From the Departments of Urology and ¶ Pathology
and the § Hamon Center for Therapeutic Oncology,
University of Texas Southwestern Medical Center,
Dallas, Texas 75390-9110
Received for publication, August 12, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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hDAB2IP (human DAB2 (also
known as DOC-2) interactive protein) is a novel
GTPase-activating protein for modulating the Ras-mediated signal
pathway. We demonstrate that the down-regulation of hDAB2IP mRNA in prostate cancer (PCa) cells is regulated by transcriptional levels. Analysis of the hDAB2IP promoter revealed that it
is a typical TATA-less promoter containing many GC-rich sequences. In
this study, we delineated the potential impact of the epigenetic control of the hDAB2IP promoter on its gene regulation in
PCa. Acetylhistone H3 was associated with the hDAB2IP
promoter, and CpG islands remained almost unmethylated in normal
prostatic epithelia, but not in PCa cell lines. Our data further
indicated that trichostatin A (histone deacetylase inhibitor) and
5'-aza-2'-deoxycytidine (DNA hypomethylation agent) acted cooperatively
in modulating hDAB2IP gene expression in PCa, whereas
histone acetylation played a more significant role in this event.
Moreover, a core promoter sequence from the hDAB2IP gene
responsible for these treatments was identified. We therefore
conclude that epigenetic regulation plays a potential role in
regulating hDAB2IP expression in PCa and that these results
also provide a new therapeutic strategy for PCa patients.
hDAB2IP (human DAB2
interactive protein) is a novel member
of the Ras GTPase-activating family (1, 2). Our recent data indicate
that it interacts directly with DAB2
(Disabled-2; also known as DOC-2
for differentially expressed in ovarian
carcinoma-2) (2), which appears to be a tumor
suppressor in cancer types (3-7). Both DAB2IP and DOC-2/DAB2
form a unique protein complex with negative regulatory activity that
modulates the Ras-mediated signal pathway (2). In the prostate gland,
this complex is detected in the basal cell population (2, 6) and may
orchestrate the differentiation and proliferation potential of these
cells during gland development. In contrast, loss of expression of
DOC-2/DAB2 and hDAB2IP proteins is often detected in metastatic
prostate cancer (PCa)1 cell
lines, and increased expression of these proteins can suppress the
growth of PCa (2, 6).
We have demonstrated that normal prostatic epithelial cells have
elevated hDAB2IP mRNA levels compared with PCa cells,
which correlate with increased hDAB2IP promoter activity
(1). These data indicate that transcriptional regulation of
hDAB2IP is responsible for the down-regulation of
hDAB2IP expression in PCa cells. However, little is known
about the underlying mechanisms for the regulation of
hDAB2IP gene expression in prostatic epithelial cells.
One of the hallmarks of the regulation of gene transcription is local
chromatin decondensation mediated by histone acetylation, which leads
to a reduced association between chromosomal DNA and histones and
subsequently increases the accession of high molecular mass protein
complexes of the transcription machinery. Conversely, histone
deacetylation can repress transcription by increasing histone-DNA
interaction (8, 9). Additionally, we have found that the
hDAB2IP promoter does not have a typical TATA box, but contains many GC-rich sequences (1, 2). DNA hypermethylation, particularly in the GC-rich promoter region, results in transcription repression that is often associated with a number of tumor suppressor gene promoters, including Rb, p15, and p16 (10, 11). In this study, we
delineated the roles of histone acetylation and DNA methylation in the
regulation of the hDAB2IP gene in normal prostatic epithelia
and PCa cells. The data presented in this work provide strong evidence
for underlying mechanisms of the down-regulation of the
hDAB2IP gene mediated by epigenetic control in PCa cells.
Cell Cultures and Treatments--
Two human prostate cancer cell
lines (LNCaP and PC-3) were maintained in T medium supplemented with
5% fetal bovine serum as previously described (2). Two normal human
prostate cell lines (PrEC, a primary prostatic epithelial cell line
derived from a 17-year-old juvenile prostate; and PZ-HPV-7, an
immortalized cell line derived from the peripheral zone of a normal
prostate) were maintained in a chemically defined medium (PrEGM)
purchased from BioWhittaker, Inc. (Walkersville, MD).
Cells were seeded at low density (6 × 105/100-mm
dish) 16 h prior to treatment with different agents at the
indicated final concentrations: 25, 100, or 200 nM
trichostatin A (TSA; Sigma) or 1, 5, or 10 µM
5'-aza-2'-deoxycytidine (5'-Aza; Sigma). For TSA treatment, medium
containing fresh agent was changed every 24 h for 48 h. For
5'-Aza treatment, medium containing fresh agent was changed every
48 h for 96 h. For combined treatment, TSA was added at
24 h and changed at 72 h, and 5'-Aza was replaced at 48 h. Cells were collected at 96 h after treatment.
