|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4347-4353
Hypermethylation of the DAP-Kinase CpG Island Is a Common
Alteration in B-Cell Malignancies
By
Rachel A. Katzenellenbogen,
Stephen B. Baylin, and
James G. Herman
From The Johns Hopkins Comprehensive Cancer Center, Baltimore, MD.
 |
ABSTRACT |
Death-associated protein kinase (DAP-Kinase) is a novel
serine/threonine kinase whose expression is required for  interferon-induced apoptosis. A previous study suggested that
DAP-Kinase expression may be lost epigenetically in cancer cell lines,
because treatment of several nonexpressing cell lines with
5-aza-2'-deoxycytidine resulted in the expression of DAP-Kinase.
Using methylation-specific polymerase chain reaction (MSP), we examined
the DAP-Kinase CpG island for hypermethylation in cancer. Normal
lymphocytes and lymphoblastoid cell lines are unmethylated in the
5' CpG island of DAP-Kinase. However, in primary tumor samples,
all Burkitt's lymphomas and 84% of the B-cell non-Hodgkin's
lymphomas were hypermethylated in the DAP-Kinase CpG island. In
contrast, none of the T-cell non-Hodgkin's lymphoma samples and 15%
or less of leukemia samples examined had hypermethylated DAP-Kinase
alleles. U937, an unmethylated, DAP-Kinase-expressing leukemia cell
line, was treated with interferon and underwent apoptosis; however,
Raji, a fully methylated, DAP-Kinase nonexpressing Burkitt's lymphoma
cell line, only did so when treated with 5-aza-2'-deoxycytidine
followed by interferon. Our findings in cell lines and primary
tumors suggest that hypermethylation of the DAP-Kinase gene and loss of
interferon-mediated apoptosis may be important in the development
of B-cell malignancies and may provide a promising biomarker for
B-cell-lineage lymphomas.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
ONE OF THE HALLMARKS of cancer is an
imbalance between cell growth and death. Whereas normal cells undergo
apoptosis after specific regulatory signals, transformed cells can lose this response and survive, accumulating genetic and phenotypic defects
leading to tumor progression. In particular, DNA damage-induced apoptosis in response to genotoxic drug treatment or ionizing radiation
may be diminished. Critical genes involved in this response include
p53,1,2 Bcl-2,3 and Bax.3,4
Alterations in each of these genes have been described in
cancers,5 including hematologic
malignancies.6,7 For example, rearrangements including
Bcl-2 are a prominent feature of follicular lymphomas.8
Apoptosis is also critical to normal B-cell function in the immune
system.9 These cells both differentiate and proliferate by
clonal selection controlled by T cells or undergo programmed cell death
when not positively selected.9 The precise signals responsible for T-cell-induced B-cell apoptosis are not completely characterized. However, in addition to direct cell-cell interactions with B cells, T cells also secrete cytokines,10 which may
be important in this regard. Such soluble signals may initiate
programmed cell death in B cells, which, if blocked, might contribute
to B-cell tumorigenesis.
Death-associated protein kinase (DAP-Kinase) is a 160-kD
serine/threonine, microfilament-bound kinase recently shown to be involved in interferon-induced apoptosis.11 It was
isolated by Deiss et al12 in 1995 by technical knock-out
(TKO) studies in which cellular resistance to interferon-induced
apoptosis was selected for after transfection with an antisense cDNA
library. DAP-Kinase was identified as an interferon-induced gene
and found to phosphorylate itself and exogenous substrates in a process regulated by a Ca2+-calmodulin binding
domain.11 DAP-Kinase contains a death domain, and its
expression is required for induction of programmed cell death in cells
treated with interferon.11 Overexpression of DAP-Kinase
induces programmed cell death, whereas a catalytically inactive mutant
protects cells from interferon-induced apoptosis.11
Several B-cell lymphoma cell lines have decreased or absent expression
of DAP-Kinase RNA or protein.13 Some of these cell lines
are able to re-express DAP-Kinase after treatment with
5-aza-2'-deoxycytidine, a DNA demethylating drug.13
This finding suggests that abnormal loss of DAP-Kinase expression could
be associated with aberrant promoter region methylation. Such
epigenetic loss of tumor-suppressor gene expression is associated with
the aberrant methylation of CpG island gene-promoter regions, serving
as an alternative to the genetic loss of a tumor-suppressor gene
function by deletion or mutation.14-16 However, the role of
inactivation of DAP-Kinase in primary malignancies has not been
examined. Therefore, we investigated the methylation status of
DAP-Kinase in normal lymphocytes, hematopoietic cancer cell lines, and
primary hematopoietic tumor samples and studied its link to DAP-Kinase
expression and interferon-induced apoptosis.
| |
MATERIALS AND METHODS |
Samples and DNA preparation.
High molecular weight DNA was obtained from leukemic samples, as
described.17 The classification of acute lymphoblastic leukemia (ALL) samples was determined by surface markers,
as described.17 Lymphoma DNA was kindly provided by Dr
Constance Griffin (Johns Hopkins University, Baltimore,
MD), and the clinical diagnosis served as the subtype classification.
Methylation-specific polymerase chain reaction (MSP).
DNA methylation patterns in the CpG island of DAP-Kinase were
determined by chemical treatment with sodium bisulfite and subsequent use of the previously described polymerase chain reaction (PCR) procedure.18 MSP distinguishes the methylation status of a
given region based on sequence changes produced by sodium bisulfite treatment18 and has been previously validated for genes,
including p16,18,19 p15,
E-cadherin, VHL,18
hMLH1,20 the estrogen receptor,21
GST ,22 and MGMT,23 and for
the diagnosis of the imprinting disorder of Prader-Willi and
Angelman's syndromes.24-27 The primer sequences designed
for DAP-Kinase spanned 6 CpGs in total within the 5' region of
the gene. Primer sequences for unmethylated reaction were 5'-GGA
GGA TAG TTG GAT TGA GTT AAT GTT-3' (sense) and 5'-CAA ATC
CCT CCC AAA CAC CAA-3' (antisense). Primer sequences for
methylated reaction were 5'-GGA TAG TCG GAT CGA GTT AAC
GTC-3' (sense) and 5'-CCC TCC CAA ACG CCG A-3'
(antisense). The 5' position of the sense unmethylated and
methylated primers corresponds to bp 2 and 5 of Genbank sequence no.
X76104, respectively. Both antisense primers originate from bp 87 of
this sequence. The annealing temperature for both the unmethylated and
methylated reactions was 60°C. All MSP reactions were performed
with positive and negative controls for both unmethylated and
methylated alleles and a no DNA control.
Restriction analysis.
Aberrant methylation of CpG dinucleotides between the primer sequences
for DAP-Kinase was determined as previously described.18 MSP products were digested by Aci I (New England BioLabs,
Beverley, MA), which cuts DNA at the sequence 5'
C ^CGC 3'/3' GGC ^G 5', were ethanol
precipitated, and were run on 8% polyacrylamide gels.
Cell culture.
Raji, a Burkitt's Lymphoma cell line, and U937, a leukemia cell line,
were grown in RPMI 1640 with glutamine, 10% fetal bovine serum (Sigma,
St Louis, MO), 100 U/mL penicillin, and 10 µg/mL streptomycin and
maintained in log phase growth. Raji cells were treated with
5-aza-2'-deoxycytidine (Sigma) in 4 different dosage and time
courses. Cells were treated with 1 µmolar on days 0 and 2, with media
changes on days 2 and 5. Cells were treated with 0.5 µmol/L either on
day 0 with media change on day 2 or were treated on days 0 and 2 with
media changes on days 2 and 5. Cells were treated with 0.3 µmol/L on
days 0, 2, 6, and 8 with media changes on days 2, 5, 6, 8, and 9 and
then were carried for 7 days in normal media. Raji cells and U937 cells
were treated with 1,000 U/mL -interferon (R&D Systems, Minneapolis,
MN) or a carrier control of 0.1% bovine serum albumin (BSA).
