Induction of Polyploidy by Histone Deacetylase Inhibitor: Abstract

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Research Article
Induction of Polyploidy by Histone Deacetylase Inhibitor:
A Pathway for Antitumor Effects
Wei-Sheng Xu, Gisela Perez, Lang Ngo, Chang-Yun Gui, and Paul A. Marks
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York
Abstract
Histone deacetylase (HDAC) inhibitors can induce various
transformed cells to undergo growth arrest and/or death.
Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor
which is in phase I/II clinical trials and has shown antitumor
activity in hematologic and solid tumors at doses well
tolerated by patients. HDAC is the target for SAHA, but the
mechanisms of the consequent induced death of transformed
cells are not completely understood. In this study, we report
that SAHA induced polyploidy in human colon cancer cell line
HCT116 and human breast cancer cell lines, MCF-7, MDA-MB231, and MBA-MD-468, but not in normal human embryonic
fibroblast SW-38 and normal mouse embryonic fibroblasts.
The polyploid cells lost the capacity for proliferation and
committed to senescence. The induction of polyploidy was
more marked in HCT116 p21WAF1 / or HCT116 p53 / cells
than in wild-type HCT116. The development of senescence of
SAHA-induced polyploidy cells was similar in all colon cell
lines. The present findings indicate that the HDAC inhibitor
could exert antitumor effects by inducing polyploidy, and this
effect is more marked in transformed cells with nonfunctioning p21WAF1 or p53 genes. (Cancer Res 2005; 65(17): 7832-9)
Introduction
Histone deacetylase (HDAC) inhibitors, a promising class of
targeted anticancer agents, can block the proliferation and induce
cell death in a wide variety of transformed cells (1). Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor which is in
phase I/II clinical trials and has shown antitumor activity in
hematologic and solid tumors at doses well tolerated by patients
(2, 3). The antitumor mechanism of HDAC inhibitors has not been
completely understood.
HDAC inhibitors selectively affect gene transcription by
acetylation of histones and components of transcription factor
complexes (4). HDAC inhibitors block the activity of class I and II
HDACs (1), leading to accumulation of acetylated histones,
relaxation of chromatin structure, which, in general, enhances
the accessibility of transcription machinery to DNA (5). The
acetylation of transcription factors alters their activities and can
alter gene transcription (6, 7). Contrary to what might be
anticipated by the widespread distribution of HDACs in chromatin,
inhibition of HDAC activity by SAHA or trichostatin A results in an
alteration in transcription of a limited number of expressed genes
(f2-10%) in various transformed cell lines (8–11). Among these
genes, p21WAF1 (to be referred to as p21) is frequently increased in
Requests for reprints: Paul A. Marks, Cell Biology Program, Memorial SloanKettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-6568;
Fax: 212-639-2861; E-mail: paula_marks@mskcc.org.
I2005 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-04-4608
Cancer Res 2005; 65: (17). September 1, 2005
expression (5, 12). p21 mediates growth arrest in the G1 phase of
the cell cycle by inhibiting cyclin-dependent kinase complexes that
regulate cell cycle progression (13). p21 can protect cells against
apoptosis (14, 15). Induction of proapoptotic proteins such as BAX,
PIG3, and NOXA via p53 acetylation contributed to HDAC inhibitor
induced cell death in cells with competent p53 (16, 17), but these
agents can induce transformed cell death in p53 null cells. The
mechanisms by which HDAC inhibitors result in cell death remain
incompletely understood.
It has been shown that HDAC inhibitors can induce mitotic
defects by causing aberrant acetylation of histones in heterochomatin and centromere domains per se (18–22). The newly replicated chromatin contains acetylated histones (23, 24). In culture
with trichostatin A, in both yeast and mammalian cells, histones
in the newly synthesized chromatin remain acetylated, and this
disrupts the structure and function of the centromere (18–22). The
mitotic defect induced by HDAC inhibitors can result in cell death
by either apoptosis or mitotic death/catastrophe (25, 26).
Generation of reactive oxygen species may play a role in HDAC
inhibitor–induced cell death (27–29). SAHA and MS-275 can induce
transformed cells (27–29), but not normal cells (29), to accumulate
reactive oxygen species, increase caspase activity and undergo cell
death. Complete inhibition of the caspase activity did not inhibit
SAHA-induced transformed cell death (28–32), but inhibition of
reactive oxygen species by N-acetylcysteine attenuated cell death
(27, 28). The resistance of normal cell to SAHA-induced death is
associated with high levels of thioredoxin, a potent scavenger of
reactive oxygen species (29).
