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INTRODUCTION |
Arsenite is a common human carcinogen found as a contaminant in
drinking water (1, 2). Long-term exposure is associated with an
increased risk for developing tumors of the skin, bladder, liver,
kidney, or lung (3-7). Studies indicate that arsenite can promote cell
transformation in vitro (8), activate mitogen-activated protein kinases (MAPKs),1
stimulate activator protein-1 (9), and induce sister chromatid exchange, chromosome aberrations, and gene amplification in a variety
of in vitro systems (10). On the other hand, arsenite is
also an extremely effective chemotherapeutic agent used to treat
certain cancer patients, especially those with acute promyelocytic leukemia (11, 12). Studies show that arsenite can induce apoptosis in
many cell types because of its ability to increase activation of the
tumor suppressor protein p53 (13). The ability of arsenic to act as
either a carcinogen or a chemotherapeutic agent is related to cell
type, arsenic species, and length and dose of exposure (1). Thus,
arsenic acts in a paradoxical manner. However, the mechanisms of
arsenic's actions as a carcinogen or as a chemotherapeutic agent are unclear.
Phosphorylation of histone H3 is known to play an important role in
chromatin remodeling and chromosome condensation (14). Phosphorylation
of histone H3 is associated with active immediate-early gene
expression, including that of proto-oncogenes c-fos and
c-jun (15, 16). We reported that UVB-induced phosphorylation
of histone H3 at serine 10 is mediated by MAPKs (17). However, the
ability of arsenite to induce phosphorylation of histone H3 at serine
10 has not been reported, and the signal transduction pathway mediating
phosphorylation of histone H3 at serine 10 also remains unclear.
To explore the mechanism of arsenite's action in carcinogenesis, we
used arsenite to induce phosphorylation of histone H3 at serine 10 in
JB6 Cl 41 cells. We found that this compound induced phosphorylation of
histone H3 at serine 10 in a time- and dose-dependent manner and increased Akt1 activation and phosphorylation of Akt1 at
serine 473. A dominant-negative mutant of Akt1 inhibited
phosphorylation of histone H3 at serine 10, and a dominant-negative
mutant of ERKs and the ERK inhibitor PD98059 also blocked the
phosphorylation of histone H3 at serine 10. In addition,
arsenite-induced phosphorylation of histone H3 at serine 10 was totally
blocked in ribosomal S6 protein kinase 2 (RSK2)-deficient cells
(Rsk2
/
). Furthermore, dominant-negative
mutant MSK1 had no effect on phosphorylation of histone H3 at serine 10 induced by arsenite. Taken together, these data indicate that Akt1,
ERKs, and RSK2, but not MSK1, are involved in the mediation of
arsenite-induced phosphorylation of histone H3 at serine 10.
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EXPERIMENTAL PROCEDURES |
Materials--
Sodium arsenite was purchased from Merck
(Darmstadt, Germany). Lowry-modified reagent was from Sigma. Eagle's
minimal essential medium (MEM), RPMI 1640 medium,
L-glutamine, and LipofectAMINETM 2000 reagent
were from Invitrogen. Plasmids of pCMV5-FLAG vector, pCMV5-FLAG-wild-type MSK1, pCMV-FLAG-MSK1 A195-N-terminal kinase-dead, and pCMV5-FLAG-MSK1-A565/C-terminal kinase-dead were kindly provided by
Dr. D. Alessi (MRC Protein Phosphorylation Unit, Dundee,
Scotland, UK). Antibodies to detect phosphorylation of histone H3 at
serine 10 and total histone H3 protein, phosphorylation of MSK1 and
RSK2, dominant-negative (DN)-Akt1 and activated-Akt1 plasmid, and pure histone H3 protein were from Upstate Biotechnology (Lake Placid, NY).
Fetal bovine serum (FBS) was from Gemini Bio-Product (Calabasas, CA).