DNA preparation.
Cells were pelleted and DNA was isolated using 1 mL/107
cells of TE9, 1% sodium dodecyl sulfate (SDS), and 0.4 mg/mL
proteinase K at 55°C for 1 to 2 hours and then 37°C overnight.
DNA was then purified using phenol-chloroform extraction and ethanol precipitated.
Protein preparation and Western analysis.
Cells were counted using a Coulter Counter (Coulter, Hialeah,
FL) and lysed (107 cells/mL) with 10 mmol/L
phosphate buffer, pH 7.5, 100 mmol/L NaCl, 1% Trition X-100, 0.5%
sodium deoxycholate, 0.1% SDS, 5 mmol/L EDTA, 1 µg/mL phenylmethyl
sulfonyl fluoride (PMSF), and 1× complete protease inhibitor
(Boehringer Mannheim, Indianapolis, IN). Cellular debris was pelleted
and protein from 106 cells were run on a 7.5%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The protein was
transferred to an immobilon-P transfer membrane (Millipore, Burlington,
MA), which was blocked with 5% BSA/TTBS for 2 hours and then incubated
with DAP-Kinase antibody (1:1,000 dilution in 1% BSA/TTBS; Sigma) for
2 hours and then with antimouse antibody-horseradish peroxidase (HRP; 1:2,000 dilution in 1% BSA/TTBS; Santa Cruz Laboratories, Santa Cruz,
CA) for 1 to 2 hours. The blot was then washed in serial TTBS washes
for 1 hour, developed with the ECL detection kit (Pierce, Rockford,
IL), and exposed to film (Eastman Kodak, Rochester, NY).
Reverse transcriptase-PCR (RT-PCR).
Total cellular RNA was obtained and reverse-transcribed as previously
described.28 Controls consisted of RNA treated identically but without the addition of reverse transcriptase and were labeled. RT-PCR was performed with the following primers for DAP-Kinase (5'-GAT AGA AAT GTC CCC AAA CCT CG-3' and 5'-TCT TCT
TG GAT CCT TGA CCA GAA-3', which amplify a 343-bp product
spanning sequence 781 to 1124 from Genbank X76104) and for
GAPDH (5'-CGG AGT CAA CGG ATT TGG TCG TAT-3' and
5'-AGC CTT CTC CAT GGT GGT GAA GAC-3'). Initial hot start
was followed by 35 cycles of amplification (95°C for 30 seconds,
58°C for 60 seconds, and 72°C for 60 seconds), and 10 µL of
product was electrophoresed on 6% nondenaturing polyacrylamide gels
and directly visualized with ethidium bromide staining under UV
illumination. GAPDH controls were amplified for 30 cycles (95°C for
30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds), and
5 µL of product was electrophoresed.
Apoptosis analysis.
Cells were fixed in 80% methanol (106 cells/150 µL) and
stored at 4°C. To stain cells for counting, cells were pelleted,
rehydrated with 150 µL phosphate-buffered saline (PBS), pelleted
again to remove the PBS, and incubated with Hoechst dye (1:500 dilution of 1 mg/mL bisbenzimide H 33342 fluorochrome trihydrochloride [Calbiochem, La Jolla, CA] in PBS). Apoptotic nuclear bodies versus nonapoptotic nuclei were counted under 40× UV microscopy in units of approximately 200 cells, counting up to 1,000 cells total. Standard
deviation was calculated when sufficient cell number allowed 2 or more
units to be counted.
| |
RESULTS |
DAP-Kinase has a region in the first 525 bp of its cDNA sequence that
qualifies as a CpG island. Within the first 525 bp of the DAP-Kinase
transcription start site, there are 46 CpGs (8.8%) and 49 GpCs, and
the sequence is 64% G+C rich. The CG:GC ratio is 0.9. Each of these is
a characteristic of a CpG island. To address the normal methylation
pattern of this region, we examined lymphocytes from 7 healthy controls
and found no evidence for methylation of the gene
(Fig 1A). We also examined Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines to determine whether
growth in cell culture led to methylation of DAP-Kinase. None of the 12 EBV-immortalized cell lines was methylated at the DAP-Kinase CpG
island, suggesting that cell culture or immortalization is not related
to DAP-Kinase methylation (Fig 1B).
View larger version (40K):
[in this window]
[in a new window]
| Fig 1.
MSP of DAP-Kinase. Primer sets used for amplification are
designated as unmethylated (U) or methylated (M). The size of the MSP
product for the unmethylated reaction is 106 bp. The size of the MSP
product for the methylated reaction is 98 bp. In all MSP reactions,
10 of product was run on a 6% polyacrylamide gel, stained with
ethidium bromide, and visualized under UV illumination. (A) MSP
analysis of DNA from lymphocytes of 6 normal individuals shows
amplification of only unmethylated alleles in the DAP-Kinase CpG
island. (B) MSP of DAP-Kinase in lymphoblastoid cell lines (cell lines
immortalized by EBV-transformation) shows amplification of only
unmethylated alleles in the DAP-Kinase CpG island. (C) MSP of
DAP-Kinase in cell lines. Raji shows amplification of only methylated
alleles for the DAP-Kinase CpG island. U937, an expressing cell line,
shows amplification of only unmethylated alleles for the DAP-Kinase CpG
island. (D) MSP of DAP-Kinase in Burkitt's lymphoma samples. Each
sample shows aberrant methylation of the DAP-Kinase CpG island. None of
these samples was microdissected, so the unmethylated alleles amplified
in each sample represent either normal tissue found within the tumor
samples or heterogeneity of aberrant methylation within the tumor
sample itself. (E) MSP of DAP-Kinase in lymphoma samples. Samples: B,
B-cell non-Hodgkin's lymphoma; T, T-cell non-Hodgkin's lymphoma; H,
Hodgkin's lymphoma. The majority of the B-cell non-Hodgkin's lymphoma
samples show aberrant methylation of the DAP-Kinase CpG island, and
none of the T-cell non-Hodgkin's lymphoma samples do.
|
|
Next, we investigated by MSP whether the methylation status of the
DAP-Kinase gene was indeed responsible for the loss of transcription
previously reported in several hematopoietic cell lines. We found that
Raji, a Burkitt's lymphoma cell line that does not express
DAP-Kinase,13 has only methylated copies of the DAP-Kinase
gene. However, U937, a leukemia cell line that expresses
DAP-Kinase,13 has only unmethylated copies of DAP-Kinase (Fig 1C). Thus, our analysis suggests that the presence of
hypermethylation of the CpG island correlates with the loss of
expression of DAP-Kinase. We studied other cancer cell lines and found
several with partial methylation of the DAP-Kinase CpG island. This mix
of methylated and unmethylated copies of DAP-Kinase represents either
intra-allelic heterogeneity of the CpG island methylation or
methylation of the island that varies from allele to allele.
Because DAP-Kinase is aberrantly methylated in hematopoietic cancer
cell lines and such methylation is linked to the transcriptional repression of the gene, we analyzed primary hematopoietic cancer samples to see if aberrant methylation of DAP-Kinase occurred in vivo.