In the present study, we showed that SAHA can induce polyploidy
and, in turn, cell senescence in transformed cells. We determined the
effect of HDAC inhibitor on human colon cancer cell line HCT116,
and p21 null (p21 / ) and p53 null (p53 / ) derivative cell lines.
The HDAC inhibitor induced the accumulation of polyploid cells in
which there was continued DNA synthesis with failure of
cytokinesis. The large polyploid cells showed loss of clonogenicity
and evidence of senescence. The effect was more marked in p21 /
cells than in the p53 / cells and in both the degree of induced
polyploids was greater than in the wild-type cells. We also found that
HDAC inhibitor induced an increased accumulation of polyploidy
cells in human breast cancer cell lines, MCF-7, MDA-MB-231, and
MDA-MB-468, but not in normal human fibroblast SW-38 and
mouse normal embryonic fibroblasts. The present findings indicate
that SAHA, in addition to causing tumor cell growth arrest,
apoptosis, mitotic cell death, or reactive oxygen species–induced
cell death, may exert antitumor effect via pathway-inducing
polyploidy, particularly in cells with nonfunctioning p21 or p53.
Materials and Methods
Cell lines. Human colon cancer cell lines HCT116 p21 / and HCT116
p53 / and the parental HCT116 cell line from which each was derived (33)
were gifts from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD)
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Polyploidy Induced by HDAC Inhibitor
and were grown in McCoy’s medium supplemented with 10% fetal bovine
serum (HyClone Laboratory Inc., Logan, UT). Human breast cancer cell lines
MCF-7, MDA-MB-231, MDA-MB-468, and human embryonic lung fibroblast
cell line SW-38 were obtained from American Type Culture Collection
(Rockville, MD), and cultured in DMEM with 10% fetal bovine serum. Three
mouse embryonic fibroblast cells lines (359, 364, and a pooled cell line) were
gifts from Dr. Andrew Koff (Memorial Sloan-Kettering Cancer Center, New
York), and grown in DMEM with 10% fetal bovine serum. The effects of SAHA
on cell growth and viability were determined as described previously (34). The
expression of p21 and p53 proteins in HCT116 cell lines were determined with
a Western blotting technique as described previously (12), using antibodies
against p21 (Ab-11; NeoMarks, Fremont, CA) and p53 (BP-53-12; Upstate, Lake
Placid, NY). The antibody to a-tubulin (Ab-1) was used to evaluate loading of
gels (EMB Biosciences Inc., San Diego, CA). The morphology of HCT116 cells
cultured without or with SAHA was examined on slides prepared with f5,000
cells by cytospin and Giemsa staining.
Cell cycle analysis. Cells were seeded in 10 cm cell culture dishes at a
density of 106 cells/dish. Cells were allowed to attach overnight, and then
treated with 0.4 to 1 Amol/L (p21+/+ and p21 / cells) or 0.8 to 4 Amol/L
(p53+/+ and p53 / cells) SAHA for 3 days. For human breast cancer cell
lines, cells were cultured with 2.5 to 20 Amol/L SAHA for 5 days. For human
and mouse embryonic fibroblast cell lines, cells were cultured with 2.5 to 20
Amol/L for 3 days. On each day, the medium, together with cells suspended
in the medium, was removed and either combined with the attached cells or
discarded. The attached cells were collected by trypsinization. The cells
were centrifuged at 1,000 rpm for 5 minutes, washed with PBS, fixed with
2 volumes of ethanol, treated with 0.2 mg/mL RNase A and 20 Ag/mL
propidium iodide for 30 minutes at 37jC, and analyzed by fluorescenceactivated cell sorter analysis. The data were analyzed with FlowJo software
(Tree Star, Inc., Ashland, OR).
