[CANCER RESEARCH 50, 4984-4990. August 15. 1990)
Presence of Cell Lineage-specific Hypomethylated Sites in the Major Breakpoint
Cluster Region
Craig E. Litz,1 Adam N. Goldfarb, John G. Strickler, and Richard D. Brunning
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
ABSTRACT
To examine the role of DNA methylation in breakpoint location of
chromosomal translocation, Hpall sites in and flanking the M-/KT on
chromosome 22 were mapped in DNA from blood granulocytes and
lymphocytes, bone marrow cells, thymic tissue, and spermatozoa from
normal individuals. Allelic 11pulìsites were identified clustered in a 600base pair genomic area of the M-bcr. Bone marrow cells and blood
granulocyte DNA showed identical allelic patterns. Thymic tissue and
blood lymphocytes showed identical allelic patterns distinct from bone
marrow cells and blood granulocytes. Spermatozoa showed a third meth
ylation pattern. In all individuals, the Hpall sites were present within
the BamHl/Bglll fragment of the M-/>o. the same area associated with
high breakpoint frequency in chronic myelogenous leukemia «All.).
Three of IS patients with chronic phase (Ml, showed fully methylated
rearranged Bglll/Bglll M-bcr restriction fragments not seen in normal
bone marrow cells. These methylation patterns of the M-/KT may be
important in (All breakpoint location and may be a marker for tissue
differentiation.
INTRODUCTION
DNA methylation is a postreplicative phenomenon whereby
methyl groups are added to nucleic acid residues by complex
nucleic acid methylase enzyme systems. It is utilized by all
living organisms providing them with several different growth
advantages. In bacteria, methylated cytosine and adenine resi
dues provide a means of recognizing and protecting bacteria
DNA from the bacteria restriction enzyme system whose pur
pose is to digest invading viral and plasmid DNA. In the
eukaryotic cell, methylated cytosine/guanine residues appear to
play an integral part in the regulation of gene expression (1-3).
Because of the important association of methylation with
gene expression, several investigators have postulated that aber
rant methylation status of potential oncogenic loci may lead to
inappropriate oncogene expression and neoplastic transforma
tion (3, 4). Specific methylation differences at specific loci
between normal and neoplastic tissues have been reported.
However, the relationship of the loci studied to the neoplasias
is not fully understood (5, 6). In addition, the neoplasias have
been shown to be genetically heterogeneous with several differ
ent mechanisms of initiation (7). As a result, the relevance of
different methylation patterns to the development of neoplasia
is uncertain.
CML2 is a hematopoietic neoplasm associated in virtually all
cases with a reciprocal rearrangement in which the c-abl onco
gene on the long arm of chromosome 9 translocates to the
major breakpoint cluster region (M-bcr), a 5.8-kilobase region
on the long arm of chromosome 22 (8-10). The resulting hybrid
BCR/c-abl gene produces a BCR/c-abl hybrid protein believed
Received 2/20/90; revised 5/2/90.
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.
1To whom requests for reprints should be addressed, at Department of
Laboratory Medicine and Pathology, Mayo Building, Box 198, University of
Minnesota Hospitals, 420 Delaware St. S.E., Minneapolis, MN 55455.
1The abbreviations used are: CML, chronic myelogenous leukemia; TNE,
0.075 M NaCI-0.01 M Tris-HCl-0.025 M disodium EDTA, pH 8.0; SDS, sodium
dodecyl sulfate; PBC, peripheral blood cell.
to play a major role in the pathogenesis of CML (11). The
breakpoint site clustering on chromosome 22 in CML is con
sidered largely due to a random event under exonal constraints
of the resulting oncogenic hybrid gene. An alternative hypoth
esis, based on studies of folate-sensitive chromosomal fragile
sites, is that the methylation status of genomic areas may be
important in determining the position of fragile sites and chro
mosomal rearrangements ( 12). To test this hypothesis the meth
ylation status of the M-bcr in various tissues from 24 normal
individuals and 15 patients with CML in chronic phase was
studied by restriction mapping with the methylation-sensitive
enzyme, //pall.
