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.
Highlights
Metallothionein (MT) proteins comprise a group of cysteinerich low-molecular-weight intracellular proteins that are classified into four groups [1,2]
Generation of MT1G overexpressing NB4 cells To clarify whether MT1G functionally contributes to myeloid cell differentiation, an MT1G cDNA was cloned into pcDNA3.1, and the vector was electroporated into NB4 cells
We checked the expression of MT1G in NB4 MT1G-overexpressing (NB4MTOE) cells and their control cells during all-trans retinoic acid (ATRA)-induced NB4 cell differentiation
Summary
Metallothionein (MT) proteins comprise a group of cysteinerich low-molecular-weight intracellular proteins that are classified into four groups [1,2]. MT1 and MT2 are the two major isoforms found in all types of tissues [3,4]. MT3 and MT4, are expressed in limited types of tissues [5,6]. MT1G is one of the major isoforms of MT, and its mRNA is abundantly expressed in various cell types [7,8]. The MT genes located in a cluster on chromosome 16 can be activated by a variety of stimuli, and the expression and induction of their encoded proteins are associated with protection against DNA damage, oxidative stress, and apoptosis [2]. The protective role of MT against oxidative stress and metal toxicity [1,2] suggests that MT may play a role in tumor cell survival and growth. Studies on MT in hematological malignancies are relatively scarce
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