Real-time Reverse Transcription-PCR Assay--
Total
cellular RNA was isolated from PC-3, LNCaP, and PZ-HPV-7 cells using
RNAzol B (Tel-Test, Inc., Friendswood, TX) according to the
manufacturer's instructions. Two micrograms of total cellular RNA were
used in each reaction. cDNA was synthesized and amplified using
either the hDAB2IP primer set (2 ng/µl) (F-hDAB2IP,
5'-TGGACGATGTGCTCTATGCC-3'; and R-hDAB2IP, 5'-GGATGGTGATGGTTTGGTAG-3')
or the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer
set (6 ng/µl) (G3P7, 5'-GAAGGTGGGTCGGAGTCAACG-3'; and G3P4,
5'-AGTGAGCTTCCCGTTCAGC-3') in a 40-µl reaction mixture containing 20 µl of platinum qPCR Supermix-UDG (Invitrogen) and 4 µl of SYBR
Green I (final dilution of 1:10,000). The reactions were carried out on
a 96-well plate, and a PCR amplification protocol was followed
(95 °C for 3 min and 40 cycles of amplification at 95 °C for
30 s, 55 °C for 30 s, and 72 °C for 1 min) using an iCycler iQ machine (Bio-Rad). A quality control was carried out using
both electrophoresis analysis on a 2% NuSieve agarose gel (3:1; FMC
Corp. BioProducts) and melting curve analysis performed immediately
after the end of amplification at 95 °C for 1 min and 55 °C for 1 min and 80 cycles of 0.5 °C increments beginning at 55 °C. We
also performed the standard curves for hDAB2IP and GAPDH to ensure the linearity and efficiency of both genes. The linear
range of both genes is from 2 × 102 to 2 × 109 copies, and the efficiency of each reaction ranges from
92 to 97%. The relative induction of hDAB2IP mRNA was
calculated as follows: Construction of Reporter Gene Vectors--
To analyze the
promoter region of hDAB2IP, a 7.6-kb fragment from
bacterial artificial chromosome (BAC) clone 298A17
(GenBankTM/EBI accession number AL365274) containing
the predicted first exon and additional 5'-upstream sequence of the
DAB2IP gene was subcloned into the EcoRI site of
pBluescript SK(
To further analyze the regulation of hDAB2IP promoters, two
sets of primers (F-PI (inner, 5'-CCTGCTTTCTGTTTCCTTCTCCTG-3') and R-PI
(inner, 5'-TTGAACCACCTCCTCCTCCCTCTC-3'); F-PII (inner, 5'-ATTCCTCCAGGTGGGTGTGG-3') and R-PII (inner,
5'-CCTAAGCCGCTGTTGCCTTG-3')) were used to amplify the PI (+768 to +873)
and PII ( Cell Transfection and Luciferase Reporter Assay--
We plated
cells at a density of 1.0 × 105 cells/well on a
six-well plate. After 16 h, we transfected the PZ-HPV-7 and PC-3 cell lines with both 0.8 µg of reporter vectors and 0.2 µg of Acid Extraction of Histone and Western
Analysis--
Cells were scraped, centrifuged at 200 × g for 10 min, and then suspended in 10 volumes of
phosphate-buffered saline. Cells were spun down; pellets were suspended
in 5 volumes of lysis buffer (10 mM HEPES (pH 7.9),
1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and 1.5 mM
phenylmethylsulfonyl fluoride); and sulfuric acid was added to a final
concentration of 0.2 M, followed by incubation on ice for
30 min. After centrifugation at 11,000 × g for 10 min at 4 °C, the cell supernatant containing the acid-soluble fraction was retained. The supernatant was dialyzed twice against 200 ml of 0.1 M acetic acid for 1-2 h and then dialyzed overnight
against 200 ml of H2O using Spectrapor®
molecular porous membrane tubing (Spectrum Medical Industries, Inc.,
Los Angeles, CA). The protein concentration was measured, and
proteins were fractionated by SDS-PAGE (15%). Western blotting was carried out with an anti-acetylhistone H3 antibody (1:3000; Upstate
Biotechnology, Inc., Lake Placid, NY). The same membrane was stripped
and reprobed with an anti-histone H3 antibody (1:1000; Upstate
Biotechnology, Inc.).
Chromatin Immunoprecipitation (ChIP)
Assay--
After treatment, formaldehyde was added to the cell medium
at a final concentration of 1% for cross-linking proteins to DNA. Cells were washed, scraped off with ice-cold phosphate-buffered saline,
and resuspended in SDS lysis buffer containing a mixture of protease
inhibitors. An equal protein concentration of cell lysate from each
sample was sonicated to reduce DNA fragments between 200 and 1000 bp.
