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Department of Radiation Oncology and the Winship Cancer Institute [K. E. C., B. B. M., C. E. B., C. D. D., P. M. V.], Departments of Biochemistry, Genetics, Pediatrics, and the Howard Hughes Medical Institute [S. T. W.], Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Despite the strong correlation between aberrant CpG island methylation and lack of gene expression at tumor suppressor loci in cancer, it remains an open question whether aberrant methylation plays a direct causal role in carcinogenesis. At present, it is not clear how genes progress from an unmethylated, active state to a methylated and inactive one, and it is not known whether it is the methylation per se that drives gene silencing in vivo. Recent studies linking methylation-specific DNA-binding proteins to repressor complexes containing histone deacetyleases and other chromatin-remodeling proteins suggest that aberrant methylation may precipitate gene silencing through induced changes in chromatin structure (9, 10, 11, 12) . Indeed, genes that are aberrantly methylated and repressed cannot be reactivated by inhibition of histone deacetylase alone without prior demethylation, suggesting that methylation may be the primary determinant in the establishment of transcriptional silencing at these loci (13) .
In this study, we sought to identify potential targets of methylation-induced gene silencing by directly screening for genes whose expression is down-regulated in response to aberrant methylation. Although the mechanism underlying aberrant methylation is not currently understood, this event can be induced in human cells by ectopic expression of DNMT14 (14) . We report the identification of TMS1, a novel CpG island-associated gene that becomes aberrantly methylated and silenced in cells overexpressing DNMT1. We further show that TMS1 is silenced in association with aberrant CpG island methylation in human breast cancer. TMS1 encodes a novel CARD protein, and promotes apoptosis in human cells. The data suggest that aberrant methylation can drive gene silencing in vivo and support a new role for methylation-mediated silencing in human breast tumorigenesis: the silencing of genes that act as positive mediators of cell death.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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DNA isolated from primary breast tissues and tumors was generously provided by Dr. Sara Sukumar (The Johns Hopkins University, Baltimore, MD). Primary breast tissue from reduction mammoplasties or from breast tumors was obtained immediately after surgical resection at Johns Hopkins University Hospital or at Duke University and frozen at -80°C. Tumors were estimated to contain at least 50% tumor cells by microscopic examination of representative tissue sections.
Representational Difference Analysis.
Total RNA was isolated from 90SV cells and HMT.1E1 cells by lysis in
guanidium isothiocyanate before acid/phenol extraction and
isopropanol precipitation. Poly(A)+ RNA was selected on oligo-dT
cellulose and used to synthesize double-stranded cDNA using a modified
oligo-dT primer and the cDNA CHOICE system (Life Technologies). Double
stranded cDNA was digested with DpnI, ligated to
linkers, and amplified by PCR using linker-specific primers to generate
the tester (90SV) and driver (HMT.1E1) cDNA amplicons. The cDNA pools
were then subjected to representational difference analysis as
described (15)
. Three rounds of subtraction and enrichment
of tester-only sequences were performed using tester:driver ratios of
1:80, 1:400, and 1:80,000. A predominant 350-bp difference product was
isolated, digested with DpnI, and subcloned into the
BamHI site of pBluescript SK+.
TMS1 Expression Constructs.
The complete TMS1 coding region was subcloned from EST H16108 into
pcDNA3.1 (Invitrogen) to generate pcDNATMS1. pcDNAmycTMS1 was
generated from pcDNATMS1 using PCR to fuse a myc-epitope tag to amino
acid 2 of TMS1.
BAC Isolation and Radiation Hybrid Mapping.
A pooled human BAC library (Release III; Research Genetics) was
screened by PCR using primers in the 3' untranslated region of TMS1
(5'-GCACTTTATAGACCAGCA-3' and 5'-ATTTGGTGGGATTGCCAG-3'), and four
positive BACs were identified. A 6-kb HindIII subclone
containing TMS1 was identified by shotgun cloning and colony
hybridization to the TMS1 cDNA. The same primers were
used in a PCR-based screen of the human/rodent somatic cell hybrid
mapping panel 2 (Coriell Cell Repository) to localize TMS1 to human
chromosome 16. Fine mapping was carried out using radiation hybrid
panels (Stanford RH Panel G3 and Stanford RH panel TNG4; Research
Genetics). Results were analyzed using the Stanford Human Genome Center
RHserver5
and indicated linkage to markers SHGC-35326 on the G3 panel (lod
score, 7.16) and SHGC-61092 on the TNG4 panel (lod score, 4.91).
