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Gene Is Associated with 5' CpG Island Hypermethylation in Human Cancers1
First Department of Internal Medicine, Sapporo Medical University, Sapporo 060-8543, Japan
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ABSTRACT |
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, was shown to be regulated by
p53 and to play a role in the G2-M-phase
checkpoint. To determine whether 14-3-3 is inactivated
in human cancers, the methylation status of the 5' region of
14-3-3 was investigated in a series of gastric,
colorectal, and hepatocellular cancer cell lines. Of 22 cell lines
examined, 6 showed aberrant methylation. The methylation status of
14-3-3 was found to be correlated with loss of
expression, which was restored by 5-aza-2'-deoxycytidine treatment.
Furthermore, normal G2 arrest after DNA damage was not
demonstrated in the cell lines with methylation. In primary gastric
cancers, 14-3-3 hypermethylation was observed frequently
in 26 of 60 (43%) cases and observed more frequently in poorly
differentiated adenocarcinomas (P = 0.0017). Our
findings suggest that 14-3-3 is inactivated by aberrant
methylation of the 5' region in various human cancers and that it might
play an important role in the development of undifferentiated gastric
cancers. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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isoform of 14-3-3
(also called stratifin or HME-1) is expressed in keratinocytes
(5)
and epithelial cells (6)
.
14-3-3 has been shown to be associated with growth
regulation and signal transduction (6
, 7)
as well as with
cellular dedifferentiation (8)
, although its function has
not been elucidated. Recently, Hermeking et al.
(9)
reported that expression of 14-3-3 mRNA
was induced in a p53-dependent manner in response to DNA
damage caused by 
-irradiation and DNA-damaging agents, and
overexpression of 14-3-3 blocks the cell cycle at the
G2 phase. More recently, 14-3-3 has been
shown to prevent mitotic catastrophe, indicating that
14-3-3 plays an important role in the G2
checkpoint (10)
. Despite the putative tumor suppressor
activity of 14-3-3, mutations or loss of the gene in human cancers
have not been reported. In the present study, we investigated the
aberrant methylation of the 5' region of the 14-3-3 gene
in a series of
GC,3
CRC, and HCC cell lines. Six of the cell lines showed aberrant
methylation, and the methylation correlated well with the loss of
14-3-3 mRNA expression. Methylation of
14-3-3 was also observed in primary GCs, and a strong
correlation between methylation and a poorly differentiated
histological phenotype was observed. To investigate methylation, we
performed a combination of bisulfite treatment and fluorescence
PCR-SSCP. PCR products from bisulfite-treated DNA were electrophoresed
in nondenaturing polyacrylamide gels, and methylated and unmethylated
alleles were readily detected due to the different mobility caused by
different conformations (11)
. This novel procedure can
provide information about the methylation level of the gene and is
quite easy to adapt to a large number of samples. |
Materials and Methods |
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Bisulfite Modification.
Bisulfite modification was performed as described previously
(12)
. Briefly, approximately 2 µg of genomic DNA were
denatured in 0.2 M NaOH for 10 min. Sodium bisulfite
(Sigma, St. Louis, MO) was added to a final concentration of 3.1
M, and hydroquinone (Sigma) was added to a final
concentration of 0.5 mM. The reaction was performed at
50°C for 16 h. Modified DNA was purified using Wizard DNA
purification resin (Promega) according to the manufacturer’s
recommendations and eluted into 50 µl of water. Modification was
completed by NaOH (final concentration, 0.3 M) treatment
for 5 min at room temperature, followed by ethanol precipitation.
Bisulfite Genomic Sequencing Analysis.
Sodium bisulfite-modified genomic DNA was PCR amplified by using
two primer pairs specific for the 5' region of the 14-3-3
gene: (a) the sequence that includes the transcription start
site (region 1); and (b) the subsequent sequence (region 2).
