Methylprednisolone Induce Terminal Differentiation in the U-937 Human Myelomonocytic Leukemia Cells
1Mersin University, Faculty of Science and Art, Department of Biology, Mersin2Hacettepe University, Faculty of Medicine, Department of Pediatrics, Ankara, Turkey
ABSTRACT
Differentiation studies have shown that methylprednisolone have some roles onto the leukemia cell line such as HL-60 andK-562. Methylprednisolone (MP), a steroid compound, binds the nuclear glucocorticoid receptor and regulates the tran-scription. In this study, differentiation effect of methylprednisolone on human monocytic leukemia (U-937) cell line wasinvestigated for two types of cluster differentiation (CD11b, CD68) markers by using flow cytometry. It was observed thatthe significant terminal differentiation of U-937 cells occured after treatment with high dose (10-3 M) MP for 24 and 48 hrs. Our data points out that MP is a differentiation inducing agent for myelomonocytic leukemic cells and could be potentialtreating compound in other type of leukemic cell differentiation. Turk J Immunol, 2008; 13: 10-14 Key Word: Acute myelomonocytic leukemia, differentiation, cell culture, flow cytometry Received: 03.05.2007 Revised: 06.11.2007 Accepted: 26.11. 2007 INTRODUCTION
has been demonstrated that other steroid compoundsuch as dexametasone and prednisone also induced differentiation on leukemic cells5-8. M ethylprednisolone (MP), a member of the family
Myelomonocytic leukemia (MMoL) is a part of the
of steroid hormones is known to play a critical
acute myeloid leukemia characterised by the accumula-
role in the cellular processes such as proliferation,
tion of malignant myelomonocytic cells9. Experimental
differentiation and apoptosis mechanisms by binding
differentiation studies of myelomonocytic leukemia
to members of the zinc-finger containing superfamily
dates back to the 1974 when Sundström and Nilsson
of nuclear hormone receptors1-2. When coupled with
established the U-937 cell line from a diffuse histiocytic
MP, these nuclear hormone receptors play a role as a
lymphoma of a 37 year old male patient in Rudbeck
transcription factor by binding directly to specific DNA
Laboratory, Uppsala-Sweden10. In this study, the U937
recognition sequences in the promoter region of target
cell line was chosen since it serves as an in-vitro model
genes, resulting in the alteration of the
for monocyte/macrophage differentiation.
transcription initiation of differentiation genes1-2. Hence,
steroid compounds suggest that they might play a role
myelomonocytic and in other types of leukemia cells
of differentiation on leukemic cells since they have
dates back to the late of 1970s and beginning of the
been shown to induce the differentiation of
1980s. The cells which contain glucocorticoid
granulocytic and monocytic stage of mouse blast
receptors are related to response of steroid molecules
cells3-6. Besides valuable data from Lotem’s group, it
In addition, the increased effect of dexametha-
purchased from Sigma Chemical Co. Fetal bovine
sone was shown in RA (Retinoic Acide) induced
differentiation of HL-60 cells to neutrophils12-13. In
Streptomycin were purchased from Biochrome
the end of 1990s, He and Jiang showed that anoth-
(Berlin, Germany). Stock solutions of the
methylprednisolone were prepared in ethanol and
differentiation of HL-60 cells in a dose dependent
diluted in fresh medium. PE-conjugated mouse
manner14. Last decade it has been shown that many
anti-human CD11b and FITC-conjugated mouse
differentiation agents had been induced the differ-
anti-human CD68 monoclonal antibodies were
entiation of HL-60 cells. Recently, a study from
purchased from Becton Dickonson (Mount View,
Turkey demonstrated that different signal
CA). The U937 cell line was kindly gifted from
Dr. Hande Canpinar from Hacettepe University
differentiation of HL-60 and K-562 cells by MP or
Institute of Oncology, (Ankara, Turkey).