Gentamicin sulfate was from Cambrex Bio Science Walkersville, Inc.
(Herndon, VA). The Akt1 Kinase Assay Kit was from Cell Signaling Technology, Inc. (Beverly, MA). Folin & Ciocalteu's phenol reagent was
from Pierce, and polyvinylidene difluoride membrane was from Millipore
(Bedford, MA).
Cell Culture--
The JB6 mouse epidermal cell line Cl 41, DN-Akt1 and activated-Akt1 plasmid stably transfected JB6 cells,
DN-ERK2 plasmid stably transfected JB6 cells, and N-terminal mutant
MSK1, wild-type-MSK1, and C-terminal mutant MSK1 plasmid stably
transfected JB6 cells were cultured as adherent monolayers in MEM
supplemented with 5% (v/v) heat-inactivated FBS, and 2 mM
glutamine at 37 °C in a humidified atmosphere of 5%
CO2. Rsk2+/+ and
Rsk2
/
cells were cultured in RPMI 1640 medium supplemented with 15% FBS and 2 mM glutamine at
37 °C in a humidified atmosphere of 5% CO2.
Extraction of Acid-soluble Proteins--
After arsenite
treatment, cells were harvested and washed twice with
phosphate-buffered saline (PBS). Extraction of the acid-soluble proteins was performed according to the protocol described by Upstate
Biotechnology. Briefly, cells were scraped from the plates after
treatment and centrifuged at 1000 rpm at 4 °C for 4 min. Cells were
washed once with PBS and resuspended with 10 volumes of lysis buffer
(10 mM HEPES, pH 7.9, 1.5 mM MgCl2,
10 mM KCl, 1.5 mM phenylmethylsulfonyl
fluoride, and 0.5 mM dithiothreitol) and then sulfuric acid
(H2SO4 for 30 min) was added to a final concentration of 0.2 M and extractions were left on ice.
Extraction solutions were centrifuged at 15,000 rpm for 10 min at
4 °C, and the acid-insoluble pellets were discarded. Supernatant
fractions were transferred to fresh tubes and precipitated on ice for
45 min with a final concentration of 20% trichloroacetic acid. Samples were then centrifuged at 14,000 rpm for 10 min at 4 °C, and the pellets were washed once with 0.1% acidic acetone and once with pure
acetone. Acid-soluble proteins were dissolved in 0.1 N NaOH solution and stored at
20 °C.
Assay of Phosphorylated H3 at Serine 10--
Acid-soluble
proteins were dissolved in SDS sample buffer and separated by 15%
SDS-PAGE and then transferred to polyvinylidene difluoride membranes.
Membranes were blocked with 5% nonfat dry milk in PBS for 1 h at
room temperature and incubated overnight at 4 °C with the primary
antibody against phosphorylation of histone H3 at serine 10, acetylation of histone H3 at lysine 9, or total histone H3. These
membranes were incubated for another 4 h at 4 °C with secondary
antibodies against rabbit IgG-conjugated alkaline phosphatase.
Membrane-bound proteins were detected with enzyme-catalyzed fluorescence (Amersham Biosciences) and analyzed using the Storm 840 PhosphorImager (Amersham Biosciences).
Establishing the Stably Transfected JB6 Cells--
Using
plasmids pCMV5-FLAG vector, pCMV5-FLAG-wild-type MSK1, pCMV-FLAG-MSK1
A195-N-terminal kinase-dead, pCMV5-FLAG-MSK1-A565/C-terminal kinase-dead, DN-ERK2, DN-Akt1, and activated-Akt1, we established these
stable transfections according to the protocol from Invitrogen. All of
these cells were selected in media containing 400 µg/ml G418 for 2 weeks; the G418 concentration was then decreased to 200 µg/ml and
maintained. Control cells were transfected with pCMV5-FLAG neo only
("mock"). G418-selected cells were tested for FLAG-epitope-tagged
Msk1 by indirect immunofluorescence staining with a monoclonal FLAG
antibody or by kinase assay for Akt1.