MATERIALS
AND METHODS
Separation of Peripheral Blood WBC. Approximately 50 ml of pe
ripheral blood from healthy volunteers was drawn into disodium EDTA.
The blood was separated on a Ficoll-Hypaque gradient (Organen Teknika. Inc.) according to the manufacturer's recommendations. The
separated mononuclear fraction was washed twice with RPMI 1640
and the monocyte fraction was adhered out in plastic Petri dishes. The
resulting lymphocytes were washed twice with TNE. Smears confirmed
that the lymphocytes were approximately 90% pure by myeloperoxidase
and nonspecific esterase stains (13, 14). The separated granulocytes
were washed twice in TNE. Smears were made and the purity of the
granulocytes was confirmed to approximately 95% with a myeloper
oxidase stain.
DNA Extraction, Restriction Enzyme Digestion, Southern Transfers,
and Hybridization. DNA was extracted from total peripheral blood,
peripheral blood lymphocytes, peripheral blood granulocytes, bone
marrow cells from volunteers without hematological malignancy, and
blood or bone marrow cells from patients with CML in chronic phase.
The lymphocyte and granulocyte DNA was obtained from the same
individuals to rule out the possibility that polymorphisms of potential
Hpa\\ sites (Mspl sites) were responsible for the allelic patterns found.
Thymic tissue was obtained from autopsies in which the patient died
due to non-hematological causes. Spermatozoa were obtained from
both living volunteers and autopsies in which the patient died due to
non-hematological causes. Nucleated cells were recovered from total
peripheral blood or separated peripheral blood granulocytes after pref
erential lysis of the RBC in 3 volumes of a cold buffer containing 0.155
M ammonium chloride, 0.005 M EDTA (free acid), and 0.01 M potas
sium bicarbonate adjusted to pH 7.4 with 1 M KOH. WBC were rinsed
in TNE buffer and crude nucleic acid solutions were obtained by direct
lysis of WBC membranes as described previously (15). Spermatozoa
and bone marrow cells were lysed in a similar fashion in TNE buffer in
the presence of 1% SDS. After histopathological confirmation of the
tissue components present, the thymus was finely minced and also
directly lysed with 1% SDS in TNE buffer. High molecular weight
DNA from the various samples was further purified by standard proteinase K treatment (Boehringer-Mannheim Biochemicals) at a final
concentration of 0.1 mg/ml. RNase treatment was followed by several
phenol-chloroform extractions and ethanol precipitations. No further
purification was required.
DNA (5-10 Mg)from each sample was digested with 8-10 units/^g
Bgfll, £coRI,and Hindlll restriction endonucleases (Bethesda Research
Laboratories, Inc.) according to the manufacturer's recommendations.
The samples were then ethanol precipitated and the restriction frag
ments redigested with 5-10 units/^g of Mspl or Hpall according to the
manufacturer's recommendations (Bethesda Research Laboratories,
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HYPOMETHYLATION OF THE MAJOR BREAKPOINT CLUSTER REGION
Methylation Status of BCR
Inc.). Conventional electrophoresis was carried out in horizontal 0.7%
agarose gels. Standard methods were used for transferring electrophoretically separated restriction fragments to Zetabind nylon membranes
(Cuno, Inc.) (16).
The following probes were used in this study: Probe 1 is the 5'-most
430-base pair Bglll/Mspl fragment of the M-bcr. Probe 1 was isolated
from the ber Pr-1 probe which is the 5'-most Bglll/Hindlll 2.0-kilobase
fragment of the M-bcr inserted into the Hindlll site of PUC 12 and
was obtained from the American Type Culture Collection (17). Once it
was established that no Hpall sites were present in the genomic 5'-Mbcr Bgl\l/Hindlll fragment of peripheral blood, the 5'-Bgl\\/Hindlll
fragment of the M-bcr was used for restriction mapping (see "Results").
Probe 2 is the 3'-Hindlll/Bgm
1.0-kilobase fragment of the M-bcr.