In 100% of the Burkitt's lymphoma samples studied (9 of 9), we
observed methylation of the DAP-Kinase CpG island (Fig 1D). In 84% of
the B-cell lymphoma samples studied (21 of 25), we found methylation of
DAP-Kinase as well (Fig 1E). Methylation of the DAP-Kinase gene did not
vary markedly between low-grade or high-grade non-Hodgkin's lymphomas.
In contrast, none of the 4 T-cell lymphoma samples we examined had
aberrant methylation of the DAP-Kinase CpG island.
In primary leukemia, there was hypermethylation in 15% or less of the
samples in each subclass (Table 1). The
involved leukemias were most often of the B-cell type. In chronic
lymphocytic leukemia, 1 of the 10 samples demonstrated methylation of
DAP-Kinase; this case was notable in presenting as a lymphocytic
lymphoma. A single chronic myeloid leukemia sample also demonstrated
DAP-Kinase hypermethylation; this occurred in a patient in a basophilic
blast crisis. In acute leukemia, 1of 26 pediatric acute myeloid
leukemias and 3 of 20 B-cell acute lymphoblastic leukemias, but none of
10 T-cell acute lymphoblastic leukemias, showed methylation of the CpG
island (data not shown). The single acute myeloid leukemia
(AML) sample with methylated DAP-Kinase CpG islands had
biphenotypic markers with a B-cell phenotype.
To address the density of methylation of the DAP-Kinase CpG island, we
also studied the methylation of selected CpG dinucleotides in this
region that did not fall under our DAP-Kinase MSP primers, as
previously described.18 This technique takes advantage of the divergence of unmethylated and methylated sodium bisulfite-modified CpG dinucleotides in restriction enzyme sites. The restriction enzyme
Aci I recognizes the sequence 5'-C ^CGC-3'
and 3'-GGC ^G-5', which is found in the DAP-Kinase
gene between the MSP primers. If the CpGs are methylated in this
sequence, the sodium bisulfite-modified 3' to 5' sequence
is unaltered by the bisulfite and Aci I cuts the DNA. If the
CpGs are unmethylated, the sodium bisulfite modifies the 3' to
5' sequence to 3'-GGTG-5' and the DNA will not be
cut. We analyzed DNA amplified by MSP for methylated and unmethylated sequences from representative MSP product samples: 2 B-cell ALL, 1 T-cell ALL, 2 chronic lymphocytic leukemia (CLL), 2 B-cell
lymphomas, 1 T-cell lymphoma, and 2 Burkitt's lymphomas as well as
Raji and normal lymphocytes. In all of the samples analyzed except 1, the unmethylated MSP product was not cut by Aci I, whereas the
methylated MSP product was (data not shown); therefore, methylation of
the DAP-Kinase CpG island was dense over this region of the CpG island. The single exception to these findings was an unmethylated CLL sample
that was partially cleaved by the restriction enzyme. This sample may
have had heterogeneous methylation of its DAP-Kinase CpG island, such
that several copies of the DAP-Kinase gene in this sample were
methylated at this restriction site sequence but were not methylated in
the region of the MSP primer sequences.
We turned our attention back to established cell lines to study the
consequences of loss of expression of DAP-Kinase in association with
hypermethylation of the gene. U937 and Raji cell lines were treated
with interferon for a total of 11 days. After interferon exposure, U937, which expresses DAP-Kinase, underwent apoptosis, with
maximal cell death occurring at days 3 through 7 of cytokine treatment
(Fig 2A). Associated with the induction of
this cell death was an upregulation of DAP-Kinase protein expression
(Fig 2C). Raji cells, on the other hand, did not express DAP-Kinase at
either the RNA or protein level (Fig 2C) and did not undergo apoptosis
after interferon treatment during this time period (Fig 2B).
View larger version (78K):
[in this window]
[in a new window]
| Fig 2.
Interferon treatment of cell lines. U937, a leukemia
cell line, and Raji, a Burkitt's lymphoma cell line, were treated with
1,000 U/mL of interferon or carrier control for 11 days. (A) U937
cell apoptosis. U937 cells were analyzed for apoptosis on days 3, 5, 7, 9, and 11. U937 cells underwent apoptosis when treated with interferon, with significant differences in cell death between treated
cells and carrier control cells on each day (P < 10 4). The inset demonstrates U937 cells treated for 5 days with interferon or the carrier control; several of the interferon-treated cells are undergoing nuclear apoptotic changes
(arrowheads point to apoptotic cells), whereas none of the carrier
control-treated cells is. (B) Raji cell apoptosis. Raji cells were
analyzed for apoptosis on days 3, 5, 7, 9, and 11. Raji cells did not
undergo apoptosis when treated with interferon or the carrier
control. Raji cells treated for 5 days with interferon show no
cells undergoing apoptosis, and there are no apoptotic nuclei. (C) DAP
kinase expression. Cells (106) were lysed on days 1, 3, 5, 7, 9, and 11 of treatment with interferon or carrier control before
Western analysis. U937 cells exposed to interferon upregulated the
expression of DAP-Kinase from days 5 through 11. Carrier control cells
expressed DAP-Kinase endogenously but did not upregulate the protein
expression. Western analysis of U937 and Raji cells and RT-PCR analysis
(far right) with GAPDH as controls demonstrate the lack of endogenous
expression of DAP-Kinase in Raji at both the RNA and protein levels.
DAP-Kinase RT-PCR amplifies a 343-bp product, whereas the GAPDH
controls are 310 bp. + and  indicate the addition or the absence
of reverse transcriptase (RT), respectively.
|
|
To further determine whether aberrant methylation of the DAP-Kinase CpG
island was directly mediating the resistance of Raji cells to interferon-induced apoptosis, we treated these cells in three different
time course experiments with the demethylating agent,
5-aza-2'-deoxycytidine, followed directly by interferon exposure. In each experiment, the DAP-Kinase CpG island was partially demethylated by 5-aza-2'-deoxycytidine within the first 2 days of
drug exposure (Fig 3A).
Although the acute toxic effects of 5-aza-2'-deoxycytidine caused
some apoptotic cell death, Raji cells in which the DAP-Kinase CpG
island had been partially demethylated underwent a higher percentage of
programmed cell death in each experiment when treated with interferon compared with the level of apoptosis in those cells exposed
only to carrier control (Fig 3). This interferon-induced apoptosis
occurred at days 5 and 7 of cytokine treatment, a time similar to that
observed for interferon-responsive U937 cells.
View larger version (39K):
[in this window]
[in a new window]
| Fig 3.
