From the Ovarian Cancer Program, Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
Received for publication, October 9, 2002, and in revised form, December 5, 2002
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ABSTRACT |
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LOT1 is a zinc-finger nuclear
transcription factor, which possesses anti-proliferative effects and is
frequently silenced in ovarian and breast cancer cells. The
LOT1 gene is localized at chromosome 6q24-25, a
chromosomal region maternally imprinted and linked to growth
retardation in several organs and progression of disease states such as
transient neonatal diabetes mellitus. Toward understanding the
molecular mechanism underlying the loss of LOT1 expression
in cancer, we have characterized the genomic structure and analyzed its
epigenetic regulation. Genome mapping of LOT1 in comparison
with the other splice variants, namely ZAC1 and
PLAGL1, revealed that its mRNA (~4.7 kb;
GenBankTM accession number U72621) is potentially spliced
using six exons spanning at least 70 kb of the human genome.
5'-RACE (rapid amplification of cDNA ends) data indicate the
presence of at least two transcription start sites. We found that
in vitro methylation of the LOT1 promoter
causes a significant loss in its ability to drive luciferase
transcription. To determine the nature of in vivo
methylation of LOT1, we used bisulfite-sequencing
strategies on genomic DNA. We show that in the ovarian and breast
cancer cell lines and/or tumors the 5'-CpG island of LOT1
is a differentially methylated region. In these cell lines the
ratio of methylated to unmethylated CpG dinucleotides in this region
ranged from 31 to 99% and the ovarian tumors have relatively higher
cytosine methylation than normal tissues. Furthermore, we show that
trichostatin A, a specific inhibitor of histone deacetylase, relieves
transcriptional silencing of LOT1 mRNA in malignantly
transformed cells. It appears that, unlike DNA methylation, histone
deacetylation does not target the promoter, and rather it is indirect
and may be elicited by a mechanism upstream of the LOT1
regulatory pathway. Taken together, the data suggest that expression of
LOT1 is under the control of two epigenetic modifications
and that, in the absence of loss of heterozygosity, the biallelic
(two-hit) or maximal silencing of LOT1 requires both processes.
LOT1 (lost-on-transformation
1)1 is a growth suppressor
gene (1, 2) localized on chromosome 6 at band q24-25, which is a
frequent site for loss of heterozygosity in many solid tumors including ovarian cancer (2, 3). The mouse ortholog of this gene was
independently identified by Spengler et al. (4) and designated as Zac1, which is highly homologous to the
hLOT1 and rLot1 genes. A splice variant of
LOT1/ZAC1 was identified by Kas et al. (5) and named as
PLAGL1 based on its homology with PLAG1 protein encoded by the gene
PLAG1 localized on chromosome 8q12 (6). The presence of
other genes with high homology to LOT1/ZAC1/PLAGL1 indicates
that this gene is a member of new family of zinc-finger proteins. Our
studies and those of others have shown that LOT1 gene
expression is frequently down-regulated in the ovarian and breast
carcinoma cells (1, 2, 7). Functional analysis of LOT1 demonstrated
that it may play a significant role as a transcription factor
modulating growth suppression through mitogenic signaling pathways
(8).
It is increasingly evident that epigenetic modification of genomic DNA
by methylation and/or histone deacetylation plays an important role in
transcriptional silencing and loss of gene function of certain tumor
suppressor genes. Methylation of DNA normally occurs at cytosine
residues within CpG dinucleotides in almost all higher eukaryotic
organisms (9-11). This type of modification can suppress gene
expression directly by interfering with the binding of transcription
factors or indirectly by initiating a complex consisting of
methyl-CpG-binding proteins (e.g. MeCP2, MBDs) and histone
deacetylases (12-16). These complexes mediate transcriptional
repression through chromatin hypoacetylation, which can be partially
reversed by TSA. In human cancers, aberrant methylation of CpG islands
silencing the promoters of some genes that function in the suppression
of malignant phenotype has been found. For example hypermethylation of
CpG islands in different cancer cells has been implicated in the
transcriptional inactivation of the Rb, p16,
estrogen receptor, INK4B/MTS2, VHL, and
H19 genes (17-24). Similarly, CpG island methylation may
play a significant role in the regulation of imprinted genes (25) and
genes located on the inactive X chromosome (26, 27).
Recently, the LOT1/ZAC1 locus at chromosome
6q24-25 was identified as a maternally imprinted region (2, 4,
28-30). These studies have established that the paternal duplication
or loss of maternal imprinting of the LOT1 locus is
associated with transient neonatal diabetes mellitus, an imprinted
disease characterized by intrauterine fetal growth retardation and
insulin dependence. However, little is known about the molecular
mechanisms that down-regulate expression of LOT1 in cancer.
In this study, we have characterized the LOT1 gene structure
with relation to different splice variants and identified the gene's
minimal promoter and a CpG island. Our results indicate that
LOT1 is subject to two epigenetic processes, methylation of
CpG islands and histone deacetylation, which may synergistically act to
regulate the transcriptional silencing of the gene.
Cells and Cell Cultures--
The ovarian cells were maintained
in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine
serum, glutamine (2 mM), insulin (10 µg/ml), penicillin
(100 units/ml), and streptomycin (100 µg/ml) in a humidified 5%
CO2 atmosphere at 37 °C (1). Normal rat ovarian surface
epithelial (ROSE) cells were obtained from the ovaries of adult Fisher
rats by selective trypsinization (31, 32). The ROSE cell line NuTu 26 was obtained as described previously (1). The breast cancer cells
MDA-MB-453 and -468 (ATCC, Manassas, VA) were cultured in Dulbecco's
modified Eagle's medium/F12 medium supplemented with 10% fetal bovine
serum, glutamine (2 mM), penicillin (100 units/ml), and
streptomycin (100 µg/ml).