Region 1 corresponds to -220 to +116, and region 2 corresponds to +93
to +350 (Fig. 1A
). PCR reactions were performed in a volume
of 50 µl containing 1 x PCR buffer (Takara, Tokyo,
Japan), 0.25 mM dNTP, 1 µM of each primer,
and 2.5 units of Taq polymerase (Takara). PCR conditions were 95°C
for 5 min and 35 cycles at 95°C for 30 s, 53°C for 30 s,
and 72°C for 30 s for region 1 amplification and 35 cycles at
95°C for 30 s, 56°C for 30 s, and 72°C for 30 s
for region 2. In all PCRs, polymerase was added after the heat block
had reached 95°C to effect a hot star of the amplification. Primer
sequences are as follows: (a) 5'-AAAGGTGTTAGTGTAGGTGGGGTT-3'
(region 1, sense primer); (b) 5'-CCTACTCTACCAACTTAACCTTCT-3'
(region 1, antisense primer); (c)
5'-AGAAGGTTAAGTTGGTAGAGTAGG-3' (region 2, sense primer); and
(d) 5'-CCTAAAACTCAATCTCCACCTTCTC-3' (region 2,
antisense primer). Amplified PCR products were cloned by using the
pGEM-T Easy Vector System (Promega, Madison, WI). Plasmid DNAs were
purified with the QIAfilter Midiplep Kit (Qiagen, Hilden, Germany).
Sequence reactions were performed by using the Auto Read Sequencing Kit
(Pharmacia Biotech, Uppsala, Sweden). Samples were loaded onto 6%
urea-polyacrylamide denaturing gels in an ALF express automated DNA
sequencer (Pharmacia Biotech). Five to 10 clones were sequenced for
each cell line.
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gene were
PCR amplified with the same primer sequences described above, of which
each sense primer was end-labeled with Cy5. One µl of fluorescent
products was diluted with 10 µl of loading dye (95% formamide, 20
mM EDTA, 0.05% xylene cyanol, and 0.05% bromphenol blue),
heat denatured for 5 min at 95°C, cooled on ice for 5 min, and loaded
onto 5% polyacrylamide nondenaturing gel (99:1 acrylamide to
bisacrylamide) containing 5% glycerol in an ALF express automated DNA
sequencer (Pharmacia Biotech). Electrophoresis was performed for 400
min, and during electrophoresis, the gel was kept at 30°C with a
circulator instrument. Obtained data were analyzed using Fragment
Manager Software (Pharmacia Biotech). Details of the protocol are as
reported previously (11)
.
Northern Blot Analysis.
A 375-bp probe specific for the 3' region of the 14-3-3
gene was generated by PCR amplification as described previously
(9)
. The PCR product was cloned and sequenced, and it was
verified to be consistent with 14-3-3. The probe was
labeled with 32P dCTP by using the Random Primer Labeling
Kit (Takara) and hybridized at 42°C, followed by analysis using an
autoradiograph instrument (BAS2000; Fuji Film, Tokyo, Japan).
5-Aza-2'-deoxycytidine Treatment.
Cell lines that did not express 14-3-3 were treated with
5-aza-2'-deoxycytidine (Sigma) that was dissolved in cold RPMI1640
immediately before use. Cells were grown in a medium containing 5
µM 5-aza-2'-deoxycytidine for 5 days, and the medium and
drug were replaced every 24 h.
mRNA-selective PCR.
RT-PCR for 14-3-3 was performed by using the
mRNA-selective PCR kit (Takara). RT reactions were performed in a
volume of 50 µl containing 1 x mRNA-selective PCR
buffer, 5 mM MgCl2, 1 mM
dNTP/analogue mixture, 0.8 unit/µl RNase inhibitor, 0.1 unit/µl
avian myeloblastosis virus reverse transcriptase XL, 1 µM
oligo (dT) primer, and 1 µg of total RNA. The RT conditions were
30°C for 10 min, 42°C for 30 min, and 5°C for 5 min. Subsequent
PCR reactions were performed in a volume of 50 µl containing
1 x mRNA-selective PCR buffer, 5 mM
MgCl2, 1 mM dNTP/analogue mixture, 0.1
unit/µl avian myeloblastosis virus-optimized Taq, 0.4
µM of each primer, and 10 µl of the RT reactant. The
PCR conditions were 25 cycles at 85°C for 1 min, 58°C for 1 min,
and 72°C for 1 min. Because cDNA generated by the RT reaction
contained dNTP analogues and was denatured at 85°C, contaminating
genomic DNA should not be amplified in the PCR reaction. For
14-3-3 mRNA amplification, the same primer sequences used
for generating the Northern blot probe were used. The integrity of RNA
was evaluated by amplification of human GAPDH mRNA. Primer
sequences for GAPDH were 5'-CAGCCGAGCCACATCG-3' (sense) and
5'-TGAGGCTGTTGTCATACTTCTC-3' (antisense).