Bromodeoxyuridine labeling assay. Cells were cultured without or
with 0.4 to 1 Amol/L (p21+/+ and p21 / cells) or 0.8 to 4 Amol/L SAHA
(p53+/+ and p53 / cells) SAHA for 24, 48, and 72 hours. At each time
point, bromodeoxyuridine (BrdUrd; Amersham Biosciences, Buckinghamshire, England) was added to the medium to a final concentration of 10
Amol/L and cells were cultured for an additional hour. Cells were harvested,
washed with PBS, fixed with 2 volumes of ethanol, treated with 2 N HCl to
denature DNA, incubated with FITC-labeled anti-BrdUrd antibody (Becton
Dickinson Immunocytometry System, San Jose, CA) followed by propidium
iodide staining according to the manufacturer’s protocol, and subjected to
fluorescence-activated cell sorter analysis.
MPM-2 labeling assay. A similar procedure for BrdUrd labeling assay,
except for HCL denaturation step, was adapted to simultaneously measure
the DNA content and MPM-2-positive cells with a fluorescein-succimidyl
ester–conjugated MPM-2 antibody (Upstate), which recognizes phosphoepitopes on proteins in mitotic cells (35).
Assay for cell division using cell membrane label, PKH2. Assay of cell
division was done using PKH2, a cell membrane label. HCT116 p21 /
cells (107) cultured without or with 0.8 Amol/L SAHA for 3 days were
trypsinized, recovered as indicated above, and labeled with PKH2 green
fluorescent cell linker kit (Sigma-Aldrich Chemical Co., St. Louis, MO)
according to the manufacturer’s protocol. The PKH2 labeled cells were
plated at 2 105 per 6 cm plate and PKH2 fluorescence signal was
measured for each of the 5 consecutive days using fluorescence-activated
cell sorter analysis with propidium iodide staining to exclude dead cells.
Clonogenicity analysis. HCT116 p21 / cells were cultured without or
with 0.8 Amol/L SAHA for 3 days, and then sorted according to size into
small and large cells using MoFlo cell sorter (DakoCytomation Inc.,
Carpinteria, CA). The cells cultured without SAHA and the sorted small
and large cells were plated in soft agar (2,000 cells/10 cm plate), and after
14 days, the colonies were stained with Giemsa and counted.
Senescence analysis. HCT116 cells were cultured without or with 0.8
Amol/L SAHA (p21+/+ and p21 / cells) or 2 Amol/L SAHA (p53+/+ and
p53 / cells) for 5 days and then grown in fresh medium without inhibitor
for up to 5 days. Cells were stained with a senescence h-galactosidase
staining kit (Cell Signaling Technology, Inc., Beverly, MA), which detects
senescence-associated h-galactosidase (SA-h-gal).
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SAHA sensitivity assay. HCT116 p21 / cells were treated with 0.8
Amol/L for 3 days and sorted as described above. The sorted small cells
were grown in SAHA-free medium for 5 days to allow for recovery from
SAHA treatment. The sensitivity to SAHA of the sorted small cells and the
untreated control cells was determined as follows: 5 104 cells were seeded
in triplicate in 24-well plate in 1 mL medium. After 24 hours, the cells were
incubated with 0.125 to 4 Amol/L SAHA. At 48 hours, 0.1 mL Alamar blue
(BioSource International Inc., Camarillo, CA) was added to the cell culture.
The cells were incubated at 37jC for an additional 4 hours. The
fluorescence signal was then measured with GeminiXS microplate reader
(Molecular Devices, Sunnyvale, CA).
Statistical analysis. The significance of differences between experimental conditions was determined using Student’s t test for unpaired
observations.
Results
SAHA inhibits cell growth and induces cell death in HCT116
cells. We first determined the effect of SAHA on the growth and
viability of HCT116 p21+/+, HCT116 p21 / , HCT116 p53+/+, and
HCT116 p53 / cells. SAHA z0.8 Amol/L inhibited cell growth of
both the HCT116 p21+/+ and the p21 knock-out cells (Fig. 1A and B).
HCT116 p21 / cells were more sensitive to SAHA-induced cell
death than HCT116 p21+/+ cells (Fig. 1E and F). For example,
1.0 Amol/L SAHA after 72 hours of culture induced 30.5 F 2.1% cell
death in p21+/+ cells compared with 55.3 F 6.1% cell death in
p21 / cells (P < 0.05). For HCT116 p53+/+ and the p53 / cells,
SAHA z2 Amol/L inhibited their growth (Fig. 1C and D), whereas
HCT116 p53 / cells were less sensitive to SAHA-induced cell
death than HCT116 p53+/+ cell (Fig. 1G and H). For example,
4 Amol/L SAHA after 72 hours of culture induced 69.2 F 5.7% cell
death in HCT116 p53+/+ cells, but caused 49.5 F 3.3% cell death in
p53 / cells (P < 0.05). This was consistent with previous reports
that wild-type p53 increased the sensitivity to HDAC inhibitorinduced cell death (16, 17).