Methylation-sensitive Restriction and Southern Blot Analysis.
Ten µg of DNA isolated from 90SV or HMT.1E1 cells were digested with
200 units of the methylation-sensitive restriction enzyme
SacII or EagI for 16 h, and then digested
for an additional 16 h with 100 units of the
methylation-insensitive enzyme HindIII. Digested DNA was
separated by electrophoresis on a 1% agarose gel, transferred to a
nylon filter (Zeta-Probe, BioRAD), and hybridized overnight with a
random prime-labeled, 1.8-kb EcoRI TMS1 genomic probe. Blots
were washed to a final stringency of 0.1x SSC-0.1% SDS at 65°C and
exposed to X-ray film using intensifying screens (BioMAX-MS; Kodak).
Northern Blot Analysis.
One µg poly(A)+ selected RNA was fractionated on a 1.5%
agarose/formaldehyde gel, transferred to nylon filters, and hybridized
with a random-prime-labeled full-length TMS1 cDNA fragment. Blots were
washed to a final stringency of 0.1x SSC-0.1% SDS at room temperature
and exposed to X-ray film using an intensifying screen (BioMAX-MS;
Kodak). Blots were stripped and rehybridized with a human ß-actin
cDNA probe.
RT-PCR.
Six µg of total RNA were pretreated with DNase I (Life Technologies)
and reverse-transcribed using random hexamer primers and MMLV-reverse
transcriptase (Life Technologies). One-thirtieth of the
reverse-transcriptase reaction (200 ng of starting RNA) was used
directly in a PCR reaction. The PCR reaction conditions were: 67
mM Tris-HCl (pH 8.8), 16.6 mM
NH4SO4, 6.7
µM EDTA, 10 mM ß-mercaptoethanol, 4.7
mM MgCl2, 10% DMSO, and 400 nM
each primer in a 25-µl reaction. A hot start was performed (5 min,
95°C), followed by the addition of 0.5 unit of Taq
polymerase (Life Technologies) and 35 cycles of PCR (95°C, 30 s;
50–55°C, 60 s; and 72°C, 60 s). TMS1 primers (5'-TGG GCC
TGC AGG AGA TG-3' and 5'-ATT TGG TGG GAT TGC CAG-3') were used at an
annealing temperature of 50°C. ß-actin primers (5'-CCT TCC TGG GCA
TGG AGT CCT G-3' and 5'-GGA GCA ATG ATC TTG ATC TTC-3') were used at an
annealing temperature of 55°C. Reaction products were separated by
electrophoresis on a 6% polyacrylamide/Tris-borate-EDTA gel, stained
with ethidium bromide, and photographed.
Bisulfite Modification and MSP.
Bisulfite modification and MSP were performed as described previously
(16)
. Approximately 50 ng of bisufite-modified DNA was
amplified by PCR with the following reaction conditions: 67
mM Tris-HCl (pH 8.8), 16.6 mM
NH4SO4, 6.7
µM EDTA, 10 mM ß-mercaptoethanol, 6.7
mM MgCl2, and 1 µM each
primer in a 25 µl reaction. A hot start was performed (5 min, 95°C)
followed by the addition of 0.5u Taq polymerase (Life
Technologies) and 35 cycles of PCR (95°C, 30 s; 58°C, 30 s; and 72°C, 30 s). Reaction products were separated by
electrophoresis on a 6% polyacrylamide/Tris-borate-EDTA gel, stained
with ethidium bromide, and photographed. Primers were designed from the
interpolated sequence after bisulfite conversion assuming the DNA was
either methylated or unmethylated at CpG sites. To ensure maximal
discrimination of unmethylated and methylated DNA, primers were
designed to overlap three potential methylation sites. Primers used
were 5'-GGT TGT AGT GGG GTG AGT GGT-3' and 5'-CAA AAC ATC CAT AAA CAA
CAA CAC A-3' for the unmethylated reaction, and 5'-TTG TAG CGG GGT GAG
CGG C-3' and 5'-AAC GTC CAT AAA CAA CAA CGC G-3' for the methylated
reaction.
