From the Epithelial Pathobiology Group, Centre For
Immunology and Cancer Research, University of Queensland Department of
Medicine, Princess Alexandra Hospital, Brisbane, Queensland 4102, the
Cancer Unit, Queensland Institute of Medical Research, Herston,
Queensland 4006, the ** Department of Pathology, University of
Queensland, Medical School, Herston, Queensland 4006, and the
Department of Physiology and Pharmacology,
University of Queensland, St. Lucia,
Queensland 4067, Australia
Received for publication, January 10, 2001, and in revised form, March 23, 2001
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ABSTRACT |
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Use of specific histone deacetylase
inhibitors has revealed critical roles for the histone deacetylases
(HDAC) in controlling proliferation. Although many studies have
correlated the function of HDAC inhibitors with the hyperacetylation of
histones, few studies have specifically addressed whether the
accumulation of acetylated histones, caused by HDAC inhibitor
treatment, is responsible for growth inhibition. In the present study
we show that HDAC inhibitors cause growth inhibition in normal and
transformed keratinocytes but not in normal dermal fibroblasts. This
was despite the observation that the HDAC inhibitor, suberic
bishydroxamate (SBHA), caused a kinetically similar accumulation of
hyperacetylated histones. This cell type-specific response to SBHA was
not due to the inactivation of SBHA by fibroblasts, nor was it due to
differences in the expression of specific HDAC family members.
Remarkably, overexpression of HDACs 1, 4, and 6 in normal human
fibroblasts resulted in cells that could be growth-inhibited by SBHA.
These data suggest that, although histone acetylation is a major target
for HDAC inhibitors, the accumulation of hyperacetylated histones is
not sufficient to cause growth inhibition in all cell types. This
suggests that growth inhibition, caused by HDAC inhibitors, may be the
culmination of histone hyperacetylation acting in concert with other
growth regulatory pathways.
Analysis of histone-modifying enzymes such as the histone
acetyltransferases (HATs)1
and deacetylases has resulted in significant advances in our understanding of transcriptional regulation (1-4). These studies have
resulted in a model of transcription in which transcriptionally competent genes are transcribed or repressed dependent upon their ability to recruit either HATs or histone deacetylases to the promoter
(4). In these models, recruited histone acetyltransferases associate
with transcription factor complexes (5-8), resulting in the
acetylation of nucleosomal histones, relaxation of nucleosomal integrity, and hence transcription. Conversely, transcriptional repression occurs when histone deacetylases (and cofactors) are recruited to DNA-bound transcription factors, resulting in the removal
of acetyl groups from NH2-terminal lysines causing a
"tightening" of nucleosomal integrity and a suppression of
transcription (9-13).
The isolation and synthesis of new and potent inhibitors of histone
deacetylase enzymes (HDACs) has allowed us to identify some of the
biological outcomes resulting from manipulation of histone deacetylase
activity. For example, it is now established that treatment of cells
in vitro and in vivo with HDAC inhibitors can
result in specific functional outcomes such as cell cycle arrest
(14-16), apoptosis/cell death (17-19), or differentiation (19-21).
These outcomes to a large extent are cell type-specific and have raised
the potential that the HDAC inhibitors may represent a new and
important class of anticancer therapeutic agents (4).
HDAC inhibitors comprise a diverse range of unrelated compounds that
all induce an accumulation of hyperacetylated histones (21). The
biological effects of these compounds (e.g. cell cycle arrest, cell death, or differentiation) are thought to result from the
accumulation of acetylated histones and transcriptional activation that
results from the use of these compounds. For instance, sodium butyrate
(NaB), suberic bishydroxamate (SBHA), and trichostatin A (TSA) are all
HDAC inhibitors that induce an accumulation of acetylated histone H4
(21, 22). These agents are also able to induce growth inhibition, which
has been shown to be associated causally with an induction of the
cyclin-dependent kinase inhibitor p21 in colon carcinoma
cells (15).
Although the data for the colon carcinoma cells used in the p21 studies
appear compelling, there are several pieces of evidence that suggest
that the biological outcomes, in response to the HDAC inhibitors in
other cell types, may not be explained simply by an accumulation of
acetylated histones. For instance, 1) it has been shown that certain
cell types (human dermal fibroblasts or murine erythroleukemia cells)
are able to grow in the presence of HDAC inhibitors and in the presence
of hyperacetylated histones (6, 18, 22). 2) We have shown previously
that the accumulation of acetylated histone H4 in response to NaB or
TSA in keratinocytes and the squamous cell carcinoma cell line, SCC25,
is transient and is temporally uncoupled from the process of growth
inhibition (21). 3) Recently it has been suggested that the cell cycle regulator, Rb, mediates its growth inhibitory effects by associating with an HDAC and thus suppressing E2F activity (23-25). Although this
has been convincingly shown in vitro, it should be noted that the E2F activation caused by HDAC inhibitors (23-25) is
inconsistent with the growth inhibition observed in cells following
HDAC inhibitor treatment. 4) Recent studies have shown that nonhistone
proteins such as E2F1 (26) and p53 (27, 28) are subject to both
acetylation and deacetylation, and more importantly these modifications
result in alterations in functional activity of these important
transcriptional controllers of growth and differentiation. 5) HDAC
inhibitors do not lead to global deregulation of transcription (29).
These observations raise the possibility that biological effects of HDAC inhibitors may be, in part, mediated by events independent of the
acetylation of histones (30).