Raji cells were treated with 5-aza-2'-deoxycytidine
in 3 experiments to demethylate the DAP-Kinase CpG island and then
exposed to interferon. (A) Raji cell apoptosis. Raji cells were
treated with 1 µmol/L 5-aza-2'-deoxycytidine for 5 days and
then with 1,000 U/mL interferon for 7 days. Cells were analyzed for
apoptosis on days 3, 5, and 7 of inteferon or carrier control
treatment. By day 7, Raji cells exposed to interferon were
undergoing more apoptosis than Raji cells exposed only to carrier
control. (B) Raji cell apoptosis. Raji cells were treated with 0.5 µmol/L 5-aza-2'-deoxycytidine for 5 days and then with 1,000 U/mL interferon for 7 days. Cells were analyzed for apoptosis on
days 3, 5, and 7 of inteferon or carrier control treatment. By day
7, Raji cells exposed to interferon were undergoing more apoptosis
than Raji cells exposed only to carrier control. (C) Raji cell
apoptosis. Raji cells were treated with 0.5 µmol/L
5-aza-2'-deoxycytidine for 2 days and then with 1,000 U/mL interferon for 7 days. Cells were analyzed for apoptosis on days 3, 5, and 7 of inteferon or carrier control treatment. By day 5, a trend
had developed in which Raji cells exposed to interferon underwent
more apoptosis than those exposed with carrier control alone.
|
|
To minimize the toxic effects of 5-aza-2'-deoxycytidine and the
apoptosis related to acute 5-aza-2'-deoxycytidine treatment, we
conducted a fourth experiment in which Raji cells were exposed to a
lower concentration of 5-aza-2'-deoxycytidine in a more chronic treatment, were allowed to recover for 7 days in normal media, and were
then treated with interferon for 11 days. In this experiment, we
again were able to partially demethylate the DAP-Kinase gene (Fig 4A). This treatment protocol now
produced a statistically significant increase in interferon-induced
apoptosis over the carrier control (Fig 4B). During the time course of
interferon treatment, we observed a loss of sensitivity to interferon exposure in this experiment, which coincided with an
increase in methylation of the DAP-Kinase CpG island (Fig 4A). This may
represent remethylation of the gene by DNA methyltransferase or the
outgrowth of cells that had not demethylated the DAP-Kinase gene and
thus were protected from interferon-induced apoptosis. All of these
data suggest that Raji undergoes interferon-induced cell death only
after demethylation of the DAP-Kinase gene.
View larger version (35K):
[in this window]
[in a new window]
| Fig 4.
Raji cells were treated with 0.3 µmol/L
5-aza-2'-deoxycytidine for 9 days to demethylate the DAP-Kinase
CpG island, placed in normal media for 7 days, and then treated with
1,000 U/mL interferon for 11 days. (A) MSP of DAP-Kinase in Raji
cells. The methylation status of the DAP-Kinase CpG island was checked
on days 0, 2, 5, and 9 of 5-aza-2'-deoxycytidine treatment
(D0-9-A) and then after the combined 7 days of normal media
and either 11 days of interferon (D11-) or carrier
control (D11-C) media. (B) Raji cell apoptosis. By days 5 and 7, Raji cells exposed to interferon underwent significantly
more apoptosis than those exposed with carrier control alone (P
= .0005 and P = .001, respectively). By days 9 and 11, Raji
cells were losing their unmethylated copies of DAP-Kinase and also
their sensitivity to interferon treatment.
|
|
| |
DISCUSSION |
DAP-Kinase is a novel protein whose expression is required in interferon-induced apoptosis of cells. The gene may be important biologically in normal cell differentiation and selection in the immune
system. Recent reports demonstrate expression of DAP-Kinase to be
decreased or absent in several cancer cell lines.13 A decrease in expression of this gene imparts resistance to interferon-induced apoptosis in cells,11,12 and a link
between the loss of DAP-Kinase expression and cellular apoptosis may
facilitate metastasis in an experimental system.29 However,
despite these compelling data, no involvement of DAP-Kinase in primary
malignancy has been investigated.
Our results suggest that the loss of expression of DAP-Kinase observed
in cell lines is indeed due to promoter region hypermethylation. This
pattern of hypermethylation was also very frequent in primary B-cell
malignancies, although not observed in T-cell malignancies or in
myelogenous leukemia. Although we examined a smaller number of T-cell
malignancies (ALL and non-Hodgkin's lymphoma) than those with a B-cell
phenotype, the incidence of hypermethylation of DAP-Kinase present in
B-cell malignancies suggests that we would have easily detected a
similar incidence in T-cell malignancies if present. However,
DAP-Kinase methylation is not part of the normal differentiation of B
cells, because normal peripheral lymphocytes (predominantly B cell) and
immortalized lymphoblasts (B cells) do not have hypermethylation of the
CpG island of the gene. Thus, hypermethylation of DAP-Kinase is
characteristic of the transformed B cell, but not of normal B cells or
those immortalized in vitro.
We have previously examined methylation of other tumor-suppressor genes
and DNA repair enzymes in many of these same samples. p15 is frequently
methylated in ALL and AML samples, p15 and/or p16 are frequently
methylated in the Burkitt's samples, and high-grade non-Hodgkin's
lymphoma frequently has hypermethylation of p16.17 We have
also recently examined DNA repair enzymes and find that only rarely is
the GST gene methylated in these samples,22 whereas MGMT
is often methylated in the high-grade lymphomas.23 In this
latter case, whereas DAP-Kinase methylation was associated with only
the B-cell phenotype (being present in both high-grade and low-grade
non-Hodgkin's lymphoma), MGMT was very highly associated with the
diffuse large-cell lymphoma phenotype.23 Thus, multiple epigenetic events, ie, the hypermethylation and silencing of many genes, along with genetic alterations, such as translocation, deletion,
and point mutations, coexist in the constellation of changes associated
with the transformed phenotype.
The functional consequences of hypermethylation associated loss of
DAP-Kinase expression are demonstrated in transformed cell lines. Raji,
a nonexpressing and hypermethylated cell line, did not respond to interferon treatment, unlike U937, an expressing and unmethylated cell
line. When we treated Raji cells with 5-aza-2'-deoxycytidine and
partially demethylated the DAP-Kinase CpG island, these cells became
sensitive to interferon treatment. This sensitivity was lost as
passaged Raji cells remethylated the DAP-Kinase CpG island. Further
emphasizing the role of DAP-Kinase in transformation rather than
immortalization is the finding that, in vivo, re-expression of
DAP-Kinase in cancer cells lowers tumorigenicity but has no effect on
cell growth in culture.13,29
DAP-Kinase is a novel protein required for programmed cell death in the
presence of interferon,11,12 and its lack of expression
and concordant hypermethylation protects cells from interferon-induced apoptosis. The high frequency of abnormal methylation of DAP-Kinase in primary B-cell cancers suggests not only
an important role for this pathway in the development of B-cell
malignancies, but also that this DNA modification may serve as a
biomarker for these cancers. Because in an experimental model, the loss
of DAP-Kinase expression aids in metastatic potential of lung cancer
cells,29 loss of expression of this gene may be important
in the progression of other cancer types as well.
| |
ACKNOWLEDGMENT |
The authors thank Dr Christof Lengauer for assistance in the apoptosis
analysis, Drs Connie Griffen and Mike Grever for primary tumor samples,
and Dr Paul Corn for cDNA samples.
| |
FOOTNOTES |
Submitted August 4, 1998; accepted February 9, 1999.
Supported in part by grants from the Howard Hughes Medical Institute
and the V Foundation and National Institutes of Health Grants No.
CA43318 and P30CA06973. R.A.K. was a Howard Hughes Medical Institute
Medical Student Research Training Fellow. S.B.B. and J.G.H. receive
research funding and are entitled to sales royalties from ONCOR, which
is developing products related to research described in this report.
The terms of this arrangement have been reviewed and approved by the
Johns Hopkins University in accordance with its conflict of interest policies.
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, Johns Hopkins Medical
Institutions, Department of Oncology, Tumor Biology, 424 N Bond St,
Baltimore, MD 21231.
| |
REFERENCES |
1.
Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB:
Wild-type p53 is a cell cycle checkpoint determinant following irradiation.
Proc Natl Acad Sci USA
89:7491, 1992[Abstract/Free Full Text]
2.