Akt1 Kinase Assay--
The ability of arsenite to induce
phosphorylation of Akt1 was tested using an Akt1 kinase assay kit. In
brief, cells were treated with arsenite at various concentrations for
desired times. Cells were washed once with ice-cold PBS after removing
the media, and then 0.5 ml of 1× ice-cold cell lysis buffer plus 1 mM phenylmethylsulfonyl fluoride was added to each plate
and kept on ice for 5 min, scraped, and transferred to fresh tubes.
Cells were sonicated on ice four times for 5 s each and
centrifuged for 10 min at 4 °C, and the supernatant fractions were
transferred to other fresh tubes. Cell lysate protein (200 µg) and
beads (20 µl) with immobilized Akt1 1G1 monoclonal antibody were
added together by gently rocking for 2 h at 4 °C. These tubes
were centrifuged for 30 s at 4 °C, washed twice with 500 µl
of 1× cell lysis buffer, and then washed twice with 500 µl of 1×
kinase buffer. The pellets were suspended in 40 µl of 1× kinase
buffer supplemented with 200 µM ATP and 2 µg of GSK-3
fusion protein and incubated for 30 min at 30 °C. The reaction was
terminated with 20 µl of 3× SDS sample buffer. The samples were
denatured at 95-100 °C for 5 min before they were separated by 8%
SDS-PAGE. The proteins were transferred to polyvinylidene difluoride
membranes. Akt1 kinase activity and Akt1 phosphorylation were analyzed
by Western blotting using a phosphor-GSK-3
/
at serine 21/9
antibody and a phosphor-Akt1 (serine 473) antibody, respectively.
Protein Phosphorylation Assay in Vitro--
Phosphorylation of
pure histone H3 or chromatin by active Akt1 kinase was carried out
according to the manufacturer's instructions (Cell Signaling Co.). In
brief, 8 µg of pure histone H3 or chromatin, extracted from JB6 Cl 41 cells, were incubated for 1 h at 30 °C with the active Akt1
kinase and 200 µM ATP in 20 µl of kinase reaction buffer (25 mM Tris, pH 7.5, 5 mM
-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, and 10 mM
MgCl2). The samples were added to 7 µl of 4× SDS sample
buffer and separated by 15% SDS-PAGE. The phosphorylation of histone
H3 at serine 10 and total histone H3 protein was detected by Western
blotting with specific antibodies from Cell Signaling Co.
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RESULTS |
Arsenite Induces Phosphorylation of Histone H3 at Serine 10 in a
Time- and Dose-dependent Manner--
JB6 Cl 41 cells were
employed to analyze the arsenite-induced phosphorylation of histone H3
at serine 10. The time-response study indicates that phosphorylation of
histone H3 at serine 10 gradually increases from 15 min to 1 h
after treatment with arsenite (10 µM) (Fig.
1, A and B). The
level of total histone H3 protein did not change (Fig. 1C).
The dose course study shows that phosphorylation of histone H3
gradually increases after treatment with increasing amounts of arsenite
(1, 5, or 10 µM) (Fig. 2,
A and B), with no effect on total histone H3
protein levels (Fig. 2C). These results indicate that
phosphorylation of histone H3 at serine 10 is induced by arsenite in a
dose- and time-dependent manner.

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Fig. 1.
Arsenite induces phosphorylation of histone
H3 at serine 10 in a time-dependent manner. JB6 Cl 41 cells were starved for 48 h in 0.1% FBS/MEM at 37 °C in a 5%
CO2 atmosphere. Cells were then incubated in fresh 0.1%
FBS/MEM for another 2 h before being treated with arsenite (10 µM) for the indicated time periods. Acidic proteins were
extracted as described under "Experimental Procedures." Using
Western blot analysis, phosphorylation of histone H3 at serine 10 was
detected (A) and analyzed (B) using the Storm
PhosphorImager analysis system (Amersham Biosciences). Total histone H3
protein (C) was determined as described under
"Experimental Procedures."