Cloning of the Full-length hLOT1 and 5'-RACE--
To obtain
additional sequence information of the 5'-untranslated region (5'-UTR)
of hLOT1 cDNA, which we previously reported (2), a
normal human fetal brain cDNA library in
We predicted the LOT1 transcription start site using
GeneRacer cDNA Kit (Invitrogen). The cDNA for 5'-RACE was
derived from human ovary and generated based on oligo-capping and RNA
ligase-mediated RACE methods (33, 34). The PCR was performed
using GeneRacer 5'1 adaptor forward primer (5'-GCACGAGGACACUGA
CAUGGACUGA-3') and gene-specific reverse primer
(5'-TTCTAAGTGAGGTACAGATGAGTTTCAGATG-3'). The gene-specific primer
was designed from the 3' end of the LOT1 exon II and at the
junction of this exon and exon III. The PCR reaction contained the
cDNA, primers, buffer, dNTPs, Me2SO, and Taq
polymerase in a volume of 50 µl. The amplification reaction consisted
of denaturation at 94 °C for 3 min, 35 cycles of 94 °C for
30 s, 60 °C for 30 s, 72 °C for 30 s, followed by
extending at 72 °C for 7 min. We analyzed the PCR products by
cloning into pGEMT vector using the TA cloning kit (Promega, Madison,
WI). The clones were sequenced using both T7 and Sp6 primers. All the primers used in this study were synthesized by the Fox Chase Cancer Center DNA synthesis facility.
Genomic Structure and Cloning of hLOT1 Promoter Region--
To
obtain the human LOT1 promoter, a human placental genomic
DNA library (EMBL-3-SP6/T7, Clontech, Palo Alto,
CA) was screened using the following fragments in the hLOT1
cDNA (GenBank accession number U72621.3) as probes: a) a fragment
containing the complete open reading frame; b) a 271-bp fragment
upstream of the LOT1 translation start codon; and c) a fragment
containing bases Construction of Luciferase Expression Vectors--
Reporter
constructs containing luciferase gene under the control of the
LOT1 promoter were created by amplifying and subcloning the
5'-flanking regions of the LOT1 promoter at the nucleotide positions as depicted in Fig. 4, using DNA from the phage or P1 clone
17513 as a template. Primers used to amplify the 5'-flanking region
of the LOT1 promoter included: TH1645,
5'-CTAGTGGGGTAGGGATAGCATT-3'; TH1647, 5'-CAGGAGGTAAGTTAGTTTGGCC-3'; and
TH1648, 5'-CTGCCCCGTCCGTCCGTCCGT-3'. The primers TH1645 and TH1647 were
linked to the XhoI recognition sequence on their 5' ends,
and the primer TH1648 contained a HindIII recognition site
at its 5' end. The plasmids constructed with these PCR products were
designated as pGL3-45 and pGL3-47, respectively. Another plasmid
designated as PGL3-60 was constructed using a PCR fragment obtained by
amplifying human normal ovarian genomic DNA as template with the
primers IMP4, 5'-GGCCCGTTGGCGAGGTTAGAGCGC-3' and IMP5,
5'-ACGGCATCTGCCATTTGTCA-3'. The PCR products were subcloned into
pGEMT vector using a TA cloning kit (Promega, Madison, WI), and the
insert fragments were purified. The DNA fragments obtained by above
procedures were ligated into pGL3-Basic reporter vector (Promega)
containing the luciferase gene, which was previously digested with
either XhoI alone (pGL3-60) or XhoI and
HindIII (pGL3-45, pGL3-47) restriction enzymes. The
ligation reactions were used to transform DH5 In Vitro DNA Methylation--
The promoter construct pGL3-45
was in vitro methylated by the bacterial methylase,
SssI (New England Biolabs), or mock-treated in the absence
of enzyme. The DNA was incubated for 24 h at 37 °C with
SssI methylase at 1 unit/µg DNA in 50 mM NaCl,
10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol (pH 7.9) supplemented with 160 µM S-adenosylmethionine.
Construction of GFP Fusion Proteins, Fluorescence Microscopy, and
FACS Analysis--
GFP-tagged LOT1 mammalian expression vector was
constructed as described previously (8). Briefly, the GFP coding
region was amplified using forward (5'-CGCCATGGCTAGCAAGGGCCAG-3') and reverse (5'-CTTGTACAGCTCGTCCATGCC-3') primers. The PCR product was ligated with XbaI linker to the LOT1 coding
region, and the resulting fragment (GFL) was introduced downstream of
the cytomegalovirus promoter into the XhoI site of
pcDNA3 plasmid. A2780 cells were cultured in RPMI 1 day before
transfection. Transient transfections were performed using
TransIT-LT1 transfection reagent (PanVera, Madison, WI)
according to the manufacturer's instructions. Cells (2 × 105/well) were seeded in 6-well plates and transfected with
4 µg/well of pcDNA/GFL vector carrying GFP-LOT1 fusion protein.
The cells were then monitored 18 h later and imaged with a Quantix
12-bit cooled charge-coupled device camera (Roper, Inc., Tuscon,
AZ). Fluorescent GFP and phase contrast images were generated
simultaneously using Isee software (Inovision Corp., Durham, NC) to
drive the charge-coupled device, a Ludl filter wheel, and a shutter
attached to a Nikon TE300 inverted microscope. The cells were observed at near-physiological conditions using a forced-air incubator, which
encompasses the microscope. For FACS analysis the transfected cells
were collected by trypsinization, washed, and resuspended in PBS. The
cell suspensions or GFP-transfectants were sorted using a Becton
Dickinson FACS VintageSE flow cytometer (Becton Dickinson, Inc., San
Jose, CA). 10,000 cells were subjected to FACS analysis.
Transient Transfection and Luciferase Assay--
Ovarian cancer
cells at a density of 4 × 105/well were cultured in
6-well plates in RPMI 1640 medium 1 day prior to transfection with the
reporter plasmid as indicated. Transient transfections were performed
using TransIT-LT1 polyamine transfection reagent (PanVera,
Madison, WI) according to the manufacturer's instructions. Briefly,
the cells were washed once with serum-free medium and incubated with
the DNA (2 µl)/(6 µl) reagent mixture for 5 h in serum-free
medium followed by additional incubation for 17 h (overnight) in
fresh medium containing 10% fetal bovine serum. Luciferase enzyme was
assayed using the Dual-Luciferase Reporter Assay System (Promega). The
cells were washed with 1× PBS, harvested with the lysis buffer, and
centrifuged, and 5-20 µl of the cell lysate (supernatant) was added
to 100 µl of the luciferase substrate. The luciferase enzyme activity
was measured immediately or within 15 s of adding the substrate,
luciferin, using the Monolight (R) 2010 Luminometer (Analytical
Luminescence Laboratory, San Diego, CA).