Probe 2 was isolated from the p22H probe which is the 1.2-kilobase
Hindlll/EcoRl fragment inserted into PSP 64 and was kindly provided
by Dr. David Leibowitz (18). Probe pSPT/pGK is the 800-base pair
BamHl/EcoRl fragment inserted into the BamHl/EcoRl sites of PSP
64-identifying alÃ-elesat the PGK locus on chromosome X and was
kindly provided by Dr. Bert Vogelstein (19).
All probes were 32P-radiolabeled using the random primer reaction
following the manufacturer's recommendations (Pharmacia, Inc.). Ze
tabind filters were prehybridized, hybridized, and rehybridized, if nec
essary, under standard conditions. The hybridization buffer contains
Ix Denhardt's solution, 5x standard saline citrate (0.75 M NaCl,
0.0075 M sodium citrate), 0.05 M Tris-HCl, pH 7.5, 0.1% sodium
pyrophosphate, 10% dextran sulfate, 50% formamide, 0.1% SDS, and
100 iig/ml sonicated salmon sperm DNA. All hybridizations were
carried out at 38-42°C for approximately 16-20 h. The length, the
temperature, and the stringency of all washes were adjusted to the
background radioactivity with a final wash usually in 0.1 x standard
saline citrate (0.015 M NaCl, 0.0075 M sodium citrate) and 0.1% SDS
at 60-65°C.The filters were exposed to Kodak XAR-5 film (Eastman
Kodak Co.) at -85°Cfor 24 h to several days.
Completeness of Hpall digestion was confirmed by three procedures:
(a) concurrent monitoring of the digestion using bacteriophage X as a
DNA marker as published previously (20), (b) repeating the initial
digests with twice the amount of enzyme and observing no change in
restriction fragment lengths, and (c) reprobing the initial Bglll/Hpall
blots with a X-linked pGK probe and observing loss of all germline
alÃ-elesin all DNA samples from males.
In order for Hpall to cut DNA, two conditions must be satisfied: the
appropriate DNA sequence (CCGG) must be present and the internal
cytosine residue of this sequence must be unmethylated. Mspl, the
methylation-insensitive isoschizomer of Hpall, was used to ascertain
the presence of potential Hpall sites in the M-bcr. Restriction mapping
of the Bglll/Hindlll fragment of the 5' portion of the M-bcr revealed
BgE Bg
—¿H \-
it
10kb
Hpall
Hpall
i tb
Probe 1
Probe 2
Bg1
Ba
B<Hpall
H
BgE
G, B, T. L
3.1)Hpall
(3.0.
Tkb
'H
G B(2.5)
and
# of Cases
Area 1
Area 2
283
16%
80%
Area 3
Fig. 1. Methylation status of major breakpoint cluster region (M-bcr). A, a
partial restriction map of the M-bcr and flanking regions. EcoRl, Hindlll. Hpall.
and select Bglll sites of the M-bcr are shown. B. a partial restriction map of the
M-bcr. Hatched boxes, probes as referenced in text. ///«Il sites are noted under
neath the map along with cellular subset in which they are found (G. granulocytes:
L. lymphocytes: B. bone marrow cells: T. thymus). Germ cells (spermatozoa)
have no Hpall sites present. The distance (in kilobase) of the Hpa\\ sile from the
5'-Bglll site of the M-bcr is given in parentheses. Below the map is a compilation
of published breakpoint locations in CML patients (10, 18, 22-25); areas I, 2,
and 3 are the Bgl\\/BamH\, BamH\/Bgl\\. and Bgl\\/Bgl\\ fragments of the Mbcr, respectively. The number of cases studied and the percentage of patients with
M-bcr breakpoints in respective area are presented. Bg. /¡i;/ll:E. EcoRl; H,
Hindlil; Ba, AamHI sites; kb, kilobases.
at least 6 Mspl sites. Restriction mapping of the Hind\\\/BgI\\ fragment
of the 3' portion of the M-bcr revealed at least 4 Mspl sites. Mspl was
of cells indicates that the cells are allelically heterogeneous.
