Effect of Valproate on histone acetylation
and cancer proliferation
Johann Mar Gudbergsson, Katrine Konggaard Madsen, Line Brøns Jensen, Nicoline Hedemann Mortensen,
Pernille Guldbæk Christensen & Stinne B. Søgaard
Medicine with industrial specialization & Medicine, 4th semester, Aalborg University
Abstract
It has long been known that epigenetic regulation of gene expression is crucial for cell differentiation,
proliferation and programmed cell death. Mutations in or disturbance of the balance of the enzymes
(i.e. HDAC) that control these processes have been proved to be involved in various diseases, including
cancer, and are therefore potential targets in cancer therapy. In this study we focus on methylation of
cytosine residues in the DNA sequence and acetylation of histone 4(H4) in relation to cancer, which
was tested in two cell lines: the malignant glioma cells U87MG and human astrocytes (HA). In accordance to this we study the effects of Valproate as a cancer therapeutic drug since Valproate contains
HDAC inhibiting properties. Our results came out quite inconclusive as the primary antibody for DNA
methylation from Abcam was defect. The H4 acetylation staining came out fine, although they did not
turn out as expected. We found more acetylation in HA cells than in the U87MGs which is not consistent with other studies. Comparing our U87MG pictures, we detected more acetylation in the cells
treated with Valproate compared to the controls, which is in accordance to the hypothesis.
Keywords
Histone modifications, DNA methylation, glioma, cancer, valproate, histone deacetylase inhibitors,
Abbreviations
CpGs: cytosine-phosphat-guanine. DMEM-F12: Dulbecco’s Modified Essential Medium and Ham’s F12.
DMEM Sigma: Dulbecco’s Modified Eagle’s Medium. DNMT: DNA methyltransferase. FCS: foetal calf serum. H3K4me: H3K4 methylation. HA: human astrocytes HAT: acetyltransferases. HDAC: histone
deacetylases. HDACi: Histone deacetylase inhibitors. HMT: histone methyltransferase. HP1: Heterochromatin Protein 1. KPBS: potassium phosphate buffered saline. MBD: methyl-CpG-binding domain. MeOH:
methanol. miRNAs: microRNA. PBS: phosphate buffered saline. PFA: paraformaldehyde. Rb: retinoblastoma protein. R-point: restriction point. SIRT1: sirtuins1. VEGF: Vascular endothelial growth factor.
VPA: valproate.
Introduction
occurring epigenetic events taking place in the
mammalian genome (4).
Epigenetic
DNA methylation
Genetic mutations are a fundamental cause in
the initiation and progression of human cancers. It is also now apparent that epigenetic
changes may be equally important in tumor
development. Epigenetics refers to heritable
changes in gene expression in somatic cells
that are determined by factors other than alterations in the primary base sequence of DNA.
Just as DNA mutations can mediate individual
stages of tumor development by fostering over, or under-, function of key genes, epigenetic
abnormalities can heritably allow similar gene
expression aberrations (1). Failure of the proper maintenance of heritable epigenetic marks
can result in inappropriate activation or inhibition of various signaling pathways and lead to
disease states such as cancer (2).
Epigenetic mechanisms that modify chromatin
structure can be divided into four main categories: DNA methylation, covalent histone modifications, non-covalent mechanisms such as
incorporation of histone variants and nucleosome remodeling and non-coding RNAs including microRNAs (miRNAs) (2).
The nucleotides cytosine and adenine can undergo enzymatic methylation without affecting
Watson-Crick pairing (5). The methylated cytosine residues are almost always found associated with a guanine, known as the dinucleotide
CG. In the double-stranded DNA the complementary cytosine is also methylated, giving rise
to:
5´ mCpG 3´
3´ GpCm 5´ (6)
DNA methylation is a covalent chemical modification (Fig. 1) (4) which affects the transcription of genes by this particular adding of a methyl-group on the fifth carbon atom of cytosine,
resulting in 5-methyl cytosine.
DNA methylation is generally associated with
transcriptional inactive regions, which must be
seen in connection with the fact that not all
genes are active at the same time. Therefore,
increased methylation in the promoter region
of a gene leads to inhibited expression.