Northern and Western Blot Analysis--
Total RNA was isolated
from the cells by the guanidinium isothiocyanate extraction method (35)
or Trizol (Invitrogen) and was separated on 1% agarose gels containing
2.2 M formaldehyde. The RNA was transferred to Nylon
membranes (Micron Separations, Inc.) by capillary action and hybridized
using a procedure described before (1, 2). The probes for visualization
of rat Lot1 and human LOT1 transcripts were the
same as described previously (1, 2). The probe for DNA Isolation, Bisulfite Sequencing, and Methylation-sensitive
Restriction Digest-PCR--
Genomic DNA was isolated by digestion with
proteinase K (Invitrogen) followed by phenol/chloroform extraction
(36). After precipitation by the addition of 2 volumes of ethanol and
0.5 volumes of ammonium acetate (7.5 M), the DNA was washed
twice with ethanol (80%, v/v), and dissolved in water. Normal and
tumor DNA samples were obtained from Biosample Repository Core Facility (Andrew Godwin and Joellen Dangel) and Tumor Bank Facility (Andre Klein-Szanto and Robert Page) at Fox Chase Cancer Center. Genomic DNA
(1 µg) was treated with sodium bisulfite and used for
methylation-sensitive PCR as previously described (37, 38). Briefly,
the DNA in a volume of 50 µl was denatured by 0.2 M NaOH
and then treated with hydroquinone (Sigma) and sodium bisulfite (Sigma)
at 50 °C for 16 h. Bisulfite-modified DNA was purified using
Wizard DNA Clean-Up system (Promega). Modification was completed by
NaOH (0.3 M) for 5 min at room temperature.
The bisulfite-treated DNA was precipitated by glycogen, ammonium
acetate, and 3 volumes of ethanol, and the pellets were washed once
with 70% ethanol and dissolved in 20 µl of water. The modified DNA
(4 µl) was amplified by PCR under the following reaction conditions: 1× buffer, 125 µM dNTPs, 7.5 mM
MgCl2, and 1 µM of each primer in a 50-µl
reaction. The reaction was carried out with the cycling condition of
denaturation at 95 °C for 10 min followed by the addition of 0.5 unit of Gold Taq polymerase (Invitrogen) and 32 cycles of
PCR (95 °C, 30 s; 58 °C, 30 s; 72 °C, 30 s)
with a final extension of 4 min at 72 °C. Second round of PCR was
performed using 2 µl of the first PCR and the same reagents and
conditions as above. Primers were designed from the interpolated
sequence after bisulfite conversion assuming DNA was either methylated or unmethylated at 14 CpG sites or the imprinting control region (28, 29). Primers used for first PCR were UM-S1:
5'-GGGGTAGTTGTGTTTATAGTTTAGTA-3', UM-AS1: 5'-CAAACACCCAAACACCTACCCTA-3'
and M-S1: 5'-GGGGTAGTCGTGTTTATAGTTTAGTA-3', M-AS1:
5'-CGAACACCCAAACACCTACCCTA-3. Primers used for the second PCR were
UM-S2: 5'-ATAGTTTAGTAGTGTGGGGT-3', UM-AS2: 5'-CCTACCCTACAAAACAACAA-3', M-S2: 5'-ATAGTTTAGTAGCGCGGGGT-3', and M-AS2: 5'-CCTACCCTACGAAACGACGA-3. Reaction products were separated by electrophoresis on a 2% gel, stained with ethidium bromide, and photographed. The resulting amplification pools were cloned into the pGEMT vector using the TA
cloning kit (Promega, Madison, WI). Four to 10 individual clones per
PCR reaction were isolated and sequenced. The clones were sequenced
using both T7 and Sp6 primers.
The methylation-sensitive restriction digest-PCR was performed on 200 ng of genomic DNA samples. The DNA was digested overnight at 37 °C
with 20 units of HpaII or MspI in a total volume
of 20 µl. These restriction enzymes both cleave at CCGG sites.
However, HpaII will not cut the DNA when the internal C is
methylated. The DNA was purified with Wizard column (Promega) and
eluted in 50 µl water, and 5 µl of the product was subjected to PCR
using the primers IMP4 and IMP5 (1 µM) and reagents as
mentioned above. The PCR condition consisted of denaturation at
95 °C for 2 min followed by the addition of 0.5 unit of
Taq polymerase and 29 cycles of PCR (96 °C, 30 s;
60 °C, 30 s; 72 °C, 1 min) with a final extension of 4 min
at 72 °C. The reaction contained the same reagents as mentioned above.
Chromatin Immunoprecipitation (ChIP)--
ChIP assay was
performed according to the manufacturer's instructions (Upstate
Biotech, Lake Placid, NY). Briefly, 1 × 106 cells in
100-mm dish were directly treated with 1% formaldehyde (Fisher
Scientific) to form protein-DNA cross-links. The cells were then
collected in ice-cold 1× PBS containing protease inhibitors and
subjected to centrifugation at 4 °C. The cell pellets were resuspended in SDS lysis buffer and incubated on ice for 10 min. The
samples were sonicated on ice with an Ultrasonics sonicator at setting
10 for 10-s pulses and then microcentrifuged. The supernatant or
chromatin solution from each sample was diluted 10× with ChIP dilution
buffer, and an aliquot was removed and used as input after reversing
cross-link. The diluted chromatin solution was precleared for 30 min
with Salmon Sperm DNA/protein A-Agarose-50% slurry and incubated
overnight at 4 °C with or without acetyl-histone H4 antibody. The
samples were then incubated for 1 h at 4 °C with Salmon Sperm
DNA/protein A-Agarose-50% Slurry with rotation and washed with
different wash buffers supplied in the kit. The chromatin-antibody complexes were eluted, and cross-links were reversed and treated with
proteinase K. The DNA was recovered by phenol/chloroform extractions,
precipitated, and used in PCR amplification. The primers used
for PCR were IMP3: 5'-GCGAGGAGGGTGTGCCTTTG-3' and IMP4 as shown
above. The amplification conditions for the ChIP assay were the same
described above for reactions using IMP4 and IMP5 primer set.