In the present study we wished to examine the relationship between the
accumulation of acetylated histones in response to HDAC inhibitors and
the induction of growth arrest in keratinocytes, fibroblasts, and SCC25
cells. We report that the induction of acetylated histones in
fibroblasts is not associated with growth inhibition. Our data are
consistent with a model in which growth inhibition in response to HDAC
inhibitors is cell type-specific and dictated by the activity of
pre-existing regulatory pathways in the cells.
Cells, Tissue Culture, and Reagents--
Human epidermal
keratinocytes (HEKs) were isolated from neonatal foreskins following
circumcision and cultured in keratinocyte serum-free medium (Life
Technologies Inc, Sydney, New South Wales (NSW), Australia) as
described (31). Human dermal fibroblasts (HDFs) were also isolated from
foreskin samples following circumcision and were cultured in
Dulbecco's modified Eagle's medium as described (32). The
keratinocyte-derived squamous cell carcinoma cell line, SCC25, was
obtained from the American Type Tissue Culture Collection and cultured
as described (31). All cells in this study were used under
subconfluent, proliferative conditions. The histone deacetylase
inhibitors sodium butyrate (NaB), the R (R-PB) and S (S-PB)
enantiomeric forms of phenylbutyrate, and the hybrid polar
compound hexamethylene bisacetamide (HMBA) were purchased from Sigma
(Sydney, NSW, Australia). The synthesis of azelaic-1-hydroxyamate-9-anilide (AAHA) has been described previously (18) as has the synthesis of suberic bishydroxamate (SBHA, Ref. 33).
Plasmids, Transfections, and Cell Line Selection--
Human
histone deacetylase 1 (HDAC1; Ref. 34) and histone deacetylases 4, 5, and 6 (HDACs 4-6; Ref. 35) in the pBJ5 expression plasmid were a
generous gift from Prof. Schreiber (Harvard University, Cambridge, MA).
A glutathione S-transferase-tagged murine HDAC2 plasmid (36)
and a human HDAC3 expression plasmid (in the pGEX expression plasmid;
Ref. 37) were a generous gift from Prof. Seto (H. Lee Moffitt Cancer
Center and Research Institute, University of San Francisco, San
Francisco, CA). To establish stably expressing HDF cell lines,
75-cm2 flasks of passage 2 HDFs (50% confluent) were
transfected with 9 µg of HDAC expression vector + 3 µg of the
pSV2neo neomycin expression vector followed by selection with 500 µg/ml G418 (31). Transfection used a similar protocol to that
described for keratinocytes (38) in the presence of LipofectAMINE Plus
reagent as per manufacturer's instructions (Life Technologies,
Inc.).
Apoptosis Assay--
Estimates of apoptosis induced by NaB (3 mM), AAHA (3 µM), or SBHA (100 µM) were determined following a 24-h treatment with the
compounds. Apoptosis was estimated using the TUNEL assay as per the
manufacturer's protocol (Roche, Brisbane, Queensland (QLD), Australia). Briefly, cells were cultured in 25-cm2 flasks,
trypsinized, and then subjected to cytospinning (100 × g for 2 min) before being used in the TUNEL assay. An
apoptotic index was then derived (apoptotic cells/total cells *
100/1).
Western Blotting and Histone Isolation--
Protein expression
and histone acetylation status were determined by immunoblotting. For
Rb, p21, acetyllysine, or FLAG-tagged HDAC protein analysis, cells were
trypsinized and rinsed with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM
KH2PO4). An aliquot was removed for protein
determination, and the remaining protein solubilized in 2× sample
buffer (25 mM Tris-Cl, 5% glycerol, 5% SDS, 5%
RNA Isolation and Northern Blotting--
Total cellular RNA was
isolated from cells following disruption in Trizol (Life Technologies,
Inc.) using previously described protocols (38, 39). Enrichment for
poly(A)+ RNA by oligo(dT)-cellulose (Collaborative Research
Inc., Bedford, MA) chromatography was then performed as described (40).
One microgram of poly(A)+ RNA (HEKs and HDFs) was then
electrophoresed, transferred to nylon membrane (Hybond N; Amersham
Pharmacia Biotech), and hybridized with probes specific for HDACs 1-6
and actin. Probes for specific HDACs were made from restriction digests
of the expression plasmids using the following restriction enzymes;
HDAC1 = NotI/EcoRI, HDAC2 = XhoI, HDAC3 = EcoRI, HDAC4
=SalI/SacI, HDAC5 = SacI/SacII, HDAC6 = SphI/AvrII. All inserts were gel-purified. Probe
labeling with [32P]dCTP (Geneworks, Adelaide, South
Australia, Australia), hybridizing, and washing were performed as
described previously (41). A probe against glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used to normalize for loading inequalities
and has been described previously (42). All blots were visualized by
autoradiography with Kodak XAR5 film. Images were then captured using a
laser densitometer (Molecular Dynamics, Sydney, NSW, Australia) and
imported into Adobe Photoshop.
Proliferation Assay and Cell Cycle Analysis--
Proliferation
was measured directly either by assaying for
[3H]thymidine incorporation (43) or by an analysis of the
cell cycle in propidium iodide-stained cells as described (44).
Kinetic Analysis and Statistics--
Dose response analysis for
proliferation suppression by histone deacetylase inhibitors was
estimated by nonlinear regression analysis with the following equation
P = P0e Growth Inhibition by Histone Deacetylase Inhibitors Is
Cell-selective--
We have shown previously that the histone
deacetylase inhibitors NaB and TSA are able to inhibit DNA synthesis in
keratinocytes and a number of keratinocyte-derived squamous cell
carcinoma cell lines (21). Treatment of keratinocytes and SCC25 cells
with varying concentrations of NaB, R-PB, S-PB, AAHA, SBHA, or the hybrid polar compound, HMBA, indicated that only NaB, AAHA, and SBHA
induced growth inhibition (Table I).