arsenic tri-oxide (As2O3) (15). Unlike As2O3, MP-induced granulocytic differentiation was related with
Cell Culture
serine/threonine protein phosphatases type 2A
subunit upregulation. In addition, the combination
medium containing 15% heat inactivated fetal
of As2O3 and MP has also a synergistic effect on
bovine serum, 2 mM L-glutamine, 10.000 units of
penicilin per ml, 10 mg/ml of streptomycin at 37oC
and in 5% CO2. Cells in logaritmic growth phase
methyleprednisolone (HDMP) (20-30 mg/kg/day)
were used for the study. U-937 cells (1 x 106
treatment has been shown to induce in vivo
cell/ml) were treated with 10-6 M and 10-3 M
differentiation of myeloid leukemia cells to mature
concentration of MP for 24 and 48 hours, in six well
granulocytes and apoptosis of myeloid leukemic
cells in children with different subtypes (AML-M1,
Cytotoxicity Assay
-M2, -M3, -M4, M7) of AML16-19. Moreover HDMP
In order to determine viability and toxicity of
has been shown to effect the differentiation of pri-
MP, the cells were seeded 1x106 cell/ml in 6 well
plates. After adding high (10-3 M) and low (10-6 M)
The objective of the present study was to evaluate
dose of MP, U-937 cells were incubated under the
the in-vitro differentiation effect of 6α-methylpredni-
humidified atmosphere and 5% CO2 conditions at
solone in U-937 (human myelomonocytic leukemia)
desired periods of time. Cell viability was
cells which is one of the rare cell lines displaying
measured by either trypan blue dye exclusion
many monocytic characteristics and has thus served
assay and CBC. The concentration of drug which
affected less than 50% of cell population were
differentiation experiment. The U-937 cells are com-
considered as non-toxic dose and used for the
mitted to the macrophage branch of the myeloid lineage and can be induced by a variety of agents to
mature from a promonocytic into a monocytic stage
Determination of Differentiation
of development. In this study, cell surface
expression of myelomonocytic and macrophage like
determination of differentiated cells was assessed
markers, CD11b and CD68 were analysed to
by the measurement of cell surface antigens
demonstrate the efffect of MP on the differentiation
CD11b and CD68 by flow cytometry. For testing
of U937 cells in to a further stage of development.
CD11b and CD68 cell surface antigens, the U-937cells were stained with PE-conjugated mouse
MATERIALS AND METHODS
anti-human CD11b and with FITC-conjugatedmouse anti-human CD68 antibodies respectively. Reagents
After incubation at +4oC in dark atmosphere for 30
minutes, the stained cells were diluted with 1 ml
dimethylsulfoxide (DMSO), trypan blue, were
Statistical Analysis
CD11b expression was dramatically reduced with
The cell surface markers experiments were
10-3M MP in both 24 and 48 hours (p<0.001) while
performed in triplicates and the data were
slight decrease was detected with 10-6 M MP.
evaluated as the mean plus or minus standart
On the other hand, it was observed that CD68
deviation (mean±S.D.). Two ratio comparing
expression levels on U-937 cells in response to MP
Z- test in flow cytometric assays were used for the
were elevated time and dose dependently. CD68
statistical analysis. A “p value” less than 0.05 was
expression was increased with 10-3 M MP in both
considered as statistically significant.
24 and 48 hours (p<0.001) while statistically insignificant increase was detected with 10-6 M MP.
RESULTS The cytotoxic effect of MP on U-937 cells DISCUSSION AND CONCLUSION
We observed that high dose MP reduced the
The role of corticosteroids on differentiation
viability of U937 cells to almost 50% in 48 hours
mechanisms has first been reported by Lotem and
whereas MP led to more than 75% viability in other
Sachs4. They demonstrated that myeloid leukemic
conditions (Table 1). The cultures with greater than
cells in-vivo can differentiate into macrophages
50% viability were considered in the study.
and granulocytes when treated with dexametha-sone. Since then, in-vitro and in-vivo differentiation
Induction of terminal differentiation of
effect of some steroid compounds such as dexam-
U-937 cells by MP
ethasone and prednisolone have been reported by
MP significantly decreased CD11b expression
different groups5,21. In Turkey, high dose methyl-
in time and dose dependent manner (Figure 1).
prednisolone (20-30 mg/kg/day) has been used in
Table 1: Effect of MP on U937 cell viability. CBC (x 106 /ml) TBE (x106 /ml) Viability (%) Figure 1. Dose and time dependency of Figure 2. Dose and time dependency of MP-induced changes of CD11-b expression on MP-mediated CD68 expression on U-937 U-937 myelomonocytic cells. Two concentrations of myelomonocytic cells. Two concentrations of MP (10-6 M and 10-3 M) were applied into the U937 MP (10-6 M and 10-3 M) were applied into the U937 cells for 24 and 48 hours. CD11b cell surface marker cells for 24 and 48 hours. CD68 cell surface marker was tested on U937 cells by flow cytometry. The was tested on U937 cells by flow cytometry. The concentration of 10-3 M MP was observed concentration of 10-3 M MP was observed decreasing of the level of CD11b. increasing of the level of CD68.
children with AML since 19879 and clinically suc-
We only showed the CD68 marker expression. This
cessful results were observed in the past two
situation could be explaned by the accumulation of
decades. It was shown for the first time, that
macrophage-like cells in the whole population.