HCT116 p21+/+ cells (parent cell line of p21 / cells) are more
sensitive to SAHA-induced cell death than HCT116 p53+/+ cells
(parent cell line of p53 / cells; Fig. 1A, C, E, G). The difference
between the two parental cells HCT116 p21+/+ and HCT116 p53+/+
may reflect the genomic instability of HCT116 cell line, which is
defective in DNA mismatch repair machinery (36). To minimize the
effect of possible genetic drift between the parental and p21 or p53
knock-out cells, only early passages (<20) of cells were used in this
study, and knock-out cell lines were compared with its own
parental line cells. We determined the expression of p21 and p53
in each cell cultured without or with SAHA. As expected, p21
and p53 proteins were expressed in the parental cells, but not in
p21 / and p53 / cells, respectively (Fig 1I). SAHA dramatically
induced expression of p21 in both parental cells and in the p53 /
cell line but not in p21 / cells. Knockout of p53 reduced the
basal level of p21 expression, but did not affect p21 induction by
SAHA. This is consistent with the finding that SAHA can induce
p21 expression in a p53-independent manner (37).
SAHA-induced polyploidy in HCT116 cells. We next determined the effects of SAHA on cell cycle progression of the HCT116
p21+/+ and p21 / cells (Table 1). With increasing concentrations
of SAHA from 0.4 to 1.0 Amol/L, there was an increasing proportion
of cells with 4 N DNA content (4 N cells). With increasing SAHA
concentration and time in culture, there was an increase in the
percentage of polyploid cells (>4 N), which was more marked in
p21 / cells, >80% by day 3 at z0.8 Amol/L SAHA. The
accumulation of 4 N cells decreased with time with the increase
in proportion of polyploid cells.
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Figure 1. Effect of SAHA on cell growth and viability of HCT116 p21+/+, p21 / , p53+/+, and p53 / cells. Cells were cultured without or with SAHA in (Amol/L)
concentrations indicated for 72 hours. A-D, effect of SAHA on cell growth of HCT116 p21+/+ (A), p21 / (B), p53+/+ (C ), and p53 / cells (D ). E-H, effect of
SAHA on viability of HCT116 p21+/+ (E ), p21 / (F ), p53+/+ (G), and p53 / cells (H). I, the expression of p21 and p53 proteins in HCT116 cell lines cultured without
or with 0.8 Amol/L SAHA for HCT116 p21+/+ and p21 / cells, or 2 Amol/L SAHA for p53+/+ and p53 / cells for 24 hours. a-Tubulin was used as a loading control.
We then determined the effect of SAHA on cell cycle progression
of HCT116 p53+/+ and p53 / cells (Table 1). SAHA induced a
marked G1 arrest at low concentrations (0.8 and 1 Amol/L) in
HCT116 p53+/+ cells with a slight accumulation of 4 N cells and
polyploid cells, whereas it induced an accumulation of 4 N cells at
higher concentrations (2 and 4 Amol/L) with increased polyploidy
accumulation. SAHA induced an accumulation of 4 N cells in
HCT116p53 / cells with accumulation of polyploid cells, but no
G1 arrest, suggesting that p53 may be required for SAHA-induced
G1 arrest. SAHA induced more polyploid cells in HCT116 p53 /
cells than in p53+/+ cells, but this difference was not as great as
that between HCT116 p21 / and p21+/+ cells. The proportion of
4 N cells in p53 / cells decreased with time.
We determined if the relative increase in polyploid cells in
culture with SAHA could result from a loss of diploid cells. p21+/+
and p21 / cells cultured with 0.4 to 0.6 Amol/L SAHA or p53+/+
and p53 / cells cultured with 0.8 to 1.0 Amol/L SAHA caused
little cell death of the four cell lines (<10%), but induced several
Cancer Res 2005; 65: (17). September 1, 2005
fold more polyploidy cells in the knock-out cells than in the wildtype (Table 1).
