The Role of Estradiol in modulating B-cell receptor (BCR)
signalling in B-cell Lymphomas and Leukaemias
Eleni E Ladikou
Supervisors: Prof Martin JS Dyer, Dr Sandrine Jayne
Department of Cancer Studies and Molecular Medicine Medical Research
Council (MRC) Toxicology Unit, University of Leicester
INTRODUCTION
Chronic Lymphocytic Leukaemia (CLL) is the most common form of leukaemia in the
western world. In the UK only, 3000 new cases are diagnosed every year while in the
United States it comprises 7% of all lymphomas (1). CLL is the accumulation of
mature monoclonal CD5+ B cells in the blood, bone marrow and lymphoid tissues
(2). Accumulating evidence demonstrates that male patients have a significantly
higher incidence in CLL than females (ratio 1.8:1) whereas the latter have a much
better prognosis and response to treatment (3). Therefore, we raised the hypothesis
that this sex-dependent difference in incidence and prognosis could be the result of
sex hormone differences.
We decided to investigate the primary female sex hormone, estrogen. Estrogen
exists in three forms in our body: Estradiol, Estrone and Estriol. Estradiol is the most
abundant one in reproductive age (4). All these three estrogens are steroid
hormones that enter the cell through the cell membrane and bind to intracellular
receptors. Estrogen Receptor alpha (ERα) and Estrogen Receptor beta (ERβ) are
transcription factors that will modulate gene expression of target genes (genomic
action of ERα and ERβ). They are also involved in several signaling pathways that can
affect the cell more rapidly through calcium influx (5). A third Estrogen receptor,
GPR30, was recently discovered (5). This receptor is a G-protein coupled receptor
with its own distinct molecular action and can act synergistically or antagonistically
with the other two ERs. Estrogen is known to have a fundamental role in
autoimmunity, predisposing females to it, and B-cell development.
Estrogen Receptors and selective estrogen receptor ligands SERLs have been shown
to play a role in B cell lymphoid malignancies. However, data on their role in primary
CLL cells, to our knowledge, do not exist. This led us to investigate the role of
estrogen and other SERLs in CLL. The aims of the present study were to investigate:
a) the ERα and ERβ expression (at mRNA and protein level) in primary CLL B cells, b)
the spontaneous apoptosis of CLL cells in absence and in presence of several stimuli
(CpG ODN2216, CD40L, IgM) and c) the effect of Estradiol and SERLs on various
cellular processes (e.g. apoptosis, proliferation) of primary CLL B cells.
METHODS
Patients and Cell lines
Blood samples were obtained from patients from the Haematological Clinic at the
Leicester Royal Infirmary, after informed consent. B cells were isolated from 36 CLL
patients and 2 healthy volunteers by ficolling. EBV-transformed B cell lines (HRC5,
GM14183, GM14295) and breast cancer cell line (MCF7) were used.
Cell Cultures
Cells were cultured in phenol red-free RPMI 1640 and stimulated with goat F(ab’)2
anti-human IgM, CpG-ODN/IL-2 or soluble CD40-L/IL4 in absence/presence of
Estradiol , selective ERα, ERβ and GPR30 agonists (PPT, DPN, G1) , ERβ antagonists
(PHTPP) and Tamoxifen at 1nM (selective estrogen receptor ligands- SERLS).
Real time qPCR
RNA were extracted using the RNEasy extraction kit (Qiagen), mRNA were reverse
transcribed using SuperScriptTM III RT (Invitrogen). qPCR were performed using SYBR
green kit (Bioline) and primers specific for ERα and ERβ.
SDS-PAGE/Western Blot
Cell lysates were run in 10% or 12% SDS-PAGE. Primary antibodies used were rabbit
polyclonal anti-human ERα (HC-20, Santa Cruz), rabbit anti-ERβ antibody (PA1-311,
Pierce), rabbit anti-phospho-ERk (91015, Cell Signalling) and rabbit anti-phospho-Syk
(2701, Cell Signalling).