Once the cell debris was removed, the supernatant was diluted in ChIP
dilution buffer (1:10), and 1% of this supernatant (as input DNA) was
collected, purified, and subjected to genomic PCR with the primer sets
described in Table I. Samples were
precleared with salmon sperm DNA/protein A-agarose slurry (Upstate
Biotechnology, Inc.) and incubated overnight at 4 °C with or without
(as a negative control) antibody. Immune complexes were collected by
adding salmon sperm DNA/protein A-agarose slurry and incubated with 20 µl of 5 M NaCl at 65 °C to reverse DNA-protein
cross-linking. DNA was then purified by proteinase K digestion, phenol
extraction, and ethanol precipitation.
The strand-specific nested PCR primers used for amplifying the
hDAB2IP gene are indicated in Table I. PCR amplifications were performed in a 50-µl reaction mixture containing 2 µl of DNA
by the addition of 2 units of ThermalAceTM DNA polymerase
(Invitrogen). A hot start was performed (98 °C for 3 min), followed
by 30 cycles at 98 °C for 30 s, 62 °C for 30 s, and
72 °C for 45 s. The PCR product from the PI region (~110 bp)
was separated on 4% E-GelTM (Invitrogen), and that from
the PII region (~230 bp) was separated on a 2% NuSieve agarose gel
(3:1).
Bisulfite Genomic Sequencing--
High molecular mass genomic
DNA was obtained from PrEC, PZ-HPV-7, LNCaP, and PC-3 cell lines and
subjected to bisulfite modification (13, 14). Briefly, 1-2 µg (5-10
µl) of genomic DNA were denatured with NaOH (final concentration of
0.2 M), 30 µl of 10 mM hydroquinone (Sigma),
and 520 µl of 3 M sodium bisulfite (Sigma) at pH 5 for 16 h at 50 °C. The modified samples were purified using Wizard DNA Clean-Up system desalting columns (Promega), followed by ethanol precipitation. Bisulfite-modified DNA (100 ng) was amplified by PCR in
a 25-µl reaction mixture containing the primers indicated in Table I.
A hot start was performed (95 °C for 5 min) by adding 0.5 unit of
HotStar Taq DNA polymerase (QIAGEN Inc., Valencia, CA). The
PCR products were cloned into the TA cloning vector pCR2.1-TOPO. Four
to eight individual clones were sequenced using reverse and forward M13 primers.
Identification of Two hDAB2IP Promoters in Prostatic Epithelial
Cell Lines--
Two putative promoters, P1 (+229 to +981, located
within the first intron) and P2 (
To analyze the basal activity of each hDAB2IP promoter in
various prostate cells, luciferase reporter vector constructs
were generated. Using pGL3-1.6S, we detected the highest luciferase activity in both PrEC and PZ-HPV-7 cells, an intermediate level in
LNCaP cells, and the lowest level in PC-3 cells (Fig. 1C), correlating with the steady-state levels of hDAB2IP mRNA
in each cell line (1). Similar patterns of reporter gene activity were detected in these four cell lines using either the P1 or P2
promoter (Fig. 1C).
Induction of hDAB2IP Gene Expression by a Hypomethylation Agent
(5'-Aza) and/or a Histone Deacetylase Inhibitor
(TSA)--
Apparently, the decreased hDAB2IP mRNA
levels detected in many human PCa cells (1) could be caused by its
reduced gene promoter activity (Fig. 1C). To understand the
mechanism leading to the down-regulation of the hDAB2IP gene
in human PCa cells, we first examined the role of epigenetic regulation
of the hDAB2IP gene. The data from a ChIP assay demonstrated
that the presence of acetylhistone H3 was associated with the PI and
PII regions of hDAB2IP in both PrEC and PZ-HPV-7 cells, but
not in PC-3 and LNCaP cells (Fig. 1D), suggesting that
histone acetylation may play a role in modulating hDAB2IP
gene expression. Recent data also indicate that epigenetic controls
such as histone acetylation and/or DNA methylation play cooperative
roles in modulating gene expression, particularly genes involved in
tumor suppression (15, 16). We therefore treated these cells with TSA,
5'-Aza, or a combination of both. The levels of hDAB2IP
mRNA expression were evaluated by real-time reverse
transcriptase-PCR using GAPDH as an internal control. As shown in Table
II, TSA and/or 5'-Aza failed to elicit
any elevation of hDAB2IP mRNA because the basal activity of the hDAB2IP promoter was very high in PZ-HPV-7 cells
(Fig. 1C). However, in PC-3 cells (Table II), either TSA or
5'-Aza was able to induce hDAB2IP mRNA expression. In
contrast, the increased hDAB2IP mRNA levels in LNCaP
cells treated with a single agent were lower than those in PC-3 cells
(Table II) because LNCaP cells had higher endogenous hDAB2IP
mRNA levels compared with PC-3 cells (1). For the combination of
both agents, the level of induction exhibited an additive effect only
in the PC-3 and LNCaP cell lines. In some cases, we noticed that the
mRNA levels after the combination treatment were lower than those
after the single-agent treatment, which was caused by the toxicity of
the drug combination.