Cloning of the Full-length LOT1 cDNA and 5'-RACE--
As a
first step toward analysis of the relationship between the
LOT1 gene structure, promoter activity, and the presence of differential splicing and regulation of expression, we began to obtain
additional sequence information of the gene's 5'-UTR cDNA region,
which we previously reported (2). A normal human fetal brain cDNA
library in the
We identified the transcription start site (TSS) of LOT1
using 5'-RACE technique. The cDNA for 5'-RACE was derived from
human ovary and generated based on oligo-capping and RNA
ligase-mediated RACE methods (33, 34) as described in the GeneRacer
cDNA Kit (Invitrogen). We detected a strong PCR band corresponding
to about 390 nucleotides in size and weaker bands that are smaller or
larger (Fig. 1). Two of the smaller bands
are shown with arrows (about 280 and 200 bp). The RACE
product was cloned and sequenced. Nine of 35 clones contained the
LOT1 sequence, and the others were unrelated. Analysis of
the amplified 5' ends predicted two potential transcription start
sites, TSS1 and TSS2, as are shown in Fig. 1. Two of the positive
clones had the 5' ends at nucleotide 53080 (TSS1) and six clones
started at nucleotide 52919 (TSS2) of clone RP3-340H11 on chromosome
6q (GenBank accession number AL109755), resulting in an alternative
size of exon 1 (326 and 165 nucleotides, respectively). Interestingly,
an expressed sequence tag clone (accession number BI551851)
constructed using the Cap-trapper method also showed the 5' end at
TSS2. These data suggest that the LOT1 message is
preferentially transcribed from these start sites, at least in the
ovary.
Analysis of the RACE-PCR sequences also revealed that all the positive
clones (100%) were extended 5' upstream from the gene-specific reverse
primer into the LOT1 splice variant at the junction of exons
1 and 2, as opposed to the ZAC1 splice variant, which
diverges from LOT1 at the 5' end of exon 2 in
LOT1 (exon 3, ZAC1) (Fig. 1) (see below). In
addition, sizes of the bands on the gel (Fig. 1), as shown by
arrows, correspond to the fragments (234 and 396 bp) from
the LOT1 splice variant at this 5' upstream region. Taken together, the sequence analysis and hybridization results using different regions of hLOT1 or rLot1 as probes
(data not shown) suggest that human LOT1 (accession number
U72621) and rat Lot1 (accession number U72620) represent the
major transcripts for this gene in the human and rat, respectively (1,
2, 8).
Analysis of the LOT1 Splice Variants--
To obtain the genomic
structure of hLOT1 we first began analyzing the open reading
frame (ORF). Several sets of primers were designed in the coding region
and used to amplify normal genomic DNA. The primers p63 and p69
generated a PCR product that encompassed the majority of the ORF. The
sequence data did not reveal any evidence of introns from zinc-finger
four to the 3' end of the ORF (data not shown). Screening a placental
DNA library indicated that the sequence between +153 (relative to ATG
site) and the last polyadenylation signal is devoid of introns,
confirming the PCR results. However, analysis of different
phage-derived clones and their subclones showed presence of an intron
in zinc-finger two and identified additional exon/intron boundaries
upstream. In addition, we screened a human genomic P1 library with the
FB2-5' fragment in the 5' UTR of LOT1, and a clone
(accession number 17512) was identified containing the LOT1
gene. Several subclones were prepared using PCR or restriction
fragments generated from this clone and sequenced. We determined the
genomic organization by comparing the LOT1 cDNA with
corresponding genomic DNA and by intron-spanning PCR using primers
specific to cDNA sequences. These experiments as well as BLAST
search of sequences in the GenBank data base allowed us to determine
the intron/exon boundaries of the LOT1 gene (Fig.
2), which generally follow the GT-AG rule (39).
As depicted in Fig. 2, the gene consists of six exons and spans at
least 70 kb. The introns range from about 2.6 to 39 kb in size. The
size of exon 1 was predicted by determination of the transcription
start site. Noticeably, exons 1 and 2 are separated from each other by
a large intron of ~39 kb in size. A CpG island is present in exon 1 and extends into the 5' flanking region of the gene, which does not
display a TATA box element. The LOT1 peptide is encoded by exons 5 and
6, resulting in a large 5' UTR. The translation initiation codon (ATG)
is mapped at the 326-bp from the beginning (5' end) of exon 5, and the
stop codon (TAA) is located at 1238 bp from the 5' end of exon 6 followed by about 1.2 kb of nucleotides including the poly(A) tail.
Sequence analysis of the coding region in different cancer cell lines
and specimens by PCR did not reveal any nucleotide mutation.
Two variants of the LOT1 gene (U72621) (2) have been
identified and deposited in the GenBank database as ZAC1
(AJ311395) (40) and PLAGL1 (U81992) (5). Therefore, to
better understand the genomic structure of LOT1 in relation
with the other variants, we determined precisely where these variants
diverge. The LOT1 cDNA was compared with the cDNA
sequences from ZAC1 and PLAGL1 (5, 40-42) and
subsequently with the genomic clones in the GenBank (AL109755 and
AL049844). The data are shown in Fig. 3,
which depicts the relative location and size of exons. These variants have a common 3' end on exon 1; however, the subsequent exons are
differentially spliced, resulting in six, nine, and five exons for
LOT1, ZAC1, and PLAGL1, respectively
(Fig. 3). Interestingly, LOT1 and ZAC1 variants
have an identical open reading frame. However, the open reading frame
of PLAGL1 lacks all the amino acid sequences upstream of the
second methionine in the LOT1 and ZAC1 sequence (5).
Characterization of the Human LOT1 Promoter--
We then cloned
the 5' flanking segments of the LOT1 gene into the
pGL3-Basic vector (Promega, Madison, WI) upstream of the luciferase
gene to determine whether this region possesses transcription activity.
The structures of the different constructs are schematically shown in
Fig. 4A, and the results of a
representative experiment from at least two similar experiments using
these plasmids are presented in Fig. 4B. As is shown in Fig.