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Fig. 2.
Arsenite induces phosphorylation of histone
H3 at serine 10 in a dose-dependent manner. JB6 cells
were treated with arsenite (1, 5, or 10 µM) after cells
were starved for 48 h. Phosphorylation of histone H3 at serine 10 was detected (A) and analyzed (B) as described
under Fig. 1. (C) indicates total histone H3.
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Arsenite Increases Akt1 Kinase Activity and Phosphorylation of Akt1
at Serine 473 in JB6 Cl 41 Cells--
Akt1 kinase activity was
analyzed by Western blotting using a specific antibody against
phosphorylation of GSK
/
, which is a target of Akt1 kinase. Our
results show that arsenite induced phosphorylation of GSK
/
via
activation of Akt1 kinase (Fig. 3,
A and B) without changing the total GSK protein
level (Fig. 3C). In addition, we found that arsenite induced
phosphorylation of Akt1 at serine 473 (Fig. 3D), whereas the
non-phosphorylated level of Akt1 was unchanged (Fig.
3E).

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Fig. 3.
Arsenite increases Akt1 kinase activity and
phosphorylation at serine 473. JB6 cells were treated with
arsenite (1, 5, or 10 µM) for 1 h. GSK3 fusion
protein was used as a substrate for Akt1. Akt1 kinase activity was
assessed by determining phosphorylation of GSK3, which was detected by
a specific phospho-GSK3 / antibody. A shows the level
of phosphorylation of GSK / , B shows densitometer
analysis, and C shows total GSK3 protein level. For
arsenite-induced phosphorylation of Akt1 in vivo, JB6 Cl 41 cells were treated with arsenite. Phosphorylation of Akt1 was detected
(D) by Western blot using a specific antibody against
phosphorylation of Akt1 at serine 473. Total non-phosphorylated Akt1
(E) was also detected.
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DN Mutant Akt1 Inhibits Phosphorylation of Histone H3 at Serine
10--
To further explore the role of Akt1 in arsenite-induced
phosphorylation of histone H3 at serine 10, we established stably expressed DN-Akt1, activated-Akt1, and empty vector plasmid-transfected JB6 cells. Our data show that phosphorylation of histone H3 at serine
10 induced by arsenite was still observed in all of the stably
transfected cells (Fig. 4, A
and C). However, DN-Akt1 distinctly inhibited
phosphorylation of histone H3 (serine 10) and activated-Akt1 greatly
increased phosphorylation of histone H3 (serine 10) compared with the
empty vector-transfected cells (Fig. 4, A and C).
The non-phosphorylated levels of histone H3 proteins remained at the same level (Fig. 4, B and D). Moreover, the
phosphorylation of histone H3 (serine 10) was also
dose-dependent in all cell lines (Fig. 4C).
These results indicate that Akt1 plays a very important role in
mediating the phosphorylation of histone H3 at serine 10 induced by
arsenite.

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Fig. 4.
Phosphorylation of histone H3 at serine 10 induced by arsenite is inhibited by DN-Akt1. Stably expressed
DN-Akt1 and activated Akt1 plasmid-transfected JB6 Cl 41 cells were
starved in 0.1% FBS/MEM for 48 h and then treated with arsenite.
Phosphorylation of histone H3 at serine 10 was detected with an
antibody against phosphorylation of histone H3 at serine 10 (A and C), and total histone protein was detected
with a rabbit anti-histone H3 antibody (B and
D).
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Active Akt1 Phosphorylates Histone H3 at Serine 10 In
Vitro--
Furthermore, we wanted to know whether Akt1 kinase can
directly phosphorylate histone H3 at serine 10 in vitro. We
used pure histone H3 protein or chromatin extracted from JB6 cells as
Akt1 substrates and incubated each with active Akt1 kinase and then analyzed phosphorylation of histone H3 at serine 10 by Western blot.