Dose-response analysis indicated that NaB and AAHA were slightly more
potent at inducing growth arrest in keratinocytes than in SCC25 cells
(Table I). In contrast, the EC50 value for the inhibition
of DNA synthesis by SBHA in HEKs and SCC25 cells was similar (Table I).
A dose-response profile for the inhibition of DNA synthesis by R-PB or
S-PB in HEKs and SCC25 cells was not possible despite analysis out to 6 mM. A modest inhibition of DNA synthesis with R-PB and S-PB was noted only at the 6 mM concentration in HEKs (R-PB,
40 ± 13%; and S-PB, 53 ± 24% that of untreated controls)
and SCC25 cells (R-PB, 112 ± 14%; and S-PB, 122 ± 6% that
of untreated controls). In contrast, these agents were not able to
inhibit DNA synthesis in HDFs (Table II).
In fact, NaB and AAHA, at high concentration, significantly increased
DNA synthesis in the HDFs (Table II). However, growth inhibition of
HDFs can eventually be induced by prolonged exposure (48 h) to high
concentrations of SBHA (300 µM). Furthermore, the
insensitivity of HDFs to SBHA could be altered by changing media
composition (data not shown). Combined, these data clearly show that
keratinocytes and the keratinocyte-derived carcinoma cell line are
sensitive to the growth inhibitory properties of histone deacetylase
inhibitors whereas fibroblasts remain insensitive under similar
conditions.
Cell cycle analysis of keratinocytes treated with varying
concentrations of SBHA for 24 h indicated that the decrease in DNA synthesis observed in these cells (Table I) was associated with an
accumulation in G0/G1 phase (Table
III). Consistent with the results of the
DNA synthesis assay in HDFs, the cell cycle analysis did not reveal any
specific blockade of the cell cycle (Table III). There was a decrease
in the percentage of HDFs in G2/M phase following treatment
with 100 µM SBHA, which suggests that SBHA may reduce
transit time through G2/M phase and may be consistent with
the modest increase in DNA synthesis observed in HDFs following SBHA
treatment (Table II). Interestingly, the cell cycle profile observed in
SCC25 cells treated with 100 µM SBHA showed an
accumulation in the G2/M phase with a lesser, if any,
accumulation of cells in G0/G1 (Table III).
This contrasts with the findings observed in HEKs and suggests that
different mechanisms of action may be invoked by SBHA treatment of
keratinocytes and transformed keratinocytes. It should be noted that,
following treatment with SBHA, ~30-50% of SCC25 became detached,
which is consistent with cell death. A similar result has been
described previously for these cells (44). Thus, the findings in the
SCC25 cells most likely represent a combination of a G2/M
accumulation of SBHA-treated cells and the induction of cell death.
A well characterized target of HDAC inhibitor action is the induction
of p21 (15) and/or the accumulation of hypophosphorylated Rb (16), both
of which are thought to be mediated by alterations in transcriptional
control mediated by HDACs (45-47). Following a 24-h treatment of HEKs,
HDFs, and SCC25 cells with SBHA (100 µM), there was an
induction of p21 protein expression in all the cell types (Fig.
1). However, the induction of p21 was
associated with a corresponding alteration in the phosphorylation
status of the pocket proteins Rb or p107 only in keratinocytes but not in HDFs or growth-inhibited SCC25 cells (Fig. 1). Moreover, significant levels of expression of the pocket protein p130 were observed only in
keratinocytes but were not altered by SBHA treatment (Fig. 1). The lack
of an association between SBHA-induced growth inhibition and the
phosphorylation status of Rb and p107 in SCC25 cells suggests that a
mechanism distinct from E2F suppression may be involved in SBHA-induced
growth suppression in SCC25 cells. These findings are consistent with
the cell cycle analysis (Table III), which indicated that SBHA
selectively induced an accumulation of SCC25 cells in the
G2/M phase and selectively induced an accumulation of
keratinocytes in the G0/G1 phase. Combined,
these data show that the ability of SBHA to induce growth inhibition
may be mediated by events in G0/G1 or
G2/M dependent upon the cell type or transformation status.
An alternative explanation for the decrease in DNA synthesis induced by
HDAC inhibitors in keratinocytes and SCC25 cells may be attributable to
apoptosis. Previously, agents such as NaB or TSA have been reported to
induce apoptosis in colon carcinoma cell lines (48) and C3H10T1/2 cells
(19). Furthermore, it has been shown that ABHA treatment of SCC25 cells
is associated with detachment and cell death (44). In the present
study, we found no evidence of apoptosis, by TUNEL assay, in adherent
HEKs and SCC25 cells following NaB, AAHA, or SBHA treatment (data not shown). However, NaB, AAHA, and SBHA treatment did cause a significant proportion of the adherent SCC25 cells to lift off the culture dish.
These data are consistent with a non-apoptotic/necrotic mechanism of
cell death in SCC25 cells (data not shown). These data indicate that
the decrease in DNA synthesis observed in the adherent HEKs following
HDAC inhibitor treatment was not due to apoptosis or cell death,
whereas the G2/M phase accumulation, observed in SCC25
cells, may be a prelude to detachment and cell death.