4B(a), insertion of the ~1.5-kb human LOT1
genomic fragment (nucleotides
We also confirmed the promoter activity in other cell lines
including the human ovarian cell line OVCAR3 and the ROSE cell line
NuTu 26. Transfection of these cells with the expression vectors
pGL3-45 and pGL3-60 showed a considerable level of reporter activity
(Fig. 4C), although the transcription activity was lower in
the ROSE cells than OVCAR3 cells. Activation of the human
LOT1 promoter in ROSE cells was expected since we found the
hLOT1 promoter sequence to be highly homologous to the mouse
Lot1 promoter (and therefore likely to the rat
Lot1 promoter) by aligning different segments of the
hLOT1 promoter with that of the mouse sequences in GenBank
(accession number AF314094) (data not shown).
Analysis of LOT1 Gene Methylation in Ovarian and Breast
Cancer--
It is increasingly evident that methylation of CpG-dense
islands in the promoter of specific tumor suppressor genes is
associated with their transcriptional silencing in human cancer.
Therefore, we tested whether in vitro modification of the
LOT1 promoter by SssI methylase is capable of
transcriptional repression. As shown in Fig. 4D, the
methylated pGL3-45 construct has significantly less promoter activity.
The 5'-upstream region of the LOT1 gene contains a CpG
island that spans the nucleotides 54333-52440 of clone RP3-340H11 on
chromosome 6q (GenBank accession number AL109755) (Fig.
5). The sequences between nucleotides
53160 and 52440 or the region on exon 1 and part of the first intron
have the highest content of CpG dinucleotides (Figs. 1 and 5).
Therefore, we examined the methylation status of specific sites within
this CpG island or the imprinting control region (28, 29) near the
transcription start site (Fig. 5) by bisulfite-PCR-sequencing analysis.
The genomic DNA isolated from cancer cell lines and tissues were
bisulfite-treated, which converts unmethylated but not methylated
cytosine into uracil, as is shown in Fig.
6. The bisulfite-treated DNA was
subjected to PCR and subcloned into a TA-cloning vector, and
independent clones were sequenced. Completion of the bisulfite reaction
was judged by the efficient conversion of all the cytosine residues that do not precede guanines. We then determined the ratio of methylated sites versus unmethylated sites, and the data
were recorded as percent. As shown in Fig.
7A, the ovarian cancer cell lines (seven cell lines) had between 31 and 52% methylation, and the
cell line CAOV3 had 99% methylation of the CpG sites on the LOT1 locus. In the two breast cancer cell lines, MDA-MB-453
and -468, the methylation levels were 93 and 58% (Fig. 7B),
respectively. Hence, in these ovarian and breast cancer cell lines the
alleles containing one or more methylated CpG dinucleotides in the
differentially methylated region ranged from 50 to 99%. It is worth
mentioning that we also subjected the DNA from MDA-MB-453 cells to
bisulfite/PCR/restriction enzyme (Taq
We examined the methylation status in paired cancerous and
non-cancerous ovarian tissues and found that in three sample pairs (59, 151, 001) the tumor DNA had higher methylation level than in the normal
DNA and in another pair (1002582) the difference was insignificant
(Fig. 7C). We also examined the methylation status of normal
ovary DNA with methylation-sensitive restriction enzyme-PCR. The DNA
was digested with HpaII or MspI. These
restriction enzymes both cleave at CCGG sites. However,
HpaII does not cut the DNA when the internal C is
methylated, and MspI digests the DNA regardless of
methylation status. The DNA was then subjected to PCR using the primers
IMP4 and IMP5 spanning the nucleotides 53002-52441 in the genomic
clone RP3-340H11 on chromosome 6q (GenBank accession number AL109755).
A PCR product (561 bp) was visible for the HpaII digest but
not for the MspI digest, indicating that one allele is
methylated in normal ovary DNA (data not shown). These results and the
evidence of methylated cytosines in normal ovary DNA (see above)
suggest that imprinting or methylation of the maternal allele is
maintained in this tissue, and possibly in breast or other somatic
cells, during development.
Inhibition of Histone Deacetylase Activates the Transcription of
LOT1--
Recent studies have revealed that histone deacetylation is a
major mechanism of DNA silencing due to its action on core histones and
alterations of chromatin structure (43). Therefore, we tested the
effect of TSA, a potent and specific inhibitor of histone deacetylase
(HDAC) (44), on LOT1 gene expression. We have previously shown that the NuTu 26 cancer cell line has lost or decreased Lot1 expression in comparison with its normal progenitor
cells (1) (Fig. 8A).
Therefore, we tested whether deacetylation mechanism plays a role in
the repression of Lot1 in this malignantly transformed cell
line and found that TSA treatment reverses the gene silencing (Fig. 8).
The transcription is induced in an early fashion (2 h) upon treatment
with the HDAC inhibitor (Fig. 8B). These results suggest
that HDAC activity plays a significant role, at least in part, in
regulating Lot1 gene. However, it appears that up-regulation of the gene by TSA is transient, suggesting involvement of
additional regulatory mechanism(s) (Fig. 8).
We then asked whether reactivation of LOT1 by the inhibition
of histone deacetylation is due to direct activation of the promoter. We tested the active 1.5-kb LOT1 promoter and found that it
was not responsive to TSA and the histone deacetylase inhibition failed to relieve methylation-mediated repression of transcription from this
promoter fragment (data not shown). In addition, we performed ChIP
assay of cross-linked chromatin isolated from the control and
TSA-treated A2780 (non-transfected) cells with antibodies against
acetylated H4 histone and PCR amplified the LOT1 promoter. The data as shown in Fig. 9A
indicate that the association of acetylated histone with the promoter
is not significantly changed in response to TSA treatment. Therefore,
we argued that the TSA treatment may target the LOT1 message
due to an indirect effect originated upstream of the LOT1
gene. To test this hypothesis, we fused the LOT1 cDNA
downstream of the green fluorescent gene and used the construct to
transfect A2780 ovarian cancer cells. The data presented in Fig.
9B show that the number of positive cells expressing
GFP-LOT1 fusion protein is amplified following the inhibition of HDAC,
as determined by fluorescence image analysis. Similar results were
obtained with the NuTu 26 cell line, which has lost or decreased
expression of the gene (Fig. 8) (data not shown). Western blot analysis
indicated that the GFP-LOT1 protein level, compared with GFP alone, is
significantly increased in the cells treated with TSA (Fig.
9C).
The effect of inhibition of histone deacetylase observed in Fig. 9,
B and C was also confirmed by FACS analysis and
cell sorting of the GFP-expressing cells. As is shown in Fig.