Genomic PCR Amplification.
Twenty-two cell lines listed above were examined for mutation by DNA
sequencing. The PCR reaction was performed in a volume of 50 µl
containing 1 x PCR buffer, 0.25 mM dNTP, 1
µM of each primer, and 2.5 units of Taq polymerase at
95°C for 5 min and 35 cycles at 95°C for 1 min, 68°C for 1 min,
and 72°C for 1 min. Primer sequences specific for the
14-3-3 gene were
5'-AGAGACACAGAGTCCGGCATTGGTCCCAGGCAGCA-3' (sense) and
5'-ACCCCATACTAGTCCTCTCGGCAGGGTGGGGGACT-3' (antisense). Amplified
PCR products were cloned by using the pGEM-T Easy Vector System
(Promega) and sequenced as described above.
Flow Cytometry Analysis.
Cells were rinsed in PBS buffer, trypsinized, collected by
centrifugation, and stained by propidium iodide (Sigma). Flow cytometry
was performed with a FACScan instrument (Becton Dickinson, San Jose,
CA).
Mutation Analysis of p53.
To detect mutations of p53, SSCP was performed for exons
5–8. PCR was performed as described previously (13)
.
Sense primers were end labeled with Cy5. For SSCP, 1 µl of PCR
products was mixed with 10 µl of loading buffer, denatured at 95°C
for 5 min, cooled on ice for 5 min, and electrophoresed in 5%
polyacrylamide nondenaturing gels as described above. The presence of
abnormally migrating bands was confirmed by four different conditions
(with 5% glycerol at 15°C, 20°C, 25°C, and 30°C).
Statistical Analysis.
Statistical comparisons were performed using Fisher’s exact test.
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Results |
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5' CpG Island in
Tumor Cell Lines. gene has a CpG island that spans approximately
850 bp. This area has a 65% GC content, and the CpG:GpC ratio is 0.53,
which fulfills the criteria for a CpG island (14)
. This
island is unique in the sense that the transcription start site is
located at the 5' edge of the island, and the most CpG-rich area is in
the coding region of the gene. We investigated the methylation status
of the 5' region of 14-3-3 in 7 GC, 8 CRC, and 7 HCC
cancer cell lines. Genomic DNA extracted from the cell lines was
treated with sodium bisulfite, followed by PCR amplification of the 5'
region of the 14-3-3 gene. Because unmethylated cytosine
residues were converted to thymine, whereas methylated cytosine
residues were resistant to bisulfite modification, different
sequences should be created according to the methylation status. The 5'
region that encompasses the transcription start site and the subsequent
region (regions 1 and 2 in Fig. 1A
) were amplified and analyzed by bisulfite-SSCP (Fig. 1B
). Two
GC cell lines (MKN28 and MKN74), one CRC cell line (Colo320DM), and
three HCC cell lines (Chang, HLE, and HuH7) showed aberrant peaks
compared with other cell lines. The PCR products were cloned, and
cumulative data derived from sequence information from multiple clones
revealed that the 5' CpG island of 14-3-3 was densely
methylated in these cell lines except for MKN28, which was
heterogeneously methylated (Fig. 1C
).
Expression and Genomic Sequence Analysis of 14-3-3.
We investigated the expression level of the 14-3-3 mRNA
in 8 GC and 5 HCC cell lines (Fig. 2A
). Five cell lines methylated for 14-3-3 showed no or only a
negligible level of expression. Seven cell lines that are not
methylated expressed 14-3-3 at various levels. To
demonstrate the reexpression of 14-3-3 mRNA by a
demethylating agent, we performed mRNA-selective PCR. Reduced
expression and lack of expression were found in MKN28 and MKN74,
respectively, and the expression of 14-3-3 was restored
in both cell lines after 5-aza-2'-deoxycytidine treatment (Fig. 2B
). Demethylation of regions 1 and 2 was observed by
bisulfite-SSCP and bisulfite sequencing, confirming that loss of
expression is associated with the 5' CpG island hypermethylation (Fig. 2C
; data not shown). PCR amplifications specific for the
genomic coding region revealed that no deletion of the
14-3-3 gene was detected in the 7 GC and 7 HCC cell lines
(data not shown). The PCR products were cloned and sequenced, and no
mutation was demonstrated.