Histone Deacetylase Inhibitors Induce an Accumulation of Acetylated
Histone H4 in HEKs, HDFs, and SCC25 Cells--
The simplest
explanation for the resistance of fibroblasts to the histone
deacetylase inhibitors is that the inhibitors are unable to induce an
accumulation of acetylated histones due to the inactivity of these
compounds in fibroblasts. We have shown previously that accumulation of
acetylated histones in keratinocytes and SCC25 cells is transient and
is maximal at 8 h in response to NaB or TSA. Therefore, we treated
HEKs, HDFs, and SCC25 cells with the various HDAC inhibitors for 8 h and then isolated the histone proteins from these cells. A Western
blot detecting acetylated histone H4 is shown in Fig.
2. These data show that NaB, SBHA, and
AAHA are able to induce an hyperacetylation of histone H4 in all cell
types (H4 acetylation caused by AAHA is visible in HDFs with a longer
gel exposure time). These data also show that the inability of the HDAC
inhibitors to cause growth inhibition of HDFs is not due to an
inability to induce histone H4 acetylation (a similar result was noted
with H3 acetylation; data not shown). It is also of interest to note
that HMBA, R-PB, and S-PB did not induce an accumulation of acetylated
H4 and were unable to produce significant growth arrest in any of the
cells.
The inability to induce growth arrest in the HDFs, in response to SBHA,
was examined in more detail to determine whether there were differences
in the kinetics for the inhibition of HDACs that may explain the
inability of SBHA to cause growth inhibition of HDFs (Fig.
3). Dose-response analysis for the
inhibition of growth (Fig. 3A) and the accumulation of
acetylated histone H4 (Fig. 3B) was examined in HEKs and
HDFs. SBHA induced growth inhibition in HEKs with an IC50
value of 11.7 ± 8.5 µM (Fig. 3A).
Similarly, SBHA induced an accumulation of acetylated histone H4 in
HEKs with an EC50 value 7.9 ± 7.8 µM.
In contrast, HDFs were resistant to the growth inhibitory properties of
SBHA (Fig. 3A) but accumulated acetylated histone H4 with an
EC50 value similar to that for HEKs (1.0 ± 1.8 µM). These data clearly show that, although HDFs are resistant to the growth inhibitory properties of SBHA, both HEKs and
HDFs are sensitive to the HDAC inhibitory action of SBHA.
An explanation for the resistance of HDFs to SBHA-mediated growth
inhibition could be due to the degradation/metabolism of SBHA by HDFs
or HDF media. This possibility was formally tested by taking media from
HDFs treated with SBHA for 24 h and placing it on SCC25 cells for
an additional 24 h (Fig. 3C). Media from HDFs treated
with SBHA (300 µM) for 24 h was able to cause growth inhibition in SCC25 cells of similar magnitude to that of SCC25 cells
treated directly with SBHA (Fig. 3C). This observation
clearly indicates that SBHA is not selectively inactivated by HDFs and suggests that another explanation for the growth inhibitor resistance of HDFs exists. This may include the possibility that 1) the HDAC family member (e.g. HDACs 1-8; Refs. 34-52) responsible
for mediating growth inhibition, in response to SBHA, may be absent or
poorly expressed in fibroblasts compared with keratinocytes or SCC
cells; 2) the growth inhibitory action of the HDAC inhibitors may be mediated by the hyperacetylation of non-histone proteins; or 3) the
HDAC complexes involved in SBHA-induced growth inhibition may be cell
type-specific or gene-specific.
HEKs and HDFs Have a Different Complement of HDAC mRNA
Expression--
The lack of an association between the
hyperacetylation of histones and the inhibition of growth of HDFs, in
response to HDAC inhibitors, may be attributable to cell type-specific
expression of different HDAC family members. We therefore screened the
HEKs, HDFs, and SCC25 cells by Northern analysis to determine whether the growth inhibitory response to HDAC inhibitors was associated with
the expression of specific HDAC family members (HDACs 1-6; Fig.
4). There are currently eight reported
HDAC family members comprising type 1 HDACs (HDACs 1-3 and 8) and type
2 HDACs (HDACs 4-7); HDAC1 (34), HDAC2 (36), HDAC3 (37, 49), HDAC4,
HDAC5, HDAC6 (35), HDAC7 (50, 51), and HDAC8 (52). Analysis of HDACs
1-6 indicated that mRNA expression for the type 1 HDACs 1 and 5 were similar between untreated keratinocytes and fibroblasts when
normalized for GAPDH expression (Fig. 4). In contrast, expression of
HDAC2 and HDAC6 was greater in untreated keratinocytes than in
fibroblasts. Furthermore, the expression of HDAC6 in HEKs was restricted to one highly expressed transcript of ~6 kilobase pairs, whereas both HDFs and SCC25 cells (data not shown) expressed very little of this transcript but did express two smaller transcripts at
lower expression levels (Fig. 4). The expression of HDAC4 was difficult
to assess, due to its high molecular mass (approximately 9 kilobase
pairs; Ref. 35). However, keratinocytes expressed two large transcripts
whereas fibroblasts expressed two smaller transcripts (Fig. 4). As with
HDAC6, we did not determine whether the difference in transcripts was
due to alternate spliced variants, different promoter usage, or
different closely related gene transcripts. Interestingly, the
expression of HDACs 1, 2, and 5 in HDFs appeared to be induced
following treatment of cells with 100 µM SBHA for 24 h (Fig. 4). The data for HDAC mRNA expression in SCC25 cells resembled that of the fibroblasts (data not shown). Although similar amounts of poly(A)+ RNA were used for HEKs and HDFs, the
HDFs consistently showed lower levels of GAPDH mRNA expression.
This most likely reflects differences in GAPDH mRNA expression
between HEKs and HDFs. Densitometric analysis in which normalization
for GAPDH expression is estimated is shown in Fig. 4B.
