Carcinogenesis vol.27 no.7 pp.1481–1488, 2006
doi:10.1093/carcin/bgl004
Advance Access publication March 7, 2006
Alterations of histone modifications and transgene silencing by nickel chloride
Qingdong Ke, Todd Davidson, Haobin Chen,
Thomas Kluz and Max Costa
Nelson Institute of Environmental Medicine, New York University School of
Medicine, 57 Old Forge Road, Tuxedo, NY 10987, USA
To whom correspondence should be addressed. Tel: +1 845 731 3515;
Fax: +1 845 351 2118;
Email: [email protected]
Although it has been well established that insoluble nickel
compounds are potent carcinogens and soluble nickel
compounds are less potent, the mechanisms remain
unclear. Nickel compounds are weakly mutagenic, but
cause epigenetic effects in cells. Previous studies have
shown that insoluble nickel compounds enter cells by phagocytosis and silence gene expression, but the entry of soluble nickel compounds and their effects on gene silencing
have not been well studied. Here, we have demonstrated,
using a dye that fluoresces when nickel ions bind, that
soluble nickel compounds were taken up by cells. Nickel
ions localized initially in the cytoplasm, but later entered
the nucleus and eventually silenced a transgene. In addition, we described three major changes in histone modification of cells exposed to soluble nickel compounds:
(i) loss of acetylation of H2A, H2B, H3 and H4;
(ii) increases of H3K9 dimethylation; and (iii) substantial
increases of the ubiquitination of H2A and H2B. These
effects were observed at nickel exposure conditions that
had minimum effects on cell cytotoxicity. Moreover, we
demonstrated that nickel-induced transgene silencing
was associated with similar changes of histone modifications in their nuclesomes. This study is the first to show that
nickel compounds increase histone ubiquitination in cells.
These new findings will further our understanding of the
epigenetic mechanisms of nickel-mediated carcinogenesis.
Introduction
Nickel compounds are carcinogenic to both humans and
experimental animals (1). Occupational exposure to nickel
compounds has been linked to lung and nasal cancers (2–4);
however, the mechanisms remain unclear. Since nickel compounds are generally not mutagenic in most conventional
assays (5–8), an epigenetic mechanism has been hypothesized
in nickel carcinogenesis (9–11).
Epigenetic dysregulation of gene expression has been found
to play an important role in cancer etiology (12). The major
epigenetic changes include aberrant DNA methylation and
alterations of histone modifications in chromatin. These
epigenetic events may act in concert to dysregulate the expression of genes that play important roles in normal cellular
functions (12). Alterations in gene expression occurred in
Abbreviations: ChIP, chromatin immunoprecipitation;
acetyltransferase; 6-TG, 6-thioguanine.
#
HAT,
histone
nickel-exposed cells as well as in nickel-transformed cell
lines (13–16).
The fundamental unit of eukaryotic chromatin is the nucleosome, which contains 146 bp of DNA wrapped around an
octamer histone core (17). Two copies of each of histones
H2A, H2B, H3 and H4 form the histone core. It has been
suggested that post-translational modifications such as acetylation, methylation and ubiquitination, on the N- and C-terminal
tails of the histones, influence chromatin folding and subsequently gene expression (18). In general, an increased
level of histone acetylation contributes to the formation of
an ‘open’ chromatin state and gene transcription, whereas
decreased histone acetylation contributes to a ‘closed’ chromatin and transcriptional repression (19,20). By comparison,
histone methylation can have multiple effects on chromatin
function, depending on the specific residue and the level of
modification. Nevertheless, it is now well accepted that
di-methylation of H3 lysine (K) 9 is largely associated with
gene silencing and heterochromatin formation (18). The roles
of histone ubiquitination are less well explored, although
recent studies have linked the ubiquitination of histones to
both gene inactivation and transcription (21–28). In response
to developmental or environmental signals, these histone
modifications may be altered by a variety of mechanisms.
Carcinogenic nickel compounds can be roughly classified
into two categories: water-soluble (i.e. nickel chloride, NiCl2)
and water-insoluble (i.e. nickel sulfide, NiS). Insoluble nickel
compounds have been shown to enter cells through phagocytosis and cause silencing of gpt (the bacterial xanthine guanine
phosphoribosyl transferase) in the transgenic gpt+ Chinese
hamster cell line, G12 (29,30). Interestingly, the silencing
of gpt gene expression did not involve mutation or deletion
of any portion of the transgene (9). Instead, increased DNA
methylation and chromatin condensation and decreased histone H3 and H4 acetylation, as well as increased histone H3K9
dimethylation, were observed at the silenced gpt locus (9,31).