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in Primary Gastric
Cancers. were examined in a
series of 60 GC cases by bisulfite-SSCP. Tissue samples that showed the
same peaks as those of methylated cell lines were presumed to have
14-3-3 methylation (see the examples in Fig. 3A
). Twenty-six of 60 (43%) cases showed aberrant methylation of regions 1
and 2. Five additional cases (two poorly and three moderately
differentiated carcinomas) showed methylation only in region 1. We also
performed bisulfite sequencing for 5 of the 26 methylated cases and
obtained concordant results (data not shown). We then analyzed the
correlation of 14-3-3 methylation and the
clinicopathological features of the tumors. For statistical analysis,
we defined those tumors with both region 1 and 2 methylation as
methylated cases. Methylation of 14-3-3 was found
preferentially in undifferentiated tumor types (1 of 12
well-differentiated adenocarcinomas versus 17 of 31 poorly
differentiated adenocarcinomas, P = 0.0017;
Fig. 3B
). The mean age of patients with methylated tissues
was 59.37 ± 7.95 years, and the mean age of patients
without methylation was 69.50 ± 8.52 years,
demonstrating that 14-3-3 methylation is not merely an
age-related phenomenon. Because good-quality RNA was not available from
our primary tumor samples, we investigated 14-3-3 mRNA
expression in five GC xenograft tissues established in our laboratory.
Examples of the methylation analysis of the xenografts are also shown
in Fig. 3A
. To avoid amplification from contaminated genomic
DNA, we performed mRNA-selective PCR. As shown in Fig. 3C
,
all three xenograft tissues with 14-3-3 methylation
showed no expression, and two xenografts without 14-3-3
methylation expressed the gene.
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). Five cases that had
no methylation in the cancer tissues did not show 14-3-3
methylation in their noncancerous gastric tissues. In the remaining
five cases with methylated GC tissues, a slight amount of methylation
was observed in their noncancerous tissues, although the methylation
levels were lower than those seen in the cancer tissues.
A small number of CRC tissue samples (n = 17)
were also investigated. However, we found no methylation of
14-3-3 in these cases (data not shown).
14-3-3 Expression and Cell Cycle Analysis.
It was shown previously that loss of the 14-3-3 gene is
correlated with impairment of the G2 checkpoint in CRC cell
lines (9
, 10) . To elucidate the role of aberrant
methylation of 14-3-3 in GC cell lines, we performed cell
cycle analyses in the cell lines with and without 14-3-3
methylation. The cell lines examined were MKN45 (p53 wild
type and 14-3-3 unmethylated), MKN7 (p53
mutated and 14-3-3 unmethylated), MKN74 (p53
wild type and 14-3-3 methylated), and MKN28
(p53 mutated and 14-3-3 methylated). The cell
lines were treated with 0.25 µg/ml Adriamycin, followed by Northern
blot and flow cytometry analyses at 0–72 h after treatment. As shown
in Fig. 4 and B
, strong induction of the
14-3-3 mRNA and accumulation in G2 phase was
demonstrated in MKN45. MKN7 also showed increased expression of
14-3-3 at 12 and 24 h after Adriamycin treatment and
showed partial accumulation in G2 phase. MKN28 did not
express 14-3-3 and did not show G2
accumulation until 72 h after treatment. MKN74 was quite fragile
to Adriamycin treatment. A small portion of MKN74 cells began to
accumulate in the G2 phase, but apoptosis occurred in most
of the cells, and they became extinct after 72 h (Fig. 4B
).
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in GC cells, MKN28 and MKN74 were treated with
5-aza-2'-deoxycytidine, followed by treatment with Adriamycin (Fig. 4C
). After treatment with 5-aza-2'-deoxycytidine, the
14-3-3 mRNA was reexpressed, and the population of cells
in G2 phase after Adriamycin treatment was increased (13%
to 29% in MKN28 and 28% to 39% in MKN74), indicating that
G2 arrest induced by DNA damage was partially restored in
these cell lines. These results indicated the involvement of
14-3-3 methylation in the impairment of the
G2 checkpoint. Consistent results were obtained by the
independent, repeated experiments.
Association Between 14-3-3 Methylation and
p53 Mutation.