Colony Formation Assays.
Breast cancer cells (3 x 105
)
were transfected with 1 µg of pcDNA3.1 or TMS1 expression constructs
using 5 µl of Lipofectamine reagent (Life Technologies). Twenty-four
h after transfection, cells were diluted and seeded into medium
containing 400 µg/ml G418. After 14 days of selection, stable
G418-resistant colonies were fixed and stained with 0.25% crystal
violet in 50% methanol and counted. The total number of colonies
recovered per transfection was extrapolated from counting at least 300
G418-resistant colonies per experiment.
Apoptosis Assays.
The 293 human embryonic kidney cells (1 x 105
) plated on glass coverslips in 24-well dishes
were transfected with 0.4 µg of pcDNA3.1 or the TMS1 expression
constructs plus 0.1 µg of ß-galactosidase expression vector
(pCMVßgal; Clonetech) using the calcium phosphate precipitation
method. Where indicated, 40 µM zVADfmk (Enzyme System
Products, Livermore, CA) was included during the recovery period.
Forty-eight h after transfection, coverslips were fixed in 4%
paraformaldehyde and stained for ß-galactosidase activity using X-gal
and for nuclear morphology using Hoechst 33528 dye. Apoptotic cells
were distinguished on the basis of morphological features typical of
adherent cells undergoing apoptosis, including becoming condensed,
rounded, and detached from the growth surface, as described
(17)
. At least 200 ß-galactosidase-positive cells from
randomly selected fields were counted from each transfection.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Sequence analysis and BLAST homology searches of
the GenBank and dbEST databases indicated that RDA-2.15 was derived
from a novel gene represented by multiple human ESTs defining tentative
human consensus (THC253346; TIGR Human Gene Index) and a unique Unigene
cluster (Hs.71869; NCBI Unigene project). A 770-bp
cDNA6
was assembled from the complete sequence of two EST clones (H16108 and
AI368975) that overlapped the entire Unigene cluster. The assembled
cDNA sequence contained a 588-bp open reading frame with 5' and
3' UTRs of 74 bp and 97 bp, respectively, and included the poly(A)
tail. No additional 5' sequence was obtained using a 5' rapid
amplification of cDNA ends protocol (data not shown), indicating that
the cDNA sequence was full-length. The cDNA hybridized to an 
0.8-kb
message, which was expressed at low, but clearly detectable, levels in
IMR90 normal human diploid fibroblasts and their immortalized
derivatives, the 90SV cells (Fig. 1, B and C)
.
The same message was undetectable by Northern blot or by RT-PCR
analysis in the DNMT1-overexpressing cell line, HMT.1E1, confirming
that the gene identified by RDA was silenced in HMT.1E1 cells (Fig. 1, B and C)
. A less abundant mRNA species was also
detected by RT-PCR (see Fig. 1C
) and was found to be an
alternatively spliced form lacking exon 2 (see below). In all studies,
the expression status of the two mRNA forms were concordant. We named
the gene TMS1 for "target of methylation-induced
silencing-1."
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1.4 kb of genomic DNA and was localized to
chromosome 16p11.2–12.1 by radiation hybrid mapping (Fig. 2A)
. An 600-bp CpG island was identified in the 5' end of
TMS1 surrounding exon 1. This region showed a high C + G content (69%), a CpG:GpC ratio of 0.82, and the presence of
multiple sites for methylation-sensitive, CpG-recognizing restriction
enzymes that often cluster within CpG islands (SacII,
EagI, BssHII, SmaI; Ref.