Our results showed that active Akt1 strongly phosphorylated histone H3
at serine 10 in vitro (Fig.
5A) and chromatin (Fig. 5C) in an apparent dose-dependent manner. The
non-phosphorylated level of total histone H3 protein was unchanged
(Fig. 5, B and D). These data indicate that Akt1
can phosphorylate histone H3 protein at serine 10 of chromatin in
vitro.

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Fig. 5.
Active Akt1 kinase phosphorylates histone H3
at serine 10 in vitro. An Akt1 kinase assay was
carried out according to the manufacturer's instructions. Pure histone
H3 (8 µg) or chromatin (8 µg) were used as the Akt1 kinase
substrate. Defined units of Akt1 kinase (one unit is defined as the
amount of Akt1 required to catalyze the transfer of 1 pmol of phosphate
to protein substrate in 1 min at 30 °C in kinase buffer in a 30-µl
reaction volume) were used to analyze the dose-response for
phosphorylation of histone H3 at serine 10 by Akt1 kinase (A
and C). Total histone H3 was detected with a
non-phospho-histone H3 antibody (B and D).
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Dominant-Negative Mutant ERK2 and PD98059 Inhibit Phosphorylation
of Histone H3 at Serine 10 Induced by Arsenite--
In DN-ERK2
plasmid-transfected cells compared with empty vector
plasmid-transfected cells, we found that DN-ERK2 almost completely blocks the phosphorylation of histone H3 at serine 10 (Fig.
6A), but the expression level
of non-phosphorylated histone H3 (Fig. 6C) and acetylation
of histone H3 at lysine 9 are unchanged (Fig. 6B). PD98059,
a MEK1 inhibitor, was used to pretreat cells for 1 h, and then
arsenite was added and cells were incubated for another 1 h. We
found that a relatively low concentration of PD98059 inhibits
phosphorylation of histone H3 (serine 10) in a
dose-dependent manner (Fig. 6D). As before, the
expression of non-phosphorylated histone H3 (serine 10) and acetylation
of histone H3 (lysine 9) were unchanged. These results indicate that
ERK2 may mediate the phosphorylation of histone H3 at serine 10 induced
by arsenite.

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Fig. 6.
Phosphorylation of histone H3 at serine 10 is
blocked by DN-ERK2 and inhibited by PD98059. DN-Erk2 cells were
treated with arsenite (1, 5, or 10 µM) for 1 h after
cells were starved for 48 h. Phosphorylation of histone H3 at
serine 10 was detected with a phospho-histone H3 (serine 10) antibody
(A). Acetylated protein level was detected with an antibody
against acetylation of histone H3 at lysine 9 (B). Total
histone H3 protein was detected with non-phospho-histone H3 antibody
(C). Inhibition of phosphorylation of histone H3 at serine
10 by PD98059 was performed by pre-treating JB6 Cl 41 cells with
PD98059 at the indicated concentrations for 1 h. Arsenite was then
added and cells were incubated for another 1 h. Cells were
harvested and the acid-soluble proteins were extracted as described
under "Experimental Procedures." Phosphorylation of histone H3 at
serine 10 (D), acetylation of histone H3 at lysine 9 (E), and total histone H3 protein (F) were
detected.
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Mutants of MSK1 and the MSK1 Inhibitor H89 Have no Effect on
Arsenite-induced Phosphorylation of Histone H3 at Serine 10--
MSK1
has already been reported to be a mediator in the phosphorylation of
histone H3 at serine 10 by the Ras-MAPK signal transduction pathway
(14). In this study, we first determined whether DN-MSK1 inhibited
UVB-induced phosphorylation of histone H3 at serine 10. Our data
corresponded with that of previous studies (18) and indicated that N-
and C-terminal MSK1 kinase-dead mutants inhibited UVB-induced
phosphorylation of histone H3 at serine 28 as expected (Fig.
