CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells
Background: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregu...
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| Zitieren: | CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells / I.M. Gordiienko, L.M. Shlapatska, V.M. Kholodniuk, L.M. Kovalevska, T.S. Ivanivskaya, S.P. Sidorenko // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 291–298. — Бібліогр.: 36 назв. — англ. |
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irk-123456789-1385492018-06-20T03:05:29Z CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells Gordiienko, I.M. Shlapatska, L.M. Kholodniuk, V.M. Kovalevska, L.M. Ivanivskaya, T.S. Sidorenko, S.P. Original contributions Background: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell surface receptors. Materials and Methods: Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Results: Median of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150⁺) compared to csCD150⁻ CLL cases or normal CD19⁺ and CD19⁺CD5⁺ B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150⁻ and csCD150⁺ CLL cases. PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells. Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA expression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was observed after CD150, CD180 or CD150 and CD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells caused increase of CD150 mRNA level that is a one of the EBF1 target genes. Conclusions: Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels 2017 Article CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells / I.M. Gordiienko, L.M. Shlapatska, V.M. Kholodniuk, L.M. Kovalevska, T.S. Ivanivskaya, S.P. Sidorenko // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 291–298. — Бібліогр.: 36 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138549 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Gordiienko, I.M. Shlapatska, L.M. Kholodniuk, V.M. Kovalevska, L.M. Ivanivskaya, T.S. Sidorenko, S.P. CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells Experimental Oncology |
| description |
Background: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell surface receptors. Materials and Methods: Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Results: Median of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150⁺) compared to csCD150⁻ CLL cases or normal CD19⁺ and CD19⁺CD5⁺ B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150⁻ and csCD150⁺ CLL cases. PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells. Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA expression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was observed after CD150, CD180 or CD150 and CD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells caused increase of CD150 mRNA level that is a one of the EBF1 target genes. Conclusions: Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels |
| format |
Article |
| author |
Gordiienko, I.M. Shlapatska, L.M. Kholodniuk, V.M. Kovalevska, L.M. Ivanivskaya, T.S. Sidorenko, S.P. |
| author_facet |
Gordiienko, I.M. Shlapatska, L.M. Kholodniuk, V.M. Kovalevska, L.M. Ivanivskaya, T.S. Sidorenko, S.P. |
| author_sort |
Gordiienko, I.M. |
| title |
CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells |
| title_short |
CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells |
| title_full |
CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells |
| title_fullStr |
CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells |
| title_full_unstemmed |
CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells |
| title_sort |
cd150 and cd180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138549 |
| citation_txt |
CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells / I.M. Gordiienko, L.M. Shlapatska, V.M. Kholodniuk, L.M. Kovalevska, T.S. Ivanivskaya, S.P. Sidorenko // Experimental Oncology. — 2017 — Т. 39, № 4. — С. 291–298. — Бібліогр.: 36 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
Experimental Oncology 39, 291–298, 2017 (December) 291
CD150 AND CD180 ARE INVOLVED IN REGULATION
OF TRANSCRIPTION FACTORS EXPRESSION IN CHRONIC
LYMPHOCYTIC LEUKEMIA CELLS
I.M. Gordiienko1, L.M. Shlapatska1, V.M. Kholodniuk1, L.M. Kovalevska1, T.S. Ivanivskaya2,
S.P. Sidorenko1, *
1Department of Molecular and Cellular Pathobiology, R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
2Department of Oncohematology, R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
Background: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that
include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5)
and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs
in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into con-
sideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell
surface receptors. Materials and Methods: Studies were performed on normal peripheral blood B-cell subpopulations and
chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation
of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. Results: Median
of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150+) compared to csCD150- CLL cases
or normal CD19+ and CD19+CD5+ B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied
groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and
BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL
cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150- and csCD150+ CLL cases.
PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells.
Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA ex-
pression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was
observed after CD150, CD180 or CD150 and CD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative
regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells
caused increase of CD150 mRNA level that is a one of the EBF1 target genes. Conclusions: Analysis of TFs expression profile
revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate
transcriptional program in CLL cells by regulating the TFs expression levels.
Key Words: transcription factors, chronic lymphocytic leukemia cells, CD150, CD180.