1
; Fig. 2A
). Examination of the methylation
status of the CpG island by methylation-sensitive restriction analysis
in TMS1-expressing (IMR90 and 90SV) and -nonexpressing
(HMT.1E1) cells indicated that silencing of TMS1 correlated
with hypermethylation of the CpG island (Fig. 2B)
. IMR90
normal diploid fibroblasts were unmethylated at SacII and
EagI sites within the TMS1 CpG island. The
TMS1 CpG island exhibited a partially methylated pattern in
the 90SV cells, with both methylated and unmethylated alleles present
in the population (Fig. 2B)
. This result was similar to some
other CpG islands we have studied in this model system which have
accumulated some methylation as a result of immortalization before
insertion of DNMT1 (14)
. In contrast, HMT.1E1
cells exhibited complete methylation of SacII and
EagI sites in the TMS1 CpG island (Fig. 2B)
. The methylation status of the TMS1 CpG
island was analyzed additionally by a more sensitive MSP technique in
which methylated and unmethylated alleles can be specifically amplified
after chemical modification of DNA with sodium bisulfite
(16)
. Data obtained from this analysis (Fig. 2C)
and from direct sequencing of bisulfite-modified DNA
(data not shown) agreed well with that obtained by
methylation-sensitive restriction analysis and confirmed that the IMR90
cells were unmethylated and the 90SV cells were partially methylated,
whereas the HMT.1E1 cells were fully methylated at the TMS1
CpG island. These data indicated that overexpression of
DNMT1 in the HMT.1E1 cells promoted both hypermethylation
and silencing of TMS1.
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. In contrast, four of nine breast
carcinoma cell lines were completely methylated at the TMS1
CpG island locus and did not express TMS1 (Fig. 3, A and B)
. As seen in the fibroblast system,
breast cancer cell lines that were either completely unmethylated
(MCF-7) or only partially methylated (T47D, MDA MB468, and CAMA-1) at
the TMS1 CpG island retained TMS1 expression
(Fig. 3, A and B)
. The only exception was SKBR3
cells which, although only partially methylated, did not express
TMS1, suggesting that these cells are above a critical
threshold level of methylation necessary to affect gene expression.
Alternately, there may be another mechanism for
TMS1-silencing in this cell line. Sequencing of the
TMS1 promoter and coding region in SKBR3 cells did not
reveal any mutations (data not shown).
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. The absence of TMS1 expression in these cell
lines is therefore not attributable to abnormalities at the gene level
or to the inability to express TMS1 (e.g. because
of a lack of necessary transcription factors), but rather is directly
related to the methylation of TMS1.
To determine whether aberrant methylation also played a role in primary
tumors, we examined primary breast tissues for TMS1
methylation. The CpG island of TMS1 was unmethylated in
normal breast tissue derived from reduction mammoplasty (Fig. 4A)
. Likewise, short-term cultures of breast epithelial
organoids derived from reduction mammoplasty (four of four) or benign
fibrocystic disease (two of two) were also completely unmethylated at
TMS1 (data not shown). In contrast, 11 of 27 (40%) primary
breast carcinomas showed aberrant de novo methylation of the
TMS1 CpG island (Fig. 4B)
. In some cases the
degree of methylation was substantial, particularly considering that
resected breast tumors consist of a heterogeneous mixture of tumor
cells and normal stromal and lymphocytic cells. Adjacent
normal-appearing breast tissue was also available for 18 of the tumor
samples. Eight of 18 pairs showed hypermethylation of TMS1
in the tumor relative to adjacent normal tissue (e.g., see
samples 1–7 in Fig. 4C
). Although a few of the adjacent
normal tissue samples showed some methylation of TMS1, these
represented normal-appearing tissue adjacent to a methylated tumor, and
methylated DNA was enriched in the tumor sample relative to the normal
(e.g., see samples 5–8 in Fig. 4C
). Tumors that
were unmethylated at TMS1 were also unmethylated in the
corresponding normal tissue (samples 9–12, Fig. 4C
).
Therefore, aberrant methylation of the TMS1 CpG island in
the breast cancer cell lines reflects aberrant methylation occurring in
primary tumors, and methylation of TMS1 is a frequent event
in human breast cancer.
