulin Receptor. The insulin receptor gene was also exam
ined for the level of DNA methylation at the 5'-CCGG-3'
sequences by using a complementary DNA probe. The results
of the //pall or Mspl analyses in the DNAs from several human
tumor cell lines revealed different levels of methylation (Fig.
5a).
l .¡iniinC. The DNAs of most of the human lymphoma and
leukemia cell lines were methylated at the 5'-CCGG-3' se
quences in the lamin C gene, except for the DNAs from cell
lines Jur and CEM which were less methylated (Fig. 5¿>).
The
DNAs from normal human lymphocytes derived from different
individuals was completely methylated in the lamin C gene
segment investigated (data not shown).
Summary of Methylation Patterns in Human Tumor Cell Lines
and in Normal Human Lymphocytes. The levels of DNA meth
ylation were determined at 5'-CCGG-3' sequences in segments
of the human genome as indicated for various human leukemia
or lymphoma cell lines or for unstimulated or lymphokinestimulated human lymphocytes (Table 3). When cleavage pat
terns in a particular gene or gene segment were identical for
//pall and Mspl, the segment was designated nonmethylated
(-). When //pall did not cut DNA in the region of a specific
probe at all, but Mspl cleaved the DNA, the segment was
considered completely methylated (+++). In most instances,
complex //pall cleavage patterns were observed, and the legend
to Table 3 described graphically what types of //pall cleavage
patterns were correlated with the approximations +/-, +, or
++. To ascertain that the amount of //pall restriction endonuclease used in individual experiments was sufficient, all frag
fragment were always unmethylated. The probed regions of the
genes c-raf, K\-ras, N-ras, and p53 were completely methylated
in all of the cell lines tested, except for cell line L428 in which
the p53 segment was partly methylated (Table 3). For many of
the gene segments probed, the status of methylation in the
DNA from normal human lymphocytes was similar to those in
the various established human leukemia or lymphoma cell lines.
In other genome segments, a tendency toward a decrease in
DNA methylation was observed in some of the tumor cell lines,
in yet others an increase in DNA methylation was apparent
(Tables 3 and 4). Differences in the methylation patterns of
unstimulated and lymphokine-stimulated
lymphocytes could
not be detected. A comparison of DNA methylation patterns
in many segments of the human genome revealed striking
interindividual similarities (6, 7, 60). The compilation of data
in Table 4 lists the differences in states of DNA methylation
for all the probed segments between normal lymphocytes and
the different cell lines investigated.
State of Methylation and Expression of Several Oncogenes in
Human Cell Lines. In a previous communication, the transcrip
tion of a number of oncogenes in the cell lines used for meth
ylation analyses were determined by RNA transfer experiments
(Northern blotting) (23). The present study was not intended
to correlate DNA methylation patterns with the extent of gene
expression. Such a comparison would have been feasible only
if methylation patterns had been elucidated mainly in the pro
moter and upstream regions of the investigated genes. Instead
we wanted to study patterns of methylation in human DNA.
However, for a few of the probes used the expression of cytoplasmic RNA was also scanned by RNA transfer experiments.
These data will not be shown, but briefly summarized. As
evidenced by the appearance of//pall fragments of 1066 and
820 base pairs in Fig. 1, the c-myc promoter was not methylated
at 5'-CCGG-3' sequences in the cell lines tested. In cell lines
KB, L591, Jur, HL60, L660, DEV, CEM, and in human
lymphocytes, the c-myc gene was expressed, giving rise to a
strong 2.2-kilobase RNA band in RNA transfer experiments
(data not shown). These findings corroborated the expected
inverse relationship between promoter methylation and gene
expression (1-4, 10). In cell line 293, however, the c-myc gene
was not found to be expressed, and its promoter was not
methylated. The p53 gene was expressed as a 2.7-kilobase RNA
band in human lymphocytes and in cell lines CEM, L660,
L591, Jur, KB, and 293, but was not expressed in cell lines
DEV and HL60. The gene body was found to be extensively
methylated (Table 3); the promoter region was not investigated
for 5'-CCGG-3' methylation. Similarly, the TNF-a gene was
expressed as a 1.8-kilobase RNA in cell lines CEM, L660,
L591, and in human lymphocytes, and was not expressed in
cell lines DEV, Jur, KB, and 293. Lastly, the lamin C gene in
cell lines HeLa and DEV was transcribed into a 2.1-kilobase
RNA, but was not detectably transcribed in cell line CEM.
