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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2445-2451
p15INK4B CpG Island Methylation in Primary
Acute Leukemia Is Heterogeneous and Suggests Density as a Critical
Factor for Transcriptional Silencing
By
Elizabeth E. Cameron,
Stephen B. Baylin, and
James G. Herman
From The Oncology Center, Department of Medicine and the Predoctoral
Training Program in Human Genetics, The Johns Hopkins University School
of Medicine, Baltimore, MD.
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ABSTRACT |
The promoter region of the cyclin-dependent kinase inhibitor
p15INK4B contains a CpG island that is
hypermethylated in many hematologic malignancies. To explore the
relationship between patterns of methylation and gene transcription, we
used bisulfite genomic sequencing to obtain a detailed analysis of
methylation in acute leukemia, leukemia cell lines, and normal
lymphocytes. The entire CpG island region of p15 was largely
devoid of methylation in normal lymphocytes, but methylation of varying
density was found in primary acute leukemia. Methylation density was
generally conserved between the alleles from each sample, but marked
heterogeneity for the specific CpG sites methylated was observed.
Patterns of methylation were compared and expression assessed with
reverse-transcriptase polymerase chain reaction (RT-PCR). The density
of methylation within the CpG island, and not any specific location,
correlates best with transcriptional loss. Leukemias with methylation
of approximately 40% of the CpG dinucleotides on each allele had complete gene silencing, with variable, but diminished expression with
less dense CpG island methylation. Our results suggest that the
transcriptional silencing of p15 in conjunction with aberrant hypermethylation is best understood as an evolutionary process that
involves progressively increasing methylation of the entire p15
CpG island.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PROMOTER REGION CpG island
hypermethylation has been reported for tumor-suppressor genes,
including both p15 and its functional homolog p16.
While p16 is inactivated by this mechanism in many
epithelial-derived cancers,1-5 CpG island hypermethylation of p15 occurs almost exclusively in the hematologic
malignancies.6-9 This division is most striking in acute
myelogenous leukemia (AML), where p15 is frequently inactivated
by hypermethylation, but p16 is not inactivated by either
homozygous deletion or methylation.7 Hypermethylation of
p15 is also observed in myelodysplasia, and this process
appears to be involved in disease progression.10,11 The
conservation of p15 hypermethylation in a murine model of T-cell malignancy12 further supports the functional
significance of this neoplastic change.
CpG island methylation associated with transcriptional silencing is an
important alternative mechanism of silencing genes involved in the
pathogenesis of neoplasia.1,2,13-16 However, despite the
importance and prevalence of this process in neoplasia, little detail
of specific patterns of methylation has been reported. Most analyses of
CpG island hypermethylation employ techniques capable of studying only
2 to 4 CpG sites within an island region. CpG island hypermethylation
assayed by Southern blot analysis using methylation-sensitive
restriction enzymes or, more recently, with polymerase chain reaction
(PCR)-based approaches based on restriction enzyme cleavage are
examples of this. These techniques depend on the presence or absence of
methylation at a small number of CpG sites relative to the total number
found across the entire island region. With this approach, CpG islands
are often described as either "unmethylated" or
"hypermethylated." Thus, the importance of hypermethylation at 1 or 2 CpG sites and their location relative to transcription start sites
remain to be determined. The density and placement of methylation
necessary for gene silencing and the issue of allelic heterogeneity of
methylation and its relationship to transcription remain unresolved.
The relatively recent addition of bisulfite genomic
sequencing17 has markedly improved our ability to assess
the methylation status of CpG islands in detail. Such analysis provides
the methylation status for each CpG site within a given region, and
cloning can provide this information for individual alleles of a given
gene. Recent studies have used this technique to demonstrate
methylation patterns for critical genes involved in neoplasia,
including Rb18, p1619,
MGMT,20,21 and BRCA1.22 While
each of these studies contributes to our understanding of the
methylation-associated silencing of tumor-suppressor genes, the
analysis of p16 and MGMT includes only cell lines and
the transcriptional consequences of hypermethylation were not studied
for BRCA1 or Rb.
In this study, we chose p15 as a model for an analysis of
allelic patterns of CpG island methylation for a number of reasons. We
had previously observed7 that not all primary acute
leukemias were as completely methylated at this CpG island as one would have expected based on the percentage of blast cells within the sample.