The interplay of histone modification and DNA
methylation creates an ‘epigenetic landscape’
that regulates the way the mammalian genome
manifests itself in different cell types, developmental stages and disease states, including
cancer (2).
Most epigenetic changes only occur within the
course of one individual organism’s lifetime
and are thereby independent of heredity, but if
a mutation in the DNA has been caused in
sperm or egg cell that results in fertilization,
then some epigenetic changes are inherited
from one generation to the next (3).
Epigenetics can be described as a stable alteration on gene expression potential that takes
place during development and cell proliferation, without any change in gene sequence.
DNA methylation is one of the most commonly
Fig. 1: DNA Methylation (7)
DNA is not uniformly methylated and contains
unmethylated regions intermixed by methylated segments.
In humans about 50% of all genes possess what
is called CpG-islands in the region of their promoter, and these are present in both housekeeping genes and genes with tissue-specific
patterns
of
expression
(4).
In contrast to the rest of the human genome,
CpG-islands which are ranging from 0, 5 to 5 kb
and occurring on average every 100 kb are
unmethylated.
The mechanisms that control DNA methylation
carry out the inactivation of genes at higher
rates per cell generation than those involving
somatic mutations. This leads to the obvious
conclusion that the functions of tumor suppressor genes and DNA repair genes are likely
to be lost more frequently through DNA methylation than mutation, a notion that is borne
out by extensive studies of the genomes of human tumor cells (1). The methylation of CpGislands can cause specific pathological cases
and cancer.
The initiation of DNA methylation, its maintenance, and its role in transcriptional repression
are all dependent on its interaction with chromatin (1).
The methylation is carried out by specific enzymes called DNA methyltransferase (DNMT).
The DNMTs known to date are DNMT1,
DNMT1b,
DNMT1o,
DNMT1p,
DNMT2,
DNMT3a, DNMTb with its isoforms and
DNMT3L (4).
DNA methylation can be de novo or
maintenance
The DNA methylation pattern is erased in the
early embryo and then re-established in each
individual at approximately the time of implantation (8). De novo methylation is a significant
change which initially methylates DNA sequences on both DNA strands and occurs in
early development. This repression occurs at
the time of gastrulation – when the embryo
begins to separate into germ layers and con-
comitantly loses the ability to maintain a pluripotent state (8).
Soon after the daughter strand is formed,
“maintenance” DNA methylase DNMT1 recognizes the hemi-methylated DNA and attaches a
methyl group to the recently formed CpG residue, thereby ensuring that both CpGs are now
methylated (1).
DNMT1 has de novo as well as maintenance
methyltransferase activity , and DNMT3a and
DNMT3b are powerful de novo methyltransferases (4). DNMT3a and DNMT3b are complexed with DNMT3L, a closely related homologue that lacks methyltransferase activity, but
recruits the methyltransferases to DNA (8).
In addition, a series of proteins, called methylCpG-binding domain (MBDs), and the protein
complexes in which they reside, can bind to
methylated CpG sites to help relay a silencing
signal (1). These MBD proteins can then recruit
repressors or co-repressors which often contain histone deacetylases (HDACs) leading to
histone deacetylation and repression of transcription, which will be elaborated later (9).
The DNMTs also interact with HDACs to help
target these enzymes to sites of DNA methylation (1).
The MBDs can also recruit histone methyltransferases (HMTs), which methylate histones
thereby further repressing transcription (9).
Consequently, the MBDs contribute to further
repression of gene transcription through histone deacetylation or histone methylation.
Moreover, it is believed that DNA methylation
and histone methylation are tied together in a
reinforcing loop where one modification depends on the other (10).
The DNA methylation can be brought to an
unbalanced state by hypomethylation or hypermethylation. Hypomethylation of the promoter regions of oncogenes can activate these
oncogenes and possibly result in carcinogenesis. Contrary to this, hypermethylation can be
linked to methylation of CpGs in the promoter
region of tumor suppressor genes silencing
these tumor suppressor genes and possibly
result in carcinogenesis.
