Korean J Physiol Pharmacol.  2012 Aug;16(4):265-271. 10.4196/kjpp.2012.16.4.265.

Increased HoxB4 Inhibits Apoptotic Cell Death in Pro-B Cells

• Park SW
• Won KJ
• Lee YS
• Kim HS
• Kim YK
• Lee HW
• Kim B
• Lee BH
• Kim JH
• Kim DK

• KMID: 2011161
• DOI: http://doi.org/10.4196/kjpp.2012.16.4.265

Abstract

HoxB4, a homeodomain-containing transcription factor, is involved in the expansion of hematopoietic stem cells and progenitor cells in vivo and in vitro, and plays a key role in regulating the balance between hematopoietic stem cell renewal and cell differentiation. However, the biological activity of HoxB4 in other cells has not been reported. In this study, we investigated the effect of overexpressed HoxB4 on cell survival under various conditions that induce death, using the Ba/F3 cell line. Analysis of phenotypical characteristics showed that HoxB4 overexpression in Ba/F3 cells reduced cell size, death, and proliferation rate. Moreover, the progression from early to late apoptotic stages was inhibited in Ba/F3 cells subjected to HoxB4 overexpression under removal of interleukin-3-mediated signal, leading to the induction of cell cycle arrest at the G2/M phase and attenuated cell death by Fas protein stimulation in vitro. Furthermore, apoptotic cell death induced by doxorubicin-treated G2/M phase cell-cycle arrest also decreased with HoxB4 overexpression in Ba/F3 cells. From these data, we suggest that HoxB4 may play an important role in the regulation of pro-B cell survival under various apoptotic death environments.

Keyword

Apoptosis; Ba/F3; Cell cycle arrest; Cell proliferation; HoxB4

MeSH Terms

Apoptosis
Cell Cycle Checkpoints
Cell Death
Cell Differentiation
Cell Line
Cell Proliferation
Cell Size
Cell Survival
Hematopoietic Stem Cells
Precursor Cells, B-Lymphoid
Stem Cells
Transcription Factors
Transcription Factors

Figure

• 

  Fig. 1 Retroviral vectors and phenotype induced by HoxB4 in Ba/F3 cells. (A) Constructs of MSCV-GFP and MSCV-HoxB4 retroviral vectors. Schematic representation of the MSCV-HoxB4-IRES-GFP construct and the MSCV-IRES-GFP control vector are shown. (B) Confirmation of HoxB4 expression in Ba/F3 cells. Using cDNA from both infected cells, we examined the effect of HoxB4-expressing Ba/F3 cells using reverse transcription-polymerase chain reaction (RT-PCR). (C) Phenotype of Ba/F3-HoxB4 cells. The morphology of the HoxB4-infected cells was demonstrated via FSC (forward scatter) and SSC (side scatter) in fluorescence-activated cell sorting (FACS) analysis. (D) Photomicrograph of Ba/F3 cells with HOXB4 overexpression. Upper panels, cell images observed under condition of phase contrast illumination; Lower panel, GFP fluorescence images in cells corresponding to phase contrast images. Representative pictures are shown from at least three independent experiments. Magnification ×400.

• 

  Fig. 2 Biological activities of HoxB4 in Ba/F3 cells. (A) Proliferation rate of Ba/F3-HoxB4 cells. 2×103 Ba/F3-HoxB4 cells were cultured with 5 ng/ml IL-3 (survival cytokine) in 96-well plates for 3 days and cell proliferation activity was assessed via MTT assay. Data are expressed as mean±standard deviation (SD). *p<0.05 in comparison with control cells. (B) Alteration of the cell cycle by HoxB4. Ba/F3-GFP and Ba/F3-HOXB4 cells were stained with PI and detected by fluorescence-activated cell sorting (FACS) after incubation in normal culture conditions. Data are expressed as mean±SD. (C) Inhibitory effect of apoptotic cell death by HoxB4. Ba/F3-GFP and Ba/F3-HoxB4 cells were incubated without IL-3 (-) for 24 h, and then the apoptotic cell death of Ba/F3-HoxB4 cells was measured using the Annexin V and PI assay with FACS. The data express the rate of double-negative (live), single-positive (early apoptosis and necrosis), and double-positive (late apoptosis) cells.

• 

  Fig. 3 Biological effects of HoxB4 in cell cycle arrest at G2/M phase. (A) HoxB4 in the cell cycle. Ba/F3-HoxB4 cells were incubated with 100 nM DOX for 24 h, and then the DNA content was determined by PI staining with flow cytometry. Representative flow cytometry data and the percentage of cells at each stage of the cell cycle are shown. These data represent the mean±standard deviation (SD) from 3 independent experiments. *p<0.05 in comparison with control cells. (B) Apoptosis assay of Ba/F3 cells infected with HoxB4. Ba/F3-HoxB4 cells were incubated with media containing 100 nM DOX. After staining with Annexin V-PE and PI, we performed flow cytometry. The data represent the proportions of double-negative (live), single-positive (early apoptosis and necrosis), and double-positive (late apoptosis) cells.

• 

  Fig. 4 Biological activities of HoxB4 in conditions that stimulate the Fas (CD95) apoptotic signaling pathway. (A) Ba/F3-GFP and Ba/F3-HoxB4 cells were cultured with 100 nM DOX for 24 h, recovered, and stained with anti-Fas (CD95) antibody, and immune fluorescence was quantified by flow cytometric analysis. Numbers in the upper parts of histogram curves indicate the positive intensity values of Fas expression in cells. The graph was obtained from left two panels and represents fold changes between results from Ba/F3-GFP and Ba/F3-HoxB4 cells in the presence of DOX. *p<0.05 in comparison with control cells. (B) Stimulation of the Fas apoptotic signal pathway. Ba/F3-GFP and Ba/F3-HoxB4 cells were treated with 1 µg/ml agonistic antibody (clone Jo2) for 24 h and analyzed by flow cytometry after staining with Annexin V-PE and PI. The data represent the proportions of double-negative (live), single-positive (early apoptosis and necrosis), and double-positive (late apoptosis) cells. (C) Stimulation of Fas induced by DOX. Ba/F3-GFP and Ba/F3-HoxB4 cells were treated with 1 µg/ml agonistic antibody (clone Jo2) and 100 nM DOX for 24 h and analyzed by flow cytometry after staining with Annexin V-PE and PI. The data express the proportions of double-negative (live), single-positive (early apoptosis and necrosis), and double-positive (late apoptosis) cells. (D) Photomicrograph of cells incubated with DOX and Jo2 antibody. Ba/F3-GFP and Ba/F3-HoxB4 cells were seeded in 24-well plates (5×105 cells in each well) and incubated with complete media, 100 nM DOX or 1 µg/ml Jo2 antibody and after 24 h, the morphology change of cells were observed in each well. Magnification ×40.

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