Because a detailed analysis of the relationship of transcriptional silencing to the methylation pattern of this island has not been reported, such a study might clarify functional questions concerning hypermethylation of p15. Primary leukemia samples provide an
especially pure neoplastic sample, minimizing the influence of normal
cell contamination. This allowed us to address the transcriptional consequences of specific patterns of promoter region hypermethylation in a primary malignancy, rather than in an established cell culture system.
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MATERIALS AND METHODS |
Sodium bisulfite DNA treatment.
DNA was extracted from frozen leukemia cell pellets using standard
phenol chloroform extraction with ethanol precipitation. In the case of
normal control individuals, peripheral blood lymphocytes were extracted
from human blood using gradient centrifugation on Histopaque (Sigma
Chemical, St Louis, MO). One to 2 µg of DNA was then treated with
sodium bisulfite17 according to established methods.23 DNA was resuspended in 15 to 24 µL of
distilled water before PCR.
PCR, cloning and sequencing of individual alleles.
Using 4 µL of resuspended, sodium bisulfite-treated DNA, PCR was
performed in a 50-µL reaction using 400 nmol/L each primer (GIBCO/BRL, Rockville, MD), 1X PCR
Buffer-Mg++ (GIBCO/BRL), 1.5 mmol/L MgCl2
(GIBCO/BRL), and 200 µmol/L dATP, 200 µmol/L dCTP, 200 µmol/L
dGTP, and 200 µmol/L dTTP (Pharmacia, Piscataway,
NJ). Reactions were hot-started at 96°C for 3 minutes and held at 80°C before addition of 1.25 U of Taq
(GIBCO/BRL). Temperature conditions for PCR were as follows: 38 cycles
of 96°C for 20 seconds, 56°C for 20 seconds,
and 72°C for 1 minute followed by 1 cycle of 72°C for 5 minutes. Primers used were 5'-TGAAGGAATAGAAATTTTTTGTTT-3' and 5'-AAGCAAGCTTAAACCCTAAAACCCCAACTACCTAA-3'.
After this PCR, products were diluted 100-fold and a nested PCR was
performed as follows. Either 5 or 10 µL of 100-fold dilution of
initial PCR was used in a PCR reaction of 50 µL total volume. Reactions were performed exactly as above except either 2.0 mmol/L or
2.5 mmol/L MgCl2 (GIBCO/BRL) was used. Temperature
conditions for PCR were as follows: 30 cycles of 96°C for 20 seconds, 56°C for 20 seconds, and 72°C for 1 minute followed by
1 cycle of 72°C for 5 minutes. Primers used were
5'-GGGGATTAGGAGTTGAG-3' and
5'-ACCCTAAAACCCCAACTACC-3'.
A 1-µL quantity of this PCR reaction was ligated into pCR 2.1 or pCR
2.1-TOPO (Invitrogen, Carlsbad, CA) vector and cloned exactly according to protocol. Clones were single-colony-purified and
cloned. Plasmid was purified using Wizard mini-prep protocol (Promega,
Madison, WI); approximately 1 µg of cloned DNA was
subjected to automated sequence analysis. The sequencing primer used
was 5'-CACCTTCTCCACTAATCCC-3' and is located at position
+424 relative to the p15 transcription start.24 Our
sequenced products include 63 CpG sites between positions  215
and +421 relative to the p15 transcription start site.
RNA extraction and reverse-transcriptase PCR of p15.