This indicated that an epigenetic, rather than a mutagenic
mechanism was involved in the loss of gpt activity by insoluble
nickel compounds (9,31). In contrast, the mechanism of entry
for soluble nickel compounds into cells and subsequent effects
on transgene silencing have not been studied, although it
was thought that soluble nickel compounds could not readily
enter cells and thus, should have little effects on gene silencing.
Despite the difference between insoluble and soluble nickel
compounds found in the previous studies, recent work has
shown that these two forms of nickel compounds share many
similarities. For instance, studies on the epigenetic effects of
nickel have shown that both insoluble and soluble nickel compounds affected genomic DNA methylation and decreased
acetylation of histone H3 and H4 at a global level (30,32–34).
The possible effects of nickel compounds on other histone
modifications though have not been investigated.
To explore the epigenetic changes induced by soluble nickel
compounds, we first studied the delivery of NiCl2 into cells
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Q. Ke et al.
using a dye that fluoresces when it binds nickel ions. Then
the global effects of NiCl2 on histone modifications including
acetylation, methylation and ubiquitination were measured
in nickel-exposed cells. In addition, the effects of NiCl2
on transgene silencing were explored further using chromatin
immunoprecipitation (ChIP) assays to investigate whether
changes of histone modifications were involved in the
silencing of the gpt transgene.
dishes and monitoring the colony formation after 1 week of growth without
selection. The mutant frequency was calculated from the number of mutant
colonies growing in selection medium as corrected by the number of clonable
cells determined as above, to give the mutant frequency as the number of
mutants per clonable cell.
Materials and methods
ChIP assay and PCR of the transgenic gpt gene
ChlP assay was performed according to the protocol of the ChIP assay kit
(Upstate) using antibodies against pan-acetylated-H2A, acetylated-H2B,
acetylated-H3 or acetylated H4 (Upstate). PCR amplication of the gpt gene
was performed using the primers as follows: 50 -TGG CGC GTG AAC TGG
GTA T-30 (sense) and 50 -TGC GAA GAT GGT GAC AAA G-30 (antisense).
[a-32P]dCTP (0.1 ml; 3000 Ci/mmol; 10 mCi/ml, Perkin Elmer Life Sciences,
Boston, MA) was added into each reaction. The conditions of PCR amplification were as follows: 94 C for 2 min, 30 cycles at 95 C for 45 s, 55 C for 45 s,
72 C for 1 min and 72 C for 5 min. The PCR products were then separated on
8% polyacrylamide gels and visualized by autoradiography.
Cell culture
Cells were grown at 37 C as monolayers in a humidified atmosphere containing 5% CO2. Human lung bronchoepithelial A549 cells were cultured in Ham’s
F-12K medium supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin. Human hepatoma Hep3B cells were grown in MEM
medium supplemented with 10% FBS and 1% penicillin/streptomycin. Mouse
epidermal JB6 C141 cells were cultured in MEM medium supplemented 5%
FBS, 2 mM L-glutamine and 1% penicillin/streptomycin. The Chinese hamster
transgenic gpt+ cell line G12 and the nickel-silenced gpt clones (N24, N37
and N96) were grown in F12 medium supplemented with 5% FBS, 2 mM
L-glutamine and 1% penicillin/streptomycin. Loss of the function of the endogenous hprt in G12 cells makes it possible to select the activation or inactivation of the transgene gpt by resistance to HAT (100 mM hypoxanthine, 1 mM
aminopterin and 100 mM thymidine; Sigma, St Louis, MO) or 6-TG
(6-thioguanine; Sigma), respectively. Therefore, spontaneous gpt cells
were purged from the G12 culture by supplementing the F12 medium with
HAT; whereas, spontaneous gpt+ revertants were routinely purged from the
nickel-silenced cell lines by supplementing the medium with 10 mg/ml 6-TG.
The cells were removed from HAT or 6-TG selection one day prior to treatment
or collection for the ChIP assay.