Symonds H, Krall L, Remington L, Saenz-Robles M, Lowe S, Jacks T, Van Dyke T:
p53-dependent apoptosis suppresses tumor growth and progression in vivo.
Cell
78:703, 1994[Medline]
[Order article via Infotrieve]
3.
Knudson CM, Korsmeyer SJ:
Bcl-2 and Bax function independently to regulate cell death.
Nat Genet
16:358, 1997[Medline]
[Order article via Infotrieve]
4.
Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T:
Bax suppresses tumorigenesis and stimulates apoptosis in vivo.
Nature
385:637, 1997[Medline]
[Order article via Infotrieve]
5.
Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC, Perucho M:
Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype.
Science
275:967, 1997[Abstract/Free Full Text]
6.
Findley HW, Gu L, Yeager AM, Zhou M:
Expression and regulation of Bcl-2, Bcl-xl, and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia.
Blood
89:2986, 1997[Abstract/Free Full Text]
7.
Meijerink JP, Mensink EJ, Wang K, Sedlak TW, Sloetjes AW, de Witte T, Waksman G, Korsmeyer SJ:
Hematopoietic malignancies demonstrate loss-of-function mutations of BAX.
Blood
91:2991, 1998[Abstract/Free Full Text]
8.
Tsujimoto Y, Cossman J, Jaffe E, Croce CM:
Involvement of the bcl-2 gene in human follicular lymphoma.
Science
228:1440, 1985[Abstract/Free Full Text]
9.
Janeway CA Jr, Travers P:
Immunobiology: The Immune System in Health and Disease. London, UK, Current Biology, 1994.
10.
Parker DC:
T cell-dependent B cell activation.
Annu Rev Immunol
11:331, 1993[Medline]
[Order article via Infotrieve]
11.
Cohen O, Feinstein E, Kimchi A:
DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity.
EMBO J
16:998, 1997[Medline]
[Order article via Infotrieve]
12.
Deiss LP, Feinstein E, Berissi H, Cohen O, Kimchi A:
Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death.
Genes Dev
9:15, 1995[Abstract/Free Full Text]
13.
Kissil JL, Feinstein E, Cohen O, Jones PA, Tsai YC, Knowles MA, Eydmann ME, Kimchi A:
DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: Possible implications for role as tumor suppressor gene.
Oncogene
15:403, 1997[Medline]
[Order article via Infotrieve]
14.
Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB, Sidransky D:
5' CPG island methylation is associated with transcriptional silencing of the tumour suppressor P16/CDKN2/MTS1 in human cancers.
Nat Med
1:686, 1995[Medline]
[Order article via Infotrieve]
15.
Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, Samid D, Duan DS, Gnarra JR, Linehan WM, Baylin SB:
Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.
Proc Natl Acad Sci USA
91:9700, 1994[Abstract/Free Full Text]
16.
Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP:
Alterations in DNA methylation: A fundamental aspect of neoplasia.
Adv Cancer Res
72:141, 1998[Medline]
[Order article via Infotrieve]
17.
Herman JG, Civin CI, Issa JP, Collector MI, Sharkis SJ, Baylin SB:
Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies.
Cancer Res
57:837, 1997[Abstract/Free Full Text]
18.
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB:
Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands.
Proc Natl Acad Sci USA
93:9821, 1996[Abstract/Free Full Text]
19.
Gonzalgo ML, Bender CM, You EH, Glendening JM, Flores JF, Walker GJ, Hayward NK, Jones PA, Fountain JW:
Low frequency of p16/CDKN2A methylation in sporadic melanoma: Comparative approaches for methylation analysis of primary tumors.
Cancer Res
57:5336, 1997[Abstract/Free Full Text]
20.
Herman JG, Umar A, Polyak K, Graff JR, Ahuja N, Issa JPJ, Markowitz S, Willson JKV, Hamilton SR, Kinzler KW, Kane MF, Kolodner RD, Vogelstein B, Kunkel TA, Baylin SB:
Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.
Proc Natl Acad Sci USA
95:6870, 1998[Abstract/Free Full Text]
21.
Lapidus RG, Nass SJ, Butash KA, Parl FF, Weitzman SA, Graff JG, Herman JG, Davidson NE:
Mapping of ER gene CpG island methylation-specific polymerase chain reaction.
Cancer Res
58:2515, 1998[Abstract/Free Full Text]
22.
Esteller M, Corn PG, Urena JM, Gabrielson E, Baylin SB, Herman JG:
Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia.
Cancer Res
58:4515, 1998[Abstract/Free Full Text]
23.
Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG:
Inactivation of the DNA repair gene O6-Methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in human neoplasia.
Cancer Res
59:793, 1999[Abstract/Free Full Text]
24.
Kubota T, Das S, Christian SL, Baylin SB, Herman JG, Ledbetter DH:
Methylation-specific PCR simplifies imprinting analysis.
Nat Genet
16:16, 1997[Medline]
[Order article via Infotrieve]
25.
Zeschnigk M, Lich C, Buiting K, Doerfler W, Horsthemke B:
A single-tube PCR test for the diagnosis of Angelman and Prader-Willi syndrome based on allelic methylation differences at the SNRPN locus.
Eur J Hum Genet
5:94, 1997[Medline]
[Order article via Infotrieve]
26.
Jacobsen J, King BH, Leventhal BL, Christian SL, Ledbetter DH, Cook EH Jr:
Molecular screening for proximal 15q abnormalities in a mentally retarded population.
J Med Genet
35:534, 1998[Abstract/Free Full Text]
27.
Huerta Rivas C, Barabash Bustelo A, Gallego Merlo J, Ramos Corrales C, Osorio Cabrero A, Robledo Batanero M, Benitez Ortiz J:
Quick diagnosis of Prader-Willi and Angelman syndromes by means of methylation test by PCR.
An Esp Pediatr
48:583, 1998[Medline]
[Order article via Infotrieve]
28.
Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB:
Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.
Nat Genet
21:103, 1999[Medline]
[Order article via Infotrieve]
29.
Inbal B, Cohen O, Polak-Charcon S, Kopolovic J, Vadai E, Eisenbach L, Kimchi A:
DAP kinase links the control of apoptosis to metastasis.
Nature
390:180, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K. Amara, M. Trimeche, S. Ziadi, A. Laatiri, M. Hachana, and S. Korbi
Prognostic significance of aberrant promoter hypermethylation of CpG islands in patients with diffuse large B-cell lymphomas
Ann. Onc.,
October 1, 2008;
19(10):
1774 - 1786.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C S Chim, W W L Chan, and Y L Kwong
Epigenetic dysregulation of the DAP kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in acute leukaemias
J. Clin. Pathol.,
July 1, 2008;
61(7):
844 - 847.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-S. Chim, R. Liang, T.-K. Fung, C.-L. Choi, and Y.-L. Kwong
Epigenetic dysregulation of the death-associated protein kinase/p14/HDM2/p53/Apaf-1 apoptosis pathway in multiple myeloma
J. Clin. Pathol.,
June 1, 2007;
60(6):
664 - 669.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Rizzi, M. P. Tschan, C. Britschgi, A. Britschgi, B. Hugli, T. J. Grob, N. Leupin, B. U. Mueller, H.-U. Simon, A. Ziemiecki, et al.