This alleile heterogeneity was further studied by examining
DNA from separated cellular subsets of normal peripheral
blood (see below).
3' Portion of the M-bcr. Bgl\\/Hpa\l digests of PBC DNA
from seven individuals were probed with the 3'-Hindlll/BgHl
fragment of the M-bcr (probe 2; Fig. IB). In all specimens this
revealed a 1.7- and 4.8-kilobase restriction fragment length.
Bglll/Mspl digests similarly probed revealed restriction frag
ment lengths of 1.0 kilobase or smaller in PBC DNA.
used with other restriction endonucleases as described in the text to
ascertain the presence of CCGG sequences.
Southern blot techniques in this study do not allow reliable resolution
of Hpall sites clustered within a 100- to 200-base pair genomic area.
M-bcr Methylation Patterns of Peripheral Blood Granulocyte
and Lymphocyte DNA
5' Portion of the M-bcr. Bglll////wll-digested
lymphocyte
RESULTS
M-bcr Methylation Patterns of PBC DNA
5' Portion of the M-bcr. The Ag/II///paII-digested PBC DNA
was probed with the 5'-most 430-base pair Bgl\\/Msp\ fragment
of the M-bcr (probe 1; Fig. Iß).Since there are no potential
internal Hpall sites in this probe, all Bglll/Hpall genomic
fragments less than 4.8 kilobases in length recognized by this
probe are defined by the 5'-Bgfll site in the M-bcr and a 3'Hpall site in the M-bcr. Probing of PBC DNA with probe 1
revealed four major restriction fragment lengths of 4.8, 3.1, 3.0,
and 2.5 kilobases (Fig. 2; lane /). Each of these restriction
fragment lengths is defined by a different 3'-Hpall site and
each fragment is, thus, allelic to the other. The identification
of at least four M-bcr Bglll/Hpall alÃ-elesin diploid populations
DNA from four individuals studied with probe 1 demonstrated
three major restriction fragment lengths at 4.8, 3.1, and 3.0
kilobases (Fig. IB and Fig. 2, lane 3). Similarly treated granulocyte DNA from the same four individuals showed three major
restriction fragment lengths at 3.1, 3.0, and 2.5 kilobases (Fig.
IB and Fig. 2, lane 2) and a minor population of 4.8-kilobase
fragments; whether these 4.8-kilobase fragments were due to
contaminant lymphocytes or heterogeneity of the granulocyte
population was not ascertained.
3' Portion of the M-bcr. Bglll/Hpall digests of lymphocyte
and granulocyte DNA from four individuals were probed with
the 3'-Hind\l\/BgHl fragment of the M-bcr (probe 2; Fig. IB).
In all DNA digests the 4.8-kilobase restriction fragment length
segregated to the lymphocyte DNA and was minimally present
or absent in the granulocyte DNA (Fig. 2, lanes 4 and 5). The
1.7-kilobase restriction fragment length was present in lympho
cyte DNA and was the only major fragment seen in granulocyte
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HYPOMETHYLATION OF THE MAJOR BREAKPOINT CLUSTER REGION
Probe 1
WB
Probe 1
Probe 2
G
G
L
L
4.8kb
4.8
3.0,3.1
3.1kb
3.0kb
—¿
2.5
2.5kb
1.7kb
Fig. 2. Southern blots of cellular subset DNA. Lanes 1, 2, and 3, whole blood (WB), granulocyte (G), and lymphocyte (i) DNA samples digested with BgH\/Hpa\\
and probed with probe 1, respectively. Lanes 4 and 5, the same granulocyte and lymphocyte digests reprobed with probe 2. kb, kilobases.
DNA. The 4.8-kilobase fragment represents a fully methylated
Bglll/Bglll fragment of the M-bcr and the 1.7-kilobase frag
ment represents the 3'-Hpall/Bglll
M-bcr fragment comple
mentary to the 2.5-, 3.0-, and 3.1-kilobase 5'-BgHl/Hpall frag
ments seen in the granulocytes and lymphocytes.