Characterization of the hDAB2IP Promoter Regulated by Histone
Acetylation and DNA Methylation--
To delineate which promoter could
be induced by TSA, 5'-Aza, or a combination of both drugs and the
underlying mechanism of the induction, we transiently transfected PCa
cells with pGL3-P1 or pGL3-P2 under the same treatment conditions. In
PC-3 cells, TSA could induce P1 promoter activity in a
dose-dependent manner; however, 5'-Aza only slightly
induced this promoter activity (Fig. 2A). The combination treatment
exhibited an additive effect only on P1 activity. In contrast, a very
different induction pattern was observed in LNCaP cells transfected
with pGL3-P1; only marginal induction of P1 activity was observed in
these cells after the different treatments (Fig. 2B).
By transfecting pGL3-P2 into PC-3 cells, a dose-dependent
induction pattern of P2 activity was observed in these cells treated with either TSA or 5'-Aza (Fig. 2C). In LNCaP cells, P2
activity could also be induced by either TSA or 5'-Aza in a
dose-dependent manner (Fig. 2D), which differed
from P1 activity induced by these drugs (Fig. 2B). Again, in
PC-3 and LNCaP cells, the combined treatment with TSA and 5'-Aza
exhibited an additive effect only on P2 activity. In addition, we
determined both P1 and P2 activities in PC-3 and LNCaP cells
with a different transfection protocol; the overall induction pattern
was consistent (see Fig. 1 in the Supplemental Material). Taken
together, these results indicate that the P2 promoter is responsible
for both TSA- and 5'-Aza-induced hDAB2IP gene expression in
PC-3 and LNCaP cell lines.
To evaluate the possibility of a global gene induction effect of
TSA or 5'-Aza on PCa cells, we examined the activity of the prostate-specific antigen (PSA) gene promoter in LNCaP and PC-3 cells
treated with either agent. As shown in Fig. 2E, no induction of PSA reporter activity was detected in both cell lines treated with a
single agent or a combination of both agents. In contrast, androgen
could induce PSA reporter activity dramatically in LNCaP cells
(androgen receptor-positive), but not in PC-3 cells (androgen receptor-negative). Therefore, we believe that TSA or 5'-Aza has a
specific effect on regulating hDAB2IP gene expression
in PCa cell lines.
Increased Levels of Acetylhistone H3 in the hDAB2IP Promoter
Induced by TSA--
To determine whether the TSA-induced
hDAB2IP gene expression correlated with the levels of
histone acetylation associated with the hDAB2IP promoter
region, we analyzed the steady-state levels of acetylhistone H3 in both
PC-3 and LNCaP cells after TSA treatment. As shown in Fig.
3A, Western blot analysis of
PC-3 cells indicated that the basal level of acetylhistone H3 was very low, whereas TSA induced a dramatic elevation of the ratio between acetylhistone H3 and total histone H3. Comparing this with the no-treatment control, TSA induced a dose-dependent (ranging
from 8- to 88-fold) elevation of acetylhistone H3. In contrast, the basal level of acetylhistone H3 was very high in LNCaP cells (Fig. 3B). Therefore, we failed to detect any changes in the
steady-state levels of acetylhistone H3 in LNCaP cells treated with
TSA. Nevertheless, it is still possible that TSA increases the
acetylhistone H3 levels associated with the hDAB2IP promoter
region.
To analyze the status of acetylhistone associated with the
hDAB2IP promoter, a ChIP assay was performed using the
sequences corresponding to the PI (+768 to +873) and PII (
Although the ChIP assay provides a unique analysis of the specific
chromatin DNA region that associates with acetylhistone proteins, the
results need to be confirmed by function assays such as a reporter gene
assay. Therefore, we investigated the luciferase activity of two
constructs, pGL3-PI (+768 to +873) and pGL3-PII (
We observed a dramatic induction of PII activity in PC-3 cells after
treatment (10-18-fold increase with TSA and 14-30-fold increase with
the combination) (Fig. 5C). Using 5'-Aza, an ~3-fold induction of PII activity was detected in PC-3 cells. A similar induction profile of PII activity was detected in LNCaP cells (Fig.