Multistep B-cell development is stringently co-
ordinated by numerous input signals from the cell
surface receptors that lead to activation of unique
transcriptional programs [1]. Exclusive gene expres-
sion profile is determined by interplay of specific
transcriptional factors (TFs) at the every stage of B-cell
development started from B-lineage diversification
until terminally differentiated plasma cells [2, 3]. Co-
operation of E2A, EBF1, PAX5 and LEF1 TFs moves
common lymphoid progenitors to pro-B cell; tandem
of IRF4 and IRF8 regulates pre-B cell stage; pheno-
type of transitional B cells is maintained by OBF1 and
NF-κB; expression of BCL6, PAX5 and MITF is essential
for germinal centre development and finally plasma
cell differentiation is regulated by IRF4, BLIMP1, and
XBP1 [2–4]. TFs often create mutual suppression
loops that regulate alternative differentiation of B-cell
subsets [2]. The block of malignant B cell maturation,
as a rule, occurs at different stages of differentiation
that bear phenotypic characteristic including TFs pro-
file of corresponding normal B-cell counterparts [1].
However, TFs expression and functions are often
dysregulated in B-cell derived leukaemia and lym-
phoma. For example, in approximately 30% of diffuse
large B-cell lymphoma (DLBCL) and 15% of follicular
lymphoma (FL) genetic alteration of BCL6 gene leads
to BCL6 constitutive expression and activation [4].
Overexpression of IRF4 was observed in multiple my-
eloma and activated B-cell like (ABC) DLBCL [5, 6].
However, it was shown that cooperation of PU.1 with
IRF4 and IRF8 prevents pre-B acute lymphoblastic
leukemia development [7]. Thus, IRF4 may act both
as oncogene and tumor suppressor [8]. The block
of terminal differentiation of malignant B cells in ABC
DLBCL is linked to downregulation of BLIMP1 expres-
sion [1]. That is why finding new approaches that could
modify stability, expression and activity of dysregulated
TFs is a perspective direction for optimising treatment
of B-cell malignancies.
Submitted: November 09, 2017.
*Correspondence: E-mail: svitasyd@yahoo.com
Fax: +380442581656;
Abbreviations used: CLL — chronic lymphocytic leukemia;
DLBCL — diffuse large B-cell lymphoma; FL — follicular lymphoma;
HL — Hodgkin’s lymphoma; PBMCs — peripheral blood mono-
nuclear cells; TFs — transcription factors.
Exp Oncol 2017
39, 4, 291–298
mailto:svitasyd@yahoo.com
292 Experimental Oncology 39, 291–298, 2017 (December) Experimental Oncology 39, 291–298, 2017 (December) 293
The present study is focused on evaluation
of expression levels of key B-cell regulatory TFs
in chronic lymphocytic leukemia (CLL) cells com-
pared to their normal counterparts. Previously,
we showed that cell surface receptors CD150 and
CD180 are involved in activation of pro-survival Akt
and MAPK signaling pathways in CLL cells. How-
ever, stimulation of both receptors on CLL cells led
to strong inhibition of Akt-mTOR, p38MAPK and JNK
pathways [9]. Since, many of TFs are downstream
targets of Akt and MAPK pathways we also tested
a hypothesis whether it is possible to regulate TFs
expression via CD150 and CD180 cell surface recep-
tors in CLL cells.
MATERIALS AND METHODS
Isolation of normal B-cell subsets and ma-
lignant CLL cells. Population of CD19+ B cells and
CD19+CD5+ B-cell subpopulation were enriched
from T-cell depleted peripheral blood mononuclear
cells (PBMCs) of healthy individuals by magnetic
separation using Dynabeads coated with sheep anti-
mouse IgG (Dynal Biotech ASA, Norway) according
to manufacturer’s protocols. Mouse anti-CD5 (10.2)
and anti-CD19 (HD37) mAbs were kindly provided
by Prof. Edward A. Clark (University of Washington,
Seattle, WA, USA). Peripheral blood of four healthy
individuals was obtained from the Blood Transfusion
Centre (Kyiv, Ukraine). PBMCs of previously untreated
67 CLL patients were obtained from the Department
of Oncohematology of R.E. Kavetsky Institute of Ex-
perimental Pathology, Oncology and Radiobiology
of National Academy of Sciences of Ukraine (IEPOR
NASU), Kyiv, Ukraine. All patients were thoroughly
informed about the study that was approved by the
Committee on Bioethics at IEPOR.