Total cellular RNA was extracted from pelleted leukemia blastic cells,
normal peripheral blood lymphocytes, and cell lines using standard acid
guanidium thiocyanate-phenol-chloroform extraction.25 A
3-µg quantity of total RNA was used for reverse-transcriptase (RT)
reactions, which were performed in a 15.75-µL final volume. Conditions for RT reactions were as follows: 10.4 µL of a mix containing 6 µL 5X first-strand buffer (Gibco/BRL), 3 µL of
0.1-mol/L DTT (GIBCO/BRL), 0.6 µL of a 25-mmol/L dNTPS mix ( 500
µmol/L each dNTP) (Pharmacia), 0.5 µL random hexamer mix
(GIBCO/BRL), and 12 U RNasin (Promega) was added to 18.1 µL of RNA
mixed in distilled water. Reactions were mixed, incubated for 5 minutes at 65°C, and placed on ice. A total of 14.25 µL was moved to a new tube (the remainder was used for the RT minus reaction) and 300 U
of Moloney Murine Leukemia Virus (MMLV) RT enzyme
(GIBCO/BRL) enzyme was then added to this new tube. All reactions (both
RT-positive and RT-negative) were then mixed and incubated for 1 hour
at 37°C. Reactions were then heated for approximately 5 minutes at
95°C and were frozen at 20°C. In some cases, 6 µg of
starting RNA was used and all reaction mixtures were doubled with the
exception of a constant amount of MMLV RT enzyme (GIBCO/BRL).
RT reactions were then diluted 5-fold. PCR was performed in a 50-µL
final volume. A 10-µL quantity of diluted RT reaction was added to
either 400 nmol/L each primer (GIBCO/BRL), 1X PCR buffer,23 1.25 mmol/L dATP, 1.25 mmol/L
dCTP, 1.25 mmol/L dGTP, 1.25 mmol/L dTTP (Pharmacia), 3% dimethyl
sulfoxide (p15 conditions); or to 400 nmol/L each primer, 1X
PCR buffer-Mg++ (GIBCO/BRL), 1.5 mmol/L MgCl2
(GIBCO/BRL), 200 µmol/L dATP, 200 µmol/L dCTP, 200 µmol/L dGTP,
and 200 µmol/L dTTP (Pharmacia) (GAPDH conditions). Reactions
were hot-started at 98°C for 3 minutes and held at 80°C before
addition of 1.25 U of Taq enzyme (GIBCO/BRL). Temperature conditions
for p15 PCR were as follows: 35 cycles of 98°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1.5 minutes followed
by 1 cycle of 72°C for 10 minutes. Temperature conditions for
GAPDH PCR were as follows: 23 cycles of 98°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Primers used were, for p15, 5'-TGGGGGCGGCAGCGATGAG-3' and
5'-AGGTGGGTGGGGGTGGGAAAT-3'; and, for GAPDH,
5'-CGGAGTCAACGGATTTGGTCGTAT-3' and
5'-AGCCTTCTCCATGGTGGTGAAGAC-3'. Either 10 µL
(p15) or 5 µL (GAPDH) was run on a 3% agarose gel and post-stained with ethidium bromide.
For relative expression quantitation (as shown in Table
1), all RT-PCR results were taken into
account. Expression was derived based on ethidium staining, as well as
blotting and hybridization for most samples. For blotting experiments,
28 cycles of p15 or 20 cycles of GAPDH RT-PCR were
performed. Products were blotted to Zeta-probe
(Bio-Rad, Hercules, CA), hybridized with a labeled p15 or GAPDH cDNA probe, and the results quantitated
via phosphorimager analysis. L117 and L157 were only assayed by
ethidium bromide staining analysis due to limited sample.
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Table 1.
Methylation Density, Expression Levels, and Leukemia
Type for Each Sample Where Both Methylation and RNA Could Be
Analyzed
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RESULTS |
The p15 promoter region CpG island is normally devoid of
hypermethylation.
To obtain more 5' sequence of p15 and better characterize
the full extent of the p15 CpG island, we cloned the
p15 promoter region from a P1 clone24 (data not
shown). Using a primer from the reported promoter region of
p15,24 we then obtained additional 5' sequence.
Southern blot analysis previously showed that the EagI site
located +167 relative to transcription start of p15 was not
methylated in normal tissues,6,8 a result confirmed by
methylation-specific PCR analysis of normal bone marrow.7
To further detail the patterns of methylation within this region in
normal tissues, we performed bisulfite genomic sequencing of 63 CpG
sites in the promoter region of p15 in lymphocytes from 3 normal individuals (Fig 1A). At least 7 individual alleles were sequenced for each normal sample studied,
revealing an almost completely unmethylated CpG island. However, the
existence of a few methylated CpG sites per allele in each individual
was found. Interestingly, normal individuals varied slightly in the
number of methylated CpG sites found within this otherwise protected
region. Although there was great variation between alleles, these
methylated sites tended to be more frequent in the far 5' and
3' portions of the CpG island.