Several studies suggest that the organization of
the DNA methylation profile throughout early
development may be carried out by histone
modifications. According to this theory, the
pattern of methylation of H3K4, including
mono-, di- and trimethylation (H3K4me),
across the genome might be formed in the embryo before de novo DNA methylation.
Sequence-directed RNA polymerase II might
direct H3K4 methylation through specific H3K4
methyltransferases. In the early embryo RNA
polymerase II particually binds to CpG-islands,
whereby these sections are marked by
H3K4me, while the rest of the genome is en-
folded around unmethylated H3K4 nucleosomes.
All types of H3K4 methylation will inhibit the
contact between DNMT3L and the nucleosome.
As a consequence to this, de novo methylation
in the embryo is situated at the majority of CpG
sites, but may be inhibited and prevented at
CpG-islands given the presence of H3K4me (8)
Histone acetylation and deacetylation
Histones consist of two H2a/H2b dimers and
one H3/H4 tetramer, which construct an octamer that enfolds DNA around its structure.
This complex is called a nucleosome. The Nterminal tails of each histone protruding the
nucleosome contain lysine residues, which are
targets of posttranslational modifications such
as methylation and acetylation, and are particularly found on H3 and H4 (Fig. 2) (6). K9,
K14, K18, K23, K27 on H3 and K5, K8, K12, K16
on H4, are well examined lysine residues in the
context of acetylation (11), (8).
Fig. 2: Shows the difference between an acetylated transcriptionally active nucleosome and a deacetylated inactive nucleosome (12).
DNA is linked to histones by a negatively
charged phosphate group, which binds to a
positively charged lysine residue on the Nterminal of histones. When the histones are
acetylated the positive charge of the lysines is
neutralized whereas the attraction to the negative phosphate group decline and consequently
makes DNA exposed for transcription by loosening the structure of the chromatin, thus
forming euchromatin. The enzymes responsible for acetylation are histone acetyltransferases (HAT). In the contrary situation when the
histones are deacetylated the chromatin condenses into heterochromatin, which impedes
transcription factors to access the promoter
region, and thereby resulting in gene silencing
occurs. This process is catalyzed by the enzyme
HDAC. There are eighteen different HDAC enzymes classified in four main groups based on
their homology to yeast proteins. Class I, II and
Fig. 3. The role of histone methylation in formation of heterochromatin. G9a is a histone
methyltransferase which methylates H3K9 (8).
Generally in human cancer DNA methylation
and posttranslational histone modifications are
misregulated, especially histone acetylation,
which in turn represses gene transcription.
Studies indicate that an upregulation of SIRT1
occurs in many cancer types. SIRT1 belongs to
HDAC class III and is the key regulator of the
acetylation of H4K16 and H3K9. Increased
SIRT1 would therefore result in loss of monoacetylated H4K16 (13). One of the effects of
IV HDACs contain zinc in their catalytic site,
whereas class III (Sirtuins, SIRT1-7) requires
NAD+ as a cofactor (13).
As mentioned in the passage above, there are
several ways in which gene expression is modified. Methylation of CpG complexes is performed by DNMT. DNMT recruit HDACs that
remove the acetyl groups from the histones
thus repressing transcription. DNMT can also
recruit HMT that methylates H3K9 and this
newly methylated site binds to Heterochromatin Protein 1 (HP1) and forms heterochromatin
(Fig. 3). Gene repression is a consequence of
the dense heterochromatin. When the cytosine
residues on DNA are methylated, MBDs recruit
HMT, which methylates H3K9. Once again this
results in the binding of HP1 and the formation
of heterochromatin. In addition to this, MBD
also
recruits
HDAC
(8).
increased SIRT1 is the deacetylation of the tumor suppressor p53. This particular deacetylation inhibits stress induced cell death and allows long-term survival of irreparable cancer
cells (13).
HDAC1 and HDAC2 interact with transcription
factors, e.g. E2F, mediated by physical binding
of the retinoblastoma protein (Rb) to E2F,
which is a cell-cycle regulating transcription
factor bound to specific promoters (13). The
binding of Rb to E2F recruits HDACs and represses gene transcription and apoptotic functions.