Flowcytometry analysis
Flowcytometry was used to assess cell apoptosis (using Annexin V-FITC staining (to
detect phosphatidyl serine on cells in early apoptosis) and Propidium Iodide (that
stains DNA and detect cells in late apoptotis) and cell proliferation (using Ki67
staining).
RESULTS
1. Investigation of the Estrogen Receptors expression
Total RNA were isolated from purified CLL B cells from 18 female and 18 male
patients as well as EBV transformed normal B cell line (HRC). After reverse
transcription, ERα and ERβ mRNA levels were investigated by real-time PCR using
levels found in the HRC cell line as a reference. ERα mRNA expression was increased
in all CLL patients with females showing higher mean expression when compared
with male patients (p=0.0138) (Figure 1); ERβ gene expression was also increased to
a lesser extent for all CLL patients, with females showing higher mean expression
when compared with male patients (p=0.0515) (data not shown); the ERα: ERβ ratio
between males and females was calculated for all patients. The mean ± SD of ratios
showed no significant difference between the two sexes (p=0.3379).
256
ERα mRNA levels of Female Patients
Fold difference
128
64
32
16
8
4
2
1
HRC WAR HOL MOC MOA PTJ PME HJC
256
FLJ
SEJ SHA AMU SMO STJ
WK HEJ SPO PMR EMP
ERα mRNA levels of Male Patients
128
64
Fold difference
Figure 1: Relative ERα
gene expression ± SD
(fold
difference
as
compared to HRC) in 18
female CLL patients (top
panel) and 18 male CLL
patients (bottom panel)
HRC level is set at 1 and
serve as a reference
32
16
8
4
2
1
HRC SDE HU LJN GKI PC WJP GMJ NRT NRO SPJ VIA POM AGD MST STO BFM HTG BJR WLD VAK
Whole cell protein extracts were prepared in parallel in the same patient samples.
ERβ protein was expressed in both male and female CLL patients as well as in healthy
volunteers (Figure 3). ERα levels on the other hand were very low, even beyond
detection in some CLL patients when compared to MCF7, a breast cancer cell line
(Figure 2). Interestingly, the ERα antibody recognized three unspecific bands at 68,
74 and 90 kDa. The band at 74kDa is B cell specific as it is present in all CLL patients
and HRC but not in HEK293T cells transfected with ERα and MCF7. It could either be
a different splicing isoform of ERα or a post-translational modification (e.g.
monoubiquitination or sumoylation). Further investigation involving the transfection
of ERα siRNA would confirm whether this band is related to ERα or not.
Figure 12: ERα protein levels in
CLL patients and 2 healthy
volunteers (female TG, male
GN). 20µg of total proteins from
B cells of 18 CLL patients, HRC,
MCF7 and ERα transfected cells
(T) were loaded. Bands with
asterisks (*, **, ***) represent
unspecific bands.
Figure 32: ERβ protein levels in
the same patients and cell lines
as in Figure2 except the addition
of 183 (male GM14183), and 295
(female GM14295) which are cell
lines of EBV transformed normal
B cells.
2. Spontaneous apoptosis
In contrast to CLL cells in the lymph node, CLL cells in the bloodstream (or cultured
on a plastic dish) are subject to spontaneous apoptosis. Taking into consideration the
sex dependent difference in CLL incidence and prognosis, we investigated the
spontaneous apoptosis of both male and female CLL cells. After isolation from blood,
cells were cultured for 1 or 4 days and analysed for the presence of Annexin V (a
marker of early apoptosis) and for DNA content (staining with propidium iodide)
using Flow Cytometry. Analysis of 43 patients reveals that female cells are more
prone to spontaneous apoptosis when compared to male patients (p<000.1). Male
patients had a significantly higher percentage of viable cells (42.17±6.9 at day1,
32.83±5.4 at day4) when compared to females (19.27±2.1 at day1, 14.09±2.3 at
day4) (day1: p=0.006, day4: p=0.0057) (Figure 4). The difference in % of viable cells
between Day 1 and Day 4 for each patient was also significant (-7.348 ± 9.345)
(p=0.011) (data not shown).