5D). For example, TSA alone induced an ~6-14-fold
increase in PII activity, whereas the combination treatment induced an 8-44-fold increase in reporter gene activity. An ~2-fold induction of PII activity was observed in LNCaP cells treated with 5 µM 5'-Aza. In addition, we repeated these experiments
with a different transfection protocol; the overall induction pattern
was consistent (see Fig. 2 in the Supplemental Material). Taken
together, these data suggest that PII ( Characterization of the Methylation Status of the hDAB2IP Promoters
in Prostate Cell Lines--
It is known that aberrant methylation
(which is associated with gene silencing) in the promoters of tumor
suppressor genes is commonly detected in cancer cells (18-20). CpG
islands appear to be critical sites modulated by DNA methylation
(21-23). Because the 5'-regulatory region in the hDAB2IP
promoter is GC-rich and the DNA hypomethylation agent (5'-Aza) can
induce hDAB2IP gene expression, determining the methylation
profile of the promoter region in normal and cancerous cells could
provide additional evidence for the role of DNA methylation in the
regulation of hDAB2IP during PCa development. In this
experiment, two PCa cell lines (PC-3 and LNCaP) and two normal prostate
cell lines (PrEC and PZ-HPV-7) were subjected to bisulfite genomic
sequencing. With respect to the high GC content in the
hDAB2IP promoter, primers were designed to avoid potential
methylation sites (e.g. CpG) such that both
methylated and unmethylated DNAs would be amplified equally. For the P1
region, PmI (spanning 35 CpG sites) was designed; and for the P2
region, PmIIa (spanning 30 CpG sites) and PmIIb (spanning 56 CpG sites)
were designed (Fig. 6A)
because we found more CpG sites in the P2 region (86 sites) than in the
P1 region (35 sites). The detailed primer information is summarized in
Table I.
In the PmI region, PC-3 cells showed a partial methylation pattern, and
LNCaP cells showed an almost completed methylation pattern. In
contrast, PZ-HPV-7 cells showed a completed unmethylation pattern, and
PrEC cells contained very few methylation sites (Fig. 6B).
The density of methylation of this region correlated inversely with the
basal activity of the P1 promoter in all cells examined (Fig.
1C).
In the PmIIa region, both normal prostate cell lines showed an almost
completed unmethylated pattern. However, LNCaP cells contained low
densities of methylation, whereas PC-3 showed a significantly higher
degree of methylation pattern (Fig. 6C). This evidence
indicated that methylation density in the PmIIa region inversely
correlated with the basal activity of P2 in these cells (Fig.
1C). Interestingly, in the PmIIb region, PC-3, PZ-HPV-7, and
PrEC cells showed almost completed unmethylated patterns, and LNCaP
cells contained few methylation sites (data not shown). Taken together,
these data clearly indicate that the PmIIa region ( The higher levels of hDAB2IP mRNA detected in
normal prostatic epithelia compared with PCa cells are mainly regulated
at the transcriptional level (1). In this study, we further
demonstrated that the activity of the hDAB2IP promoter is
more active in normal prostatic epithelia than in PCa cells (Fig.
1C). We also noticed that the 5'-upstream regulatory region
of the hDAB2IP gene has GC-rich sequences, but no
canonical TATA boxes. This is a typical feature of the promoters of
many housekeeping genes and of ~40% of tissue-specific genes (24).
Although various mechanisms may underlie this repression in PCa cells,
our data demonstrate that histone acetylation and/or DNA methylation
plays a crucial role in modulating hDAB2IP gene expression
in PCa cells. The treatment of PCa cell lines such as PC-3 and LNCaP
with TSA, 5'-Aza, or a combination of both significantly increased the
steady-state levels of hDAB2IP mRNA (Table II). In
contrast, TSA or 5'-Aza could not induce hDAB2IP gene
expression in normal prostatic epithelia (Table II). These data
indicate that both DNA methylation and histone deacetylation act
cooperatively to silence the hDAB2IP gene in PCa cells. Such
action is presumably mediated through a complex chromatin structure in
which methyl-CpG-binding proteins are associated with histone
deacetylases (HDACs) (25, 26).
Eukaryotic DNA is packed into a highly organized structure (27). It has
become increasingly clear that gene transcription from this tightly
packed DNA is regulated by chromatin-remodeling events, which can
render DNA either more or less accessible to transcription factors. One
of the key events in the regulation of eukaryotic gene expression is
the post-translational modification of nucleosomal histones, which
convert regions of chromosomes to transcriptionally active or
inactive. The most well studied post-translational modification of
histones is the acetylation of DNA hypermethylation has been implicated in parental gene imprinting, X
chromosome inactivation, and endogenous retrovirus silencing (42-46)
as well as in the transcriptional silencing of tumor suppressor genes
(47, 48). Hypermethylation of CpG islands is also found in the
3'-ends of some genes; however, the density of DNA methylation in
promoter or first exon regions correlates inversely with gene
transcription (22, 49). It has also been shown that transcription
repression mediated by methyl-CpG-binding proteins involves an HDAC
complex (50, 51), indicating that there is a close relationship between
DNA methylation and histone deacetylation.