Methylation Status of the Regions Flanking the M-bcr in PBC
DNA
5'-Flanking region of the M-bcr. To determine the position
of the flanking Hpall sites 5' of the M-bcr, Hpall/EcoRl digests
of PBC DNA were hybridized with the 5'-BgH\/Hindlll frag
ment of the M-bcr. In 10 of 10 individuals studied, at least
three restriction fragments were identified. The longest was 10
kilobases in length. The other fragments varied from approxi
mately 8-9 kilobases (Fig. 3). Since the EcoRl/EcoRl fragment
recognized by this probe is approximately 20 kilobases, the
fragments seen in the Hpall/EcoRl blot represent Hpall/EcoRl
orHpall/Hpall fragments (Fig. \A). ///»all-only digests of PBC
DNA similarly probed produced restriction fragments between
8 and 9 kilobases in length. The 10-kilobase fragment was no
longer present. This indicates that the 8- to 9-kilobase frag
ments are Hpall/Hpall restriction fragments and the 10-kilo
base fragment is a Hpall/EcoRl fragment. Since the probed
sequence is located more than 10 kilobases downstream from
the 5'-flanking EcoRl site (Fig. 1/1), the EcoRl site bounding
this 10-kilobase Hpall/EcoRl restriction fragment is the 3'EcoRl site flanking the probed sequence. The 5'-Hpall site of
this fragment is located 10 kilobases upstream from this 3'-
EcoRl site and this fragment includes the 4.8-kilobase BgHl/
Bglll fragment of the M-bcr (Fig. IA). This indicates that the
10-kilobase Hpall/EcoRl fragment includes the 4.8-kilobase
hypermethylated fragment seen on the Bgfll/Hpall digests of
lymphocyte DNA probed with the same sequence. This was
confirmed on further analysis; the 10-kilobase Hpall/EcoRl
fragment segregated to the lymphocyte DNA and was mini
mally present in the granulocyte DNA similar to the 4.8kilobase hypermethylated fragment from the Bgfll/Hpall di
gests (Fig. 3; top).
The 8- to 9-kilobase Hpall/Hpall fragments seen in both the
granulocyte and lymphocyte DNA include the Bgill/Hpall
fragments with the 3'-Hpall sites being located between 2.5
and 3.1 kilobases downstream from the 5'-M-bcr Bglll site as
described previously. This indicates that the 5'-flanking Hpall
sites are located at least 7 kilobases upstream of the M-bcr
Hpall sites in PBC DNA (Fig. IA).
3'-Flanking region of the M-bcr. To determine the position
of the 3'-Hpall sites flanking the M-bcr, Hindlll/Hpall digests
of granulocyte and lymphocyte DNA were probed with the 3'Hindlll/Bgni fragment of the M-bcr (probe 2; Fig. 1). In two
of two cases both granulocyte and lymphocyte DNA revealed
only a 4.6-kilobase fragment (Fig. 4; bottom). This size corre
sponds to an intact Hindlll/Hindlll
fragment of the 3' portion
of the M-bcr. This indicates the absence of Hpall sites in
granulocyte and lymphocyte DNA at least 5 kilobases 3' of the
M-bcr Hpall sites (Fig. IA).
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HYPOMETHYLATION
OF THE MAJOR BREAKPOINT CLUSTER REGION
M-bcr Methylation Pattern of Spermatozoa, Thymus, and Bone
Marrow Cells DNA
Probe 1
Bglll/Hpall digests of spermatozoa (germ cell) DNA, thymus
DNA, and bone marrow cell DNA were probed with probe 1
(Fig. 4). Two of two DNA samples of spermatozoa revealed a
single major alÃ-eleat 4.8 kilobases indicating no Hpall sites
Thymus
Bone
Marrow
—¿
4.8kb
Probe Bglll/Hindlll
E/Hp
—¿
3.0,3.1kb
E/Hp
8-9kb
—¿
2.5kb
10kb
9kb
Lane 1
Lane 2
Lane 3
Fig. 4. Southern blots of tissue DNA. Lanes I, 2, and 3, spermatozoa (S),
thymus, and bone marrow DNA digested with Bgl\\/Hpa\\ and probed with probe
1, respectively, kb, kilobases.