DISCUSSION
The human genome is characterized by complex patterns of
DNA methylation in specific segments. Some segments are
3707
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DNA METHYLATION PATTERNS IN HUMAN LYMPHOMA AND LEUKEMIA CELL LINES
completely methylated, others are hypomethylated, perhaps
nonmethylated, and many areas of the genome seem to exhibit
complex arrays in the distribution of 5-mC residues. The data
presented here, which are based on Hpall/Mspl restriction
analyses, have inherent limitations, since only a subset of all
5'-CG-3' sequences can be screened by this method. On the
other hand, an extensive investigation into major parts of the
human genome with the genomic sequencing technique (55, 56)
would be unrealistic at present. Moreover, there is evidence
that Hpall (5'-CCGG-3') methylation patterns provide a useful
guidance to the actual status of methylation, as demonstrated
for parts of integrated adenovirus genomes (57, 58) and for
parts of the human genes for TNF-« and -fi (6, 7). In these
latter experiments, the exact distributions of 5-mC residues
have been determined by the genomic sequencing method. It is
conceivable that patterns of DNA methylation in the human
genome would reflect the levels of different genetic activities,
such as transcription, replication, repair, recombination, trans
position, mutability, and others.
Patterns of DNA methylation, i.e., the distribution of 5-mC
as the fifth nucleotide in DNA, are cell type specific, highly
conserved, inherited, and show a surprising concordance, e.g.,
among different individuals in the human population. These
patterns are probably different in every gene and DNA segment.
Thus, significant information is imprinted upon DNA se
quences by the methylation of specific 5'-CG-3' sequences.
Long-term promoter silencing (1-4) is probably only one of
several functional implications of specific patterns of DNA
methylation. As the methyl group on the deoxycytidines ex
tends into the major groove of DNA and can modulate the
interactions of many different proteins with specific DNA seg
ments, distinct patterns of DNA methylation must have farreaching structural and functional implications.
With this background information, it has become mandatory
to investigate the possibility that specific changes in patterns of
methylation exist in specific parts of the human genome be
tween normal and tumor cells. We have chosen normal human
lymphocytes and a series of human cell lines derived from
lymphomas and leukemias. Alterations in patterns of DNA
methylation are postulated to have significant functional con
sequences. Before these implications can be elucidated, it will
be necessary to document these alterations between normal and
tumor cells. The present report contributes to the notion that,
depending on the human DNA segment investigated and on
the particular tumor cell line chosen for the study, specific
alterations in patterns of DNA methylation can be observed in
some instances but not in others (Tables 3 and 4). Our findings
suggest that these changes are highly specific. A comparison of
the data on DNA methylation in various, rather randomly
selected coding regions of the human genome between the
DNAs of normal human lymphocytes and several human lymphoma or leukemia cell lines (Tables 3 and 4), reveals the
complexity of these patterns. In some instances, there is no
striking difference in the extent of 5'-CCGG-3' methylation
but have been also observed in tumor cells that have been
directly isolated from patients.
The present study can be viewed as a beginning with several
experimental limitations, (a) The cell lines included in this
study have been of quite different malignant origins (Table 1).
Although it will eventually be interesting to ask questions about
changes in methylation patterns for specific segments of the
human genome and their significance for the malignant status
of a cell (13), many fundamental problems about the nature,
stability, and functional meaning of methylation patterns in the
human genome have still to be solved, (b) Many of the very
small Hpall/Mspl fragments (<250 base pairs) cannot be re
solved by electrophoresis on the agarose gel system. A more
complete analysis could be accomplished by the genomic se
quencing procedure, (c) The selection of hybridization probes
for the present study has been purposefully random. Of course,
we have concentrated on the protooncogenes, for which a large
body of information is available, and on the genes for p53,
TNF-«, TNF-/3, lamin C, and the insulin receptor (Tables 3
and 4) for some of which we have previously studied methyla
tion patterns in normal human cells under different conditions
(6, 7). It will be a demanding task for the future to include an
ever increasing part of the human genome in these analyses.
ACKNOWLEDGMENTS
We are indebted to Hanna Mansi-Wothke for the preparation of
media, and to Petra Böhmfor expert editorial work. We gratefully
acknowledge gifts of the cloned DNA fragments, which have been
summarized in Table 2, from the references cited therein.
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Patterns of DNA Methylation in Selected Human Genes in
Different Hodgkin's Lymphoma and Leukemia Cell Lines and in
Normal Human Lymphocytes
Sabine Achten, Annett Behn-Krappa, Manfred Jücker, et al.
Cancer Res 1991;51:3702-3709.
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