ABSTRACT
Downregulation of the myeloid master regulator Spi1/PU.1 plays a pivotal role in leukemogenesis, and we previously showed that Spi1/PU.1 directly represses metallothionein (MT)-1G through the epigenetic activity of PU.1. Furthermore, we recently demonstrated that overexpression of MT-1G inhibits retinoic acid-induced differentiation of acute promyelocytic leukemia NB4 cells. As PU.1 is a master regulator of growth and differentiation in myeloid cells, we examined its effects on cell proliferation of MT-1G-overexpressing NB4 (NB4MTOE) cells in the present study. Although there were no significant differences in total viable cell numbers between NB4MTOE cells and control cells during the time course examined, the proportion of S-phase cells was obviously increased in all NB4MTOE cells at 16-24 h after serum stimulation. Consistent with these findings, real-time PCR analyses revealed marked increases in the expression of cyclin E (G1/S-phase cyclin) and cyclin A (S-phase cyclin) in NB4MTOE cells during the same time period. Furthermore, NB4MTOE cells were significantly resistant to cytosine arabinoside (Ara-C), an S-phase-specific chemotherapeutic drug. Collectively, these findings suggest a role for MT-1G in G1/S transition during the growth phase of NB4 cells.
Subject(s)
Leukemia, Promyelocytic, Acute/metabolism , Metallothionein/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , G1 Phase Cell Cycle Checkpoints/genetics , Humans , Leukemia, Promyelocytic, Acute/genetics , Metallothionein/genetics , Metallothionein/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Tretinoin/pharmacologyABSTRACT
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.