Flow cytometry. PBMCs of all CLL patients were
immunophenotyped on the subject of CD150 and
CD180 expression as well as common B cell mark-
ers (CD19, CD20, CD22, CD37, CD38, CD40, CD48,
CD95) and CLL marker (CD5, CD23, CD43). Detailed
description of immunophenotyping procedure and im-
munophenotyping results were published earlier [9].
PBMCs from CLL patients were used for in vitro
stimulation assay only when the level of CD19+ cells
was more than 95% and these cells coexpressed
CD150 and CD180 receptors.
In vitro cell stimulation. To initiate signaling via
CD150 and CD180 receptors 10•106 of CD150+CD180+
PBMCs from CLL patients were incubated with
anti-CD150 (IPO3, IEPOR NASU) or/and anti-
CD180 (G28-8, kindly provided by Prof. Edward Clark,
University of Washington, Seattle, WA, USA) mAbs both
at final concentration 10 µg/ml in RPMI-1640 medium
supplemented with 10% FCS. In experiments with
CD150 mRNA upregulation CD150-CD180+ PBMCs
from CLL patients were used. PBMCs cultivated in me-
dium alone were used as a negative control. Stimula-
tion was stopped after 4 h of incubation with ice cold
PBS + 0.01% NaN3.
Western blot analysis. Cell lysis, gel electro-
phoresis, western blot analysis were performed
as described early [10]. The list of followed anti-
bodies was used for western blot analysis: goat
anti-IRF4, anti-IRF8, anti-Actin, anti-BLIMP1, rabbit
anti-BCL6 (all from Santa Cruz Biotechnology, USA),
rabbit anti-PU.1 (Cell Signaling Technology, Beverly,
MA, USA). Secondary goat anti-rabbit and donkey
anti-goat HRP-conjugated antibodies were from
Santa Cruz Biotechnology (USA). Clarity Western
ECL substrate (Immunо-Star HRP, BioRad, USA)
and medical X-ray film (AGFA, Belgium) were used
for visualization.
Quantitative RT-PCR. A detailed description of to-
tal RNA isolation, cDNA synthesis and real-time PCR
were reported elsewhere [10]. The following forward
(For) and reverse (Rev) primers for real-time PCR were
used: IRF4: For 5ʹ-CCACTACCTCCTTTCCTATC-3ʹ,
Rev 5ʹ-CCGTTCCTTTTCAGAGTCCT-3ʹ; IRF8: For
5ʹ-CCAACAGATCACCGTCTAA-3ʹ, Rev 5ʹ-AAGTG-
CAAAGTAAGGCATC-3ʹ; PU.1: For 5ʹ-CTTCCAGTTCTC-
GTCCAA-3ʹ, Rev 5ʹ-GAGCTTCTTCTTCACCTTC-3ʹ;
SPIB: For 5ʹ-GCATACCCCACGGAGAACT-3ʹ; Rev
5ʹ-GGCTGTCCAACGGTAAGTCT-3ʹ, BCL6: For 5ʹ-CTC-
CGTGCCCATGTGCTTA-3ʹ; Rev 5ʹ-GAGTCTGAAGGT-
GCCGGAAA-3ʹ, EBF1: For 5ʹ-GTACCATGCTGGTCTG-
GAGTG-3ʹ; Rev 5ʹ-GTGTGACTTCCACAACACCAGG-3ʹ,
PAX5: For 5ʹ-GTCCCAGCTTCCAGTCACAG-3ʹ; Rev
5ʹ-CGGAGACTCCTGAATACCTTCG-3ʹ. As internal
control gene for normalization was chosen TATA-box
binding protein (TBP): For 5ʹ-CCACTCACAGACTCT-
CACAAC-3ʹ; Rev 5ʹ-CTGCGGTACAATCCCAGAACT-3ʹ.