HDAC inhibitors
Histone deacetylase inhibitors (HDACi) are
currently under investigation for their potential anti-cancer effects. It is acknowledged that
HDACi changes the acetylation balance of
chromatin and other non-histone proteins (14),
which induces modification of tumor cells,
apoptosis and growth arrest (15).
As mentioned earlier an increase in HDAC levels leads to hypoacetylation of target proteins
e.g. histones. This hypoacetylation represses
transcription of the DNA. An increase in HDACi
results in hyperacetylation and thereby enhances the transcription.
HDACis have an impact on 5 % to 20 % of
genes expressed. HDACis only influences a minor part of these per cents, whereas the rest of
the 5 % to 20 % are affected by the secondary
effects of HDACis (16).
HDACi affects Zn2+-dependent HDACs. The
HDAC has an active site forming a tubular
pocket that consists of amino acids and a Zn2+ion. HDACi binds to the Zn2+-ion at the bottom
of this pocket. A cap group on the HDACi interacts with the external surface of the HDAC and
thereby blocking the active site. Depending on
their chemical Zn2+-binding group, HDACi belong to different classes (16). At present seven
structural distinct classes of inhibitors are
identified but only four of them are used in
clinical trials. These four structural groups are
categorised as hydroxamic acids, cyclic
tetrapeptides, carboxylic acids and benzamides
(15). HDACis’ isoform selectivity is a topic that
is examined, however not yet fully understood.
The question of whether HDACis are selective
for HDAC class I only or selective for all Zn2+dependent HDACs remains unanswered (16).
Valproate (VPA) (Fig. 4) is a carboxylic acid
selective to HDAC class I and IIa with relatively
weak inhibitory effects on HDACs (17). Originally VPA is used as treatment of epilepsy, mi-
graine headaches and as a mood stabilizer, but
has recently shown effects as a HDAC inhibitor
(15). Although VPA is a weak inhibitor, it is an
interesting inhibitor to investigate because of
its already known adverse effects and toxicity
rates.
Fig. 4 Valproate (14)
It has been shown that VPA and HDACi in general induces reversible acetylation of histones,
thus upregulating gene transcription (18) and
cell cycle arrest in G1 and/or G2 phase (14).
HDACi stimulate apoptosis and besides Burgess et al (19) suggests that HDACi kill nonproliferating tumor cells, thereby preventing
relapse (14).
Transformed cells express higher sensitivity to
HDACi-induced cell death than normal cells.
Normal cells are relatively resistant to the inhibitors (17). In a study by Osuka et al VPA
inhibited angiogenesis in glioma by two mechanisms: an indirect pathway and a direct pathway (20). Vascular endothelial growth factor
(VEGF) secretion is inhibited by the indirect
pathway of VPA, which leads to a decrease in
angiogenesis and in that way decreasing the
metastatic spread (14). The direct pathway
affects the proliferation and tube formation of
the endothelial cells (20). HDACi have furthermore immunomodulatory effects, which stimulate upregulation of major histocompatibility
complex (MHC) I and II and surface antigens,
thereby resulting in recognition of malignant
cells (14).
Clinical trials investigating the effects of VPA
indicate that use of this inhibitor in cancer
combination therapy may possibly be the best
therapeutic method (20).
Cancer
Weinberg et al 2007 presents the characteristics of cancer cells as the six hallmarks of cancer. Those characteristics are 1) the cells resistance to inhibitory growth signals, 2) the
cells ability to stimulate their own growth, 3)
their immunity to apoptosis and thereby 4)
their immortality, 5) their ability to stimulate
angiogenesis and 6) their ability to invade other tissues by metastasis (21). All these hallmarks are interesting factors in the search of
possible cancer treatments and several studies
suggest that cancer characteristics are potential targets for treatment.
The majority of normal cells are at arrest in G0
phase, while cancer cells always are in the active phases of the cell cycle. During cell division
the cells are more sensitive to treatment. The
restriction point (R point) regulates proliferation in cells and deregulation of this accompanies the formation of most cancer cells. At the R
point the cell decides whether it is going to stay
in G1 phase, go to the non-active G0 phase or
will continue to the S phase (1).