10, the number of positive
GFP-LOT1-expressing cells was lower than that of GFP-expressing cells
and that the number increased significantly upon the stimulation with
TSA. Taken together, these data and the Northern analysis (above) show
clear correlation between the transcriptional responses of the
LOT1 gene to TSA, exogenously and endogenously.
The candidate tumor suppressor gene LOT1 encodes a
nuclear transcription factor and is strongly regulated by the
activation of the epidermal growth factor receptor signaling pathway
(1, 2, 8). In this paper, we have reported cloning of the full-length LOT1 cDNA, characterized the genomic structure, and
analyzed its epigenetic regulation. We have also determined the
exon/intron splice junctions of LOT1 and its splice
variants, namely ZAC1 and PLAGL1. The
LOT1 gene consists of six exons and spans at least 70 kb.
The introns range from about 2.6 to 39 kb in size. The LOT1 peptide is
encoded by exons 5 and 6, resulting in a large 5' UTR. A CpG-rich
island is present in exon 1 and extends into the 5' flanking region of
the gene, which does not display a TATA box element. We predicted the
TSS of LOT1 using the 5'-RACE technique. Analysis of the
amplified 5' ends using cDNA obtained from whole normal ovary
predicted two potential transcription start sites, TSS1 and TSS2.
However, the absence of a TATA box within the 5'-upstream sequences of
LOT1 suggests that the actual TSS of transcription may be
heterogeneous. We have identified a putative promoter region within 1.5 kb 5'-upstream of LOT1. Insertion of the ~1.5-kb fragment into pGL3 resulted in a strong induction (up to ~35-fold) of the luciferase gene in the ovarian cancer cell lines. Interestingly, however, a second but weaker promoter activity (~10-fold) was observed with the exon 1 proximal region (Fig. 5), suggesting the
presence of at least two potentially active promoter region in the
LOT1 gene.
We previously hypothesized that the LOT1 gene expression may
be regulated by mechanism(s) that may involve histone
acetylation/deacetylation, DNA methylation, and/or mRNA stability
(8). We have identified CpG islands in the upstream sequences of exon 1 and into the promoter of LOT1, which suggest that DNA
methylation of CpG nucleotides may play a role in silencing the gene
expression. In addition, recent reports suggested that
LOT1/ZAC1 is part of a maternally imprinted chromosomal
region (28-30). Therefore, we have analyzed the imprinting based on
methylation of the CpG-region and found that DNA from both normal ovary
and ovarian cancer cell lines exhibit methylation of the cytosine
residues. It appears that ovarian tumors and cancer cells demonstrate
relatively increased levels of methylation in CpG dinucleotides. The
breast cancer cell lines also had high levels of methylation. The
presence of methylated alleles in normal ovary DNA suggests that
imprinting of the maternal allele of the actual LOT1 gene is
maintained in this tissue, and possibly in breast or other somatic
cells, during mammalian development. Thus, based on these results, it
is rational to believe that the LOT1 gene may be subject to
two-hit inactivation or silencing by loss of heterozygosity and methylation.
Furthermore, in this study we have presented evidence for the possible
role of histone deacetylation in repressing the LOT1 gene
expression. Treatment of the cells with trichostatin A, a potent
inhibitor of histone deacetylase, reverses the gene's silencing in
NuTu 26 and A2780 ovarian cancer cell lines. These results suggest that
HDAC activity plays a role, at least in part, in regulating the
LOT1 gene. Direct interaction of HDACs with either the
acetylated histones (H2A, H2B, H3, and H4) or non-histone proteins may
result in deacetylation of these proteins and regulation of gene
transcription. Deacetylation of histones has been found to stabilize
the genomic DNA/histone complex, hindering accessibility of the
promoter to the transcription machinery (43). Apparently, this process
is further enhanced by DNA methylation, which may promote histone
deacetylation and/or may assist in recruiting methylated DNA-binding
protein MeCP2 to the regions of DNA by the HDACs/co-repressor complex
(45-48). However, in this study we found that TSA treatment does not
reactivate the in vitro methylated LOT1 promoter
and does not significantly change the promoter DNA-acetylated histone
association, suggesting that there exists a histone
deacetylase-independent mechanism(s) for gene silencing of this locus
by DNA methylation, as has also been described for other loci (49).
Consistent with our findings, it has been recently reported that
treatment of maternal uniparental mouse embryonic fibroblasts with TSA
does not result in the inheritable expression of the imprinted
Lot1/Zac1 gene (50). This notion is supported by other
reports that in cancer the hypermethylated genes such as
MLH1, TIMP3, CDKN2B/INK4B/p15, and
CDKN2A/INK4/p16 can not be transcriptionally reactivated
with TSA alone (51). Therefore, it is possible that re-expression of
LOT1 by the inhibition of histone deacetylase may be due to the effect on the unmethylated or hypomethylated allele of the gene and
be elicited by a process originating elsewhere upstream in the
LOT1 pathway. We conclude that distinct mechanisms other than imprinting or methylation may play major roles in regulating the
LOT1 expression, particularly when the expression needs to be completely blocked, e.g. expression from the paternal
allele or from the maternal allele when imprinting may be erased during development. Perhaps, promotion of cell survival and positive selection
for development and maturation in a cell-type and stage-specific manner
require repressed activity of LOT1. Studies are currently underway to elucidate in detail the mechanism of LOT1 gene
regulation by different genetic and epigenetic factors, which may be
useful in determining precisely the role of this gene in cancer and/or development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ZAPII vector (Stratagene, La Jolla, CA) was used. A positive clone, FB2, which hybridized to the hLOT1 probe, gave extended 5'-UTR
sequences. Insert sequences of all plasmids were determined by
automated ABI PRISM dye terminator cycle sequencing with FS AmpliTaq
DNA polymerase and run through an Applied Biosystem 373/377 sequencer (ABI/PerkinElmer Life Sciences). The data were analyzed using FASTA and
PILEUP DNA sequence homology searches in the Wisconsin Genetics
Computer Group package (Madison, WI).