Regarding the potential role of histone acetylation, data from the ChIP
assays indicated that acetylhistone H3 was associated with the
hDAB2IP promoter in normal epithelial cell lines
(PrEC and PZ-HPV-7) expressing hDAB2IP proteins (Fig. 1D). A
dramatic increase in the levels of acetylhistone H3 associated with the hDAB2IP promoter was detected in PCa cells in the presence
of TSA (Fig. 4, A and B). We further demonstrated
that the DNA fragment identified in the ChIP assay had promoter
activity and could respond to TSA treatment (Fig. 5, C and
D). Based on these results, we conclude that the status of
acetylhistone in the hDAB2IP promoter is critical for its
gene regulation. We also noticed that several potential transcription
factor-binding sites such as AP-1, AP-2, interferon-stimulated
response element, CCAAT box-binding transcription factor-nuclear factor 1 (CTF-NF1), and adenovirus early region 4 promoter transcription factor 1 (E4TF1) and a cluster of Sp1-binding sites located in this region (Fig. 1B). In particular,
members of the Sp1 family have been shown to act as positive or
negative regulators of gene transcription. This mechanism is dependent on the competition between the transcription repressor HDAC1 and the
transcription factor E2F1, which actives histone acetyltransferase (52). The presence of Sp1-binding elements in the proximal
hDAB2IP gene promoter could underlie the basis of gene
repression mediated by histone deacetylation (53, 54). Further
investigation is warranted.
Regarding the role of DNA methylation in regulating hDAB2IP
gene transcription, bisulfite sequencing data indicated that CpG islands remained almost unmethylated in normal prostate cell lines (PrEC and PZ-HPV-7) expressing the transcriptionally active
hDAB2IP gene (Fig. 1C) (1). However, in PCa cells
(PC-3 and LNCaP), hypermethylation of CpG islands was commonly
associated with the hDAB2IP promoter region (Fig. 6). 5'-Aza
could induce the expression of hDAB2IP mRNA in PCa cells
(Table II). Our results are consistent with the promoter activity
determined by the reporter gene assay (Fig. 2).
Regarding the regulation of the hDAB2IP gene, our data
clearly demonstrate that DNA methylation and histone deacetylation can
act cooperatively in silencing the hDAB2IP gene (Table II and Fig. 4). It has been shown that DNA methyltransferases recruited by
an oncogene to a gene promoter suppress the expression of this gene (55). Also, the binding of the methyl-CpG-binding protein complex (21) to methyl-CpG islands competes with transcription factors
and prevents them from binding to the promoter region. Recent data
indicate that the methyl-CpG-binding protein can recruit HDACs,
leading to condensation of the local chromatin structure and thereby
rendering the methylated DNA less accessible to transcription factors (25).
In the hDAB2IP gene, there are two regions with potential
promoter activity: P1 and P2. In this study, we found that P2 promoter activity has a better correlation with the induction of
hDAB2IP mRNA in every tested cell line (Fig.
1C). The methylation profile of the P2 promoter in each cell
line exhibited a reciprocal relationship between P2 reporter gene
activity (Fig. 5C) and the density of methylated cytosine
residues (Fig. 6C). Furthermore, the P2 (but not P1)
promoter was able to respond to both TSA and 5'-Aza treatment (Fig. 2).
Nevertheless, TSA seemed more potent than 5'-Aza in eliciting P2
promoter activity (Figs. 2 and 5). Therefore, we believe that the P2
region in the hDAB2IP gene represents a core promoter in
prostatic epithelia. Our results also suggest that the
deacetylhistone-mediated transcriptional silencing of the hDAB2IP gene may be a critical event during the
carcinogenesis of PCa.
In summary, cytosine methylation and histone deacetylation in the
hDAB2IP regulatory regions associated with the silencing of
hDAB2IP gene expression have been observed in PCa cells
(PC-3 and LNCaP). Such a phenomenon seems to be specific to cancer
because it was not detected in normal prostate cells (PZ-HPV-7 and
PrEC). Therefore, this gene could potentially serve as a surrogate
marker for early cancer detection. The outcome of this study also
indicates that histone deacetylase and DNA methyltransferase can be
novel targets for PCa therapy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ct (threshold cycle) of
each sample = mean of
Ct(hDAB2IP)
mean
of Ct(GAPDH). The -fold induction of
each sample = 1/2
Ct(sample)
Ct(control).
) (Stratagene) (1). pGL3-1.6S, a 1.6-kb
KpnI-XhoI fragment, was subcloned from this
7.6-kb element into the pGL3-Basic vector (Promega). Two putative
promoter regions (P1, a 0.8-kb SfiI-XhoI fragment from +229 to +981; and P2, a 0.6-kb
KpnI-Kpn2I fragment from
598 to +44)
were subcloned into the pGL3-Basic vector (see Fig. 1A).
520 to
287) fragments. PCR fragments were cloned into
pCR2.1-TOPO (Invitrogen), sequenced, and then subcloned into the
pGL3-Basic vector using HindIII-XhoI sites.