HDAC1, HDAC4, and HDAC6 Restores SBHA-induced Growth Inhibition in
HDFs--
The data in Fig. 4A indicate that there are some
differences in the mRNA expression levels and transcript species
between HEKs and HDFs. This raises the possibility that different HDAC family members contribute to the difference in response between HEKs
and HDFs. To test this we transfected HDFs with expression plasmids for
the neomycin resistance gene (pSV2neo) and either pBJ5 (control vector)
or plasmids coding for HDAC1, HDAC4, HDAC5, or HDAC6. Pooled colonies
were enriched in media containing 500 µg/ml G418 and then treated
with vehicle or 100 µM SBHA. Fig. 5A shows that the selected
HDFs express the relevant FLAG-tagged HDAC with which they were
transfected. Of the selected cells, HDAC1 appeared to have the highest
expression followed by HDAC6 and then HDAC4 (Fig. 5A).
HDAC5-transfected cell lines could not be established and appeared to
undergo premature senescence (data not shown). Analysis of DNA
synthesis in the cell lines indicated that the control selected cells
were still resistant to the growth inhibitory properties of SBHA
whereas HDAC1-, HDAC4-, and HDAC6-expressing cells had become
profoundly sensitive to SBHA (Fig. 5B). Furthermore, the DNA
synthesis between the untreated cell lines was similar, indicating that
HDAC1, -4, and -6 expression did not alter constitutive proliferation
of the HDFs. It was interesting to note that, although there was no
difference in DNA synthesis between the untreated control and HDAC1-,
HDAC4-, and HDAC6-expressing cells, there were some marked differences
in their cell cycle profile (Fig. 5C). For instance,
HDAC4-expressing cells have an increased percentage of cells in the
G2/M phase (Fig. 5C). Thus, although our data revealed little change in DNA synthesis in the HDAC-expressing cells,
there were profound changes in the distribution of HDAC4-expressing cells in the cell cycle, suggesting that transit through the cell cycle
phases can be altered by overexpressing HDAC4. Furthermore, these data
would suggest that the ability to induce growth inhibition in response
to SBHA is not restricted to a particular HDAC but is a property of
type 1 (HDAC1) and type 2 HDACs (HDACs 4 and 6).
Recent studies have shown that non-histone proteins are the target for
acetylation/deacetylation (30). We considered the possibility that the
growth inhibition of HEKs and SCC25 cells, in response to SBHA, may be
associated with a hyperacetylation of proteins that were not
hyperacetylated in HDFs (Fig. 6). Western blot analysis, using a generic anti-acetyllysine antibody, revealed four protein bands in HEKs and SCC25 cells that were hyperacetylated in
response to SBHA and were not hyperacetylated in HDFs (Fig. 6).
However, a similar analysis of HDFs overexpressing HDAC1, HDAC4, or
HDAC 6 did not show any corresponding hyperacetylation of proteins, in
response to SBHA, that were similar to HEKs (data not shown). These
data indicate that, although there are increases in proteins in HEKs
and SCC25 cells, that are immunoreactive to an anti-acetyllysine
antibody, following SBHA treatment, these same protein bands were not
increased in HDFs or in HDFs overexpressing HDAC1, HDAC4, or HDAC6.
In the present study we have examined the ability of various HDAC
inhibitors to induce histone acetylation and growth inhibition in
normal keratinocytes, transformed keratinocytes, and normal fibroblasts. Our results indicate that, although all three cell types
were similarly responsive to the HDAC inhibitors, with respect to the
accumulation of acetylated histone H4, they were dissimilar in their
ability to respond to the growth inhibitory properties of the HDAC
inhibitors. Overall, our data provide evidence that the action of
specific HDACs and HDAC inhibitors is complex and may involve
mechanisms in addition to histone acetylation.
Histone H4 Acetylation Is Not Sufficient to Cause HDAC
Inhibitor-mediated Growth Inhibition--
There is a growing body of
evidence suggesting that HDAC inhibitor-induced growth inhibition may
be independent of the effects of the HDAC inhibitors on global histone
acetylation status (30). Clearly, our data with HDFs indicate that
increases in histone H4 acetylation status can occur in response to
HDAC inhibitors in the absence of growth inhibition. Further support
for the discordance between HDAC inhibitor-induced histone
hyperacetylation and growth inhibition can be seen in cells in which
both growth inhibition and histone hyperacetylation occur. For
instance, the accumulation of acetylated histones, induced by HDAC
inhibitors, in keratinocytes, transformed keratinocytes (21), and colon
carcinoma cells (53) is transient. Moreover, in keratinocytes, the time
at which growth inhibition occurs is not associated with
hyperacetylation of histone H4 (21). Although it remains a formal
possibility that the transient hyperacetylation of histones that
precedes growth inhibition is sufficient to initiate growth inhibition,
it is unlikely because HDAC inhibitor-induced growth suppression
requires the continued presence of inhibitor at a time at which histone
H4 acetylation has returned to basal levels (21). A similar finding for
HT-29 cells has also been reported (15). These data indicate that the
temporal events associated with histone hyperacetylation and growth
inhibition, in response to HDAC inhibitors, are not consistent with
histone acetylation being the sole mediator of growth inhibition. Furthermore, we show that SBHA-induced H4 acetylation in normal fibroblasts is maximal at a dose that is not associated with growth inhibition. A similar lack of correlation between histone acetylation and growth inhibition has been reported previously for fibroblasts (18,
20), murine erythroleukemia cells (22), and MCF-7 cells (54) treated
with HDAC inhibitors. Thus, HDAC-mediated growth inhibition in HDFs and
HEKs has been shown to be temporally and kinetically independent of
histone acetylation status.