1547 to
567 with respect to the LOT1
translation start codon. Phage inserts were sequenced using both the
polylinker-specific and LOT1-specific primers. These primers
as well as cDNA-specific primers were used in PCR to amplify
regions of the phage insert and to gain additional insight into the
genomic structure of LOT1. We also screened a human P1
library (Genome Systems, St. Louis, MO), which resulted in
identification of a new genomic clone designated as clone 17513. This
P1 clone was purified by Qiagen Plasmid Purification Kit (Qiagen Inc.,
Valencia, CA) and used in subsequent promoter analyses. The
forward and reverse primer, p63 (5'-TGGGTGTGAGGAGTGTGGGAAGA-3') and p69 (5'-GCAGTTTGATTACAGAACACGCG-3') were used to sequence most of the LOT1 open reading frame using genomic DNA as template.
Escherichia coli competent cells and the clones
were confirmed by sequencing their insert. All plasmids used for the
transfections were purified using the Qiagen kit.
-actin was from
Clontech, Inc. (Palo Alto, CA). Western blot
analysis was performed with lysates obtained from the cells plated on
6-well dishes in RPMI 1640 containing 10% FBS. After incubation with
the transfection reagent supplemented with the appropriate DNAs,
whole-cell lysates were prepared using 250 µl/well of M-PER Protein
Extraction Reagent (PIERCE, Rockford, IL) containing
phenylmethylsulfonyl fluoride and dithiothreitol. Equal volumes of
protein (15 µl) from each sample were electrophoresed on SDS-PAGE
(10%) followed by transfer to a Hybond nylon membrane (Amersham Life
Science, UK) for Western blot analysis. Immunoblots were blocked
overnight in 5% nonfat dry milk (w/v) in TBST containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1%
Tween 20. Blots were incubated for 1 h with anti-GFP monoclonal
antibody (Clontech, Palo Alto, CA) diluted with 1%
nonfat dry milk in TBST. The blots were washed with TBST three times,
and incubated for 1 h with the second antibody (anti-mouse IgG
horseradish peroxidase, Amersham Biosciences).
Anti-glyceraldehyde-3-phosphate dehydrogenase antibody was obtained
from NeoMarkers (Fremont, CA). The protein bands were detected by the
Western Lightning Chemiluminescence Reagent (PerkinElmer Life Sciences)
and exposure to x-ray films (Kodak Co, Rochester, NY).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ZAPII vector (Stratagene) was used for screening, and
a positive clone, FB2, hybridized to the hLOT1 probe and
produced an extension of the 5'-UTR sequence. This newly identified
nucleotide sequence of the LOT1 gene was added to the previously published sequence that is stored in GenBankTM
with the accession number U72621.
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Fig. 1.
5' RACE and prediction of the transcription
start site. Nucleotide sequence of a portion of the
LOT1 5' UTR as obtained by the rapid amplification of 5'
cDNA ends. Underline indicates the reverse gene-specific
primer used for amplification. The two potential transcription start
sites (TSS1 and TSS2) are shown with forward arrows. The
numbers in the parenthesis indicate the number of RACE clones with
either TSS1 or TSS2. The open triangle indicates the
LOT1 sequence diverged from ZAC1 splice variant.
U72621 is the previously published GenBank accession number for LOT1
(2). 100% (at 3' end of exon 1) indicates that all the positive RACE
clones had the LOT1 sequence and not the ZAC1
sequence at this diverging point. The RACE clones for TSS1 are 396 nucleotides in size and for TSS2 are 234 nucleotides in size. The gel
on the right shows the position of RACE clones compared with
the 100-bp ladder (MW).
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Fig. 2.
Genomic structure of
LOT1. A, the six exons in
LOT1 and the introns are shown (q = long arm
of chromosome 6; TEL = telomere; CEN = centromere). B, exon/intron boundaries were determined by
subcloning different fragments obtained from a phage DNA, P1 genomic
clone, and total genomic DNA using PCR with LOT1-specific
primers or restriction digest analysis. The nucleotide sequences of
these subclones were aligned to the LOT1 cDNA sequence
(GenBank accession number U72621). The numbers on each intron indicate
the approximate size of nucleotide sequence.
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Fig. 3.
Schematic representation of
LOT1/ZAC1/PLAGL1 splice variants. Location and
size of each exon in LOT1 in relation to the splice variants
ZAC1 and PLAGL1 are depicted. The 5' end of exon
1 in LOT1 was predicted by RACE technique and is shown for
the longer transcript (TSS1). The 5' end of PLAGL1 has not
yet been identified and therefore is shown as open box;
according to the nucleotide sequences deposited in GenBank, the last
exon of the three splice variants have some nucleotide
variations.
1456 to
1 with respect to the 5' end
of LOT1 sequence in GenBank accession number U72621) into
pGL3, designated as pGL3-45, resulted in a strong increase (up to
~35-fold) in the activation of the luciferase gene in the A2780
ovarian cancer cell line. The transcription activity was abolished
following the 5' truncation of the promoter, as is shown for pGL3-47
(nucleotides
525 to
1) plasmid. Interestingly, however, a second
but weaker promoter activity (~10-fold compared with ~35-fold for
pGL3-45) was observed with the exon 1 proximal region (nucleotides
123 to +450; vector pGL3-60) as is apparent from Fig.
4B(b), suggesting the presence of at least two
potentially active promoter region in the LOT1 gene.
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Fig. 4.
Analysis of the LOT1
promoter. A, the strategy for cloning the LOT1
promoter-luciferase constructs used in transient transfection assays.
The constructs that were PCR-amplified from the human normal ovary
genomic DNA (pGL3-60) or P1 clone 17513 (pGL3-45; pGL3-47) are
indicated. Position of each construct is shown relative to the
nucleotides (54333-52440) in clone RP3-340 HII (GenBank accession
number AL109755). B and C, transcriptional
activity of the LOT1 promoter region. The ovarian cells were
transfected with the luciferase constructs using TransIT-LT1
reagent (PanVera, Madison, WI), and fresh medium was added to the
cultures 5 h after transfection and incubated for an additional
18 h. Duplicate wells were transiently transfected with the DNA,
and the luciferase activity for each sample was determined. The
relative activity of each promoter-pGL3 vector is presented as a
percent of pGL3-Basic luciferase activity (arbitrarily assigned as
100%). Each bar graph represents the mean ± S.E.
D, transcriptional silencing of pGL3-45 promoter construct
after treatment with SssI methylase enzyme; C,
control, M, methylated.