-galactosidase vector (pCH110) using FuGENE 6 (Roche Molecular Biochemicals). The LNCaP and PrEC cells were transfected with the same
amount of DNA with LipofectAMINE Plus transfection reagent (Invitrogen). Twenty-four hours after incubation, the transfected cells
were treated with TSA for 24 h, 5'-Aza for 48 h, or a
combination of both drugs by incubating with 5'-Aza for 24 h and
then adding TSA for an additional 24 h. After washing twice with
cold phosphate-buffered saline, the cells were harvested with lysis
buffer (Promega). Both luciferase and
-galactosidase
activities were assayed as previously described (1, 12). The protein
concentration of each extract was measured using the Bio-Rad protein
assay. The relative luciferase activity (RLA) was calculated by
normalizing both
-galactosidase and protein concentrations in each
sample, and the data were averaged from RLA in triplicate.
PCR primers used on bisulfite-treated DNA and in ChIP assays
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
598 to +44, located 5'-upstream of
exon Ia) (Fig. 1A), were
identified using both the TSSW program (human PII recognition using the
TRANSFAC Database)2 and
experimental deletion analysis (1) of a 1662-bp
hDAB2IP locus surrounding the transcription
initiation site (+1). As shown in Fig. 1B, there are many
GC-rich sequences, and potential transcription factor-binding sites
were detected within this region using MacVector Version 6.5.3.
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Fig. 1.
Characterization of hDAB2IP
gene promoters. A, schematic
representation of potential hDAB2IP promoters. The
transcription start site (TSS) at +1 was predicted by
MacVector Version 6.5.3. P1 (+229 to +981) is within the first intron;
P2 ( 598 to +44) is located 5'-upstream of exon Ia. The depicted
restriction endonucleases sites were used in subsequent cloning. PI
(+768 to +873) and PII (
520 to
287) were used for ChIP assay.
B, potential regulatory sequences of the hDAB2IP
gene. Exon Ia of the hDAB2IP gene is boxed, and
the putative cis-acting elements are underlined.
ISRE, interferon-stimulated response element.
C, differential promoter activities of the
hDAB2IP gene in PrEC, PZ-HPV-7, PC-3, and LNCaP cell. The
-fold RLA was calculated from the pGL3-Basic vector (taken as 1).
D, determination of the levels of acetylhistone H3
associated with the hDAB2IP gene promoter in normal and
malignant prostatic epithelia. The ChIP assay was carried out to
determine the status of acetylhistone H3 associated with either the PI
or PII region of the hDAB2IP gene promoter in PrEC,
PZ-HPV-7, PC-3, and LNCaP cells. M, molecular mass markers;
NC, negative control for PCR.
Determination of hDAB2IP mRNA expression induced by TSA and 5'-Aza
by real-time reverse transcriptase-PCR
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Fig. 2.
Effect of TSA and/or 5'-Aza on
hDAB2IP or PSA promoter activity in PCa cell
lines. Either PC-3 or LNCaP cells were transfected with
pGL3-P1 (A and B), pGL3-P2 (C and
D), or pPSA6.1 (E) under different treatment
conditions, and luciferase activity was determined as described under
"Experimental Procedures." The -fold RLA was calculated using the
pGL3-Basic vector (taken as 1). DHT,
dihydrotestosterone.
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Fig. 3.
Steady-state levels of histone H3 acetylation
in PCa cells induced by TSA. After TSA treatment, the
acid extract of nuclear protein from PC-3 cells (A)
or LNCaP cells (B) was subjected to Western blot analysis
and probed with an anti-acetylhistone H3 antibody (1:3000). The same
membrane was stripped and reprobed with an anti-histone H3 antibody
(1:1000) as an internal control. The values depicted beneath each lane
represent the relative levels of acetylhistone H3 determined by
normalizing the amount of acetylhistone H3 proteins to that of total
histone H3 proteins.
520 to
287) regions (Fig. 1A). Elevated levels of acetylhistone
H3 were clearly associated with the PI region in both PC-3 and LNCaP
cells treated with TSA or the combination, but not with 5'-Aza (Fig.
4A). Similarly, an
accumulation of acetylhistone H3 levels associated with the PII region
was also detected in both cell lines treated with TSA or the
combination (Fig. 4B). Interestingly, we also found that 5'-Aza treatment could induce the accumulation of acetylhistone H3 in
the PII region, but not in the PI region (Fig. 4B), because P2 (but not P1) activity could be induced in both PCa cell lines treated with 5'-Aza (Fig. 2). A similar phenomenon has also been observed in several different genes treated with 5'-Aza (10, 17). Also,
some data suggest that DNA methylation and histone deacetylation can
act cooperatively to silence tumor suppressor genes in cancer cells
(18, 20).
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Fig. 4.
