Public Health Advancement of global health: key messages from the Disease Control Priorities Project Ramanan Laxminarayan, Anne J Mills, Joel G Breman, Anthony R Measham, George Alleyne, Mariam Claeson, Prabhat Jha, Philip Musgrove, Jeﬀ rey Chow, Sonbol Shahid-Salles, Dean T Jamison The Disease Control Priorities Project (DCPP), a joint project of the Fogarty International Center o
ImmunolojiMethylprednisolone 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
Accepted: 26.11. 2007
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.
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)
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 show the CD11b cell surface antigen expression.
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Quantitative evaluation of drug transport and metabolism in the body Research Department of Genetic, Evolution and Environment, University College London (UCL), London, United Kingdom Professor Yuichi Sugiyama Associate Professor Hiroyuki Kusuhara Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo Abstract