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That TMS1 was aberrantly methylated and silenced in a
substantial proportion of human breast cancer cells suggested that
TMS1 may play a novel tumor suppressor function.
TMS1 exhibited widespread low-level expression in human
tissues, with the greatest expression in colon, spleen, small
intestine, lung, and peripheral blood leukocytes (Fig. 5A)
. Interestingly, TMS1 was also silent in other
human tumor cell lines, including Molt 4 lymphocytic leukemia cells and
HeLa cervical carcinoma cells (Fig. 5A)
. The cDNA sequence
predicted that TMS1 encodes a protein of 195 amino acids and
22,000 Mr. Through a
combination of BLAST homology searches of GenBank and the SMART
domain recognition database (20)
, we found that TMS1
contained a COOH-terminal CARD (Fig. 5B)
. Similar in
structure and function to the death domain and the death effector
domain, the CARD is a homotypic protein interaction domain found in
proteins that function in the regulation and execution of apoptosis
(21)
. An alignment of TMS1 with other human CARDs
indicated that the TMS1 CARD was most similar to that of the
serine/threonine kinase RICK/CARDIAK/RIP2 (25% identity, 51%
similarity; Refs. 17
, 22
) and the CED-4/apoptotic protease
activating factor-1 family member CARD4/Nod1 (24% identity, 44%
similarity; Refs. 23
, 24
; Fig. 5B
).
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.
Expression of wild-type, or myc-tagged TMS1 induced a 6-fold
increase in apoptosis (Fig. 5C)
. The apoptotic activity of
TMS1 was blocked by the general caspase inhibitor zVAD,
indicating that TMS1-induced cell death requires caspase
activation (Fig. 5C)
. We investigated further the effects of
TMS1 on breast cancer cells. Stable expression of wild-type
or myc-tagged TMS1 in the TMS1-negative Hs578t
cells or MDA MB231 cells reduced the number of surviving colonies by
3–4-fold (Fig. 5D)
. Not surprisingly given the proapoptotic
activity of TMS1, overexpression of TMS1 had similar
growth-inhibitory effects on both TMS1-negative (Hs578t and
MB231) and TMS1-positive (MCF7) breast cancer cells (Fig. 5D)
. Taken together, these data indicate that
TMS1 functions in the promotion of caspase-dependent
apoptosis and overexpression of TMS1 inhibits the growth of
breast cancer cells. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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TMS1 belongs to a growing family of apoptotic signaling molecules that
contain a CARD. The CARD is found within the prodomain of a number of
caspases, and oligomerization with upstream CARD-containing regulatory
proteins mediates their cleavage and activation (21)
.
Other CARD-containing proteins with known roles in apoptosis include
the Caenorhabditis elegans CED-3 and CED-4, the human
homologue of CED-4, apoptotic protease activating factor-1, the
cellular and viral inhibitors of apoptosis, the cellular homologue of
herpesvirus EHV2 E10 protein, BCL10, and several proteins involved in
the activation of NF-
B (28)
. By analogy to other CARDs,
TMS1 is likely to function as an adapter protein, acting in the
initiation phase of an apoptotic pathway by coupling death receptors at
the cell surface or intrinsic death signals to the activation of the
caspase cascade. Consistent with this hypothesis, we show here that
TMS1 promotes apoptosis directly, and that this apoptosis
requires the activation of downstream caspases. Our additional studies
indicate that apoptosis is triggered by a CARD-mediated cytoplasmic
redistribution of TMS1 and requires the activity of
caspase-9 (B. McConnell and P. Vertino; see accompanying article).
Thus, the data are most consistent with a role for TMS1 as a positive
mediator of apoptosis.