gran
present in the M-bcr, a relatively minor population of 3.0- and
3.1-kilobase alÃ-eleswas present presumably due to lymphocytes
(21). Thyrnic DNA showed 3.0-, 3.1-, and 4.8-kiIobase alÃ-eles
similar to peripheral blood lymphocytes. Bglll/Hpall digests
of bone marrow cell DNA showed 2.5-, 3.0-, and 3.1-kilobase
alÃ-elessimilar to the peripheral blood granulocytes.
The composite restriction map (Fig. Iß)shows a bone mar
row and granulocyte Hpall site 2.5 kilobases downstream of
the 5'-M-bcr Bgl\\ site. The 3.0- and 3.1-kilobase Hpall sites
lymph
are common to the lymphocytes, thymus, bone marrow cells,
and granulocytes. Only lymphocytes, spermatozoa, and thymus
contain alÃ-eleswith none of the above M-bcr Hpall sites.
Probe 2
H/Hp
Minor AlÃ-elesof the 5' M-bcr
H/Hp
Three minor populations of restriction fragments were noted
on the Bglll/Hpall digests probed with probe 1. The 1.0-, 2.7-,
and 2.8-kilobase restriction fragments were noted in the bone
marrow, granulocyte, and PBC specimens only on long autoradiogram exposure; their significance was undetermined in
this study.
4.6kb
gran
^ 4.6kb
M-bcr Methylation Pattern in Chronic Phase CML
To examine the M-bcr methylation pattern in CML, BgHl/
Hpall digests of blood or bone marrow DNA from 15 patients
with chronic phase CML were probed with the S'-Bgfll/Hindlll
fragment of the M-bcr. There were two distinct M-bcr methyl
ation patterns. The first pattern, found in 12 patients, demon
strated 3.0-, 3.1-, and 2.5-kilobase alÃ-elessimilar to the normal
bone marrow pattern (Fig. 5; lane 1). The second pattern, found
in 3 patients, demonstrated a fully methylated rearranged Bgfll/
Bgl\\ fragment in addition to the 3.0-, 3.1-, and 2.5-kilobase
alÃ-eles(Fig. 5; lane 2).
lymph
Fig. 3. Methylation status of flanking regions of the M-bcr. Top, autoradiograms of granulocyte and lymphocyte DNA digested with EcoRl/HpaU (E/Hp)
and probed with the 5'-Bgl\l/Hindlll fragment of the M-bcr. Bottom, autoradiograms of granulocyte and lymphocyte DNA digested with Hind\l\/Hpa\\ and
probed with probe 2. Gran, peripheral blood granulocytes; lymph, lymphocytes;
E, EcoRl; H, Hinälll; Hp, Hpali; kb, kilobases. Probes are as referenced in the
text.
DISCUSSION
The biology of the t(9:22) chromosome translocation in CML
is not well understood. The reported data of M-bcr breakpoints
in CML indicate that there is a clustering of breakpoints in the
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HYPOMETHYLATION
B/H
B
B/H
OF THE MAJOR BREAKPOINT CLUSTER REGION
important in the determination of the site to which the c-abl
oncogene translocates in CML. This hypothesis is alluded to
by earlier investigators working on chromosomal fragile sites.
Increased expression of certain fragile sites has been observed
in cell culture conditions that do not allow normal cellular
methylation processes to occur, suggesting that such processes
are important to chromosomal integrity (12). Furthermore,
chromosomal fragile sites have been found to occur in areas
frequently rearranged in malignancy, including CML, suggest
ing that the etiology of fragile sites may be related to carcinogenesis (28).
The results of the present study suggest that an isolated
cluster of Hpall sites is present 2.5-3.1 kilobases downstream
of the 5'-M-bcr Bglll site in nucleated peripheral blood cells.