To address whether the accumulation of 4 N cells induced by
SAHA represented a G2-M arrest, we analyzed the 4 N cells in
cultures without or with SAHA (2 Amol/L for p53+/+ and p53 /
cells, 0.8 Amol/L for p21+/+ and p21 / cells) using a mitosisspecific antibody, MPM-2, which recognizes a subset of mitotic
phosphoproteins (35). SAHA induced a transient accumulation of
MPM-2-positive mitotic cells in all four cell lines within 12 to 24
hours, with 26% in p53+/+, 32% in p53 / , 28% in p21+/+ and 21%
in p21 / cells being MPM-2-positive at 12 hours (Fig. 2A). By
24 hours, the percentage of MPM-2-positive cells in SAHA-treated
cells returned to the control level. Cells (4 N) accumulated at 24
hours in all four cell lines cultured with SAHA (Table 1). Taken
together, the data suggests that after 24 hours in culture with SAHA,
most 4 N cells are not in mitosis. Morphologic analysis showed that
cells cultured with SAHA became polymorphic, and the polyploid
cells contained giant nuclei and/or multiple varied size nuclei,
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Table 1. Percentage of the cells in different phases of cell cycle in HCT116 cells cultured without or with SAHA
HCT116 p21+/+ G1
day 0
Control
25
0.4 Amol/L SAHA
0.6 Amol/L SAHA
0.8 Amol/L SAHA
1 Amol/L SAHA
S
4N
>4 N
day 1 day 2 day 3 day 0 day 1 day 2 day 3 day 0 day 1 day 2 day 3 day 0 day 1
day 2
day 3
19
30
24
14
17
36
32
22
14
14
50
39
20
11
11
59
50
27
18
11
7
37
23
15
11
4
32
26
23
9
6
13
28
33
47
60
65
25
33
41
43
59
17
24
36
45
47
3
4
10
11
15
11
3
12
22
32
23
1
11
21
35
35
25
14
6
5
5
29
14
6
3
1
35
19
7
2
1
55
53
28
13
14
10
41
29
13
9
5
35
31
17
6
4
23
20
33
49
53
58
26
25
20
15
15
26
23
16
10
9
2
3
25
32
28
27
4
33
60
74
79
4
28
60
82
86
35
48
55
20
11
58
60
52
24
13
80
15
47
22
12
36
32
20
11
8
9
22
15
20
1
0
11
17
21
9
0
37
32
27
27
60
67
19
22
23
56
77
7
23
26
41
76
1
1
1
1
5
3
3
9 F 4* 7 F 1* 8 F 2*
12
19
28
12
11
12
25
26
18
6
7
50
27
17
9
8
74
28
19
5
8
44
39
10
6
1
2
26
27
19
2
2
13
24
20
7
0
29
32
46
58
77
74
22
25
31
78
79
12
26
27
52
79
3
4
3
2
18
22
23
19 F 4* 37 F 7* 34 F 9*
17
12
35
17
11
13
HCT116 p21 /
Control
20
0.4 Amol/L SAHA
0.6 Amol/L SAHA
0.8 Amol/L SAHA
1 Amol/L SAHA
HCT116 p53+/+
Control
25
0.8 Amol/L SAHA
1 Amol/L SAHA
2 Amol/L SAHA
4 Amol/L SAHA
HCT116 p53 /
Control
24
0.8 Amol/L SAHA
1 Amol/L SAHA
2 Amol/L SAHA
4 Amol/L SAHA
*The values represent the average and SD from three independent experiments and the difference between the HCT116 p53+/+ and p53 /
significant (P < 0.001).
including micronuclei (Fig. 2C). These are features of abnormal
mitosis which can be associated with failure of cytokinesis.
Induction of polyploidy by SAHA is associated with DNA
replication but without cell division. To confirm that the
polyploidy cells were induced by SAHA, we evaluated DNA replication with BrdUrd labeling in SAHA-treated HCT116 p21+/+,
p21 / , p53+/+, and p53 / cells. A portion of cells in each of
these cell lines cultured with SAHA continued to replicate DNA
without cell division and became polyploid cells (Fig. 2H-K),
whereas few cells cultured without SAHA became polyploid
(Fig. 2D-G). This is indicated by the incorporation of BrdUrd into
DNA of cells with >4 N DNA content.