The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells
J. Leukoc. Biol.,
June 1, 2007;
81(6):
1599 - 1608.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Agrawal, W.-K. Hofmann, N. Tidow, M. Ehrich, D. v. d. Boom, S. Koschmieder, W. E. Berdel, H. Serve, and C. Muller-Tidow
The C/EBP{delta} tumor suppressor is silenced by hypermethylation in acute myeloid leukemia
Blood,
May 1, 2007;
109(9):
3895 - 3905.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. M. Brena and J. F. Costello
Genome-epigenome interactions in cancer
Hum. Mol. Genet.,
April 15, 2007;
16(R1):
R96 - R105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. R. L. Young, C. Mumaw, J. A. Marrs, and D. G. Skalnik
Antisense Targeting of CXXC Finger Protein 1 Inhibits Genomic Cytosine Methylation and Primitive Hematopoiesis in Zebrafish
J. Biol. Chem.,
December 1, 2006;
281(48):
37034 - 37044.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. J. Reu, S. I. Bae, L. Cherkassky, D. W. Leaman, D. Lindner, N. Beaulieu, A. R. MacLeod, and E. C. Borden
Overcoming Resistance to Interferon-Induced Apoptosis of Renal Carcinoma and Melanoma Cells by DNA Demethylation
J. Clin. Oncol.,
August 10, 2006;
24(23):
3771 - 3779.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. O. Machida, M. V. Brock, C. M. Hooker, J. Nakayama, A. Ishida, J. Amano, M. A. Picchi, S. A. Belinsky, J. G. Herman, S. Taniguchi, et al.
Hypermethylation of ASC/TMS1 Is a Sputum Marker for Late-Stage Lung Cancer.
Cancer Res.,
June 15, 2006;
66(12):
6210 - 6218.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. L. Russo, A. Thiagalingam, H. Pan, J. Califano, K.-h. Cheng, J. F. Ponte, D. Chinnappan, P. Nemani, D. Sidransky, and S. Thiagalingam
Differential DNA Hypermethylation of Critical Genes Mediates the Stage-Specific Tobacco Smoke-Induced Neoplastic Progression of Lung Cancer
Clin. Cancer Res.,
April 1, 2005;
11(7):
2466 - 2470.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Murai, M. Toyota, H. Suzuki, A. Satoh, Y. Sasaki, K. Akino, M. Ueno, F. Takahashi, M. Kusano, H. Mita, et al.
Aberrant Methylation and Silencing of the BNIP3 Gene in Colorectal and Gastric Cancer
Clin. Cancer Res.,
February 1, 2005;
11(3):
1021 - 1027.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Fujiwara, N. Fujimoto, M. Tabata, K. Nishii, K. Matsuo, K. Hotta, T. Kozuki, M. Aoe, K. Kiura, H. Ueoka, et al.
Identification of Epigenetic Aberrant Promoter Methylation in Serum DNA Is Useful for Early Detection of Lung Cancer
Clin. Cancer Res.,
February 1, 2005;
11(3):
1219 - 1225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Tang, W. Wu, S.-y. Sun, I. I. Wistuba, W. K. Hong, and L. Mao
Hypermethylation of the Death-Associated Protein Kinase Promoter Attenuates the Sensitivity to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Non-Small Cell Lung Cancer Cells
Mol. Cancer Res.,
December 1, 2004;
2(12):
685 - 691.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ropero, F. Setien, J. Espada, M. F. Fraga, M. Herranz, J. Asp, M. S. Benassi, A. Franchi, A. Patino, L. S. Ward, et al.
Epigenetic loss of the familial tumor-suppressor gene exostosin-1 (EXT1) disrupts heparan sulfate synthesis in cancer cells
Hum. Mol. Genet.,
November 15, 2004;
13(22):
2753 - 2765.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Blum, G. Lozanski, and J. C. Byrd
Adult Burkitt leukemia and lymphoma
Blood,
November 15, 2004;
104(10):
3009 - 3020.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Roman-Gomez, A. Jimenez-Velasco, J. A. Castillejo, X. Agirre, M. Barrios, G. Navarro, F. J. Molina, M. J. Calasanz, F. Prosper, A. Heiniger, et al.
Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia
Blood,
October 15, 2004;
104(8):
2492 - 2498.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. I. de Caceres, C. Battagli, M. Esteller, J. G. Herman, E. Dulaimi, M. I. Edelson, C. Bergman, H. Ehya, B. L. Eisenberg, and P. Cairns
Tumor Cell-Specific BRCA1 and RASSF1A Hypermethylation in Serum, Plasma, and Peritoneal Fluid from Ovarian Cancer Patients
Cancer Res.,
September 15, 2004;
64(18):
6476 - 6481.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Dulaimi, J. Hillinck, I. I. de Caceres, T. Al-Saleem, and P. Cairns
Tumor Suppressor Gene Promoter Hypermethylation in Serum of Breast Cancer Patients
Clin. Cancer Res.,
September 15, 2004;
10(18):
6189 - 6193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Brakensiek, F. Langer, H. Kreipe, U. Lehmann, M. T. Voso, A. Scardocci, L. Valentini, F. Guidi, S. Hohaus, and G. Leone
Low level of DAP-kinase DNA methylation in myelodysplastic syndrome
Blood,
September 1, 2004;
104(5):
1586 - 1588.
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Kishino, K. Yukawa, K. Hoshino, A. Kimura, N. Shirasawa, H. Otani, T. Tanaka, K. Owada-Makabe, Y. Tsubota, M. Maeda, et al.
Deletion of the Kinase Domain in Death-Associated Protein Kinase Attenuates Tubular Cell Apoptosis in Renal Ischemia-Reperfusion Injury
J. Am. Soc. Nephrol.,
July 1, 2004;
15(7):
1826 - 1834.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. C. Pulling, B. R. Vuillemenot, J. A. Hutt, T. R. Devereux, and S. A. Belinsky
Aberrant Promoter Hypermethylation of the Death-Associated Protein Kinase Gene Is Early and Frequent in Murine Lung Tumors Induced by Cigarette Smoke and Tobacco Carcinogens
Cancer Res.,
June 1, 2004;
64(11):
3844 - 3848.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-i. Maruya, J.-P. J. Issa, R. S. Weber, D. I. Rosenthal, J. C. Haviland, R. Lotan, and A. K. El-Naggar
Differential Methylation Status of Tumor-Associated Genes in Head and Neck Squamous Carcinoma: Incidence and Potential Implications
Clin. Cancer Res.,
June 1, 2004;
10(11):
3825 - 3830.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Takahashi, N. Shivapurkar, J. Reddy, H. Shigematsu, K. Miyajima, M. Suzuki, S. Toyooka, S. Zochbauer-Muller, J. Drach, G. Parikh, et al.
DNA Methylation Profiles of Lymphoid and Hematopoietic Malignancies
Clin. Cancer Res.,
May 1, 2004;
10(9):
2928 - 2935.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Levy, G. Plu-Bureau, Y. Decroix, D. Hugol, W. Rostene, A. Kimchi, and A. Gompel
Death-Associated Protein Kinase Loss of Expression Is a New Marker for Breast Cancer Prognosis
Clin. Cancer Res.,
May 1, 2004;
10(9):
3124 - 3130.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Terasawa, S. Sagae, M. Toyota, K. Tsukada, K. Ogi, A. Satoh, H. Mita, K. Imai, T. Tokino, and R. Kudo
Epigenetic Inactivation of TMS1/ASC in Ovarian Cancer
Clin. Cancer Res.,
March 15, 2004;
10(6):
2000 - 2006.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. B. Douglas, Y. Akiyama, H. Carraway, S. A. Belinsky, M. Esteller, E. Gabrielson, S. Weitzman, T. Williams, J. G. Herman, and S. B. Baylin
Hypermethylation of a Small CpGuanine-Rich Region Correlates with Loss of Activator Protein-2{alpha} Expression during Progression of Breast Cancer
Cancer Res.,
March 1, 2004;
64(5):
1611 - 1620.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. T. Voso, A. Scardocci, F. Guidi, G. Zini, A. Di Mario, L. Pagano, S. Hohaus, and G. Leone
Aberrant methylation of DAP-kinase in therapy-related acute myeloid leukemia and myelodysplastic syndromes
Blood,
January 15, 2004;
103(2):
698 - 700.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Obata, M. Toyota, A. Satoh, Y. Sasaki, K. Ogi, K. Akino, H. Suzuki, M. Murai, T. Kikuchi, H. Mita, et al.