Gene silencing in cancer selectively occurs at
sites coding for tumor suppressor genes but do
not necessarily occur at other sites of the DNA,
thereby not turning off the oncogenes. This
induces uncontrolled proliferation of cancer
cells (21).
Hypothesis
The article is based upon the academic and
scientific literature on DNA methylation and
histone acetylation in connection to the pathological state of cancer. In the light of the attained knowledge, an experimental setup was
prepared in order to test and study the effects
of VPA on histone acetylation and the associa-
tion to DNA methylation. VPA is added to two
diverse cell cultures, HA and U87MG.
The hypothesis is that dissimilarity amongst
the two cell lines concerning DNA methylation,
histone 4-acetylation and cell growth will be
detectable. VPA is expected to inhibit the proliferation and gene transcription of cancer
cells. The hypothesis is based on previous studies.
Materials and methods
Culture of HA
The majority of the cells in the human brain
consist of astrocytes, which are a subset of glial
cells. They modulate synaptic activity in combination with providing structural, metabolic
and trophic support to neurons. The HA are
isolated from human cerebral cortex and delivered by ScienCell Research Laboratories (22).
HA were received frozen in passage 5 and were
afterwards thawed. On day 1, the cells were
passaged to two T75 bottles (Sarstedt) coated
with poly-l-lysine and a basic medium was
added (10 % foetal calf serum (FCS) and 1 %
penicillin-streptomycin mixed with Dulbecco’s
Modified Essential Medium and Ham’s F12
(DMEM-F12) (Biochom AG)). At day 3 the cells
were 50 % confluent. Medium was changed
every day. At day 9 the medium was removed
from one of the T75 bottles and the cells were
washed three times with phosphate buffered
saline (hyclone HYCL SH30258.01 - VWR).
Trypsin (10X) (Gibco) was then added in order
to detach the cells from the bottle surface and
the cells were then incubated for five minutes
at 37C. Afterwards medium was added with
the purpose of inactivating the trypsin. The
cells from the T75 bottle were distributed into
one T75 bottle and one T25 bottle (Sarstedt).
Basic medium was added. On day 13, the cells
in the T25 bottle were 100 % confluent and the
cells were detached by the process of trypsi-
nation (see procedure above). One fourth of the
T25 bottle was sowed in a new T75 bottle with
new medium and the remains from both the
T25 bottle and the T75 bottle were pooled in a
centrifuge tube. A cell count was performed
and the cells were seeded in two 24-well
plates, containing a poly-l-lysine coated coverslip in each well, in a suspension of 33,000
cells per well. On day 16, 1 mM VPA was added
to nineteen wells in a 24-well plate (1mL in
each well) and incubated for 24 hours at 37C.
The remaining five wells were used as controls
and were added the basic medium. On day 17,
the cells were chemically fixed. The fixatives
used were 4 % paraformaldehyde (PFA) and
methanol (MeOH). First, medium was removed
from all wells. Then, all the wells were washed
three times with PBS. Twelve wells in each
plate were fixed in five minutes with 250 l 4
% PFA and twelve were fixated in five minutes
with 250 l 100 % MeOH. Fixation process
situates the cells in an irreversible enzymatic
arrest without damaging the permeability of
the cell membrane and thereby maintaining the
morphology. The fixatives were removed from
the wells and the wells were washed once
again three times with PBS. The last quantity of
PBS was left in overnight in the refrigerator,
now ready for immunocytochemistry.
Culture of U87MG
The malignant cells used in this study are
U87MG (malignant gliomas) and are a highly
differentiated type of human astrocytic brain
tumor cells. They derive from a grade IV cancer
patient and have epithelial-like morphology.
The cells are used to study potential cancer
treatment in vitro (ATCC) (23).
The cells were received at passage 11 in a T75
bottle and basic medium was added. The basic
medium was a mixture of 10 % FCS and 1 %
penicillin-streptomycin mixed with Dulbecco’s
Modified Eagle’s Medium (DMEM Sigma) (Sigma-Aldrich). First, the medium was removed
and then trypsin (10X) (Gibco) was added in
order to detach the cells from the bottle surface
and then the cells were incubated for five
minutes at 37C. Afterwards medium was added to inactivate the trypsin. The U87MGs were
counted and seeded on coverslips in a 24-well
plate in a suspension of 35,000 cells per cm2.