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| Fig 1.
p15 CpG island methylation state in normal
individuals. Representation of the CpG island of p15 showing
CpG site methylation. A schematic of the p15 CpG island is
shown at the top for reference, and transcription start is denoted by
the arrow. The symbols above the schematic represent potential
trans-acting factor binding sites. ( ) Sp1 binding
sites.34 Sunbursts downstream of
transcription start ( ) represent several additional potential Sp1
binding sites. ( ) represents a potential G/T box. ( ) represents a
purine/pyrimidine tract that binds an unidentified potential
trans-acting factor on gel shift analysis (data not shown). For
each sequenced leukemia, each row represents an individual cloned and
sequenced allele following sodium bisulfite DNA modification. CpG sites
are marked as circles and drawn to accurately reflect CpG density of
the region. ( ) Methylated CpG sites; ( ) unmethylated sites.
"x" represents a CpG site for which sequence data was ambiguous.
(A) The p15 CpG island is largely unmethylated in normal
lymphocytes taken from 3 individuals. (B) Individual cloned and
sequenced alleles from 3 leukemia cell lines: KG1a, Raji, and HL60.
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p15 hypermethylation encompasses CpG sites throughout the
entire promoter region in primary leukemia and leukemic cell lines.
We first examined the allelic methylation patterns of cell lines
already characterized for p15 methylation by Southern analysis (Fig 1B).6 KG1a demonstrated methylation of nearly all CpG sites on every allele sequenced. Raji had dense methylation of the
majority of alleles, but also maintained alleles that were nearly
devoid of methylated CpG sites. HL60, like the unmethylated primary
leukemias, had complete absence of p15 CpG island hypermethylation.
DNA from 13 primary acute leukemias (both AML and acute lymphoblastic
leukemia [ALL]) was treated with sodium bisulfite,
cloned, and multiple alleles from each sample sequenced. Although most primary AMLs and ALLs are completely methylated by Southern analysis at
the EagI site located at position +167 in exon 1 of
p15,7 we chose to analyze several of the more
unusual primary leukemia samples previously found to have incomplete
methylation at this Eag1 site by Southern blot to investigate
the consequences of methylation heterogeneity at the level of
individual alleles.
The patterns of CpG island methylation in these primary leukemias were
markedly different from normal lymphocytes. This methylation in primary
leukemias was found to encompass the entire sequenced CpG island
region, and was not restrained to the few sites previously analyzed by
Southern blot (Fig 2). Patterns of the
individual CpG sites methylated in each leukemia varied greatly, which
suggests the absence of site-specific methylation of p15 in
this disorder. Rather, the alteration occurred heterogeneously
throughout the entire p15 CpG island and often involved many
CpG sites.
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| Fig 2.
Methylation status for individual alleles of p15
from 13 primary acute leukemias, depicted as in Fig 1. Primary acute
leukemias show varying degrees of methylation density, as well as
allelic heterogeneity of methylation. No site-specific methylation
patterns were seen.
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A subset of primary leukemia samples does not have hypermethylation of
p15 based on Southern analysis.6-8 We found these to be unmethylated throughout the CpG island region by bisulfite sequencing analysis as well. In these 3 primary leukemia samples (L161,
L117, and L3), all alleles are completely devoid of hypermethylation. These primary leukemia samples are hypomethylated compared with normal
lymphocytes, where infrequent methylated CpG sites are observed (Fig
1A).
Overall methylation density for each sample was derived by calculating
the number of methylated CpG sites per sample divided by the total
number of CpG sites sequenced per sample. All alleles were included in
this analysis, and these data are summarized in Table
1.
Allelic heterogeneity of p15 CpG methylation is pronounced in
primary acute leukemia and the Raji cell line.
Although many CpG sites throughout the p15 CpG island were
affected by methylation, we found that the exact location of methylated sites varied not only between samples, but also between alleles from
each leukemia. Two distinct patterns of allelic heterogeneity were
apparent. Frequently, we observed cases where the majority of alleles
contained hypermethylation of CpG sites, but the individual CpG sites
methylated varied from allele to allele. Such was the case for leukemia
samples L56, L41, L63, L226, and L129 (Fig 2). This type of
heterogeneity suggests instability or infidelity in the process of
maintenance methylation at the p15 locus.