To elucidate whether methylation of the 14-3-3 gene plays
a role in inactivating the p53-dependent pathway, we examined the
correlation between 14-3-3 methylation and p53
mutations. Four of five cell lines with 14-3-3
methylation had p53 mutations (MKN74, wild type
p53; MKN28, Colo320DM, HLE, and HuH7, mutated
p53), and six of seven cell lines without
14-3-3 methylation had p53 mutations (MKN45,
wild-type p53; MKN7, NUGC3, DLD1, HT29, Colo201, and
PLC/PRF/5, mutated p53). There was no difference in the
frequency of p53 mutations between methylated and
unmethylated cell lines. However, in primary GCs, p53
mutations were found at a lower frequency in 14-3-3
methylated cases (1 of 16 versus 6 of 16,
P = 0.03; data not shown).
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Discussion |
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was detected
in cell lines from various tissue types and in a subset of primary GC
tissues. Aberrant methylation of the 5' region of 14-3-3
correlated well with loss of expression, and treatment with the
methyltransferase inhibitor 5-aza-2'-deoxycytidine induced
demethylation and reexpression of the gene. Loss of expression of
14-3-3 mRNA was also found in xenografts from GC with
methylation, suggesting that aberrant methylation of the 5' CpG island
of 14-3-3 plays a role in silencing this gene.
Much evidence has shown that 14-3-3 proteins are involved in
G2-M-phase progression as well as in signal transductions
that lead to apoptosis, and it is strongly postulated that the
inactivation of 14-3-3 might play an important role in
tumor progression. Ostergaard et al. (8)
showed
that less-differentiated bladder squamous cell carcinoma is
characterized by decreased expression of some proteins, including
14-3-3. In the present study, 14-3-3
methylation was frequently observed in poorly differentiated
adenocarcinomas. These results suggest that decreased expression of
14-3-3 is associated with the development of
undifferentiated GCs. Recently, Melis and White (15)
also
demonstrated decreased expression of 14-3-3 in colonic
polyps. Thus, 14-3-3 inactivation may be widely seen in
human neoplasias of various origins, although the association between
inactivation and the role in cancer development should be further
investigated. Because methylation of multiple CpG islands has often
been observed in a subset of CRCs (16)
and GCs
(17)
, methylation of 14-3-3 may be involved
in a "hypermethylator phenotype." In fact, GC cases with
14-3-3 methylation showed methylation of several other
loci, including p16, hMLH1, and
E-cadherin.4
It is also interesting that undifferentiated GC rarely showed
p53 mutations, and this could be partially explained by
epigenetic inactivation of 14-3-3, which is one of the
downstream target genes of p53 (9)
.
Inactivation of 14-3-3 by methylation in cancers may lead
to impairment of part of the p53 function. Recent studies
have indicated that aberrant G2 checkpoint control causes
genetic instability in neoplasia (18)
. Cahill et
al. (19)
demonstrated mutations of mitotic checkpoint
genes, hBUB1 and hBUBR1, in CRCs with CIN.
Although a high proportion of human cancers are likely to have a CIN
phenotype, mutations of the genes involving the G2-M-phase
checkpoint have rarely been found, and the mechanism of CIN in most of
them is still unknown. It is not clear whether 14-3-3
inactivation can cause genetic instability in cancers, but two GC cell
lines with 14-3-3 methylation showed the CIN phenotype by
fluorescence in situ hybridization analysis (data not
shown). Further study on the correlation between genetic instability
and 14-3-3 is needed.
From the results of the flow cytometry analysis, after treatment
with a DNA-damaging agent, the G2 checkpoint appears to be
impaired in cell lines with 14-3-3 methylation. MKN74,
which showed methylation of 14-3-3, failed to arrest
at G2-M phase and underwent apoptosis, which seems to be
consistent with the findings in a recent study by Chan et
al. (10)
In contrast, MKN45 and MKN7, which did not
show methylation of 14-3-3, arrested at G2,
indicating that DNA-damaging agents are more effective in cancers with
14-3-3 methylation. MKN7 (with mutated p53) initially
showed a slight increase in 14-3-3 mRNA expression after
treatment but subsequently showed reduced expression and incomplete
G2 arrest. The incomplete induction of 14-3-3
in MKN7 seems to be due to the impairment of p53 function.