7A). Then we explored the role
of MSK1 in the phosphorylation of histone H3 at serine 10 induced by
arsenite. Our results show that both MSK1-A195-N-terminal kinase-dead
and MSK1-A565-C-terminal kinase-dead mutants had no effect on
arsenite-induced phosphorylation of histone H3 at serine 10 compared
with MSK1 wild type (Fig. 7, C and F). More
interestingly, H89, a potent MSK1 inhibitor, had no effect on
phosphorylation of histone H3 at serine 10 induced by arsenite,
although the concentration of H89 used was up to 20 µM
(Fig. 7, G and H). This concentration has been
shown to inhibit phosphorylation of histone H3 at serine 28 induced by
UVB (4 kJ/m2) (18). The protein expression levels of
non-phosphorylated histone H3 and acetylation of histone H3 at
lysine 9 were unchanged (Fig. 7, B, E,
H, and J). Together, these results indicate that MSK1 does not mediate the phosphorylation of histone H3 at serine 10 induced by arsenite.

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Fig. 7.
Dominant-negative mutant MSK1 or the MSK1
inhibitor H89 have no effect on arsenite-induced phosphorylation of
histone H3 at serine 10. Stably expressed MSK1 N- or C-terminal
kinase dead plasmid-transfected JB6 Cl 41 cells were starved in 0.1%
FBS/MEM for 48 h and then treated with UVB (4 kJ/m2 as
a positive control) or arsenite. Phosphorylation of histone H3 at
serine 10 (A, C, and F), acetylation
of histone H3 at lysine 9 (D and G), and total
histone H3 (B, E, and H) were detected
with the corresponding specific antibodies. JB6 Cl 41 cells were
pretreated with H89 for 1 h and then treated with arsenite (10 µ M) for an additional 1 h. The phosphorylation of histone H3 at
serine 10 (I) and total histone H3 (J) were
detected as described above.
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RSK2-deficient Cells Block Arsenite-induced Phosphorylation of
Histone H3 at Serine 10--
Reports indicate that RSK2 is another
very important factor in mediating phosphorylation of histone H3 (19).
In the present report, Rsk2-deficient cells were used to
test its role in arsenite-induced phosphorylation of histone H3 at
serine 10. The results showed that Rsk2-deficient cells
almost completely blocked arsenite-induced phosphorylation of histone
H3 at serine 10 in a time and dose-dependent manner (Fig.
8, A and D), with
no effect on protein expression of non-phosphorylated histone H3 or
acetylation of histone H3 at lysine 9 (Fig. 8, B,
C, E, and F). This result indicates
that RSK2 also takes part in the mediation of arsenite-induced
phosphorylation of histone H3 at serine 10.

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Fig. 8.
Phosphorylation of histone H3 at serine 10 induced by arsenite is blocked by deficiency of Rsk2. In the
dose-response study, RSK wild-type (Rsk+/+) and
RSK2-deficient (Rsk2 / ) cells were cultured
in 15% FBS/RPMI 1640 medium. Cells were starved in 0.5% FBS/RPMI 1640 medium for 48 h and then treated with arsenite. Phosphorylation of
histone H3 at serine 10 (A), acetylation of histone H3
(lysine 9) (B), and total histone H3 protein (C)
were detected by Western blot analysis using corresponding antibodies.
In the time-response study, cells were treated arsenite (10 µ M) for
the indicated time periods after starvation for 48 h in 0.5%
FBS/RPMI 1640 medium. Phospho-histone H3 (Ser10) (D),
acetyl-histone H3 (lysine 9) (E), and total histone H3
protein (F) were determined as described above.
