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A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

Hirako N, Nakano H, Takahashi S - PLoS ONE (2014)

Bottom Line: Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells.Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA.Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Minami-ku, Sagamihara, Japan.

ABSTRACT
We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

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Analyses of ATRA-induced differentiation of NB4MTOE cells by NBT assay.(A) Assessment of NBT assay with or without ATRA. Examples of strongly positive (++), weakly positive (+), and negative (-) cells in the NBT reduction test. (B) Assessment of NBT assay in NB4MTOE cells and control cells. Cells were treated with 1 µM ATRA for 24 h (left), 48 h (middle), or 72 h (right panel). Black bars: NBT strongly positive cells (++); gray bars: NBT weakly positive cells (+); white bars: NBT negative (-) cells. After blind labeling of each sample, at least 200 cells were counted and the percentage of NBT-positive cells was calculated. The indicated p-value was calculated for the difference between the percentages in the (++) control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells treated with ATRA for 72 h.
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pone-0103282-g004: Analyses of ATRA-induced differentiation of NB4MTOE cells by NBT assay.(A) Assessment of NBT assay with or without ATRA. Examples of strongly positive (++), weakly positive (+), and negative (-) cells in the NBT reduction test. (B) Assessment of NBT assay in NB4MTOE cells and control cells. Cells were treated with 1 µM ATRA for 24 h (left), 48 h (middle), or 72 h (right panel). Black bars: NBT strongly positive cells (++); gray bars: NBT weakly positive cells (+); white bars: NBT negative (-) cells. After blind labeling of each sample, at least 200 cells were counted and the percentage of NBT-positive cells was calculated. The indicated p-value was calculated for the difference between the percentages in the (++) control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells treated with ATRA for 72 h.

Mentions: We further evaluated the role of MT1G in NB4 cell differentiation by performing NBT reduction assays. This assay is based on the ability of phagocytic cells to produce superoxide upon stimulation with phorbol 12-myristate 13-acetate (PMA), which is comparable to that generated by normal peripheral blood granulocytes [18]. Increased amounts of NBT-positive cells were observed in ATRA-induced control cells, even at 24 h after the addition of the reagent (Fig. 4A, B). Notably, at 72 h, there were significant reductions in NBT-positive cells in NB4MTOE cells compared with the control cells (Fig. 4A, B).


A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

Hirako N, Nakano H, Takahashi S - PLoS ONE (2014)

Analyses of ATRA-induced differentiation of NB4MTOE cells by NBT assay.(A) Assessment of NBT assay with or without ATRA. Examples of strongly positive (++), weakly positive (+), and negative (-) cells in the NBT reduction test. (B) Assessment of NBT assay in NB4MTOE cells and control cells. Cells were treated with 1 µM ATRA for 24 h (left), 48 h (middle), or 72 h (right panel). Black bars: NBT strongly positive cells (++); gray bars: NBT weakly positive cells (+); white bars: NBT negative (-) cells. After blind labeling of each sample, at least 200 cells were counted and the percentage of NBT-positive cells was calculated. The indicated p-value was calculated for the difference between the percentages in the (++) control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells treated with ATRA for 72 h.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone-0103282-g004: Analyses of ATRA-induced differentiation of NB4MTOE cells by NBT assay.(A) Assessment of NBT assay with or without ATRA. Examples of strongly positive (++), weakly positive (+), and negative (-) cells in the NBT reduction test. (B) Assessment of NBT assay in NB4MTOE cells and control cells. Cells were treated with 1 µM ATRA for 24 h (left), 48 h (middle), or 72 h (right panel). Black bars: NBT strongly positive cells (++); gray bars: NBT weakly positive cells (+); white bars: NBT negative (-) cells. After blind labeling of each sample, at least 200 cells were counted and the percentage of NBT-positive cells was calculated. The indicated p-value was calculated for the difference between the percentages in the (++) control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells treated with ATRA for 72 h.
Mentions: We further evaluated the role of MT1G in NB4 cell differentiation by performing NBT reduction assays. This assay is based on the ability of phagocytic cells to produce superoxide upon stimulation with phorbol 12-myristate 13-acetate (PMA), which is comparable to that generated by normal peripheral blood granulocytes [18]. Increased amounts of NBT-positive cells were observed in ATRA-induced control cells, even at 24 h after the addition of the reagent (Fig. 4A, B). Notably, at 72 h, there were significant reductions in NBT-positive cells in NB4MTOE cells compared with the control cells (Fig. 4A, B).

Bottom Line: Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells.Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA.Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Hematology, Kitasato University Graduate School of Medical Sciences, Minami-ku, Sagamihara, Japan.

ABSTRACT
We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

Show MeSH
Related in: MedlinePlus