The PCR cycling conditions were the following: 10 min
at 95 °C, 40 cycles of 15 s at 95 °C and 40 s at 60 °C. The
threshold cycle (Ct) values were determined for the
internal control (TBP) and the tested genes at the
same threshold level in the exponential phase of the
PCR amplification curves. The Ct method was used
to calculate test genes expression level normalized
to the endogenous control (in case of TFs expression
in CLL cells and normal B cells subsets). Experiments
with receptors ligation were additionally normalized
to untreated control cells. Results were presented
in relative units or fold change. Dissociation curve
analysis was performed after every run to check the
specificity of the reactions. For all types of cell samples
3–5 reactions (each in triplicates) were run for each
genes with internal control and standard error of the
mean (SEM) was calculated.
Statistical analysis. Obtained data were anal-
ysed by unpaired Mann — Whitney U test using Prism
software Version 4.0. Statistical significance between
groups was assessed as p ≤ 0.05. Pearson’s coef-
ficient was used for determination of correlation
between variables. Box plots showed the results
of TFs mRNA expression level where whiskers means
maximum and minimum values, the line within the
rectangle shows the median, and the top and bottom
of the rectangle represent the third and first quartile,
respectively.
Fig. 1. TFs mRNA expression levels in CLL cells (qRT-PCR). The csCD150− CLL cells (n = 6) and csCD150+ CLL cells (n = 6) were compared
to CD19+ and CD19+CD5+ PB B cells (4 donors).*p ≤ 0.05 compared to CD19+ normal B cells. **p ≤ 0.05 compared to CD19+CD5+ normal B cells
294 Experimental Oncology 39, 291–298, 2017 (December) Experimental Oncology 39, 291–298, 2017 (December) 295
ous ligation of these receptors on CLL cells resulted
in strong downregulation of IRF4 mRNA (Fig. 3, g).
Coligation of CD150 and CD180 did not have any ad-
ditive effect on IRF4 mRNA downregulation. PU.1 and
SPIB as the partners could act in cooperation with
IRF4 and IRF8 TFs [16]. Stimulation of CLL cells via
CD150 or CD180 receptors alone or their combina-
tion resulted in increased PU.1 mRNA level in CLL
cells with additive effect of CD150 and CD180 coliga-
tion (Fig. 3, e). At the same time, CD150 ligation led
to downregulation of SPIB mRNA level in malignant
B cells, while CD180 and CD150 + CD180 ligation
did not have any impact on regulation of SPIB ex-
pression level (Fig. 3, f). Taken together, CD150 and
CD180 receptors are involved in regulation of TFs
mRNA expression levels in CLL cells. CD150 and
CD180 receptors alone or their combination had
unidirectional effect on downregulation of IRF4, up-
RESULTS AND DISSCUTION
CLL cells is clonal malignant B cells that according
to gene expression signature is closely related to CD5+
peripheral blood B-cell subpopulation [11]. That
is why peripheral blood CD19+ B cells and CD19+CD5+
B cells obtained from healthy individuals were used
in our study for comprehensive analysis of TFs profile
expression between CLL cells and normal B cell coun-
terparts. The present study is focused on the TFs that
maintain B-cell identity (EBF1 and PAX5) and regulate
differentiation from naïve B cell stage toward plasma
cells (IRF4, IRF8, PU.1, SPIB, BCL6, and BLIMP1).
CLL cases were grouped according to the cell surface
CD150 expression. We tested the hypothesis that cell
surface CD150 positive (csCD150+) and CD150 nega-
tive (csCD150−) CLL cells may stop their maturation
at the different stages of B-cell development and
therefore could have diverse TFs expression profiles.
mRNA expression level of B-cell identity markers
PAX5 and EBF1 was similar in studied normal B cell
subsets and csCD150− CLL cases (Fig. 1, a, b). How-
ever, median of PAX5/EBF1 expression was higher
in csCD150+ than in csCD150− CLL cases. CLL cells
showed the similar mRNA expression levels of IRF8 and
BCL6 regardless of csCD150 expression (Fig. 1, c, d).