Newport green dye staining
A549 cells were seeded onto Lab-Tek chambered slides (Nalge Nunc International, Naperville, IL) and cultured for 24 h before being exposed to NiCl2
(0.5 mM) or NiS (2.5 mg/cm2) for another 24 h. At the end of treatment, the
cells were washed with HBSS/1 mM EDTA and HBSS/5% FBS before being
incubated in 1 ml of HBSS/5% FBS containing 7 mM of the Newport green dye
mixture (1:1 of dye: F-127 Pluoronic acid (Molecular Probes, Eugene, OR)) at
37 C for 30 min. The cells were incubated in 3 ml of HBSS/5% FBS for
another 30 min after being rinsed three times with the same buffer. At the end
of incubation, the buffer was removed from each chamber; the slide was
detached from the chamber and immersed in the buffer to prevent drying.
The slide was visualized under a Zeiss fluorescent microscope.
Cell cytotoxicity assay
A549 cells growing in 100-mm dishes were treated with NiCl2 (0.5 or 1.0 mM)
for 24 h. At the end of the treatment, the medium was removed and cells were
washed twice with PBS. The cells were then cultured in the normal medium
and the medium was changed every other day. The numbers of cells were
counted every day until Day 5.
Histone purification and Western blotting
After a 24-h exposure of the cells to NiCl2 (Sigma), histones were extracted
from the cells according to Broday et al. (32). Of the total histones, 5–15 mg
were separated on 15% SDS–PAGE gels and subjected to Western blotting
with antibodies against pan-acetylated-H2A, acetylated-H2B, acetylated-H3,
acetylated H4, dimethylated H3K9, ubiquitinated H2A or H2B (Upstate
Biotechnology, Lake Placid, NY). The gels were stained with Coomasie
Blue after being transferred in order to monitor the equal loading of histones.
Mutagenesis assay
The mutagenesis assay was performed as previously described with minor
modifications (35,36). Briefly, G12 cells were removed from the HAT selection
and cultured in F12 medium containing 5% FBS 24 h prior to exposure
with chemicals. The medium was changed and freshly prepared NiCl2 (50
or 100 mM) was added every 2 days for time courses ranging from 1 day to up to
3 weeks. In the case of NiS, 1 mg/cm2 of NiS was added at the beginning of
the treatment (Day 1), and 0.5 mg/cm2 of NiS was added in at Day 7 and 14,
when the cells were typsinazed and reseeded. At the end of the treatment, the
cells were reseeded and subjected to 6-TG selection for 2 weeks with 10 mg/ml
6-TG in the medium. The colonies formed were stained with Giemsa solution
and counted. The fraction of clonable cells at the time of reseeding into the
selection medium was determined by plating 400 cells in each of three 6-cm
1482
Northern blotting analysis of gpt gene expression
Total RNA was extracted from G12 cells using the RNeasy Mini kit (Qiagen
Inc. Valencia, CA) following the manufacturer’s instruction. Northern blot
hybridization was conducted as the standard protocol. The probe for the gpt
gene has been previously described (31).
Results
Delivery of nickel chloride into cells
Insoluble nickel compounds have been shown to enter the cells
readily, while the entry of soluble nickel compounds into the
cells has received far less attention (37). In order to investigate
whether soluble nickel compounds could enter cells, we utilized a fluorescent dye (Newport green dye), which binds to
nickel ion inside the cell and yields a fluorescent green signal.
This dye has been demonstrated to be exceptionally sensitive
to the presence of nickel ions in solution. Although it can also
detect higher concentrations of zinc and cobalt ions, it is
insensitive to other metal ions such as calcium, manganese,
iron, copper, lead and mercury in solution (38). Figure 1 shows
that exposure of A549 cells to NiCl2 (0.5 mM) resulted in
intracellular nickel ion accumulation, as indicated by the fluorescence of the green dye. When the exposure time was extended up to 72 h, nickel ions were detected in the nuclei. As
expected, exposure of the cells to insoluble NiS (2.5 mg/cm2)
resulted in nickel ion accumulation in both the cytoplasm and
the nucleus. By comparison, there was no detectable fluorescent signal when cells were exposed to other metal compounds
tested, including CaCl2, FeSO4, MnCl2, ZnCl2 and CoCl2 (data
not shown).