Identification of HRK as a Target of Epigenetic Inactivation in Colorectal and Gastric Cancer
Clin. Cancer Res.,
December 15, 2003;
9(17):
6410 - 6418.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. He, L. You, K. Uematsu, K. Zang, Z. Xu, A. Y. Lee, J. F. Costello, F. McCormick, and D. M. Jablons
SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer
PNAS,
November 25, 2003;
100(24):
14133 - 14138.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. G. Herman and S. B. Baylin
Gene Silencing in Cancer in Association with Promoter Hypermethylation
N. Engl. J. Med.,
November 20, 2003;
349(21):
2042 - 2054.
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. N. Reddy, W. W. Jiang, M. Kim, N. Benoit, R. Taylor, J. Clinger, D. Sidransky, and J. A. Califano
Death-Associated Protein Kinase Promoter Hypermethylation in Normal Human Lymphocytes
Cancer Res.,
November 15, 2003;
63(22):
7694 - 7698.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Capello, M. Cerri, G. Muti, E. Berra, P. Oreste, C. Deambrogi, D. Rossi, G. Dotti, A. Conconi, M. Vigano, et al.
Molecular histogenesis of posttransplantation lymphoproliferative disorders
Blood,
November 15, 2003;
102(10):
3775 - 3785.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Lee, H. J. Lee, J.-H. Kim, H.-S. Lee, J. J. Jang, and G. H. Kang
Aberrant CpG Island Hypermethylation Along Multistep Hepatocarcinogenesis
Am. J. Pathol.,
October 1, 2003;
163(4):
1371 - 1378.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. H. Kang, H. J. Lee, K. S. Hwang, S. Lee, J.-H. Kim, and J.-S. Kim
Aberrant CpG Island Hypermethylation of Chronic Gastritis, in Relation to Aging, Gender, Intestinal Metaplasia, and Chronic Inflammation
Am. J. Pathol.,
October 1, 2003;
163(4):
1551 - 1556.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. V. Brock, M. Gou, Y. Akiyama, A. Muller, T.-T. Wu, E. Montgomery, M. Deasel, P. Germonpre, L. Rubinson, R. F. Heitmiller, et al.
Prognostic Importance of Promoter Hypermethylation of Multiple Genes in Esophageal Adenocarcinoma
Clin. Cancer Res.,
August 1, 2003;
9(8):
2912 - 2919.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Toyooka, K. O. Toyooka, K. Miyajima, J. L. Reddy, M. Toyota, U. G. Sathyanarayana, A. Padar, M. S. Tockman, S. Lam, N. Shivapurkar, et al.
Epigenetic Down-Regulation of Death-associated Protein Kinase in Lung Cancers
Clin. Cancer Res.,
August 1, 2003;
9(8):
3034 - 3041.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kozar, R. Kaminski, T. Switaj, T. Oldak, E. Machaj, P. J. Wysocki, A. Mackiewicz, W. Lasek, M. Jakobisiak, and J. Golab
Interleukin 12-based Immunotherapy Improves the Antitumor Effectiveness of a Low-Dose 5-Aza-2'-Deoxycitidine Treatment in L1210 Leukemia and B16F10 Melanoma Models in Mice
Clin. Cancer Res.,
August 1, 2003;
9(8):
3124 - 3133.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y Kaneko, S Sakurai, M Hironaka, S Sato, S Oguni, Y Sakuma, K Sato, K Sugano, and K Saito
Distinct methylated profiles in Helicobacter pylori dependent and independent gastric MALT lymphomas
Gut,
May 1, 2003;
52(5):
641 - 646.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
O. Galm, H. Yoshikawa, M. Esteller, R. Osieka, and J. G. Herman
SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma
Blood,
April 1, 2003;
101(7):
2784 - 2788.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Sampath, B. Mazumder, V. Seshadri, and P. L. Fox
Transcript-Selective Translational Silencing by Gamma Interferon Is Directed by a Novel Structural Element in the Ceruloplasmin mRNA 3' Untranslated Region
Mol. Cell. Biol.,
March 1, 2003;
23(5):
1509 - 1519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Bhatia, A. K Siraj, A. Hussain, R. Bu, and M. I Gutierrez
The Tumor Suppressor Gene 14-3-3{sigma} Is Commonly Methylated in Normal and Malignant Lymphoid Cells
Cancer Epidemiol. Biomarkers Prev.,
February 1, 2003;
12(2):
165 - 169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Ogi, M. Toyota, M. Ohe-Toyota, N. Tanaka, M. Noguchi, T. Sonoda, G. Kohama, and T. Tokino
Aberrant Methylation of Multiple Genes and Clinicopathological Features in Oral Squamous Cell Carcinoma
Clin. Cancer Res.,
October 1, 2002;
8(10):
3164 - 3171.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S A Pileri, E Sabattini, P Rosito, P L Zinzani, S Ascani, G Fraternali-Orcioni, B Gamberi, M Piccioli, D Vivenza, B Falini, et al.
Primary follicular lymphoma of the testis in childhood: an entity with peculiar clinical and molecular characteristics
J. Clin. Pathol.,
September 1, 2002;
55(9):
684 - 688.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Tada, M. Wada, K.-i. Taguchi, Y. Mochida, N. Kinugawa, M. Tsuneyoshi, S. Naito, and M. Kuwano
The Association of Death-associated Protein Kinase Hypermethylation with Early Recurrence in Superficial Bladder Cancers
Cancer Res.,
July 15, 2002;
62(14):
4048 - 4053.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Majumder, K. Ghoshal, J. Datta, S. Bai, X. Dong, N. Quan, C. Plass, and S. T. Jacob
Role of de Novo DNA Methyltransferases and Methyl CpG-binding Proteins in Gene Silencing in a Rat Hepatoma
J. Biol. Chem.,
May 3, 2002;
277(18):
16048 - 16058.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Stimson and P. M. Vertino
Methylation-mediated Silencing of TMS1/ASC Is Accompanied by Histone Hypoacetylation and CpG Island-localized Changes in Chromatin Architecture
J. Biol. Chem.,
February 8, 2002;
277(7):
4951 - 4958.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Sze Wong, H. Wen Chang, K. Chi Tang, W. Ignace Wei, D. Lai Wen Kwong, J. Shun Tong Sham, A. Po Wing Yuen, and Y. Lam Kwong
High Frequency of Promoter Hypermethylation of the Death-associated Protein-Kinase Gene in Nasopharyngeal Carcinoma and Its Detection in the Peripheral Blood of Patients
Clin. Cancer Res.,
February 1, 2002;
8(2):
433 - 437.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. W. Y. Chan, L. W. Chan, N. L. S. Tang, J. H. M. Tong, K. W. Lo, T. L. Lee, H. Y. Cheung, W. S. Wong, P. S. F. Chan, F. M. M. Lai, et al.