The rest of the cell suspension was seeded in a
T75 bottle. On day 3 we fixed the 24-well plate
with the identical method as the HA (see
above) and used as control. On day 6 the cells
in the T75 bottle were too densely packed. The
cells were detached from the bottle surface by
trypsin and the cells were then incubated for
five minutes at 37C. Medium was added,
which inactivated the trypsin and the cells
could be counted and seeded on coverslips in
an additional 24-well plate in a suspension of
35,000 cells per cm2. On day 8, 1 mM VPA was
added to twenty wells in the 24-well plate
(1mL in each well) and was incubated for 24
hours at 37C. The remaining four wells were
used as controls. The 24-well plate with VPA
was then fixed similarly to the control plate
and the HA.
Immunocytochemistry
The two primary antibodies used were Anti-5methyl cytidine antibody (Abcam) for detecting
DNA methylation and Anti-acetyl-Histone H4
(Millipore) for detecting acetylation of the lysine residues at the histone H4. Anti-5-methyl
cytidine antibody is a monoclonal antibody
binding to one specific epitope, while Antiacetyl-Histone H4 is a polyclonal antibody
binding to an epitope on each lysine residue.
At first the PBS was removed from the plates
and potassium phosphate buffered saline
(KPBS) was added to block against unspecific
binding. Afterwards the plates were washed
with incubation buffer (3 % pig serum in KPBS
and 0,3 % Triton X-100) and placed one hour
on a rocking shaker at room temperature. The
incubation buffer was removed and the prima-
ry antibodies were added in incubation buffer
at a 1:100 and 1:200 dilution and left overnight
at 4C. The antibodies were removed the following day and the plates were washed three
times with washing buffer (incubation buffer
diluted 1:50 in KPBS). The secondary antibodies used were Alexa Flour 350 donkey antirabbit IgG antibody (Invitrogen) and Texas Red
horse anti-mouse IgG antibody (Vector Labs).
These antibodies were used in a 1:200 dilution
in a washing buffer and 250 l were added to
each well and incubated for 30 minutes at
room temperature. Once again the cells were
washed with KPBS three times. To prepare the
coverslips for microscopy they are placed upside down on a microscopy slide adding one
drop of fluorescence mounting medium
(DAKO), which will help reduce fading of immunofluorescence during microscopy. The
slides were placed to dry overnight.
Microscopy
During the microscopy we used a Nomarski
filter to study the morphology of the cells by
switching on regular light. In the study of the
Texas Red staining and thereby the DNAmethylation we used an additional fluorescence filter, the N2.1 (Excitation filter – BP
515-560 and supression filter LP 590) (Leica).
In the study of Alexa 350 and histone H4 acetylation we used the A fluorescence filter (Excitations filter – BP 360/40 and suppression filter
470/40) (Leica). The microscopy was done
with an objective at 40 times magnification.
Results
We wanted to demonstrate that VPA through
epigenetic changes, DNA methylation and histone acetylation, would inhibit the proliferation of U87MGs. To demonstrate this we performed an experiment using both U87MGs and
HA cells, some treated with VPA and some
were not. The immunocytochemistry should
clarify our hypothesis: that VPA would affect
DNA methylation and histone acetylation in the
U87MGs and thereby affect the cancer cell proliferation. We expected no influence of VPA on
the HA cells.
Furthermore some cells were fixed with 4 %
PFA, while other cells were fixed with MeOH.
As shown in fig. 5.A and fig. 6.A the two different fixatives show a difference in the morphology of the HA cells. The HA cells fixed with 4 %
PFA (fig. 5.A) have a “clumpy” appearance,
while the HA cells fixed with MeOH (fig. 6.A)
are not “clumpy” but are far away from each
other. This difference is not seen on the
U87MGs (fig. 7.A and 8.A).