Genetic alterations that lead to cellular resistance to apoptosis, such as mutational inactivation of proapoptotic genes such as TP53 and BAX, or translocation and activation of antiapoptotic genes like BCL2, promote tumorigenesis by allowing damaged or unnecessary cells to persist and accumulate additional genetic insult (29) . Our data suggest that epigenetic alterations resulting in the silencing of a proapoptotic gene such as TMS1 could provide a similar survival advantage. Reintroduction of TMS1 inhibited the growth of breast cancer cells, most likely as a result of its proapoptotic activity. Interestingly, this growth-suppressive activity appears to be p53-independent because p53-negative (Hs578t and MB231) and p53-positive (MCF7) cells were similarly affected. Together with the findings of others showing aberrant methylation of the death-associated protein kinase gene in hematological malignancies (30 , 31) and methylation-associated silencing of caspase-8 in neuroblastomas (32) , these data extend the role of aberrant methylation in carcinogenesis to include the silencing of genes that act as positive mediators of cell death.
There is accumulating evidence to suggest that altered function of
CARD-containing regulatory molecules represents an important step in
human carcinogenesis; two other CARD encoding genes, API2
and BCL10, have been identified at translocation breakpoints
in mucosal-associated lymphoid-tissue lymphomas (33, 34, 35)
.
Recently, TMS1 was independently identified (called
"ASC") and shown to be involved in drug-induced
apoptosis in HL-60 cells (36)
. Methylation-mediated
silencing of TMS1 may thus confer resistance to
chemotherapeutic agents or other genotoxic stress. Interestingly, both
our group and Masumoto et al. (36)
noted that a
number of human cell lines from multiple tumor types also lack
TMS1 expression (see Fig. 5A
). Our data are the
first to show the loss of function of a CARD protein through
methylation-mediated silencing and to implicate TMS1 as a
novel tumor suppressor for breast, and perhaps other, cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Grants 1RO1-CA77337
(to P. M. V.) and F32-CA83289 (to B. B. M.) from the National
Cancer Institute. P. M. V. is an Avon Scholar in Breast Cancer
Genomics. S. T. W. is an investigator of the Howard Hughes Medical
Institute. 
2 These authors contributed equally to this work
3 To whom requests for reprints should be
addressed, at Winship Cancer Institute, Room B5119, 1365-B Clifton Road
NE, Atlanta, GA 30322. Phone: (404) 778-3119; Fax: (404) 778-3965;
E-mail: pvertin{at}emory.edu
4 The abbreviations used are: DNMT1, DNA
cytosine-5-methyltransferase-1; BAC, bacterial artificial chromosome;
CARD, caspase recruitment domain; EST, expressed sequence tag; HMEC,
human mammary epithelial cell; MSP, methylation-specific PCR; RT-PCR,
reverse-transcriptase PCR.
5 Internet address:
www-shgc.stanford.edu/RH/rhserver_form2.
6 Sequences appearing herein have been deposited
in GenBank under accession nos. AF184072, AF184073, and AF255794.
Received 7/10/00. Accepted 10/ 3/00.
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D. J. Taxman, J. Zhang, C. Champagne, D. T. Bergstralh, H. A. Iocca, J. D. Lich, and J. P.-Y. Ting Cutting Edge: ASC Mediates the Induction of Multiple Cytokines by Porphyromonas gingivalis via Caspase-1-Dependent and -Independent Pathways J. Immunol., October 1, 2006; 177(7): 4252 - 4256. [Abstract] [Full Text] [PDF]
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 F. S. Sutterwala, Y. Ogura, D. S. Zamboni, C. R. Roy, and R. A. Flavell NALP3: a key player in caspase-1 activation Innate Immunity, August 1, 2006; 12(4): 251 - 256. [Abstract] [PDF]
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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]
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A. Sarkar, M. Duncan, J. Hart, E. Hertlein, D. C. Guttridge, and M. D. Wewers ASC Directs NF-{kappa}B Activation by Regulating Receptor Interacting Protein-2 (RIP2) Caspase-1 Interactions. J. Immunol., April 15, 2006; 176(8): 4979 - 4986. [Abstract] [Full Text] [PDF]