However, the patterns observed for leukemias L224, L5, and L157 were
different. Here, individual alleles were found to have every CpG
methylated on approximately 70% to 90% of alleles and every CpG
unmethylated in approximately 10% to 30% of alleles. Thus, instead of
showing diversity in CpG site heterogeneity within alleles, these
samples show involvement of most, but not all alleles, in extremely
dense hypermethylation. While one explanation for this is the presence
of unmethylated alleles derived from contaminating normal cells, a
similar pattern was found in our sequencing of alleles from the Raji
cell line, where normal cell contamination can be excluded. Two
distinct patterns of allelic methylation indicate that even within a
clonal population of cells, alleles can be maintained in either
methylated or unmethylated form.
Transcriptional activity of p15 correlates with overall
methylation density and not site-specific methylation placement.
We next explored the relationship of methylation heterogeneity to
p15 expression. RNA from 10 of the sequenced primary leukemias and from normal lymphocytes, Raji, HL60, and KG1a cell lines was used
for RT-PCR analysis (Fig 3). Additionally,
lower cycle RT-PCR was performed for 8 of these samples, and these
products were blotted, hybridized, and expression quantitated relative
to GAPDH via phosphorimager analysis (data not shown).
Expression levels for each sample were derived taking into account all
RT-PCR data (Table 1). In the case of L157 and L117, expression levels
were analyzed based on ethidium staining relative to normal lymphocyte expression and GAPDH.
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| Fig 3.
RT-PCR for primary acute leukemias, leukemia cell lines,
and normal lymphocytes. Total cellular RNA was recovered from 9 of the
sequenced primary acute leukemias in addition to RNA from 3 leukemia
cell lines and normal lymphocytes. RT-PCR for p15 was performed
to assess transcription in these samples. p15 expression was
found to correlate with density of methylation and with the presence of
completely unmethylated alleles in a population. KG1a, which does not
express p15,6 has a nonspecific PCR product of a size not
matching p15. The control GAPDH RT-PCR was done using low cycle
number (20 cycles) and is shown below demonstrating that intact and
relatively equal RNA was used for each sample. (+) Indicates the
addition of MMLV RT enzyme; () indicates control RT reaction to
which RT enzyme was not added. The size of p15 product is 450 bp and the size of GAPDH product is 306 bp.
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Normal lymphocytes from 4 sampled individuals express p15 mRNA
(Fig 3, Table 1, and data not shown). Similarly, primary acute leukemias that were devoid of p15 hypermethylation (L161, L117, and L3) were shown to transcribe p15 with varying expression
levels. Leukemias such as L41 and L105, which have a relatively low
density of hypermethylation per allele, express p15, but at a
lower level than normal lymphocytes or the unmethylated acute
leukemias. Samples that were more densely methylated (>30% of total
CpG sites sequenced) such as L56 and L63 did not express detectable
p15 mRNA. This result indicates that methylation density, and
not merely the presence of hypermethylation, may be of critical
importance to transcription. Interestingly, we observed 3 leukemias
(L5, L224, and L157) that were able to transcribe p15 despite
having a majority of densely methylated alleles (Fig 3 and data not
shown). However, as noted earlier, each of these samples also contains
1 or more completely unmethylated alleles.
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DISCUSSION |
Our results on the allelic methylation status of 63 CpG sites in the
p15 promoter region CpG island confirm that aberrant hypermethylation found within the central region of the p15 CpG island represents a leukemic specific process. No significant methylation of CpG sites in this area could be found in normal lymphocytes. Our study also shows pronounced intra-allelic
heterogeneity of methylation with varying degrees of methylation
density at the p15 CpG island in the leukemias analyzed.
Methylation heterogeneity, though perhaps to a smaller degree, has also
been observed for Rb in sporadic retinoblastoma.18
This variability for involvement of CpG sites in each leukemia suggests
that the DNA methyltransferase enzyme does not faithfully maintain
methylation at individual CpG sites, and could reflect a type of
epigenetic instability in these cells.