Increased levels of histone H3 acetylation
associated with the hDAB2IP promoter in PCa cells
treated with TSA and/or 5'-Aza. The ChIP assay was performed using
an anti-acetylhistone H3 antibody. Nested PCR to detect the PI
(A) and PII (B) regions was performed using the
primer sets summarized in Table I. The input DNA (lower
panels) was used as a positive control. M, 1-kb plus
marker; NC, negative control without antibody.
520 to
287), in
PC-3 and LNCaP cell lines after treatment. As shown in Fig.
5 (A and B), the
basal luciferase activity of the pGL3-PI construct was much higher than
that of the pGL3-P1 construct in both PC-3 and LNCaP cells (Fig. 2,
A and B), suggesting that the deletion of 5'- and
3'-flanking sequences from the P1 region may contain some negative
elements. Overall, we detected a slight increase in PI activity only in
PC-3 cells treated with TSA or a combination of both TSA and 5'-Aza
(Fig. 5A); however, no change in PI activity was
detected in LNCaP cells after treatment (Fig. 5B).
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Fig. 5.
Characterization of the core promoter in the
hDAB2IP gene regulated by TSA and/or 5'-Aza.
Constructs pGL3-PI (A and B) and pGL3-PII
(C and D) derived from a ChIP assay were
transfected into PC-3 or LNCaP cells under different treatment
conditions, and RLA was determined as described under "Experimental
Procedures." The -fold RLA was calculated using the pGL3-Basic vector
(taken as 1).
520 to
287) within the
hDAB2IP promoter is the core regulatory region for
modulating hDAB2IP gene transcription.
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Fig. 6.
Characterization of the methylation status of
the hDAB2IP gene promoter in human prostatic
epithelial cells. A, schematic representation of the three
separated positions in the hDAB2IP locus subjected to
bisulfite sequencing analysis. B and C,
methylation patterns in the PmI (+282 to +975) and PmIIa ( 522 to
285) regions in human prostatic epithelial cells. High molecular mass
DNA isolated from each sample was modified with sodium bisulfite and
amplified by PCR using the primer sets indicated in Table I. The PCR
product was subcloned, and each individual clone (horizontal
rows) from every sample was sequenced. The position of each CpG
dinucleotide (vertical bars) is labeled with the number
representing its location in the hDAB2IP gene.
,
unmethylated CpG;
, methylated CpG.
522 to
285) in
hDAB2IP is the key regulatory sequence operative in
prostatic epithelia.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino groups on positively charged
lysine residues in histone amino-terminal tail domains (7, 28), which
can release negatively charged DNA to interact with transcription
factors. The effect of histone acetyltransferases (29) is
counterbalanced by the presence of (HDACs) (30). Aberrant acetylation
or deacetylation leads to such diverse disorders as leukemia,
epithelial cancers, fragile X syndrome, and Rubinstein-Taybi syndrome
(31). It is also known that HDACs can function as transcriptional
corepressors and are often present in multisubunit complexes such as
Sin3 and Mi2 complexes (32-34). From recent reports, HDAC-containing
complexes are involved in DNA methylation-mediated transcriptional
silencing of various tumor suppressor genes (15, 16). Therefore,
targeting HDAC activity has become a new strategy of cancer
chemotherapy; several inhibitors have been developed and tested in
clinical trials (35). Recent studies by several groups (36-38) have
demonstrated the existence of cellular complexes containing both HDACs
and ATP-dependent nucleosome-remodeling activity,
suggesting that some chromatin remodeling is mediated by cellular
complexes with HDAC activity. In contrast, histone acetyltransferases
such as CBP (cAMP-responsive element-binding
protein-binding protein)/p300, CBP-associated
factor (PCAF), and GCN5 have been identified in the protein
complex of transcriptional activators (39-41).
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ACKNOWLEDGEMENTS |
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We thank Dr. Trapman for providing the PSA reporter gene vector and Richard Hsu for editing this manuscript.
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FOOTNOTES |
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* This work was supported by NIDDK Grant DK-47657 from the National Institutes of Health and Department of Defense Grant PC970259.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) AF367051.
The on-line version of this article (available at
http://www.jbc.org) contains Supplemental Data
and Supplemental Figs. 1 and 2.
To whom correspondence should be addressed: Dept. of Urology,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., Dallas, TX 75390-9110. Tel.: 214-648-3988; Fax: 214-648-8786; E-mail: JT.Hsieh@UTSouthwestern.edu.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208230200
2 Available at genomic.sanger.ac.uk/gf/gf.htm.
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ABBREVIATIONS |
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The abbreviations used are: PCa, prostate cancer; TSA, trichostatin A; 5'-Aza, 5'-aza-2'-deoxycytidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RLA, relative luciferase activity; ChIP, chromatin immunoprecipitation; PSA, prostate-specific antigen; HDAC, histone deacetylase.
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