BCL6 mRNA expression was higher in normal B cells
that in CLL cells (p ≤ 0.03) (Fig. 1, d). All tested CLL
cases were characterised by lower level of PU.1 mRNA
compared to normal B-cell subpopulations (p ≤ 0.02)
(Fig. 1, e). On the other hand, the level of SPIB
mRNA was significantly higher in csCD150− as well
as csCD150+ CLL cells than in normal B cell subsets
(Fig. 1, f). Expression level of IRF4 was similar in CLL
and normal B cells (Fig. 1, g). mRNA expression level
of BLIMP1 — master regulator of plasma cell develop-
ment, was low in all tested normal B cell subsets and
CLL cells (Fig. 1, h). According to obtained results CLL
cells has similar profile in mRNA expression of IRF4,
IRF8, EBF1, and PAX5 TFs to normal peripheral blood
B-cell subpopulations. Unique feature of malignant
CLL cells is upregulated mRNA expression of SPIB
and downregulated PU.1 and BCL6 TFs.
To clarify whether csCD150− and csCD150+ CLL
cells have any differences in TFs expression on protein
level we used western blot analysis. High expres-
sion levels of IRF4 and IRF8 proteins were detected
in all CLL cases regardless of csCD150 expression
(Fig. 2, a). All CLL cases were negative for BLIMP1 pro-
tein expression (data not shown). BCL6 protein was re-
vealed only in 31% of CLL cases despite the presence
of BCL6 mRNA in all studied CLL cases. Moreover,
BCL6 expression did not correlate with cell surface
CD150 expression. At the same time, PU.1 protein
was predominantly expressed in csCD150+ CLL cases
regardless of the uniform expression of PU.1 mRNA
in CLL (see Fig. 2, a, Fig. 1, e). Furthermore,
PU.1 protein level positively correlated with cell sur-
face CD150 expression (r = 0.4, p < 0.05). The level
of PU.1 protein was 14 times higher in csCD150+ than
in csCD150− CLL cases (p = 0.002) (Fig. 2, b). It was
shown that CD20 receptor is one of the PU.1 target
genes [12]. We checked whether any correlations ex-
ist between cell surface CD150 and CD20 expression
in CLL cases. Indeed we found positive correlation
between CD150 and CD20 cell surface expression
in CLL cases (r = 0.45, p ≤ 0.05). Median of CD20 cell
surface expression was higher in csCD150+ CLL
cases (Fig. 2, c) (p = 0.01). Thus, upregulated levels
of PU.1 protein expression and its target CD20 were
correlated with cell surface CD150 expression in CLL
cells.
Numerous data suggest that CD150 is involved
in activation of Akt and MAPK signaling in normal
and malignant B cells [9, 13–15]. In case of CLL,
CD150 could realize its signaling properties alone
or together with CD180 receptor [9]. Since many
of downstream targets of Akt and MAPK pathways
are TFs, we test the hypothesis whether CD150 and/
or CD180 are involved in regulation of TFs expression
in CLL cells. To check this we performed in vitro ligation
of CD150 and CD180 alone or simultaneously on CLL
cells with followed evaluation of TFs mRNA levels.
These receptors were not involved in B-lineage main-
tenance TF PAX5 expression level regulation (Fig. 3, a).
Only CD180 ligation on CLL cells leaded to upregula-
tion of EBF1 mRNA level and CD150 receptor did not
abrogate this effect of EBF1 (Fig. 3, b). Expression
level of IRF8 mRNA was significantly upregulated
in CLL cells after ligation of any if these receptors alone
or in combination (Fig. 3, c). CD150 or CD180 alone,
but not CD150 and CD180 ligation upregulated
BCL6 expression in CLL cells (Fig. 3, d). In opposite,
crosslinking of CD150 or CD180 alone and simultane-
Fig. 2. TFs protein expression in CLL cells: a — protein expres-
sion of BCL6, IRF4, IRF8, and PU.1 in csCD150− and csCD150+
CLL cases. Results of western blot analysis; b — densitometry
analysis of PU.1 expression level in csCD150- and csCD150+
CLL cases. Results were normalized to actin expression and
presented in relative units; c — cell surface CD20 expression
level in csCD150− and csCD150+ CLL cases. Flow cytometry
analyses. Results are presented as GeoMean MFI ratio of antigen
to isotype control. *p = 0.05 compared to csCD150− CLL cases
Fig. 3. CD150 and CD180 mediated regulation of PAX5 (a), EBF1 (b), IRF8 (c), BCL6 (d), PU.1 (e), SPIB (f), and IRF4 (g) mRNA
expression. Results are presented as a mean of mRNA expression in three independent CD150+CD180+ CLL cases. (h) CD150 mRNA
expression level in csCD150− CLL cells after CD180 ligation in three independent CLL cases. *p ≤ 0.05 compared to control (CLL
cells cultivated in medium alone)
296 Experimental Oncology 39, 291–298, 2017 (December) Experimental Oncology 39, 291–298, 2017 (December) 297
Described here regulation of TFs expression via cell
surface receptors is only one side of crosstalk between
TFs and cell surface receptors. Thus, number of TFs
such as EBF1, E2A, PU.1, IRF4, STAT6, and NF-κB
(RELA) could bind to SLAMF1 promoter and regulate
its activity [34]. The early B-cell factor 1 (EBF1) inter-
acts with specific binding sites both in the promoter
and in the enhancer regions of the SLAMF1 gene and
is a key regulator of SLAMF1 expression in human and
mouse B cells [34, 35]. In CLL cells CD150 cell surface
expression is significantly downregulated compared
to normal B lymphocytes [36]. Previously it was shown
that EBF1 protein expression was not detected in CLL
cases both with mutated and unmutated IGHV [11].
