2A). decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression Mouse monoclonal to XRCC5 of several myeloid differentiation-related genes (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 Demethoxycurcumin may play a role in tumor cell survival and growth. A number of studies have shown that increased MT expression is closely associated with tumor grade and proliferative activity in solid tumors [1], [2]. Compared with other tumors, Demethoxycurcumin however, studies on MT in hematological malignancies are relatively scarce. PU.1 is a hematopoietic transcription factor, encoded by the gene, expressed in granulocytic, monocytic, and B-lymphoid cells [9]. alleles that reduce PU.1 Demethoxycurcumin expression to 20% of its normal levels exhibit blockade of myeloid differentiation, leading to the development of acute myeloid leukemia (AML) [11]. We recently revealed that and are direct target genes of PU.1, and that their expressions are negatively regulated by PU.1 [12]. Thus far, no studies analyzing MT functions in myeloid cells have been published. As MT1G is one of the major isoforms in the MT family [7], [8], we analyzed the function of MT1G in myelopoiesis in the present study. As a result, we found that overexpression of inhibited the ATRA-induced myeloid differentiation of NB4 cells. Materials and Methods Plasmids To generate an MT1G expression vector, pcDNA-was constructed using the following primers, and expression vector and its parental pcDNA 3.1/myc-His(-) version A vector (Invitrogen) were transfected using a CLB-Transfection device (Lonza, Basel, Switzerland). NB4 clones stably transfected with the vectors were isolated by limiting dilution and selection with 400 g/ml of neomycin in RPMI (Gibco BRL, Rockville, MD) made up of 10% heat-inactivated fetal bovine serum (HIFBS). Cells were cultured under 5% CO2 at 37C in a humidified atmosphere. Microarray and mRNA expression analyses For RNA preparation for real-time PCR analyses, MT1G-overexpressing (NB4MTOE) cells and their control cells were seeded at a density of 1105 cells/ml and treated with 1 M all-trans retinoic acid (ATRA) or an equal volume of its solvent (ethanol). The cells were harvested after 72 h, or at specified occasions. For microarray analyses, total cellular RNA was isolated from control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells using an RNA Mini Purification Kit (Qiagen, Miami, FL) according to the manufacturer’s protocol. Aliquots made up of 10 g of RNA from each sample of control cells were mixed and used as controls. Similarly, 10 g of RNA from each sample of NB4MTOE cells were mixed and used as NB4MTOE cells. The samples were subjected to microarray analyses using a CodeLink Human 54K Whole Genome Bioarray (Filgen, Nagoya, Japan). The gene expression datasets have been deposited in the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through the GEO series accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE56739″,”term_id”:”56739″GSE56739. For mRNA expression analyses, cDNAs were prepared from your cells using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Quantitative PCR was performed using the Quantitect SYBR Green PCR Reagent (Qiagen) according to the manufacturer’s protocol and an Opticon Mini Real-time PCR Instrument (Bio-Rad, Hercules, CA) as previously explained [13]. The sequences and conditions of the primers utilized for real-time quantitative PCR are outlined in Table 1. The copy number of each sample was calculated as previously explained [14]. Table 1 Sequences and conditions for the primers utilized for real-time quantitative PCR. for 10 min, the pellets were washed with buffer B (20 mM Hepes, 420 mM NaCl, 25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 phosphatase inhibitor cocktail, 1 protease inhibitor cocktail) and resuspended. The lysates were subjected to ultrasonic sonication, followed by centrifugation at 8000for 15 min and collection of the supernatants. Aliquots of the supernatants made up of 20C30 g of protein were separated in a Tris-tricine gel (Bio-Rad), transferred to Sequi-blot PVDF membranes (Bio-Rad), and immunoblotted. To detect cell cycle-related proteins, total cellular extracts were prepared and immunoblotted as explained [16]. To examine the expression of exogenous MT1G, a rabbit polyclonal metallothionein antibody (FL-61) (Santa Cruz, Santa Cruz, CA) was used. To examine the expressions of p21, cyclin D1, and cyclin A, specific rabbit polyclonal antibodies were used (Cell Signaling Technology, Beverly, MA). To verify equivalent loading of proteins.