Abstract
BackgroundGranulocyte colony-stimulating factor (G-CSF) promotes proliferation, survival, and differentiation of myeloid-linage leukemic cells, as well as normal hematopoietic cells. Terminal granulocytic differentiation can be induced in acute promyelocytic (APL) cell line HT93A by G-CSF and all-trans retinoic acid (ATRA). Because the detailed mechanism has never been shown, we investigated the signal transduction pathway in granulocytic differentiation by G-CSF, alone or in combination with ATRA.MethodsHT93A cell viability and growth were investigated by trypan blue exclusion assay. Cell differentiation was assessed by CD11b and CD34 expressions. Intracellular protein expressions were also evaluated by flow cytometry after fixation and permeabilization.ResultsATRA (100 nM) induced granulocytic differentiation (upregulation of CD11b and downregulation of CD34) and the effect was potentiated by addition of G-CSF, while G-CSF alone had no effect on HT93A cells. The addition of G-CSF to ATRA had little or no effect on NB4 and THP-1 cells in comparison to ATRA alone. G-CSF receptor expression was reduced by ATRA treatment in a time-dependent manner. After 5 days’ incubation with ATRA, the expression levels of signal transducer and activator of transcription (STAT) 3, and phosphorylated STAT3 and STAT5, were significantly reduced. STAT5 was strongly activated by G-CSF stimulation in ATRA-pretreated cells in comparison to untreated cells. In contrast, STAT3 showed no response to G-CSF. Janus kinase (JAK) inhibitor ruxolitinib (320 nM) had little or no effect on ATRA-induced differentiation, but eliminated the enhancing effect of G-CSF, as evidenced by the levels of CD11b and CD34 expression. These results suggest G-CSF activates STAT5 through the JAK pathway in combination with ATRA, resulting in myeloid differentiation in HT93A cells.ConclusionsIn conclusion, activation of the JAK-STAT pathway is likely essential for inducting differentiation in the APL cell line HT93A; thus, monitoring its expression and activation is important for predicting clinical efficacy and understanding the mechanisms of cytokine-dependent myelopoiesis, proliferation, and differentiation of acute myeloid leukemia.
Highlights
Granulocyte colony-stimulating factor (G-CSF) promotes proliferation, survival, and differentiation of myeloid-linage leukemic cells, as well as normal hematopoietic cells
Differentiation of HT93A cells by all-trans retinoic acid (ATRA) and G-CSF After treatment with 100 nM ATRA for 7 days, expression of major differentiation marker CD11b increased, while primitive marker CD34 decreased in HT93A cells (Figure 1A)
G-CSF activates STAT5 rather than STAT3 in the presence of ATRA After treatment with 100 nM ATRA for 5 days, the expression levels of STAT3, STAT5A, STAT5B, and phosphorylated STAT3 and STAT5 were evaluated by intracellular staining followed by flow cytometry as described in the Materials and methods
Summary
Granulocyte colony-stimulating factor (G-CSF) promotes proliferation, survival, and differentiation of myeloid-linage leukemic cells, as well as normal hematopoietic cells. Terminal granulocytic differentiation can be induced in acute promyelocytic (APL) cell line HT93A by G-CSF and all-trans retinoic acid (ATRA). Because the detailed mechanism has never been shown, we investigated the signal transduction pathway in granulocytic differentiation by G-CSF, alone or in combination with ATRA. Granulocyte colony-stimulating factor (G-CSF) is a myeloid growth factor that promotes proliferation, survival, and differentiation of myeloid-lineage leukemic cells, as well as normal hematopoietic cells [1,2]. Studies in various cell lines have revealed that G-CSF functions through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway [8,9,10]. Basic studies of G-CSF in various in vitro systems have been performed [8,9,10,11,12,13,14,15,16,17], the efficacy and mechanisms of G-CSF, alone or in combination with ATRA, are not well understood in the context of differentiation induction
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