In our study EBF1 mRNA level was higher in csCD150+
CLL cells than in csCD150− CLL cells, but whether
EBF1 mRNA is translated in these CLL cases was not
addressed. Absence or low level of EBF1 expression
could result in significantly decreased cell surface
CD150 expression level in CLL cells compared to nor-
mal periphe ral blood B cells. Our study showed that
CD180 is specific positive regulator of EBF1 mRNA
expression. Moreover in normal B cells CD180 is a most
potent upregulator of CD150 expression [37]. That
is why we tested a hypothesis whether it is possible
to regulate CD150 expression via CD180 receptor in CLL
cells. Indeed, ligation of CD180 on CLL cells led to more
than two fold upregulation of CD150 mRNA expression
(Fig. 3, h). According to results published earlier cell
surface expression of CD150 and CD180 is positively
correlated and these receptors coexpression is ob-
served in 59% of CLL cases. However, 11% of CLL
cases were positive only for csCD180 expression [9].
Since, cell surface CD150 expression is associated
with favourable clinical outcome of CLL patients and
csCD150+ CLL cells are more sensitive to chemothera-
peutic drugs [36] upregulation of CD150 cell surface
expression have a good perspectives in optimization
of CLL treatment. Signaling via CD180 may be one of the
possible approaches to induce CD150 cell surface ex-
pression in CD150− CLL cells that will result in modula-
tion of CLL B-cell pathobiology.
Taking together, our study showed that CLL cells
are characterised by decreased PU.1 and elevated
SPIB mRNA levels compared to normal B-cell coun-
terparts. Protein expression of PU.1 and its direct
target CD20 positively correlated with cell surface
CD150 expression in CLL cells. Ligation of CD150 and
CD180 alone or in combination upregulated IRF8 and
PU.1 while downregulated of IRF4 mRNA expres-
sion. Signaling via CD150 or CD180 elevated the level
of BCL6 mRNA. We found that in CLL cells CD150 is a neg-
ative regulator of SPIB whereas CD180 is involved in up-
regulation of EBF1 expression level. Moreover, CD180-
mediated signaling leaded to increased CD150 mRNA
level in CLL cells and could be considered as a potential
positive regulator of CD150 expression level in malignant
CLL cells. Thus, CD150 and CD180 receptors may modu-
late transcriptional program in CLL cells by regulating the
TFs expression levels.
ACKNOWLEDGEMENTS
Authors thank to Prof. E.A. Clark, Ms. Geraldine
Shu, Dr. M.Y. Yurchenko, Dr. E.V. Kashuba, and
Mr. R.G. Vasyliev for their help in the achievement
of this study. The work was supported by scientific
grants #0113U008330 and #0116U007817 from NAS
of Ukraine.
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signaling via CD150 as well as CD180 increase the level
of BCL6 mRNA expression. In addition, CD150 recep-
tor is negative regulator of SPIB mRNA expression
while CD180 is a positive regulator of EBF1 mRNA
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CLL is a disease of CD5+CD19+CD23+ B cells,
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