HDMP treatment induces differentiation and apop-
In a valuable study, it was shown that the
tosis of leukemic cells in children with APL and in
other with different morphological subtypes of
isomers and 16-dehydroxyprogesterone have
acute myeloblastic leukemia (AML) in-vivo by
been shown to induce differentiation of myeloid
Hiçsönmez et al.9, 16-17. Especially, the effect of pure
leukemic cells. It was demonstrated that
form of MP (6α- methylprednisolone 21-hemisuc-
trans-guggulsterone highly effected HL-60 cells by
cinate) studies in primer culture from AML blasts
increasing both CD11b and CD14 cell surface anti-
has first been shown by Özbek et al.20. They suc-
cessfully demonstrated that low (10-6 M) and high
cis-guggulsterone promoted only increase in the
dose (10-3 M) 6α-MP 21-hemisuccinate induced
expression of CD14 antigen23. These results sug-
mature granulocytic form and apoptosis in the
gested that different compounds of steroids might
blast cells from AML patients at different sub-types
(M1, M2, M3, and M7) following 24 hours of incu-
The effect of high dose MP has been initially
bation20. However, showing more differentiated
shown to induce in-vivo differentiation of myeloid
and apoptotic cells after treatment with HDMP
leukemic cells to mature granulocytes in patients
(10-3 M) points out the clinical effects of high dose
with AML. We also showed the increased CD68
expression at 24 and 48 hours in U-937 cells
In the present study, we evaluated the in-vitro
treated with high-dose concentrations of MP which
effects of MP onto the differentiation of
is considered as macrophage-like differentiation.
promonocytic leukemia cells. As a result of CD11b
The results of our study suggest the importance of
analyses, low dose (10-6 M) concentration of MP
clinical use of HDMP therapy in patients with all
did not show any significant effect on U-937 cells
types of AML. Moreover, this study would be
but high dose (10-3 M) concentration of MP sharply
enlarged to work with other leukemia and lymphoma
counterpart cell lines except that U-937, HL-60 and
surface marker on U-937 cells. In other words the
K-562 by using other differentiation inducers with
myelomonocytic cells were reduced by the effect
steroid for the best curing of lymphoma and
of high dose MP. On the contrary, increased
leukemia. It would also be interesting to search if the
expression of CD68 supported the effect of MP
changes in the signaling patway of differentiation are
onto the terminal differentiation of U-937
related to the particular regulation of gene and pro-
myelo-monocytic cells. A possible explanation for
the suppressive effect of MP on CD11b expressioncould be decrease in monocytic cells since
Acknowledgement
terminal differentiated cells accumulated in the
whole cell population as it is seen CD68 terminal
Hicsonmez for her valuable contributions in the
differentiation marker accumulation (Figure 2).
final draft of the studies. We also thanks to Dr
The another differentiation study group from
Hande Canpinar who gifted the U-937 cell line to
Turkey investigated the differentiation process on
us. Finally we gratefully thank to Ms Seval KUL, Mr.
different types of myeloid leukemic cells (HL-60
Önder SUNBUL and Dr. Adnan ERKUS for valu-
and K-562) by showing signal regulation of
able help in statistics session of this work.
methyleprednisolone and arsenic trioxide compounds. They have tested the effect of arsenic
CORRESPONDENCE
trioxide and methylprednisolone alone and togeth-er. The valuable synergistic effect has been
demonstrated in terminal differentiation of HL-60
Mersin University, Faculty of Science and Art,
and K562 cells with the significant increase of
Department of Biology, 33100 Mersin, Turkey
CD11b and CD11c15,22. In contrast, we did not
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Data and Safety Monitoring During Randomized Controlled Trials of Nursing Interventions Nancy T. Artinian ▼ Erika Sivarajan Froelicher ▼ Background: Principal investigators have a responsibility to ensure and maintain the scientific integrity of their research studies and to protect the safety of the partici-pants. Data and safety monitoring is required for all types of c