This 600-base pair region is flanked by genomic regions devoid
of HpalI sites 5-7 kilobases in length. Separated populations
of peripheral blood cells manifest both cell-specific and com
mon methylation alÃ-eles.Lymphocytes have three M-bcr meth
ylation alÃ-eles.The first is a fully methylated M-bcr Bgl\\/Bgl\\
fragment, 4.8 kilobases long, the second is a hypomethylated
alÃ-elewith one or more closely clustered //pall sites between
3.0 and 3.1 kilobases from the 5'-most Bglll site of the M-bcr,
B
- 4.8kb
M
- 3.1/3.0kb
and the third alÃ-eleis a hypomethylated alÃ-elewith one or more
closely clustered Hpall sites beginning and ending at 3.1 kilobases from the 5'-most Bglll site of the M-bcr. Granulocytes
also show three M-bcr methylation alÃ-eles.The first alÃ-eleshows
the largest cluster of Hpall sites between 2.5 and 3.1 kilobases
from the 5'-most Bglll site of the M-bcr. The granulocytes and
- 2.5kb
lymphocytes share two alÃ-eleswith Hpall sites beginning at 3.0
and 3.1 kilobases from the 5'-most Bglll site of the M-bcr. This
Fig. 5. Southern blots of DNA from patients with CML. Panels I and 2, BgH\
(B) and Bgl\\/Hpa\\ (B/H) digested bone marrow DNA from two patients with
chronic phase CML probed with the 5'-Bgl\\/Hind\\\ fragment of the M-bcr. kb.
kilobases.
BamHl/Bglll fragment of the M-bcr (Fig. \B; 10, 18, 22-25).
This CML breakpoint "clustering" within the major breakpoint
cluster region originally defined by Groffen et al. (10) has not
been fully explained but several hypotheses exist. One currently
being explored is based on the exonal composition of the BCR/
c-abl oncogene hybrid gene. Studies have suggested that the
biological course of CML correlates with the position of the
M-bcr breakpoint; those CML patients with breakpoints 5'- of
the 3'-most M-bcr ///Will site appear to have longer chronic
phases than those with breakpoints 3' of this HindlU site.
Breakpoints located in the 5' M-bcr include different exons in
the final hybrid gene than do those with the 3' breakpoints;
thus, it is postulated that the translated hybrid protein products
are slightly different and presumably account for the different
clinical courses (18, 22-26). Recently, the region of the M-bcr
containing the third exon, located in the M-bcr BamHl/Bglll
fragment, has been implicated as the prognostically crucial area
based on epidemiological restriction-mapping studies in large
numbers of CML patients (26).
A second hypothesis is that Alu sequences found in proximity
to the CML breakpoints may predispose to recombinational
events. It is postulated that Alu-repetitive sequences ubiqui
tously located throughout the genome may in some fashion
serve as a nidus of recombinational activity. This concept has
been supported previously in both CML- and non-CML-related
chromosomal recombinations. An Alu consensus sequence has
been mapped to the BamHl/Bglll fragment of the M-bcr (27).
A third hypothesis which is addressed in this study is that
the methylation status of the M-bcr region may in some way be
latter finding suggests at least two possibilities: these shared
alÃ-elesare established in a pluripotential hematopoietic stem
cell before lymphocyte/myeloid differentiation or the alÃ-elesare
established in a programmed, independent fashion during both
lymphocyte and myeloid maturation. In either instance, the
reproducible difference in methylation patterns of the M-bcr in
different hematopoietic cells indicates that methylation status
may be important in the way that this genomic area evolves
during cellular differentiation, either by means of genomic
"packaging" or accessibility to transcriptional factors. This
finding is similar to the changes in methylation patterns de
scribed in the globin gene family in erythropoiesis and it is
possible that these patterns may be useful markers of differen
tiation in neoplasia (29, 30).