SAHA induced polyploidy in other tumor cells, but not in
normal cells. We next examined whether SAHA can induce
polyploidy in other transformed cells. We analyzed the cell cycle
profile of human breast cell lines MCF-7, MDA-MB-231, and MDAMB-468 cultured without or with SAHA. SAHA induced polyploidy in
each of these cell lines, although the effect in MCF-7 cells was small
(5 Amol/L SAHA at 5 days, 6% versus 3% in control), greater in MDAMB-231 (2.5 Amol/L SAHA at 5 days, 16% versus 1% in control) and
MDA-MB-468 (2.5 Amol/L SAHA at 5 days, 10% versus 6% in control).
Because MDA-MB-231 and MDA-MB-468 each contain a mutant
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cell lines is
p53 whereas MCF-7 has a wild-type p53, loss of p53 function may
contribute to the difference in these cells. SAHA did not increase
polyploidy in any of the four normal fibroblasts examined, including
human embryonic lung fibroblast SW-38 and three mouse
embryonic fibroblast cell lines (data not shown).
Polyploidy cells lose the capacity to proliferate. To evaluate
whether polyploidy cells can divide, proliferation was assayed by
labeling cells with PKH2, which is a fluorescent dye that binds to
membranes (38). HCT116 p21 / cells were cultured without or
with 0.8 Amol/L SAHA for 3 days at which time medium was
removed, cells labeled with PKH2 and cultures continued for
5 days in fresh medium without SAHA. As cells divide, the PKH2
distributes to daughter cells and the florescent signal decreases in
the progeny cells. This was observed in cells in prior culture
without SAHA (Fig. 3A). In cells in prior culture with SAHA, two
peaks of fluorescent signal were apparent, one peak remained
relatively unchanged in intensity indicating that cells were not
dividing (Fig. 3B), and a second peak lost intensity of fluorescence
with time, indicating that a portion of the cells in prior culture
with SAHA resumed proliferation or cell division (Fig. 3B).
Analysis of cell size showed that the dividing cells (low level of
PKH2) in cultures without or with prior culture with SAHA were
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smaller in size than the majority of the nondividing cells (Fig.
3C-E). These findings were consistent with the conclusion that
SAHA induced large polyploid cells that lost the capacity to divide.
SAHA-induced polyploid cells have low clonogenicity. To
evaluate the clonogenicity of polyploid cells, HCT116 p21 / cells
cultured with 0.8 Amol/L SAHA, and after 3 days of culture with the
HDAC inhibitor, cells were collected, and sorted by size to populations of relatively small and large cells. Cells from culture without
SAHA and the ‘‘small’’ and ‘‘large’’ cells sorted after culture with
SAHA were plated in soft agar without SAHA and incubated for
14 days. The large cells sorted from culture with SAHA grew
significantly fewer colonies than either the control or the small cell
populations (P < 0.05; Fig. 4).
SAHA-induced polyploid cells undergo senescence. We then
determined if the polyploid cells undergo senescence. HCT116 p21+/
+ and p21 / cells were cultured without or with 0.8 Amol/L cells
for up to 5 days and then cultured without SAHA for another
5 days, and cells were stained for SA-h-gal, which is a marker for
senescent cells (39). Cultures with SAHA contained many
polyploid cells
(Fig. 5B and D). On day 3 of culture with SAHA,
few polyploid cells were SA-h-gal-positive (Fig. 5C), whereas f60%
polyploid cells were SA-h-gal-positive on day 5 (Fig. 5F), and virtually
all of the polyploid cells became SA-h-gal-positive when cells were
cultured with SAHA for 5 days and then cultured with SAHA-free
medium for an additional 3 days (Fig. 5E). The senescent polyploid
cells became larger with more nuclei compared with the polyploid
Figure 2. HCT116 cells treated with SAHA replicated DNA without cell division and became polyploid cells with features of mitotic abnormalities and cytokinesis
failures. A, SAHA induced a transient mitotic arrest. Percentage of MPM-2-positive cells in HCT116 cell lines cultured with or without SAHA at indicated concentrations
for indicated time. B, morphology of HCT116 p21 / cells (original amplification 200). The size and shape of cells and nuclei were relatively monomorphic.