Although it may be argued that the genes responsible for
inhibiting growth in response to HDAC inhibitors are "switched
off/silenced" in fibroblasts, this would seem unlikely since the
overexpression of HDACs 1, 4, and 6 in fibroblasts is able to render
the cells sensitive to growth inhibition by SBHA. This is also
supported by the lack of further acetylation in the presence of excess
HDAC inhibitor in HDFs. This would argue that all histone H4 that could be hyperacetylated has been in the HDFs. Finally, the ability of
overexpressed HDACs 1, 4, or 6 to render the HDFs sensitive to growth
inhibition by SBHA indicates that the ability to induce growth
suppression is not restricted to specific HDACs or HDAC types.
If histone hyperacetylation is not sufficient to cause HDAC
inhibitor-induced growth arrest, what are the targets of HDAC inhibitor
action that contribute to growth inhibition? One possibility is that
HDAC inhibitors have non-histone targets such as structural proteins
(30), transcription factors (26-28, 55, 56), or cell cycle regulators
(57). Attempts to identify possible non-histone protein targets by
comparing acetyllysine profiles for HEKs, HDFs, and SCC25 cells, using
a generic anti-acetyllysine antibody, indicated that there are
non-histone proteins that are hyperacetylated in response to SBHA in
HEKs and SCC25 cells that are not hyperacetylated in HDFs. Although
these data provide tantalizing evidence of potential HDAC targets, it
will require more rigorous analysis to determine their role, if
any, in mediating SBHA-induced growth inhibition. Despite this, our
data clearly show that the regulation of any alternative regulatory
pathway(s), mediating HDAC inhibitor affects, clearly requires HDACs
since overexpressed HDACs 1, 4, and 6 reinstate growth inhibitor
sensitivity to HDFs. Although these observations do not preclude a role
for histone acetylation in HDAC-mediated growth inhibition, they do
show they are not sufficient to induce growth inhibition in HDFs.
Anti-proliferative Effects of HDAC Inhibitors Is Mechanistically
Different between Normal Keratinocytes and Transformed
Keratinocytes--
The present study has demonstrated that the
mechanism by which HDAC inhibitors induce growth inhibition is cell
type-specific and possibly transformation-specific. For instance, SBHA
treatment caused an accumulation of normal keratinocytes in the
G0/G1 phase of the cell cycle, which was
associated with an induction of the cyclin-dependent kinase
inhibitor, p21 and an accumulation of hypophosphorylated pocket
proteins pRb and p107. In transformed keratinocytes (SCC25 cells), SBHA
treatment caused an accumulation of cells in the G2/M phase
and non-apoptotic cell death, which was also associated with an
induction of p21 but no accumulation of hypophosphorylated pocket
proteins pRb or p107. In contrast, human dermal fibroblasts were not
growth-inhibited by SBHA yet still induced an increase in p21
expression. The induction of p21 by SBHA in HDFs was unexpected since
it was not associated with reduced DNA synthesis. A similar finding has
been reported by others (14). Furthermore, the p21 induced in HDFs, in
response to SBHA, appeared to be inactive since it was not associated
with a cell cycle block or an accumulation of hypophosphorylated pocket proteins. This suggests that p21 may not be a universal marker of HDAC
inhibitor-mediated actions and is consistent with the proposition that
the response to HDAC inhibitors is cell type-specific (58). In this
respect, it is important to note that epithelial cells are capable of
undergoing irreversible growth arrest and terminal differentiation
whereas fibroblasts generally undergo a reversible growth arrest and
are not thought to undergo terminal differentiation. Given the very
different biological fates of epithelial versus mesenchymal
cells, it is not unexpected that they may respond to similar stimuli in
different ways.
The cell type specificity of action of the HDAC inhibitors on the cell
cycle may help to explain the seemingly paradoxical role of HDACs and
HDAC inhibitors in regulating the p21/Rb/E2F axis. For instance, p21 is
known to inhibit phosphorylation of Rb, suppress E2F activity, and
inhibit proliferation. HDACs repress p21 transcription (45-47, 59),
and release of this repression by HDAC inhibitors leads to induction of
p21 and growth inhibition (4, 15). Conversely, HDAC inhibitors have
been reported to relieve Rb-mediated E2F inhibition, resulting in E2F
activation (23-25) and hence proliferation. Clearly, these effects of
HDAC inhibitors upon p21 transcription and Rb activity oppose one
another. This suggests that the biological consequences of HDAC
inhibitor treatment may in fact represent the sum of these two, or
more, opposing actions, which in turn may explain the differing
biological outcomes for HEKs, HDFs, and SCC25 cells treated with HDAC
inhibitors. For instance SBHA-mediated p21 induction in HEKs is
associated with pocket protein hypophosphorylation and growth arrest,
whereas in SCC25 cells, which have defective Rb/E2F regulation (31, 38), p21 induction is associated with an accumulation in
G2/M. In contrast, p21 induction in HDFs is not associated
with hypophosphorylation of pocket proteins or cell cycle block,
suggesting that a higher level of proliferative regulation predominates.