Spermatozoa (germ cells) show a single alÃ-elewith no internal
Hpall sites present. Thymic lymphocytes show the same allelic
pattern as blood lymphocytes with a small cluster of Hpall sites
present in one of the alÃ-eles.Since blood lymphocytes are
predominantly thymic derived (T) lymphocytes, this suggests
that the methylation alÃ-elesfor blood lymphocytes are estab
lished in a pre-thymic or thymic based cell. Bone marrow cells
demonstrate the same allelic pattern as blood granulocytes with
a 600-base pair cluster of Hpall sites present in one alÃ-ele,
suggesting that the methylation alÃ-elesfor the granulocytes are
established in a pre-bone marrow or bone marrow cell. The
acquisition of internal Hpall sites from germ cell to lymphocyte
and/or granulocyte suggests that during cell development the
M-bcr undergoes progressive demethylation.
The 600-base pair area of the M-bcr that undergoes progres
sive hypomethylation with cell development is located in the
BamHl/Bglll fragment of the M-bcr, the region associated with
the majority of chromosomal translocations in CML (Fig. IB).
This small area is flanked by much larger areas devoid of Hpall
sites suggesting a nonrandom association of CML breakpoint
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HYPOMETHYLATION
OF THE MAJOR BREAKPOINT CLUSTER REGION
clustering and Hpall sites. How this association relates to the
t(9:22) chromosomal translocation is undetermined. It is pos
sible that spatially or temporally aberrant methylation patterns
may occur in this region in those patients with CML. This may
in some way predispose to a chromosomal translocation event.
Supporting such a mechanism, experimentally induced DNA
hypomethylation has been shown to lead to aberrant chromatin
decondensation and, in some cell lines, chromosomal translo
cations and tumorigenesis (31, 32).
The Alu sequence and its methylation pattern may be in
volved in the chromosomal translocation associated with CML.
Approximately 3 x IO5 high repeat Alu sequences 300 base
pairs in length are located ubiquitously around the human
genome (33). The function of these sequences has yet to be
elucidated; however, studies have suggested that Alu sequences
may represent DNA replication origins (34, 35). Alu sequences
have also been repeatedly found in proximity to chromosomal
breakpoints in chromosomal rearrangements associated with
malignant and nonmalignant conditions indicating a possible
role in genomic recombination (27, 36-43). It is of interest that
the 600-base pair area of the M-bcr which undergoes cellspecific hypomethylation is located adjacent to, if not super
imposed on, the M-bcr Alu sequence described previously. This
association suggests that the M-bcr methylation status may in
some way be related to the function of the M-bcr Alu sequence.
Supporting this relationship, hypomethylated Hpall tiny frag
ments and Alu sequences have been associated with regions of
expressed DNA sequences and it has been suggested that both
reside in chromosomal metaphase R bands (44, 45). Aberrant
methylation patterns of the Alu sequence established at an
abnormal time of cell development may predispose to the
t(9:22) chromosomal translocation in CML. Comparison of the
M-bcr methylation status in CML patients to the M-bcr meth
ylation status in normal individuals described in this study may
further clarify this hypothesis. Preliminary data from our lab
oratory on 15 blood and bone marrow specimens from patients
with chronic phase CML have shown a major population of
aberrant 5'-M-bcr methylation alÃ-elesin three cases not ob
served in normal bone marrow specimens. In each case this
aberrant alÃ-elewas the fully methylated, rearranged Bgfll/Bglll
M-bcr restriction fragment. This finding may represent a basic
defect in methylation of the rearranged M-bcr alÃ-eleor may be
due to M-bcr breakpoints located 5' of the three Hpall sites.
ACKNOWLEDGMENTS
We are indebted to Dr. D. Leibowitz for generously providing probe
p22H from which probe 2 was derived, Dr. B. Vogelstein for generously
providing probe pGK, and the American Type Culture Collection for
providing pbcr P-l from which probe 1 was derived.
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Presence of Cell Lineage-specific Hypomethylated Sites in the
Major Breakpoint Cluster Region
Craig E. Litz, Adam N. Goldfarb, John G. Strickler, et al.
Cancer Res 1990;50:4984-4990.
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