I enzyme, BioLab)
analysis and found that the DNA was hypermethylated, confirming the
sequencing data. We have previously shown that the ovarian cancer cell
lines express differential levels of LOT1 message (1).
However, we were not able to find a direct association between the
expression data and the methylation status in these cell lines.
Furthermore, we treated MDA-MB-453, CAOV3, and NuTu 26 cancer cell
lines with the demethylating agent 5-aza-2' deoxycytidine to determine
whether the expression of LOT1/ZAC1 could be reactivated.
The results showed that in these cell lines LOT1 is
unresponsive to 5-aza-2' deoxycytidine treatment (data not shown).
These results suggest that additional mechanism(s) other than
methylation may be involved in the regulation of LOT1. In
other words, we hypothesize that in some cases constitutive down-regulation of LOT1 may be due to alteration elsewhere
upstream in the pathway and that direct demethylation of CpG
dinucleotides may not be sufficient to restore the normal expression
pattern of this gene.
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Fig. 5.
Schematic diagram of the LOT1
5'-upstream region containing the CpG island used for the
methylation analysis. We arbitrarily divided this region in the
clone RP3-340H11 (numbers 54333-52440 indicate the nucleotides on
this clone; see also the legend to Fig. 4) into three parts and the CpG
sites were counted as shown in the figure (24 CpG, 41 CpG, and 94 CpG,
respectively). The CpG-rich (94 CpG) region spans the first exon and
extends into intron 1 (diagram shows a portion of this intron). TSS1
and TSS2 are the approximate locations of the two transcription start
sites (Fig. 1). Lower panel in the figure depicts the
fourteen CpG sites used for bisulfite sequencing using the primers
indicated. S-1 and AS-1 indicate sense and antisense primers used for
first round of PCR for both unmethylated (UM) and methylated
(M) DNAs. Similarly, S-2 and AS-2 indicate the primers
(UM and M) used for the second PCR.
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Fig. 6.
Example of direct DNA sequencing
chromatogram for the methylated allele. A, nucleotide
sequence of the fragment used for methylation analysis depicting the
original sequence (NT, not treated with bisulfite) and the
sequence after bisulfite treatment (UM, unmethylated;
M, methylated). The numbers 1-14 indicate the position of
each CpG dinucleotide. B, the chromatogram of methylated
allele after bisulfite treatment showing that the cytosines that
precede the guanines remain unaltered, whereas all the other cytosines
are deaminated and converted to uracil.
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Fig. 7.
Methylation analysis of the CpG island of the
LOT1 gene in ovarian and breast cancer. Genomic
DNA was isolated from the indicated cell lines or tumors and analyzed
for methylation by direct bisulfite sequencing. Bisulfite-PCR products
were subcloned into pGEMT vector and sequenced by Automated Sequencer.
The ratio of total methylated sites to unmethylated sites was
determined and recorded as percent. Each circle indicates a
CpG site in the primary DNA sequence, and each line of
circles represents analysis of a single cloned allele. The numbers
of the CpG site are indicated at the top and are identical
to the numbers in Fig. 6. Open and closed circles
represent unmethylated and methylated CpG dinucleotides, respectively.
A, ovarian cancer cell lines; B, breast cancer
cell lines; C, paired cancerous and non-cancerous ovarian
tissues.
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Fig. 8.
The effect of inhibition of HDAC activity on
LOT1 expression. TSA treatment relieves the
Lot1 transcriptional silencing in NuTu 26 ROSE cells.
A-C, normal and NuTu 26 cells were cultured to
subconfluence and treated with TSA (100 ng/ml) for the indicated
time-points. C, images of the hybridization signals shown in
B were determined by AMBIS image analyzer, and the relative
intensity was plotted. RNA from the normal cells in A was
used as comparison with RNA from NuTu 26 cells hybridized with the
Lot1 probe. Total RNA was hybridized with the
isotope-labeled Lot1 probe.
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Fig. 9.
A, PCR amplification of the
LOT1 promoter from the input (non-precipitated control),
mock-precipitated ( IP), and immunoprecipitated
(+IP) DNAs using the IMP3 and IMP4 primer set (see
"Materials and Methods"). B, the fluorescence microscopy
of A2780 cells transfected with GFP-LOT1 fusion protein. The cells were
transfected and then treated with TSA for 18 h. The fluorescence
images were examined with a fluorescent microscope. C,
Western blot analysis of the proteins extracted from the transfected
cells, comparing GFP-LOT1 protein (~78 kDa) expression level with GFP
(~27 kDa) expression level in cells treated with or without TSA.
Fifteen microliters of whole-cell lysate was loaded on each lane,
blotted, and exposed to the GFP antibody as described under
"Materials and Methods."
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Fig. 10.
A, flow cytometry and cell sorting
analysis of the cells transfected with GFP or GFP-LOT1 fusion protein.
Cells were transfected with the two constructs used in Fig. 9 and
treated with or without TSA for 18 h. The treated cells were
collected, and 10,000 cells were subjected to FACS analysis.
B, the percent of the fluorescent cells was
plotted.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Andrew Godwin, Joellen Dangel, Andre Klein-Szanto, and Robert Page for the tissue samples and Robert Bast, Yinhua Yu, and Jiuhong Yuan for scientific and technical discussions. We also thank Jonathan T. Boyd and Susan Shinton for fluorescence microscopy and FACS analysis and Anita Cywinscki for DNA sequencing.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA56916 (to T. C. H. and A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U72621.
To whom correspondence should be addressed: Ovarian Cancer
Program, Dept. of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3679; Fax: 215-728-2741; E-mail: A_Abdollahi@fccc.edu.
§ Supported by National Institutes of Health Grants CA06927, CA56916, CA51228, CA84242, SPORE, and CA83638 and an appropriation from the Commonwealth of Pennsylvania, the Adler Foundation, and the Evy Lessin Fund.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M210361200
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ABBREVIATIONS |
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The abbreviations used are: LOT1, lost-on-transformation 1; TSA, trichostatin A; ROSE, rat ovarian surface epithelial; RACE, rapid amplification of cDNA ends; UTR, untranslated region; FACS, fluorescence-activated cell sorter; GFP, green fluorescence protein; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; TSS, transcription start site; ORF, open reading frame; HDAC, histone deacetylase.
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