In leukemic samples with abnormal methylation in each allele examined,
we studied the potential relationship between CpG site methylation
density and p15 mRNA. Our data suggest that methylation of 30%
to 40% of CpG sites in the CpG island of this gene correlates with a
state of complete gene silencing, while lower levels of hypermethylation do not. This level of methylation leading to complete
gene silencing is consistent with in vitro methylation of reporter
constructs, including graded loss of expression of the Rb
promoter with increasing methylation density26 and complete silencing of RSV LTR promoter activity with methylation density of 20%
to 30%.27 In 2 primary leukemias (L105, L41) with
methylation densities of 6% to 8%, the decreased p15
expression compared with normal lymphocytes suggests that this density
may be near the threshold for complete transcriptional silencing of
this promoter.
Although there was little variation in p15 expression in
lymphocytes taken from normal individuals, we found variable expression levels of p15 between leukemias that are unmethylated. For
example, we did observe a single leukemia (L161) that had diminished
expression of p15 mRNA despite its lack of methylation in the
p15 CpG island. This implicates mechanisms other than CpG
island methylation in the regulation of p15 expression. These
may include events upstream of p15 (such as disruption of the
transforming growth factor-beta [TGF ] signaling pathway), which
could result in the downregulation of p15 transcription in the
absence of promoter region hypermethylation.
We found occasional leukemias that had a large proportion of completely
methylated alleles and yet express p15 relatively well. Common
to these was the presence of a subset of alleles with an absence of any
CpG site methylation. Previous studies have not explored the presence
of such unmethylated alleles within a population of largely
hypermethylated alleles.18 Although these samples were
predominantly leukemic blasts, the unmethylated alleles could be from
normal lymphocytes and might account for the expression observed. The
presence of these alleles certainly correlates with p15
expression (L5, L224, and L157). However, it is important to note that
in L56, a leukemia for which we show lack of transcription in the
presence of a majority of heterogeneously, but densely, hypermethylated
alleles, 2 alleles in this population also show a relatively normal
methylation pattern. These alleles could simply reflect the randomness
of the cloning process, and may not really represent 2 in 12 alleles,
since this leukemia has been previously shown to be 98% methylated at
the EagI site.
Similar to L5, L224, and L157, the cell line Raji also maintains a
state of coexistent densely methylated and unmethylated alleles within
a population of cells that are clonal and transformed. This pattern
indicates the ability of 2 different allelic patterns to be maintained
within a clonal population of cells, similar to the normal processes of
allelic distinction such as X chromosome inactivation28 and
gene imprinting.29 We have also previously documented this
situation for the p16 gene in a colorectal carcinoma cell
line.30
Taken together, our data suggest how hypermethylation-associated gene
inactivation may occur with time during tumor progression. Recent
studies suggest that aberrant promoter methylation can occur early in
the progression of some tumors.31 In an experimental cell
culture system, overexpression of the DNA methyltransferase results in
a time-dependent hypermethylation of several CpG
islands.32,33 Recent work suggests that leukemias may be
evolving increasing density of methylation at the p15 CpG
island over time, since the p15 methylation progresses in the
transition of myelodysplasia to overt leukemia, as analyzed either by
Southern analysis or methylation-specific PCR.10,11 At any
point in time, there may be a wide range of density of methylation
within a population of blasts. Our data suggest that once most alleles
reach an average density of 30% to 40% of CpG sites methylated within
this CpG island, loss of expression of p15 occurs. Such a
progressive loss may be an important step in the alterations of cell
cycle control characteristic of acute leukemia, and may provide a
growth advantage leading to selection of those cells with the most
dense p15 methylation.
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ACKNOWLEDGMENT |
The authors thank Dr Curt Civin for primary acute leukemia samples, Dr
Jin Jen for providing us with a P1 clone containing the p15
promoter region, and Dr Sanna Myohanen for helpful scientific advice
and assistance.
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FOOTNOTES |
Submitted October 23, 1998; accepted May 24, 1999.
Supported in part by Grant No. CA43318 from the National Institutes of Health.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to James G. Herman, MD, The
Johns Hopkins Oncology Center, 424 N Bond St, Baltimore, MD 21231.
| |
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ABSTRACT
INTRODUCTION