These results indicated that 14-3-3 inactivation could be
achieved by either mutation of p53 or methylation of
14-3-3. Pretreatment with 5-aza-2'-deoxycytidine before
Adriamycin treatment was performed to analyze the effect of the
reexpressed 14-3-3 gene in the methylated cells.
Restoration of G2 arrest by DNA damage after
5-aza-2'-deoxycytidine treatments was present in the cell lines.
However, the genes induced by 5-aza-2'-deoxycytidine are not
limited to 14-3-3, and the involvement of many unknown
genes that were inactivated by demethylation should be considered;
further study is necessary to analyze this issue from different
aspects.
In conclusion, we demonstrated that inactivation of the
14-3-3 gene is associated with 5' CpG island
hypermethylation in human cancers. To our knowledge, this is the first
report on epigenetic silencing of the G2 checkpoint gene in
human neoplasia. Our results indicate that epigenetic changes can
induce further genetic alterations. GC cases with 14-3-3
methylation may be a good target for treatment with chemotherapeutic
agents. Although much remains to be clarified regarding aberrant
methylation of 14-3-3, further study should shed light on
the mechanism of cancer development as well as more effective cancer
therapies.
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Acknowledgments |
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FOOTNOTES |
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1 Supported by a grant-in-aid from the Ministry of
Education, Science, Sports and Culture (to F. I. and K. I.). M. T.
was a postdoctoral fellow from Japan Society for the Promotion of
Science. H. S. and F. I. contributed equally to this work. 
2 To whom requests for reprints should be
addressed, at First Department of Internal Medicine, Sapporo Medical
University, S-1, W-16, Chuo-ku, Sapporo, 060-8543, Japan. Fax:
81-011-613-1141; E-mail: fitoh{at}sapmed.ac.jp
3 The abbreviations used are: GC, gastric cancer;
CRC, colorectal cancer; HCC, hepatocellular cancer; SSCP, single-strand
conformational polymorphism; RT, reverse transcription; dNTP,
deoxynucleotide triphosphate; CIN, chromosomal instability.
4 H. Suzuki et al., Concordant
methylation of multiple genes in gastric cancer, manuscript in
preparation.
Received 12/10/99. Accepted 6/26/00.
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REFERENCES |
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is a p53-regulated inhibitor of G2/M progression. Mol. Cell, 1: 3-11, 1997.[Medline]
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 J. L. Ramirez, R. Rosell, M. Taron, M. Sanchez-Ronco, V. Alberola, R. de las Penas, J. M. Sanchez, T. Moran, C. Camps, B. Massuti, et al. 14-3-3{sigma} Methylation in Pretreatment Serum Circulating DNA of Cisplatin-Plus-Gemcitabine-Treated Advanced Non-Small-Cell Lung Cancer Patients Predicts Survival: The Spanish Lung Cancer Group J. Clin. Oncol., December 20, 2005; 23(36): 9105 - 9112. [Abstract] [Full Text] [PDF]
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K. Ito, T. Suzuki, J.-i. Akahira, M. Sakuma, S. Saitou, S. Okamoto, H. Niikura, K. Okamura, N. Yaegashi, H. Sasano, et al. 14-3-3{sigma} in Endometrial Cancer-A Possible Prognostic Marker in Early-Stage Cancer Clin. Cancer Res., October 15, 2005; 11(20): 7384 - 7391. [Abstract] [Full Text] [PDF]
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 E. W. Wilker, R. A. Grant, S. C. Artim, and M. B. Yaffe A Structural Basis for 14-3-3{sigma} Functional Specificity J. Biol. Chem., May 13, 2005; 280(19): 18891 - 18898. [Abstract] [Full Text] [PDF]
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A. Perathoner, D. Pirkebner, G. Brandacher, G. Spizzo, S. Stadlmann, P. Obrist, R. Margreiter, and A. Amberger 14-3-3{sigma} Expression Is an Independent Prognostic Parameter for Poor Survival in Colorectal Carcinoma Patients Clin. Cancer Res., May 1, 2005; 11(9): 3274 - 3279. [Abstract] [Full Text] [PDF]