Alteration of histone modifications by nickel chloride
Decreased histone acetylation of H2A, H2B, H3 and H4
induced by nickel chloride. The balance between histone
acetylation and deacetylation is critical for the dynamics of
chromatin remodeling and gene transcription, deregulation of
which has been found in various human diseases such as cancer
(39,40). By using the pan-acetylated histone antibodies, the
global levels of histone acetylation on H2A, H2B, H3 and H4
were measured after A549 cells were exposed to soluble NiCl2
(0.25–1.0 mM) for 24 h. The doses of NiCl2 used had a minimal effect on cell viability as determined in A549 cells by a
cell cytotoxicity assay shown in Figure 2A. As shown in
Figure 2B, NiCl2 was able to decrease the histone acetylation
of all four core histones. This nickel-induced loss of histone
acetylation was also observed in other cell lines tested, such as
in mouse epidermal Cl41 cells, human hepatoma Hep3B cells
and in the gpt transgenic Chinese hamster G12 cells, as shown
in Figure 2C. These findings demonstrated that the soluble
An epigenetic mechanism of nickel carcinogenesis
Fig. 1. Delivery of NiCl2 into A549 cells. A549 cells were exposed to NiCl2 (0.5 mM) or NiS (2.5 mg/cm2) for 12, 24, 48 or 72 h, incubated in the
Newport green dye mixture and then in HBSS/5% FBS for 30 min each. At the end of incubation and washing, the slide was detached from the chamber
and visualized under a Zeiss fluorescent microscope.
nickel-induced loss of histone acetylation was ubiquitous
across a number of different cell lines.
Increased histone methylation of H3K9 induced by nickel
chloride. Mounting experimental evidence indicates crosstalk among histone acetylation, histone methylation, and
DNA methylation (18). A functional linkage between DNA
methylation, histone deacetylation and H3K9 methylation has
also been implicated in gene repression and in the establishment of a heterochromatic state (41). Thus, it was of interest to
study the steady state levels of H3K9 dimethylation in the
nickel-exposed cells. The data shown in Figure 2D demonstrated that exposure of A549 cells to NiCl2 resulted in an
increase of dimethylated H3K9.
Increased histone ubiquitination of H2A and H2B induced by
nickel chloride. Histone ubiquitination predominantly occurs
on the K119 of H2A and K120 of H2B as monoubiquitinated-H2A and -H2B (42). Approximatety 10% of
total H2A, and to a lesser extent 1–1.5% of H2B, is ubiquitinated in a variety of higher eukaryotic cells. Previous studies
have reported that nickel ion could bind to the C-terminal tails
of H2A and H2B and cause cleavage of H2A. (43–45). Since
the ubiquitin molecule is conjugated at the C-terminal tails of
H2A and H2B, we investigated whether nickel affected histone
ubiquitination in the living cells. The levels of ubiquitinated
histones were measured with antibodies against ubiquitinated
H2A or H2B. As shown in Figure 2E and F, exposure of A549
cells to NiCl2 resulted in increases of uH2A and uH2B,
respectively, in a dose-dependent manner.
Silencing of the gpt transgene by nickel chloride
The G12 Chinese hamster transgenic gpt+ cell line has been
described previously (35). The cells were exposed to NiCl2 (50
or 100 mM) for time intervals ranging from 1 day to 3 weeks. In
a parallel experiment, G12 cells were exposed to insoluble NiS
(1 mg/cm2) as the positive control. As previously reported,
there was no gpt silencing when the cells were exposed to
NiCl2 for a relatively short time interval of exposure, ranging
from 1 to 3 days (data not shown). However, when the exposure time was extended, NiCl2 increased the frequency of 6-TG
resistant colonies, which suggested that the gpt transgene was
silenced in a time-dependent manner (Figure 3A). Similarly,
NiS silenced the gpt gene as well.
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An epigenetic mechanism of nickel carcinogenesis
In order to explore whether soluble nickel compounds
silenced the transgene through an epigenetic mechanism, the
G12 cells exposed to NiCl2 for 3 weeks were allowed to
continuously grow in the normal medium for either 1 week
or 5 weeks. Northern blotting was conducted to measure the
mRNA level of the gpt gene in the recovering G12 populations.