Possible Mechanisms for HDAC Inhibitor Action in Normal
Keratinocytes, Fibroblasts, and Transformed Keratinocytes--
The
lack of an effect on constitutive DNA synthesis by overexpressed HDACs
suggests that in normal HDFs the activity of HDACs may be subordinate
to preexisting pathways controlling proliferation. Although the
identity of such pathways is unknown, it is possible to speculate on
the nature of such pathways. Thus, one could speculate on the existence
of a regulatory framework in normal fibroblasts (or other cells) that
is controlled by the location or interaction of HDACs with other
regulatory proteins such that HDAC inhibitors have little effect on
proliferation. When these processes are overwhelmed through the
overexpression of HDACs, the normal regulatory interactions are
saturated leading to the availability of HDACs to interfere with cell
cycle progression. This notion gains further support from the
observation that overexpression of HDAC4 is able to disrupt the
constitutive cell cycle phases in fibroblasts, resulting in an
equivalent fraction of cells existing in the
G0/G1 phase as in the G2/M phase.
This proposition also is supported by the growth inhibition in HDFs
overexpressing HDACs 1, 4, and 6 following treatment with SBHA.
Moreover, recent reports suggest that the activity and subcellular
localization of HDACs may be critical events in regulating
differentiation control (60, 61).
The profound growth arrest of fibroblasts overexpressing HDACs 1, 4, and 6 in response to SBHA is consistent with a process in which
proliferation control by HDACs and HDAC inhibitors in normal HDFs is
selectively suppressed by homeostatic growth pathways. This implies
that certain cells may have an intrinsic resistance to HDAC
inhibitor-induced growth inhibition dependent upon the relative
activity of these regulatory pathways. Such a proposal is not
unprecedented since the discovery of interactions of HDACs with key
cellular regulators (58, 60-62) associated with growth and
differentiation control, which indicate that the available "active"
HDACs may be regulated by their interaction with, or sequestration by,
other intracellular effector molecules (60, 62). For instance, HDACs
are known to interact with E2F1 (26), p53 (27, 28), 14-3-3 (58, 62),
MEF2A (55), Sp1 (56), ERK 1/2 (61), or Hus1/Rad9 (57). These disparate
partner proteins reflect the growing complexity of our understanding of
how HDACs may work and how HDAC-dependent affects may be
determined by non-histone protein interactions. These data also
establish that histone acetylation alone may not be sufficient to
mediate HDAC inhibitor-induced actions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 0.005% bromphenol blue) and boiled for 5 min.
Twentyfive micrograms of protein were then run on a 7.5% SDS-PAGE gel
(16 cm × 18 cm), transferred to polyvinylidene difluoride
membrane, and probed with antibodies specific for Rb (sc-50; 1:1000),
p107 (sc-318; 1:1000), p130 (sc-317; 1:1000), p21 (sc-397; 1:1000;
Santa Cruz Biotechnology, Brisbane, QLD, Australia), acetyllysine
(06-933; 1:1000; Upstate Biotechnology, Parkville, Victoria,
Australia), or anti-FLAG antibody (1 µg/ml; F-3165; Sigma, Sydney,
NSW, Australia). To determine histone acetylation status, cells were
harvested by trypsinization and histones isolated as described (21).
Five micrograms of purified histones were then run on a 15% SDS-PAGE gel and blotted to PVDF membrane and probed with either an antibody specific for acetylated histone H4 (06-598) or an antibody specific for
acetylated lysines (06-933; Upstate Biotechnology Inc, Melbourne, Victoria, Australia). Purified H2A, H3, and H4 (Roche) were run in
separate lanes to confirm the identity of the purified histones. All
immunoblots were visualized using a primary antibody dilution of 1:1000
and chemiluminescent detection (ECL; Amersham, Sydney, NSW, Australia)
as described (21). Quantitation of acetylation level was determined by
densitometric analysis of the autoradiographs as described (21).
k[I], where
P = DNA synthesis, P0 = DNA
synthesis in the absence of inhibitor, k = rate
constant defining the inhibition DNA synthesis, and [I] = the
concentration of HDAC inhibitor (39). Dose-response analysis for the
accumulation of acetylated histones was estimated by nonlinear
regression analysis using the following equation: Ac = (Acmax * EC50)/(EC50 + [I]) where
Ac = the acetylation level, Acmax = the maximal amount
of histone acetylation, EC50 = the concentration at which
acetylation is half-maximal and [I] = the concentration of HDAC
inhibitor. All data were fitted using GraphPad Prism software
(Brisbane, QLD, Australia).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
IC50 values for various histone deacetylase inhibitors for the
inhibition of growth in keratinocytes or SCC25 cells
Ability of the histone deacetylase inhibitors to inhibit DNA synthesis
in keratinocytes and fibroblasts
Cell cycle analysis on human dermal fibroblasts, SCC25 cells, and human
epidermal keratinocytes treated with varying concentrations of SBHA
20 °C until used for cell cycle analysis. Data are presented as
percentage of total cells present in the different cell cycle phases.
View larger version (55K):
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Fig. 1.
Pocket protein (Rb, p107, p130) and p21
protein expression in HEKs, HDFs, and SCC25 cells in response to
SBHA. Keratinocytes (HEK), fibroblasts
(HDF), and SCC25 cells (SCC) were left untreated
( ) or were treated with 100 µM SBHA for 24 h (+).
Twenty-five µg of cellular protein were electrophoresed, blotted, and
then probed for the expression of Rb, p21, p130, and p107.
Hyperphosphorylated forms of Rb (RbPP) and p107
(P107PP) as well as hypophosphorylated forms of
Rb and p107 are shown.
View larger version (27K):
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Fig. 2.
Accumulation of acetylated histone H4 in
response to HDAC inhibitors. Histones were purified from cultured
human epidermal keratinocytes (HEK), human dermal
fibroblasts (HDF), or SCC25 cells (SCC25)
following treatment with potential HDAC inhibitors for 8 h
(lanes 2-7). Five µg of purified histone
protein were then run on a 15% SDS-PAGE gel, blotted, and probed with
an antibody specific for acetylated histone H4. NaB = 3 mM; R-PB = 6 mM; S-PB = 6 mM; AAHA = 3 µM; SBHA = 100 µM; HMBA = 1 mM.