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DISCUSSION |
Studies show that JB6 Cl 41 cells, which are derived from mouse
skin, are a well developed cell culture model for studying tumor
promotion (20, 21). Moreover, we found previously that exposure of JB6
P+ cells to low concentrations of arsenic induces cell transformation
(8, 22). In the present study, to further explore the mechanism of
arsenite's action in carcinogenesis, JB6 Cl 41 cells were employed to
study phosphorylation of histone H3 at serine 10 induced by arsenite.
Using dominant-negative mutant cells and kinase inhibitors, we found
that arsenite induced phosphorylation of histone H3 at serine 10 in a
time- and dose-dependent manner (Figs. 1 and 2). DN-ERK2
and the MEK inhibitor PD98059, DN-Akt1, and a deficiency of RSK2
distinctly inhibited arsenite-induced phosphorylation of histone H3 at
serine 10, but DN-MSK1 had little effect. To our knowledge, this is the
first report to show that arsenite can induce phosphorylation of
histone H3 at serine 10 through activation or phosphorylation of the
Akt1, ERK2, and RSK2 pathways.
Studies show that histone H3 is associated with activation,
proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration (23).
Histone H3 in situ hybridization has become an extremely
accurate technique for assessment of S-phase cell proliferation indices
and has shown that histone H3 mRNA expression levels are greatly
increased in malignant cells compared with normal cells (24).
Specifically, histone H3 at serine 10 is phosphorylated in mitotic and
meiotic chromosome condensation (25-27) and is induced by various
stimuli, including
12-O-tetradecanoylphorbol-13-acetate (TPA) (15),
epidermal growth factor (14), and UV irradiation (17). MAPKs (14), MSK1
(18), and RSK2 (28) are involved in mediation of histone H3
phosphorylation. Arsenite acts as a cocarcinogen with a second (genotoxic) agent by inhibiting DNA repair and/or enhancing positive growth signaling (29), but the relationship between arsenite and
histone H3 has not reported until now. We hypothesized that arsenite
could act to induce phosphorylation of histone H3 through MAPKs or
other factors. Our results show that phosphorylation of histone H3 at
serine 10 was induced by arsenite in a time- and
dose-dependent manner (Figs. 1 and 2). These results are in agreement with our primary hypothesis and also provide evidence to
illustrate the potent carcinogenic effect of arsenite in cell lines.
Akt1 is a serine/threonine kinase that is activated by various stimuli,
such as hormones, growth factors, and extracellular matrix components
(30). Akt1 has been shown to promote cell survival by inhibiting
apoptosis because of its ability to phosphorylate Bad, one of its
primary targets (31). A number of studies have shown that
Akt1 gene amplification and the Akt1 pathway may play a
major role in stimulating proliferation and survival in cells overexpressing erbB2 in cancer (32). In this study, we report that Akt1
kinase is involved in arsenite-induced phosphorylation of histone H3 at
serine 10 in JB6 cells. Both Akt1 kinase activity and phosphorylation
of Akt1 at serine 473 increased when JB6 Cl 41 cells were stimulated
with arsenite (Fig. 3). DN-Akt1 inhibited arsenite-induced
phosphorylation of histone H3 at serine 10 and activated-Akt1 increased
arsenite-induced phosphorylation of histone H3 at serine 10 compared
with empty vector-transfected JB6 cells (Fig. 4). Moreover, using
either pure histone H3 protein or chromatin, active Akt1 was shown to
strongly phosphorylate histone H3 at serine 10 in vitro in a
dose-dependent manner (Fig. 5). These results indicated
that Akt1 kinase plays a very important role in arsenite-induced
phosphorylation of histone H3 at serine 10.