As shown in Figure 3B, the gpt mRNA level was relatively
low after NiCl2 was removed from the medium for 1 week,
compared with that from the control G12 cells. However, when
cells were given a sufficient recovery time of 5 weeks, the gpt
mRNA level returned to basal levels. This data suggests that
NiCl2 transiently silenced the gpt gene in an epigenetic
fashion.
Changes in histone modifications at the nickel-silenced gpt
transgene locus
Our previous studies have derived nickel-silenced gpt G12
variants (i.e. N24, N37 and N96) following NiS or Ni3S2
exposure (9). Cells were cloned from single colonies and
selected in 6-TG, which purges spontaneous gpt+ revertants.
The silencing of the gpt transgene by nickel compounds in
the variants was shown to be accompanied by increased
DNA methylation and chromatin condensation (9), as well
as decreased histone H3 and H4 acetylation and increased
dimethylation of H3K9 (31). However, the status of histone
acetylation on H2A and H2B at this nickel-induced silenced
locus of the gpt gene was unknown. To determine whether the
acetylation of histone H2A or H2B was also involved in the
nickel-induced silencing of the gpt gene, ChIP assays were
performed. As shown in Figure 4, there was a loss of histone
acetylation on histone H2A and H2B, as well as on H3 and H4,
in all three selected nickel-silenced G12 variants (N24, N37
and N96). This result suggests that loss of acetylation of all
four core histones was involved in the nickel-induced gpt gene
silencing.
Discussion
Nickel compounds have been well established as carcinogens
in both humans and animals (1). Epidemiological studies have
correlated the incidence of respiratory, larynx and nasal cancers with occupational exposure to mixtures of insoluble and
soluble nickel compounds (2). Animal studies have shown that
insoluble nickel compounds could cause significant tumor
formation at the site of administration, whereas the soluble
nickel compounds exhibit poor tumor induction in animals
(46). Therefore, it is generally thought that insoluble nickel
compounds are more potent carcinogens than the soluble
compounds. However, here, we have demonstrated that, like
insoluble NiS, soluble NiCl2 was found in both the cytoplasm
and the nucleus. It is possible that soluble nickel ions enter the
cells through similar transport systems used by other essential
metals. This hypothesis is supported by the recent finding
that nickel ions compete with iron to enter cells through the
divalent metal transporter-1 (DMT-1) (47).
In addition, the capability of soluble nickel to silence the
gpt transgene in G12 cells was reassessed by optimizing the
‘mutagenesis’ assay. Previous studies have shown that exposure of G12 cells to insoluble nickel compounds for 24 h followed by extensive washing with PBS and incubation of the
cells in fresh medium for 1 week resulted in the inactivation of
the gpt gene. In contrast, soluble nickel compounds did not
show such effects within the same time course (6,30,36). We
speculated that in the previous studies, the soluble nickel
dissolved in the medium was readily removed by washing,
whereas insoluble nickel compounds persisted in the culture
dish or remained attached to cells. A change in medium and
washing with PBS might not efficiently remove insoluble
nickel compounds from the cells, which caused a continuous
exposure of the cells to the insoluble nickel compounds during
the recovery period. This effect caused the exposure time to be
longer for the insoluble nickel compounds than that had been
intended. Thus, in this study, the G12 cells were treated with
low doses of soluble or insoluble nickel compounds for 1 day
to as long as 3 weeks without a recovery period in order to
diminish the exposure time difference in the previous studies.
The data showed that, like insoluble nickel, soluble nickel
silenced the gpt transgene significantly in an epigenetic fashion
when the exposure time was extended. Therefore, it suggests
that if nickel ions remain in contact with the cell for a sufficient
time interval, both soluble and insoluble nickel compounds
can cause gene silencing. These new findings imply that
both forms of nickel compounds may have similar biological
effects in some circumstances. However, it should be noted
that in vivo soluble nickel compounds would be cleared away
from the tissue much faster than the insoluble compounds.
In support of this notion, recent findings have shown
similarities between the effects of soluble and insoluble nickel
in cell culture models. For instance, both insoluble and soluble
nickel compounds have been reported to induce the hypoxiainducible factor-1a (HIF-1a); activate signaling pathways such
as phosphatidylinositol-3-kinase/Akt-dependent (PI-3K/Akt),
nuclear factor-kappaB (NFkB), and nuclear factor activated
T cells (NFAT) (48,49); and enhance or suppress the expression of the same genes (13,14,50).