C, HCT116 p21 / cultured with 0.8 Amol/L SAHA for 3 days (original amplification 200). The cells were polymorphic with many large cells, which contained giant
and/or multiple varied size nuclei, including micronuclei. D-G, few HCT116 p21+/+ (D ), p21 / (E ), p53+/+ (F ), and p53 / (G ) cells cultured without SAHA
spontaneously became polyploid cells. H-K, HCT116 p21+/+ (H ) and p21 / (I ) cells cultured with 0.8 Amol/L SAHA, p53+/+ cells cultured with 2 Amol/L SAHA (J ) and
p53 / cells cultured with 1 Amol/L SAHA (K) for 2 days continued to replicate DNA, as indicated by BrdUrd incorporation, without cell division and formed
polyploid cells.
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Polyploidy Induced by HDAC Inhibitor
Figure 3. SAHA-induced large cells lost
proliferation capacity. HCT116 p21 /
cells cultured with 0.8 Amol/L SAHA for
3 days were labeled with PKH2 and then
cultured without SAHA for 5 days. A, PKH2
signal [1] reduced with time in control cells
previously cultured without SAHA. B, two
peaks of PKH2 signal appeared in cells
previously cultured with SAHA: one
reduced as control [2] and one remained
unchanged [3]. C-E, the control cells (C )
and cells previously cultured with SAHA
with reduced PKH2 signal were small in
size (D ), whereas cells previously cultured
with SAHA with unchanged PKH2 signal
were large (E ).
cells in culture with SAHA for day 3 (Fig. 5B and D), suggesting that
the polyploid cells kept replicating DNA without cell division, namely
endoduplication, before senescence. The senescent cells were
characterized by flattening and spreading (40). Similar results were
observed in HCT116 p53+/+ and p53 / cells cultured with 2 Amol/
L SAHA for 5 days and in fresh medium for another 5 days.
We next analyzed whether p21 / and p53 / affect the
development of senescence in SAHA-induced polyploid cells. The
proportion of the polyploid cells that are SA-h-gal-positive cells in
HCT116 p21 / or HCT116 p53 / cell culture was not
significantly different from that of their parental cells at day 5 of
culture with SAHA (P > 0.05; Fig. 5F).
The small cells that survive from SAHA treatment are less
sensitive to SAHA. We next determined if the small cells which
were not polyploid and remained viable in culture with SAHA
are different from the unselected control in sensitivity to the
HDAC inhibitor. We sorted the small cell population from HCT116
p21 / cells cultured with 0.8 Amol/L SAHA for 3 days and grew
the small cells in SAHA-free medium for 5 days to let them recover
from SAHA treatment. We found that the small cells that survived
SAHA treatment were less sensitive to SAHA, that is, 1.2 Amol/L
SAHA was required to inhibit cell growth by 50% of the small cells
by 48 hours compared with 0.7 Amol/L SAHA for the unselected
population of HCT116 p21 / cells.
Discussion
Figure 4. SAHA-induced large cells lost proliferation capacity. HCT116 p21 /
cells cultured with 0.8 Amol/L SAHA for 3 days were sorted into small and
large cells and grown in soft agar plates for 14 days. A-C, the large cells (C )
formed fewer clones than the small cells (B) and the control cells cultured without
SAHA (A). D, clone numbers of control, small, and large cells. The data
represented three experiments.
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HDAC inhibitors have been shown to induce G1 and G2-M cell
cycle arrest (12, 41), caspase-dependent apoptosis (37, 41–43), and
cell death independent of caspase activation (28, 30, 31, 44). In this
study, we showed that SAHA can induce polyploid cells which lose
proliferating capacity and become committed to senescence.
Studies have shown that HDAC inhibitors can cause aberrant
acetylation of histones in the centromere domain of chromosome,
disrupt the function of kinetochore, and cause mitotic defects
(18–22). The mitotic defects result in cell death either by apoptosis
or by mitotic death/catastrophe (25, 26). Mitotic catastrophe is a
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Cancer Research
Figure 5. SAHA-induced polyploid cells underwent senescence. A, HCT116 p21 / cells cultured without 0.8 Amol/L SAHA for 3 days were negative for SA-h-gal.
B-C, most HCT116 p21 / cells cultured with 0.8 Amol/L SAHA for 3 days became polyploid cells (B), but only few of them were positive for SA-h-gal (C ).