The morphology in the HA cells treated with
VPA (fig. 5.A and 6.A) is not significant different
compared to the HA cells not treated with VPA
(fig. 5.D and 6.D). On the other hand the
U87MGs show a different morphology dependent on VPA treatment. For both fixatives,
U87MGs treated with VPA (fig. 7.D and 8.D)
show cells that appear more compact compared to the U87MGs without VPA (fig. 7.A and
8.A).
Unfortunately the secondary antibody binding
is unspecific and therefore fig. 5.B, 5.E, 6.B, 6.E,
7.B, 7.E, 8.B, 8.E, 9.B and 9.E do not show the
DNA methylation as hoped.
The histone acetylation is not significantly different in the HA cells treated with VPA and the
HA cells without VPA (fig. 5.C, 5.F, 6.C and 6.F).
In fig. 5.F the histone 4 acetylation of the HA
cells is vague compared to the HA controls. We
expected the HA cells not to be influenced by
the VPA. As for the U87MGs fixed with 4 % PFA
there is a noticeable difference in histone 4
acetylation. In the U87MGs controls (fig. 7.C)
the nuclei are not as coloured as the U87MGs
treated with VPA (fig. 7.F). This follows our
hypothesis about VPA’s effect on histone 4
acetylation in cancer cells. For the U87MGs
fixed with MeOH (fig. 8.C and 8.F) the differ-
ence in histone 4 acetylation between the cells
treated with VPA and the cell controls is the
difference not as noticeable as the cells fixed
with 4 % PFA (fig. 7.C and 7.F).
Fig. 9 demonstrates that the secondary antibody has not bound to anything else than the
primary antibody as the negative control wells
do not have primary antibody added to it.
Fig. 5: HA, 4 % PFA 1:100
(A) Control. Nomarski.
(B) Control. N2.1-filter. Texas
Red.
(C) Control. A-filter. Alexa 350.
(D) VPA. Nomarski.
(E) VPA. N2.1-filter. Texas Red.
(F) VPA. A-filter. Alexa 350.
Fig. 6: HA, MeOH, 1:100
(A) Control. Nomarski.
(B) Control. N2.1-filter. Texas
Red.
(C) Control. A-filter. Alexa 350.
(D) VPA. Nomarski.
(E) VPA. N2.1-filter. Texas Red.
(F) VPA. A-filter. Alexa 350.
Fig. 7: U87MGs, 4 % PFA, 1:100
(A) Control. Nomarski.
(B) Control. N2.1-filter. Texas
Red.
(C) Control. A-filter. Alexa 350.
(D) VPA. Nomarski.
(E) VPA. N2.1-filter. Texas Red.
(F) VPA. A-filter. Alexa 350.
Fig. 8: U87MGs, MeOH, 1:100
(A) Control. Nomarski.
(B) Control. N2.1-filter. Texas
Red.
(C) Control. A-filter. Alexa 350.
(D) VPA. Nomarski.
(E) VPA. N2.1-filter. Texas Red.
(F) VPA. A-filter. Alexa 350.
Fig. 9: Negative control, U87MGs, MeOH, 1:100
(A) Nomarski. (B) N2.1-filter. Texas Red. (C) A-filter. Alexa 350.
Discussion
Methylation
As mentioned in the section of Materials and
Methods the secondary antibody are marked
with the fluorescence color “Texas Red”.
The obtained results revealed that the primary
antibody for 5-methylcytosine binds unspecifically in the cytoplasm of the cells. The producer
of the primary antibody for 5-methylcytosine
has admitted a defect, which explains our problems.
Acetylation
The results of the histone 4 acetylation are
moderately inconsistent and not in accordance
to our hypothesis. We expected that the effects
of VPA would result in hyperacetylation and
that VPA would have more significant effect on
the U87MG cell line. As seen on fig. 7C compared to picture fig. 7F there is a noticeable
difference in the acetylation, which explains
more acetylation in the cells with the treatment
of VPA. The difference is not as apparent in the
cells fixed with MeOH. A possible explanation
could be the fact that the MeOH can block the
antibody-epitope binding, which would affect
the immunostaining and thereby not give the
same result as cells fixed with 4 % PFA.