The enzyme-catalyzed, post-translational modification of
histones plays an important role in the transcriptional processes. The balance of histone acetylation and deacetylation,
which is regulated by the opposing actions of histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzyme
activities, is critical for the dynamics of gene transcription
(40,51). Broadly, histone acetylation contributes to gene
activation, and deacetylation mainly results in repression
and silencing (19,20). The disruption of histone acetylation
Fig. 2. Change of histone modifications by NiCl2. (A) Determination of cell cytotoxicity induced by NiCl2. A549 cells were exposed to NiCl2 (0.5 or
1.0 mM) for 24 h and then cultured in the normal medium. The numbers of the cells were counted at day 1, 2, 3, 4 and 5. (B) NiCl2 causes decreased
acetylation of histone H2A, H2B, H3 and H4 in A549 cells. A549 cells were treated with 0.25, 0.5 or 1.0 mM NiCl2 for 24 h. Five micrograms of isolated
histones was separated in 15% SDS–PAGE gels and subjected to Western blotting with antibodies against acetylated H2A (acH2A), acetylated H2B
(acH2B), acetylated H3 (acH3) or acetylated H4 (acH4). The values shown are quantitative results measured by quantitative densitometry and are expressed
relative to the control. The lower panels show the gels stained with Coomassie blue after being transferred in order to monitor the loading of histones. (C)
NiCl2 causes decreased acetylation of histone H2A, H2B, H3 and H4 in Cl41, Hep3B and G12 cells. Cl41, Hep3B and G12 cells were treated with 0.25 or
0.5 mM NiCl2 for 24 h. Same as above (Figure 1A). (D) NiCl2 causes increased dimethylated H3K9 in A549 cells. Five micrograms was separated in 15%
SDS–PAGE gels and subjected to Western blotting with the antibody against dimethylated H3K9. (E) NiCl2 causes increased histone ubiquitination of H2A
in A549 cells. Five micrograms of isolated histones was separated in 15% SDS–PAGE gels and subjected to Western blotting with the antibody against
ubiquitinated H2A. (F) NiCl2 causes increased histone ubiquitination of H2B in A549 cells. Fifteen micrograms of isolated histones was separated in 15%
SDS–PAGE gels and subjected to Western blotting with the antibody against H2B.
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NiCl2 50 µM
NiCl2 100 µM
NiS 1µg/cm2
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Time
Fig. 3. NiCl2 silences the gpt transgene in G12 cells in an epigenetic fashion. (A) NiCl2 silences the gpt transgene in G12 cells. G12 cells were treated with
NiCl2 (50 or100 mM) or NiS (1 mg/cm2) for 7–21 days. At the end of the treatment, the cells were reseeded into the selection medium containing 6-TG for
1 week to select the gpt- cells. The formed colonies were stained and counted. The data represent the values of observed colonies normalized by the cell
survival rate. The results given are the average and standard errors of three independent experiments. (B) Return of gpt mRNA levels after nickel removal.
The G12 cells were cultured in the normal medium for 1 or 5 weeks after being exposed to NiCl2 (100 mM) for 3 weeks. RNA was isolated and the blots
were hybridized with the gpt probe (the upper panel). The bottom panel is the gel strained with ethidium bromide after being transferred and shows
ribosomal RNA on the gel that reflects equivalent loading of RNA.
Fig. 4. Loss of histone acetylation of H2A, H2B, H3 and H4 at the
gpt locus in the nickel-silenced gpt transgene cell clones. The ChIP assay
was performed with antibodies against acetylated H2A (acH2A),
acetylated H2B (acH2B), acetylated H3 (acH3) or acetylated H4 (acH4)
in G12 and was compared with nickel-silenced gpt transgene G12 cell
clones: N24, N37 and N96. The lower panel shows PCR amplification of
the input DNA in order to monitor the loading of the fractionated
chromatin.
and deacetylation balance in nickel-exposed cells is likely to
affect the normal expression of genes and contribute to
nickel-mediated carcinogenesis. Among the various sites of
methylation, dimethylation of H3K9 by histone methyltransferase (HMT) has been correlated to gene repression and
silencing (18).