S p o n ta n e o u s A p o p to s is
100
M a le s
F e m a le s
80
% v ia b le c e lls
Figure 4: Mean ± SD of viable cells (% of total
cells) in 43 patients (22 males, 21 females).
Cells were cultured for 4 days receiving no
stimulation. Males: 32.83±5.4, n=22.
Females: 14.09±2.3, n=21 (t- test analysis
p<0.0057)
60
40
20
0
M a le s
F e m a le s
P a t ie n ts
This implies that differences in the internal microenvironment signals may
predispose female cells to being more susceptible to spontaneous apoptosis. No
current data on sex-dependent difference in spontaneous apoptosis exist, to our
knowledge. In order to investigate whether sex-dependent difference in
spontaneous apoptosis was linked with alterations in BCL2 and AID mRNA levels, we
evaluated the expression of these two genes in 22 patients (a subset of the 43
previously studied). The result showed no sex dependent difference in BCL2 and AID
mRNA levels (data not shown).
Due to the fact that spontaneous apoptosis has been shown to depend on
environmental pro-survival signals, we next examined the spontaneous apoptosis in
CLL patient cells when stimulated with either of these pathways: a) Soluble CD40
ligand together with IL-4 mimicking the proliferative microenvironment of lymph
nodes, b) anti-IgM antibody to mimic the in vivo engagement of BCR with antigen
(adaptive immunity) and c) CpG oligodeoxynucleotides (ODN) with IL-2 to stimulate
Toll-Like receptor 9 (innate immunity). CpG ODN and CD40L stimulation of CLL cells
abrogated this sex-dependent difference in spontaneous apoptosis (p=0.45 and
p=0.51). On the other hand, after IgM stimulation female CLL cells remain more
susceptible to spontaneous apoptosis (p=0.02) (Figure 6). Data on CD40L and IgM
stimulation have a small sample size (6 patients) and should therefore be extended.
These data imply that spontaneous apoptosis in female CLL patient cells depends on
specific environmental signals for their survival (Figure 5).
CpG stimulated cells
Unstimulated cells
100
S p o n ta n e o u s A p o p to s is
100
F e m a le s
% v ia b le c e lls
60
40
80
% v ia b le c e lls
M a le s
80
60
40
20
20
0
0
M a le s
M a le s
F e m a le s
CD40L stimulated cells
IgM stimulated cells
100
50
80
40
% v ia b le c e lls
% v ia b le c e lls
F e m a le s
P a t ie n ts
P a t ie n ts
60
40
20
30
20
10
0
0
M a le s
F e m a le s
P a t ie n ts
M a le s
F e m a le s
P a t ie n ts
Figure 5: Graph of Mean ± SD of viable cells (% of total cells) in CLL patients after 4 days in culture without/ with
either CpG ODN2216, CD40L or IgM stimulation.
3. Effect of Estrogens on cell number
We next wanted to know if the addition of Estradiol or SERLs would have an effect
on the cell number of various B cell lines (RIVA, PR1, RAMOS) and primary cells. The
addition of either Estradiol at 10nM or SERLs at a wide concentration range (1nM10µM) had no effect on cell number. These data are in contrast with those provided
by the study of Yakimchuk et al (3). In their report, the use of Estradiol or DPN at 10
nM showed a strong antiproliferative effect on lymphoma growth in culture as
compared to untreated cells. In agreement with our findings, but in contrast to those
provided by Yakimchuk et al., early reports have shown an antiproliferative effect of
Estradiol in human leukaemia cell lines and human acute leukaemia Jurkat T cells
only at high concentration (>5µM), whereas at a lower concentration, no
antiproliferative effect was seen (6,7). It is unlikely in our view that these very high
concentrations are physiologically achievable (normal range of total Estradiol in
serum: 15-350pg/ml).