D-E, most HCT116 p21 / cells cultured with 0.8 Amol/L SAHA for 5 days and without SAHA for another 3 days became polyploid cells with more nuclei, large size
and wide and fat cytoplasm (D), and virtually all polyploid cells were positive for SA-h-gal (E). F, percentage of SA-h-gal-positive polyploid cells in HCT116 p21+/+
and p21 / cells cultured with 0.8 Amol/L SAHA, and p53+/+ and p53 / cells cultured with 2 Amol/L SAHA for 5 days.
form of cell death that results from aberrant mitosis, and is
characterized by the formation of large nonviable cells with
multiple micronuclei containing uncondensed chromosome (40).
The present study showed that cells cultured with SAHA could exit
from mitosis without cell division and survive with continuing
DNA replication and become polyploid cells, and the polyploid cells
can then proceed to senescence.
We found that SAHA can induce polyploidy in transformed
cells, although not to the same extent in all transformed cell lines,
but not in normal cells. It has been reported that HDAC
inhibitors can trigger a G2 checkpoint in normal cells, which is
defective in tumor cells (25). Although the property of this
checkpoint is not understood, the present findings indicate that
normal cells are resistant to HDAC inhibitor–induced polyploidy
and cell death compared with transformed cells. This difference
may confer selectivity of the HDAC inhibitor consistent with its
use as an anticancer agent with a potentially good therapeutic
index.
The transformed cells without functioning p53 or p21 cultured
with SAHA developed a higher proportion of abnormal mitosis,
failure of cytokinesis, and polyploidy than parental wild-type cells.
This is consistent with the function of p21 and p53 to maintain
genomic integrity (33, 45).
Senescence is a physiologic process that limits the proliferative
span of normal cells. The senescence phenotype can be induced in
transformed tumor cells by overexpression of p53, pRb, p16, or p21
tumor suppressor genes (46–49), or by antitumor reagents such as
cisplatin (50), doxorubicin (38), retinoid (51), and radiation (38).
HDAC inhibitor sodium butyrate induced senescence in gynecologic cancer cells (52) and sodium butyrate, sodium dibutyrate and
trichostatin A induced senescence in human fibroblasts (53, 54).
We found that SAHA induced characteristics of senescence in the
Cancer Res 2005; 65: (17). September 1, 2005
polyploid cells which accumulated in the HDAC inhibitor–treated
HCT116 cells lines.
Induction of senescence has been shown to contribute to
the treatment outcome of chemotherapy (55). The induction of
senescence may also contribute to the antitumor efficacy of
HDAC inhibitors. In this regard, HDAC inhibitor may have
advantage over certain chemotherapy reagents. Unlike the
chemotherapy drugs which are genotoxic and induce DNA
damage and whose antitumor effects depend on p53, p21, and
p16 for senescence induction (38, 55), HDAC inhibitor–induced
senescence in tumors could be independent of p53, p21, and p16
functions (52). Because inactivation of p53 and p16 are common
defects in human tumors (56), application of HDAC inhibitors
alone or in combination with chemotherapeutic drugs may
achieve a better outcome.
An interesting finding was that the small cells in HCT116 p21 /
cells that survived from SAHA treatment are less sensitive to SAHAinduced cell growth arrest. Because cancer cells have intrinsic
genomic instability and heterogeneity, whether cells that survive
from SAHA treatment in other tumors could become less sensitive to
SAHA than the original cells remains to be determined.
In summary, the induction of polyploid cells is an antitumor
mechanism of HDAC inhibitor. The polyploid cells induced by
SAHA may commit to senescence. Lack of p21 or p53 enhances
this effect with increased polyploid formation. Because p53
abnormalities are common in human tumors, this pathway
may play an important role in the antitumor mechanisms of
SAHA.
Acknowledgments
Received 12/28/2004; revised 5/13/2005; accepted 6/22/2005.
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Polyploidy Induced by HDAC Inhibitor
Grant support: National Cancer Institute grant (CA-08748), the DeWitt Wallace
Fund for Memorial Sloan-Kettering Cancer Center, Susan and Jack Rudin Foundation,
and by the David H. Koch Prostate Cancer Research Award.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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Induction of Polyploidy by Histone Deacetylase Inhibitor: A
Pathway for Antitumor Effects
Wei-Sheng Xu, Gisela Perez, Lang Ngo, et al.
Cancer Res 2005;65:7832-7839.
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