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J. M. A. Moreira, G. Ohlsson, F. E. Rank, and J. E. Celis Down-regulation of the Tumor Suppressor Protein 14-3-3{sigma} Is a Sporadic Event in Cancer of the Breast Mol. Cell. Proteomics, April 1, 2005; 4(4): 555 - 569. [Abstract] [Full Text] [PDF]
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 E Poschl, A Fidler, B Schmidt, A Kallipolitou, E Schmid, and T Aigner DNA methylation is not likely to be responsible for aggrecan down regulation in aged or osteoarthritic cartilage Ann Rheum Dis, March 1, 2005; 64(3): 477 - 480. [Abstract] [Full Text] [PDF]
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A. Guweidhi, J. Kleeff, N. Giese, J. E. Fitori, K. Ketterer, T. Giese, M. W. Buchler, M. Korc, and H. Friess Enhanced expression of 14-3-3sigma in pancreatic cancer and its role in cell cycle regulation and apoptosis Carcinogenesis, September 1, 2004; 25(9): 1575 - 1585. [Abstract] [Full Text] [PDF]
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L. Cheng, C.-X. Pan, J.-T. Zhang, S. Zhang, M. S. Kinch, L. Li, L. A. Baldridge, C. Wade, Z. Hu, M. O. Koch, et al. Loss of 14-3-3{sigma} in Prostate Cancer and Its Precursors Clin. Cancer Res., May 1, 2004; 10(9): 3064 - 3068. [Abstract] [Full Text] [PDF]
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 M. K. Dougherty and D. K. Morrison Unlocking the code of 14-3-3 J. Cell Sci., April 15, 2004; 117(10): 1875 - 1884. [Abstract] [Full Text] [PDF]
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J.-i. Akahira, Y. Sugihashi, T. Suzuki, K. Ito, H. Niikura, T. Moriya, M. Nitta, H. Okamura, S. Inoue, H. Sasano, et al. Decreased Expression of 14-3-3{sigma} Is Associated with Advanced Disease in Human Epithelial Ovarian Cancer: Its Correlation with Aberrant DNA Methylation Clin. Cancer Res., April 15, 2004; 10(8): 2687 - 2693. [Abstract] [Full Text] [PDF]
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J. M. A. Moreira, P. Gromov, and J. E. Celis Expression of the Tumor Suppressor Protein 14-3-3{sigma} Is Down-regulated in Invasive Transitional Cell Carcinomas of the Urinary Bladder Undergoing Epithelial-to-Mesenchymal Transition Mol. Cell. Proteomics, April 1, 2004; 3(4): 410 - 419. [Abstract] [Full Text] [PDF]
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A. Satoh, M. Toyota, F. Itoh, Y. Sasaki, H. Suzuki, K. Ogi, T. Kikuchi, H. Mita, T. Yamashita, T. Kojima, et al. Epigenetic Inactivation of CHFR and Sensitivity to Microtubule Inhibitors in Gastric Cancer Cancer Res., December 15, 2003; 63(24): 8606 - 8613. [Abstract] [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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 H.-Y. Yang, Y.-Y. Wen, C.-H. Chen, G. Lozano, and M.-H. Lee 14-3-3{sigma} Positively Regulates p53 and Suppresses Tumor Growth Mol. Cell. Biol., October 15, 2003; 23(20): 7096 - 7107. [Abstract] [Full Text] [PDF]
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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]
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M. Gasco, A. K. Bell, V. Heath, A. Sullivan, P. Smith, L. Hiller, I. Yulug, G. Numico, M. Merlano, P. J. Farrell, et al. Epigenetic Inactivation of 14-3-3 {sigma} in Oral Carcinoma: Association with p16INK4a Silencing and Human Papillomavirus Negativity Cancer Res., April 1, 2002; 62(7): 2072 - 2076. [Abstract] [Full Text] [PDF]
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G. Tzivion and J. Avruch 14-3-3 Proteins: Active Cofactors in Cellular Regulation by Serine/Threonine Phosphorylation J. Biol. Chem., January 25, 2002; 277(5): 3061 - 3064. [Full Text] [PDF]
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H. Konishi, T. Nakagawa, T. Harano, K. Mizuno, H. Saito, A. Masuda, H. Matsuda, H. Osada, and T. Takahashi Identification of Frequent G2 Checkpoint Impairment and a Homozygous Deletion of 14-3-3{epsilon} at 17p13.3 in Small Cell Lung Cancers Cancer Res., January 1, 2002; 62(1): 271 - 276. [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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