To make sure that the cells were not dead before ICC we could have used a live/dead viability or cytotoxicity kit. In that way we could
prevent using antibodies on dead cells. The
live/dead viability or cytotoxicity kit cannot be
used instead of ICC in this setup.
Concentrations
In this experimental setup, two different concentrations of primary antibodies were tested,
1:100 and 1:200. This was done as a consequence of the fact that we in advance did not
know, which of the two concentrations would
end up with the best and clearest result. In the
microscope it became evident that the concentration 1:100 presented the best results. This
outcome created the basis of the results,
whereas the concentration 1:200 is hardly relevant.
Fixation
The 4% PFA fixes cells by forming partiallyreversible methylene bridges between proteins
and/or nucleic acids. Overfixation will result in
blocking of the antibody binding to the epitope,
while underfixation can cause proteolysis of
target proteins. 4% PFA is appropriate for fixing the majority of proteins, peptides and enzymes of low molecular weight (24), (25).
Generally, the HA cells exhibit more acetylation
than the U87MG’s after treatment with VPA,
which is in conflict with our hypothesis. The
HA cells ought to be less sensitive to VPA than
U87MG and therefore HA cells should be less
acetylated according other studies.
MeOH replaces water molecules in tissues and
instantly coagulates proteins. The replacement
of water molecules results in altered conformation, which can block antibody-epitope
binding. MeOH is suitable for fixing large protein antigens e.g. immunoglobulin (25).
In both cell lines black spots are detected in the
nuclei, which might indicate that the cells were
already dead prior to the immunocytochemistry (Svend Birkelund, personal communication). Furthermore, we observed various weak
acetylation in the cytoplasm of U87MG’s treated with VPA and this may indicate that the
dose of VPA was toxic and induced cell apoptosis (18).
Although the 4% PFA preserves the morphology of the cells better than MeOH, 4% PFA may
possibly kill the cells, which is why the MeOH
was used as a “back-up fixative”.
Perspectives
Kaiser et al. (2006) (18) have studied the effect
of VPA on myoloma cell lines and found an increase of acetylated histone H3, whereas we
have found a limited increase in acetylated
histone H4 in the U87MGs. They also demonstrate that proliferation of myoloma cells is
inhibited after 48 hours of treatment with VPA.
They conclude that VPA inhibits proliferation
in a dose-dependent manner, using VPA at different concentrations (0,5 mM, 2 mM and 2,5
mM). Furthermore they treated the myoloma
cells with VPA for 48 hours, whereas we treated both U87MGs and HA cells for 24 hours with
a concentration of 1 mM, which did not show
any inhibiting effects on the cell proliferation.
In relation to our study it would have been
interesting to investigate the function of VPA at
different doses and in different time intervals
to conclude if VPA affects proliferation and
apoptosis.
As we were not capable of verify our hypothesis that VPA would affect U87MGs by acetylation and methylation, others have used VPA in
combination therapy (14). As mentioned earlier VPA is a weak inhibitor and therefore treatment with VPA in combination with other
drugs might be the best therapeutic method.
Osuka et al. (2012) (20) suggest that the most
potential therapy in cancer treatment would be
VPA or another stronger HDACi combined with
another relevant drug. In relation to our study
it would have been interesting to investigate
the function of VPA as a combination drug.
In our study the effect of VPA on HA and
U87MG cells focusing on the acetylation of histones and DNA methylation were investigated.
We are incapable of concluding whether VPA
as an HDACi has an effect on the acetylation of
U87MG cells. The experiment is not statistically
significant and the results are too diverse in
order to verify our hypothesis.
Acknowledgements
We would like to thank associate professor
Jacek Lichota for his guidance through writing
this article. Also, thanks to Laboratory Techni-
cian Merete Fredsgaard for her technical help
and assistance in the laboratory and her general patience. Further, we want to thank the
Laboratory of Neurobiology (LNB) at Aalborg
University for providing the proper facilities to
perform this study. Last, thanks to professor
Svend Birkelund for teaching us how to use the
excellent microscope for fluorescence microscopy, which he generously provided.
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