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A. Loriot, E. De Plaen, T. Boon, and C. De Smet Transient Down-regulation of DNMT1 Methyltransferase Leads to Activation and Stable Hypomethylation of MAGE-A1 in Melanoma Cells J. Biol. Chem., April 14, 2006; 281(15): 10118 - 10126. [Abstract] [Full Text] [PDF]
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J. M. Teodoridis, J. Hall, S. Marsh, H. D. Kannall, C. Smyth, J. Curto, N. Siddiqui, H. Gabra, H. L. McLeod, G. Strathdee, et al. CpG Island Methylation of DNA Damage Response Genes in Advanced Ovarian Cancer Cancer Res., October 1, 2005; 65(19): 8961 - 8967. [Abstract] [Full Text] [PDF]
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 R. van Doorn, W. H. Zoutman, R. Dijkman, R. X. de Menezes, S. Commandeur, A. A. Mulder, P. A. van der Velden, M. H. Vermeer, R. Willemze, P. S. Yan, et al. Epigenetic Profiling of Cutaneous T-Cell Lymphoma: Promoter Hypermethylation of Multiple Tumor Suppressor Genes Including BCL7a, PTPRG, and p73 J. Clin. Oncol., June 10, 2005; 23(17): 3886 - 3896. [Abstract] [Full Text] [PDF]
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 A. A. Melnikov, R. B. Gartenhaus, A. S. Levenson, N. A. Motchoulskaia, and V. V. Levenson (Chernokhvostov) MSRE-PCR for analysis of gene-specific DNA methylation Nucleic Acids Res., June 8, 2005; 33(10): e93 - e93. [Abstract] [Full Text] [PDF]
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M. Hasegawa, R. Imamura, T. Kinoshita, N. Matsumoto, J. Masumoto, N. Inohara, and T. Suda ASC-mediated NF-{kappa}B Activation Leading to Interleukin-8 Production Requires Caspase-8 and Is Inhibited by CLARP J. Biol. Chem., April 15, 2005; 280(15): 15122 - 15130. [Abstract] [Full Text] [PDF]
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 Q. Feng, A. Balasubramanian, S. E. Hawes, P. Toure, P. S. Sow, A. Dem, B. Dembele, C. W. Critchlow, L. Xi, H. Lu, et al. Detection of Hypermethylated Genes in Women with and Without Cervical Neoplasia J Natl Cancer Inst, February 16, 2005; 97(4): 273 - 282. [Abstract] [Full Text] [PDF]
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 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]
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M. G. Friedrich, D. J. Weisenberger, J. C. Cheng, S. Chandrasoma, K. D. Siegmund, M. L. Gonzalgo, M. I. Toma, H. Huland, C. Yoo, Y. C. Tsai, et al. Detection of Methylated Apoptosis-Associated Genes in Urine Sediments of Bladder Cancer Patients Clin. Cancer Res., November 15, 2004; 10(22): 7457 - 7465. [Abstract] [Full Text] [PDF]
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 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]
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 A. R. Stone, W. Bobo, D. J. Brat, N. S. Devi, E. G. Van Meir, and P. M. Vertino Aberrant Methylation and Down-Regulation of TMS1/ASC in Human Glioblastoma Am. J. Pathol., October 1, 2004; 165(4): 1151 - 1161. [Abstract] [Full Text] [PDF]
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 C. Stehlik and J. C. Reed The PYRIN Connection: Novel Players in Innate Immunity and Inflammation J. Exp. Med., September 7, 2004; 200(5): 551 - 558. [Abstract] [Full Text] [PDF]
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M. Alaminos, V. Davalos, N.-K. V. Cheung, W. L. Gerald, and M. Esteller Clustering of Gene Hypermethylation Associated With Clinical Risk Groups in Neuroblastoma J Natl Cancer Inst, August 18, 2004; 96(16): 1208 - 1219. [Abstract] [Full Text] [PDF]
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T. Kubo, J. Yamamoto, Y. Shikauchi, Y. Niwa, K. Matsubara, and H. Yoshikawa Apoptotic Speck Protein-Like, a Highly Homologous Protein to Apoptotic Speck Protein in the Pyrin Domain, Is Silenced by DNA Methylation and Induces Apoptosis in Human Hepatocellular Carcinoma Cancer Res., August 1, 2004; 64(15): 5172 - 5177. [Abstract] [Full Text] [PDF]