We reported that ERK2 and JNKs are involved in UVB-induced
phosphorylation of histone H3 at serine 10. In the present research, ERK2 was also shown to mediate arsenite-induced phosphorylation of
histone H3 at serine 10. DN-ERK2 and PD98059, a MEK inhibitor, almost
totally blocked phosphorylation of histone H3 at serine 10 induced by
arsenite (Fig. 6). But unlike UVB-induced phosphorylation of histone H3
at serine 10, JNKs had no effect on arsenite-induced phosphorylation of
histone H3 at serine 10 (data not shown). These results indicated that
ERK2 but not JNKs are involved in the mediation of arsenite-induced
phosphorylation of histone H3 at serine 10.
MSK1 is involved in the phosphorylation of nucleosomal components (33)
and UVB-induced phosphorylation of histone H3 at serine 28. An
inactivation mutation in either the N- or C-terminal MSK1 kinase domain
completely annuls its activity (33). We established stably expressed N-
or C-terminal MSK1 kinase dead JB6 Cl 41 cells and found that either
one inhibited UVB-induced phosphorylation of histone H3 at serine 28. In the present study, we confirmed our previous results (Fig.
7A) but found that the N- or C-terminal MSK1 kinase dead
mutants had no effect on phosphorylation of histone 3 at serine 10 induced by arsenite (Fig. 7, C and F). H89, a
MSK1 inhibitor, suppressed
12-O-tetradecanoylphorbol-13-acetate- or epidermal growth
factor-induced phosphorylation of histone H3 at serine 10 and inhibited
phosphorylation of histone H3 at serine 28 induced by UVB. However, in
this study, H89 had no effect on phosphorylation of histone H3 at
serine 10 induced by arsenite (Fig. 7I). So, these results
indicate that MSK1 is not involved in the mediation of arsenite-induced
phosphorylation of histone H3 at serine 10.
The formation of the MAPK-RSK complex has been shown to be necessary
for activation of the RSK isoform stimulated by growth factors in
vivo (34). RSK2 has been implicated in the phosphorylation of
histone H3 in response to mitogenic stimulation by epidermal growth
factor (35). In this study, we found that arsenite-induced phosphorylation of histone H3 at serine 10 was almost totally blocked
in a time- and dose-dependent manner by deficiency of RSK2,
but deficiency of Rsk2 had no effect on total histone H3 or
acetylation of histone H3 at lysine 9 (Fig. 8). These data strongly
suggest that arsenite-induced phosphorylation of histone H3 at serine
10 is also mediated by RSK2.
In summary, this study shows that arsenite induces phosphorylation of
histone H3 at serine 10, and Akt1, ERK2, and RSK2, but not MSK1,
mediate arsenite-induced phosphorylation of histone H3 at serine 10. This pathway for arsenite-induced phosphorylation of histone H3 at
serine 10 is distinctly different from the pathway of UVB-induced
phosphorylation of histone H3 at serine 28 (Fig. 9). Akt1 is another kinase that mediates
the phosphorylation of histone H3 at serine 10 in vivo and
in vitro. The present study further provides powerful
evidence illustrating the probable mechanisms of arsenite in
carcinogenesis in cells or animal models. However, many questions,
including how arsenite enters cells, still need to be answered.

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Fig. 9.
Akt1, ERK2, and RSK2 but not MSK1 are
involved in arsenite-induced phosphorylation of histone H3 at serine
10. Unlike UVB-induced phosphorylation of histone H3 at serine 28, which is mediated through MSK1, arsenite induces phosphorylation of
histone H3 at serine 10 through Akt1, ERK2, and RSK2 but not MSK1.
Inhibition of ERK2 by dominant-negative mutant ERK2 or the MEK
inhibitor PD98058 causes inhibition of phosphorylation of histone H3
(serine 10). Deficiency of RSK2 also blocks the phosphorylation of
histone H3 (serine 10) induced by arsenite but no inhibition of
phosphorylation of histone H3 at serine 10 was detected in DN-MSK1
plasmid-transfected JB6 Cl 41 cells. In contrast, DN-MSK1 or the MSK1
inhibitor, H89, inhibits or totally blocks the phosphorylation of
histone H3 at serine 28 by UVB.
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