The present study showed that exposure of G12 cells
to nickel resulted in histone deacetylation, downregulation
of the transgene gpt expression and eventually silencing
of this transgene. The return of gpt expression after nickel
1486
removal suggested that nickel inactivated the transgene
through a transient epigenetic mechanism. In addition, the
ChIP data obtained from the nickel-silenced gpt transgene
showing a broad loss of histone acetylation of all four core
histones at the silenced locus indicated that loss of histone
acetylation was associated with the nickel-induced inactivation
of the gpt transgene. Although there is no direct evidence
showing that nickel-induced dimethylated H3K9 was correlated to histone deacetylation following nickel exposure, it is
possible that increased dimethylation of H3K9 and histone
deacetylation, as well as DNA methylation, may work in concert to trigger sequential events leading ultimately to transcription repression. Results from our previous studies support
this notion. We showed that the endogenous gene Serpina3g,
a member of the mouse serpin family, was downregulated by
nickel exposure. Loss of histone H3 and H4 acetylation and
increased dimethylated H3K9 were observed at the Serpina3g
gene locus (52).
Exposure of cells to nickel resulted in a substantial increase
of histone H2A and H2B monoubiquitination. It has been
suggested that histone ubiquitination does not result in
degradation but has multiple functional and structural effects
on chromatin regulation (42). Recent studies have correlated
H2A and H2B ubiquitination with transcriptionally active as
well as silent chromatin (23–25,28,53). Histone ubiquitination
has been linked to cancer in the early work which showed that
the levels of mono-ubiquitinated H2A were highly upregulated
in transformed human cells, when compared with their normal
counterparts (54). Although the investigation of the role of
histone ubiquitination at the gene specific locus remains
challenging due to the limitation of specific antibodies, it is
not unreasonable to postulate that nickel-induced histone
An epigenetic mechanism of nickel carcinogenesis
ubiquitination may interplay with the other nickel-induced
epigenetic changes. Such changes may cause chromatin
structure and gene transcription alterations and ultimately contribute to nickel-mediated transformation or carcinogenesis.
To the best of our knowledge, this is the first study showing
that nickel compounds increase histone ubiquitination in cells.
Future studies should investigate the mechanisms by which
nickel compounds cause such increase of histone ubiquitination and the consequences of this process in the context of the
chromatin and the cell.
Based upon the present study and previous work in the
literature, we propose a preliminary model for the epigenetic
effects of nickel. Nickel, structurally or chemically, resembles
essential metals such as zinc, copper, iron and manganese,
and it could compete with them to enter the cells. Once in
the cells, the overwhelming amount of nickel ion may replace
the metal ions that are generally required for the structure
and function of many enzymes, leading to their disruption
or inactivation. Inactivation of such histone-modifying
enzymes would contribute to nickel-induced epigenetic
changes. For example, inactivation of HAT, which may lead
to histone deacetylation; inactivation of histone demethylase
(HDM, yet to be found), which may increase histone methylation; inactivation of a histone deubiquitinase (HDUB, yet to be
found), which could cause increases of histone ubiquitination;
and inactivation of DNA demethylase (DNDM, yet to be
found), which may cause DNA hypermethylation. A previous
study supporting this notion showed that nickel inhibited
HAT activity but not HDAC, thereby contributing to
nickel-induced histone deacetylation (34). Furthermore, the
nickel-induced global loss of histone acetylation, increased
histone methylation and histone ubiquitination, as well as
DNA methylation, may all work together in various orders
to bring about an efficient repression state to specific genes.
In particular, these epigenetic silencing mechanisms may
favorably target exogenous genes placed in susceptible chromosomal positions, thereby marking chromatin in such a way
that this silencing state is inherited over many cell generations.
In summary, the present study showed that soluble NiCl2
was delivered into the cells and caused transgene silencing, and
this was associated with loss of histone acetylation. Exposure
of cells to NiCl2 led to reduced histone acetylation, increased
dimethylation of H3K9 and increased ubiquitination of H2A
and H2B. These findings will help us to understand the
epigenetic effects that soluble nickel compounds exert on
mammalian cells.
Acknowledgements
This work was supported by grant numbers ES05512, ES00260, ES10344 and
T32ES07324 from the National Institute of Environmental Health Sciences,
CA16087 from the National Cancer Institute, and FP-91641801-0 from the
Environmental Protection Agency.
Conflict of Interest Statement: None declared.
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Received January 12, 2006; revised February 22, 2006;
accepted February 28, 2006