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 K. Raymond, E. Bergeret, A. Avet-Rochex, R. Griffin-Shea, and M.-O. Fauvarque A screen for modifiers of RacGAP(84C) gain-of-function in the Drosophila eye revealed the LIM kinase Cdi/TESK1 as a downstream effector of Rac1 during spermatogenesis J. Cell Sci., June 1, 2004; 117(13): 2777 - 2789. [Abstract] [Full Text] [PDF]
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 Y. Wang, M. Hasegawa, R. Imamura, T. Kinoshita, C. Kondo, K. Konaka, and T. Suda PYNOD, a novel Apaf-1/CED4-like protein is an inhibitor of ASC and caspase-1 Int. Immunol., June 1, 2004; 16(6): 777 - 786. [Abstract] [Full Text] [PDF]
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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]
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 C Cazeneuve, S Papin, I Jeru, P Duquesnoy, and S Amselem Subcellular localisation of marenostrin/pyrin isoforms carrying the most common mutations involved in familial Mediterranean fever in the presence or absence of its binding partner ASC J. Med. Genet., March 1, 2004; 41(3): e24 - 24. [Full Text] [PDF]
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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]
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S. Papin, C. Cazeneuve, P. Duquesnoy, I. Jeru, D. Sahali, and S. Amselem The Tumor Necrosis Factor {alpha}-dependent Activation of the Human Mediterranean Fever (MEFV) Promoter Is Mediated by a Synergistic Interaction between C/EBP{beta} and NF{kappa}B p65 J. Biol. Chem., December 5, 2003; 278(49): 48839 - 48847. [Abstract] [Full Text] [PDF]
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C. Stehlik, S. H. Lee, A. Dorfleutner, A. Stassinopoulos, J. Sagara, and J. C. Reed Apoptosis-Associated Speck-Like Protein Containing a Caspase Recruitment Domain Is a Regulator of Procaspase-1 Activation J. Immunol., December 1, 2003; 171(11): 6154 - 6163. [Abstract] [Full Text] [PDF]
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C. Stehlik, L. Fiorentino, A. Dorfleutner, J.-M. Bruey, E. M. Ariza, J. Sagara, and J. C. Reed The PAAD/PYRIN-Family Protein ASC Is a Dual Regulator of a Conserved Step in Nuclear Factor {kappa}B Activation Pathways J. Exp. Med., December 16, 2002; 196(12): 1605 - 1615. [Abstract] [Full Text] [PDF]
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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]
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 J. Masumoto, S.'i. Taniguchi, J. Nakayama, M. Shiohara, E. Hidaka, T. Katsuyama, S. Murase, and J. Sagara Expression of Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain, a Pyrin N-terminal Homology Domain-containing Protein, in Normal Human Tissues J. Histochem. Cytochem., October 1, 2001; 49(10): 1269 - 1276. [Abstract] [Full Text] [PDF]
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J. F Costello and C. Plass Methylation matters J. Med. Genet., May 1, 2001; 38(5): 285 - 303. [Abstract] [Full Text]
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R. Dammann, G. Yang, and G. P. Pfeifer Hypermethylation of the CpG Island of Ras Association Domain Family 1A (RASSF1A), a Putative Tumor Suppressor Gene from the 3p21.3 Locus, Occurs in a Large Percentage of Human Breast Cancers Cancer Res., April 1, 2001; 61(7): 3105 - 3109. [Abstract] [Full Text]
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M. Esteller, P. G. Corn, S. B. Baylin, and J. G. Herman A Gene Hypermethylation Profile of Human Cancer Cancer Res., April 1, 2001; 61(8): 3225 - 3229. [Abstract] [Full Text]
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B. B. McConnell and P. M. Vertino Activation of a Caspase-9-mediated Apoptotic Pathway by Subcellular Redistribution of the Novel Caspase Recruitment Domain Protein TMS1 Cancer Res., November 1, 2000; 60(22): 6243 - 6247. [PDF]
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