4. Effect
of
Estrogens
on
cell
apoptosis,
proliferation
and
kinase
phosphorylation
One of the main tasks of this project was to investigate the effect of Estradiol and
SERLs on various functions of B cells from CLL patients. Initially, we investigated the
effect of compounds on apoptosis and proliferation of unstimulated primary B cell.
None of the compounds tested showed any significant difference in either apoptosis
or proliferation when compared with the untreated control (p>0.05) (data not
shown).
Next, we investigated the effect of Estradiol and SERLs on apoptosis, proliferation
and kinase phosphorylation of IgM-stimulated CLL cells. We found that in one
patient (out of 4 tested), Estradiol and PPT (ERα selective agonist) attenuated the
time-dependent changes in ERK and SYK phosphorylation; in contrast, DPN (ERβ
selective agonist) enhanced the time-dependent changes in ERK and SYK
phosphorylation (Figure 6).
Figure 6: Western Blot analysis of phospho-ERK and phospho-SYK in B cells isolated from a
CLL patients at various timepoints after IgM stimulation (1-30mins). Cells were pretreated
ineither Estradiol or different SERLs (Estradiol, DPN, PPT) at 10µM.
Although this result is promising, more patient samples need to be analysed to
identify groups of patients for which the BCR signaling pathway is affected by the
addition of SERLs.
In our experiments, the overall effect of Estradiol and SERLs on apoptosis and
proliferation in stimulated primary B cells was variable, e.g. ERα agonist may alter
apoptosis depending on the experimental conditions. This may be explained by the
fact that, because different signalling stimuli were used in each condition activating
different pathways, this resulted in a variable internal microenvironment, hence the
differential response to Estradiol and SERLs. Another hypothesis for the lack of
consistent results regarding response of B cells to Estradiol and SERLs could be that
Estrogen Receptors in CLL cells may already be highly phosphorylated, a condition
which would render ER unresponsive to estrogen treatment. The following facts
support this hypothesis. a) Previous studies have shown that Estrogen Receptors can
be phosphorylated by ERK and AKT, activating ERs in a ligand-independent manner
(8), b) CLL B cells are characterized by high tonic signalling having SYK and NF-κB
constitutively phosphorylated. This is additionally true for ERK in almost half of CLL
cases (9) and c) Our experiments also show that ERK and SYK are slightly
phosphorylated even at timepoint 0 before exogenous stimulation.
CONCLUSION AND FUTURE WORK
In this study I could show that CLL B cells derived from female patients are more
prone to spontaneous apoptosis than cells from male patients. Furthermore,
stimulation of the cells by CD40L/IL4 and CpG Oligodeoxynucleotides abrogated this
difference, indicating that female cells might depend more on environmental prosurvival signals than their male counterparts. Additionally, engagement of either ERα
or ERβ altered BCR signal duration. ERβ selective antagonists did not induce
apoptosis in CLL cells. My study provides novel data to explain the gender difference
in CLL incidence and prognosis as well as estrogen signalling in B-cells
Future studies should include:

Repeat the spontaneous apoptosis assay on a larger sample size and
investigate further the effect of stimulation by CD40L, IgM or CpG ODN

Determination of the mRNA and protein levels of various genes associated
with apoptosis (Bclx, Mcl1 for example) and proliferation in male and female
samples.

Effect of various stimuli on ERα, ERβ, GPR30 mRNA and protein levels effect
of SERL pre-treatment in ER levels and several signalling parameters (kinases
and transcription factors such as NF-κB), apoptosis and proliferation.
This work was presented as an oral presentation at ISCOMS- International Student
Congress of (bio)Medical Sciences –one of the world’s leading student conferences
in the (bio)medical sciences (Groningen, June 2013). It is also accepted for a poster
presentation at the 24th European Student Conference (Berlin, September 2013).
The Pathological Society is acknowledged for providing us with grants to support
this study.
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