View larger version (14K):
[in a new window]
Fig. 3.
Kinetics of growth suppression and histone H4
acetylation of HEKs and HDFs in response to varying concentrations of
SBHA. Proliferating keratinocytes (HEK) or dermal
fibroblasts (HDF) were treated with varying concentrations
of SBHA. DNA synthesis in response to a 24-h treatment with varying
concentrations of SBHA was then estimated by thymidine incorporation
(A). For DNA synthesis cells were incubated with 2.5 µCi
of [3H] thymidine for 3 h, followed by analysis of
dpm of thymidine/µg of cellular protein. Data are presented as
mean ± S.E. of triplicate determinations from two experiments and
expressed as a percentage of the value of the untreated cells. The
line represents the line of best fit determined by nonlinear
regression analysis (HEK; ) or linear regression analysis (HDF;
). B, following an 8-h treatment of HEKs and HDFs with
varying concentrations of SBHA, histone proteins were purified and 5 µg were used in a Western blot to determine histone H4 acetylation.
Autoradiograms were scanned by laser scanning densitometer and data
fitted by nonlinear regression analysis. Data for keratinocytes (HEK;
) and fibroblasts (HDF;
) represent mean ± S.E. of at least
four independent experiments. Histone acetylation is presented as
arbitrary units. C, DNA synthesis was measured in untreated
proliferating SCC25 cells (PROL) or in SCC25 cells treated
with 300 µM SBHA for 48 h (48 hr SCC25).
Alternatively, HDFs were treated with 300 µM SBHA for
24 h and the media removed and placed on SCC25 cells for another
24 h (24 hr HDF/24 hr SCC25). Data are
presented as mean ± S.E. of triplicate determinations.
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Fig. 4.
mRNA expression levels for HDACs 1-6 in
HEKs and HDFs. Keratinocytes (HEK) and fibroblasts
(HDF) were left untreated ( ) or were treated with 100 µM SBHA for 24 h (+). Total RNA was then isolated
and enriched for poly(A)+ RNA. One microgram of
poly(A)+ RNA was then electrophoresed, blotted, and probed
with cDNA probes specific for HDACs 1-6 or GAPDH (A).
Autoradiograms were scanned with a laser densitometer and imported into
Adobe Photoshop. Quantitation of mRNA expression is presented
(B). Data are normalized for GAPDH expression.
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Fig. 5.
Overexpression of HDAC1, HDAC4, and HDAC6
causes growth inhibition of fibroblasts in response to SBHA. HDFs
were transfected with expression plasmids for HDAC1, HDAC4, HDAC6, or
control plasmid (pBJ5). Cells were selected in G418 and then pooled
clones analyzed for expression of the FLAG-tagged HDAC protein in
untreated ( ) or SBHA-treated (+, 100 µM) cells
(A) or growth inhibition in response to 24-h treatment with
SBHA (100 µM) (B). Data represent the
mean ± S.E. of six determinations and are presented as dpm
incorporated [3H]thymidine/µg of cellular protein.
C, constitutive cell cycle profile of the untreated cell
lines expressing the HDACs.
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[in a new window]
Fig. 6.
SBHA causes increase in anti-acetyllysine
immunoreactive bands in HEKs and SCC25 cells but not HDFs.
Keratinocytes (HEK), fibroblasts (HDF), and SCC25
cells (SCC) were left untreated ( ) or were treated with
100 µM SBHA for 8 h (+). Twenty-five µg of
cellular protein was extracted, electrophoresed, blotted, and then
probed for the expression of acetylated lysine. Acetylation status of
histone H4 is shown at the top to verify the efficacy of
SBHA. Arrows indicate immunoreactive bands that are induced
in HEKs and SCC25 cells but not in HDFs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Schreiber for the generous gift of expression plasmids for HDAC1, HDAC4, HDAC5 and HDAC6. We also thank Dr. E. Seto for the generous gift of expression plasmids coding for HDAC2 and HDAC3.
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FOOTNOTES |
---|
* This work was supported in part by Queensland Cancer Fund Grant 98/QCFN001G, a grant from the Princess Alexandra Hospital Foundation, a grant from the Australian Government Employees Medical Defense Fund, Australian National Health and Medical Research Committee Grant 142906, and a grant from the Garnett Passe and Rodney Williams Memorial Foundation.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.
§ Supported by an Australian postgraduate award.
¶ Supported by a postgraduate scholarship awarded by the Garnett Passe and Rodney Williams Memorial Foundation.
§§ Supported by a Lions Medical Research Foundation senior research fellowship. To whom correspondence should be addressed: Epithelial Pathobiology Group, Centre for Immunology and Cancer Research, University of Queensland Department of Medicine, Princess Alexandra Hospital, Ipswich Rd., Brisbane, Queensland 4102, Australia. E-mail: nsaunders@medicine.pa.uq.edu.au.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M100206200
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ABBREVIATIONS |
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The abbreviations used are: HAT, histone acetyltransferase; HDAC, histone deacetylase; SBHA, suberic bishydroxamate; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human epidermal keratinocyte; HDF, human dermal fibroblast; R-PB, R enantiomeric form of phenylbutyrate; S-PB, S enantiomeric form of phenylbutyrate; AAHA, azelaic-1-hydroxyamate-9-anilide; HMBA, hexamethylene bisacetamide; TSA, trichostatin A; TUNEL, terminal dUTP nick-end labeling.
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