Hypermethylation of Multiple Genes in Tumor Tissues and Voided Urine in Urinary Bladder Cancer Patients
Clin. Cancer Res.,
February 1, 2002;
8(2):
464 - 470.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Esteller, G. Gaidano, S. N. Goodman, V. Zagonel, D. Capello, B. Botto, D. Rossi, A. Gloghini, U. Vitolo, A. Carbone, et al.
Hypermethylation of the DNA Repair Gene O6-Methylguanine DNA Methyltransferase and Survival of Patients With Diffuse Large B-Cell Lymphoma
J Natl Cancer Inst,
January 2, 2002;
94(1):
26 - 32.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Kwong, K.-W. Lo, K.-F. To, P. M. L. Teo, P. J. Johnson, and D. P. Huang
Promoter Hypermethylation of Multiple Genes in Nasopharyngeal Carcinoma
Clin. Cancer Res.,
January 1, 2002;
8(1):
131 - 137.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Shohat, T. Spivak-Kroizman, O. Cohen, S. Bialik, G. Shani, H. Berrisi, M. Eisenstein, and A. Kimchi
The Pro-apoptotic Function of Death-associated Protein Kinase Is Controlled by a Unique Inhibitory Autophosphorylation-based Mechanism
J. Biol. Chem.,
December 7, 2001;
276(50):
47460 - 47467.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Worm, A. F. Kirkin, K. N. Dzhandzhugazyan, and P. Guldberg
Methylation-dependent Silencing of the Reduced Folate Carrier Gene in Inherently Methotrexate-resistant Human Breast Cancer Cells
J. Biol. Chem.,
October 19, 2001;
276(43):
39990 - 40000.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. V. Velentza, A. M. Schumacher, C. Weiss, M. Egli, and D. M. Watterson
A Protein Kinase Associated with Apoptosis and Tumor Suppression. STRUCTURE, ACTIVITY, AND DISCOVERY OF PEPTIDE SUBSTRATES
J. Biol. Chem.,
October 12, 2001;
276(42):
38956 - 38965.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Sun, G. Jiang, M.-A. A. Zaydan, V. F. La Russa, H. Safah, and M. Ehrlich
ABL1 Promoter Methylation Can Exist Independently of BCR-ABL Transcription in Chronic Myeloid Leukemia Hematopoietic Progenitors
Cancer Res.,
September 1, 2001;
61(18):
6931 - 6937.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Dong, H.-S. Kim, S.-H. Rha, and D. Sidransky
Promoter Hypermethylation of Multiple Genes in Carcinoma of the Uterine Cervix
Clin. Cancer Res.,
July 1, 2001;
7(7):
1982 - 1986.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. H. L. Ng, K. W. To, K. W. Lo, S. Chan, K. S. Tsang, S. H. Cheng, and H. K. Ng
Frequent Death-associated Protein Kinase Promoter Hypermethylation in Multiple Myeloma
Clin. Cancer Res.,
June 1, 2001;
7(6):
1724 - 1729.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. B. Baylin and J. G. Herman
Promoter Hypermethylation--Can This Change Alone Ever Designate True Tumor Suppressor Gene Function?
J Natl Cancer Inst,
May 2, 2001;
93(9):
664 - 665.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. F Costello and C. Plass
Methylation matters
J. Med. Genet.,
May 1, 2001;
38(5):
285 - 303.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. Esteller, C. Cordon-Cardo, P. G. Corn, S. J. Meltzer, K. S. Pohar, D. N. Watkins, G. Capella, M. A. Peinado, X. Matias-Guiu, J. Prat, et al.
p14ARF Silencing by Promoter Hypermethylation Mediates Abnormal Intracellular Localization of MDM2
Cancer Res.,
April 1, 2001;
61(7):
2816 - 2821.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
G. Hoon Kang, Y.-H. Shim, H.-Y. Jung, W. Ho Kim, J. Y. Ro, and M.-G. Rhyu
CpG Island Methylation in Premalignant Stages of Gastric Carcinoma
Cancer Res.,
April 1, 2001;
61(7):
2847 - 2851.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. L. Guzman, D. Upchurch, B. Grimes, D. S. Howard, D. A. Rizzieri, S. M. Luger, G. L. Phillips, and C. T. Jordan
Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells
Blood,
April 1, 2001;
97(7):
2177 - 2179.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. L. B. Rosas, W. Koch, M. d. G. d. C. Carvalho, L. Wu, J. Califano, W. Westra, J. Jen, and D. Sidransky
Promoter Hypermethylation Patterns of p16, O6-Methylguanine-DNA-methyltransferase, and Death-associated Protein Kinase in Tumors and Saliva of Head and Neck Cancer Patients
Cancer Res.,
February 1, 2001;
61(3):
939 - 942.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. Zöchbauer-Müller, K. M. Fong, A. K. Virmani, J. Geradts, A. F. Gazdar, and J. D. Minna
Aberrant Promoter Methylation of Multiple Genes in Non-Small Cell Lung Cancers
Cancer Res.,
January 1, 2001;
61(1):
249 - 255.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. L. Hecht and J. C. Aster
Molecular Biology of Burkitt's Lymphoma
J. Clin. Oncol.,
November 1, 2000;
18(21):
3707 - 3721.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. E. Conway, B. B. McConnell, C. E. Bowring, C. D. Donald, S. T. Warren, and P. M. Vertino
TMS1, a Novel Proapoptotic Caspase Recruitment Domain Protein, Is a Target of Methylation-induced Gene Silencing in Human Breast Cancers
Cancer Res.,
November 1, 2000;
60(22):
6236 - 6242.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. B. Baylin, S. A. Belinsky, and J. G. Herman
Aberrant Methylation of Gene Promoters in Cancer--Concepts, Misconcepts, and Promise
J Natl Cancer Inst,
September 20, 2000;
92(18):
1460 - 1461.
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Tang, F. R. Khuri, J. J. Lee, B. L. Kemp, D. Liu, W. K. Hong, and L. Mao
Hypermethylation of the Death-Associated Protein (DAP) Kinase Promoter and Aggressiveness in Stage I Non-Small-Cell Lung Cancer
J Natl Cancer Inst,
September 20, 2000;
92(18):
1511 - 1516.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Liang, K. D. Robertson, C. Talmadge, J. Sumegi, and P. A. Jones
The Gene for a Novel Transmembrane Protein Containing Epidermal Growth Factor and Follistatin Domains Is Frequently Hypermethylated in Human Tumor Cells
Cancer Res.,
September 1, 2000;
60(17):
4907 - 4912.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
A. Aggerholm, P. Hokland, P. Guldberg;, and J. G. Herman
DAP-kinase CpG island methylation in acute myeloid leukemia: methodology versus biology?
Blood,
May 1, 2000;
95(9):
2997 - 2999.
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sanchez-Cespedes, M. Esteller, L. Wu, H. Nawroz-Danish, G. H. Yoo, W. M. Koch, J. Jen, J. G. Herman, and D. Sidransky
Gene Promoter Hypermethylation in Tumors and Serum of Head and Neck Cancer Patients
Cancer Res.,
February 1, 2000;
60(4):
892 - 895.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. Lu, W.-C. Au, W.-S. Yeow, N. Hageman, and P. M. Pitha
Regulation of the Promoter Activity of Interferon Regulatory Factor-7 Gene. ACTIVATION BY INTERFERON AND SILENCING BY HYPERMETHYLATION
J. Biol. Chem.,
October 6, 2000;
275(41):
31805 - 31812.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION