Effects of Overexpression of HBP1 Upon Growth and Differentiation of Leukemic Myeloid Cells
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Leukemia (2005) 19, 1958–1968 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Effects of overexpression of HBP1 upon growth and differentiation of leukemic myeloid cells CJ Yao1,2, K Works1, PA Romagnoli2,3 and GE Austin1,2 1Department of Pathology and Laboratory Medicine, Veterans Affairs Medical Center, Decatur, GA, USA; 2Emory University School of Medicine, Atlanta, GA, USA; and 3Department of Pediatrics, Veterans Affairs Medical Center, Decatur, GA, USA HMG-box containing protein 1 (HBP1) is a member of the high sequences: an LXCXE site and an IXCXE site. This protein also mobility group (HMG) of chromosomal proteins. Since HBP1 contains a 28 amino-acid sequence that shows 86% homology exhibits tumor-suppressor activity in nonmyeloid tissues, we with a transcriptional activation domain previously demon- examined the effects of ectopic overexpression of HBP1 upon strated in rat HBP1.11 This activation domain is flanked by two the growth and differentiation of myeloid cells. We prepared transient and stable transfectants of the myeloblast cell line putative repressor domains (identified by comparison with the K562, which overexpress HBP1 mRNA and protein. HBP1 rodent HBP1 gene). The presence of these sequences suggests transfectants displayed slower growth in cell culture and that human HBP1 may activate and/or repress transcription of reduced colony formation in soft agar, retardation of S-phase other genes, in part through interaction with RB. progression, reduced expression of cyclin D1 and D3 mRNAs Information developed by ourselves and other investigators and increased expression of p21 mRNA. HBP1 transfectants indicates that HBP1 is an important regulator of the cell cycle, also underwent increased apoptosis, as demonstrated by cell cycle exit, apoptosis and terminal differentiation in a variety morphology and binding of Annexin V. Fas ligand mRNA levels 11–22 were increased in HBP1 transfectants, suggesting involvement of tissues and cell types. In specific nonmyeloid tissues it of the Fas/Fas ligand pathway. HBP1 overexpression enhanced partners with the tumor-suppressor RB and other pocket differentiation of K562 cells towards erythroid and megakar- proteins,11–13 inhibits the p47phox gene,22 targets downstream yocyte lineages, as evidenced by increased hemoglobin and factors such as N-Myc, Histone H1, and cyclin D1,23 and alters CD41a expression. Overexpression of HBP1 modulated mRNA chromatin structure by bending DNA and recruiting histone levels for myeloid-specific transcription factors C/EBPa, c-Myb, 17 c-Myc, and JunB, as well as lineage-specific transcription deacetylase cofactors. However, its role in myeloid cell factors PU.1, GATA-1, and RUNX1. These findings suggest that growth and differentiation remains to be elucidated. in myeloid cells HBP1 may serve as a tumor suppressor and a Mounting evidence from nonmyeloid tissues suggests that general differentiation inducer and may synergize with chemi- HBP1 may be a tumor suppressor. For example, HBP1 inhibits cal differentiating agents to enhance lineage-specific differen- G1 progression in fat and muscle cells12 and blocks the Wnt tiation. pathway,21 which is important in cancer. The HBP1 gene lies Leukemia (2005) 19, 1958–1968. doi:10.1038/sj.leu.2403918; within the 7q31 chromosomal region that is frequently deleted published online 22 September 2005 10 23 Keywords: HBP1; high mobility group proteins; transcription; or translocated in cancers. Yee et al have recently isolated myeloid differentiation; cell cycle variants of HBP1 in breast cancers and myeloid leukemias, suggesting a possible role of HBP1 in human neoplasia. Introduction However, the role of HBP1 in pathogenesis of myelodysplasias and myeloid leukemias remains to be elucidated. HMG-box containing protein 1 (HBP1) was first identified in rat To explore the role of HBP1 in regulating the growth of myeloid brain, on the basis of its ability to suppress the potassium cells and to study its possible tumor-suppressor activity we transport-defective phenotype of mutant Saccharomyces cere- overexpressed HBP1 in the myeloblast cell line K562 and studied visiae.1 HBP1 was recognized to be a member of the high its effect upon cell growth and differentiation. Our results show mobility group (HMG) protein family, since it contains a 70 that overexpression of HBP1 in myeloblasts leads to growth arrest amino-acid DNA-binding domain known as an HMG-box, and apoptosis and acts as a general inducer of differentiation. characteristic of these proteins.2 HMG proteins are transcription factors that are widely distributed among eukaryotic cells and Materials and methods have diverse functions.2–9 In the course of a yeast one-hybrid search for novel factors that bind to the MPO promoter and Materials modulate MPO promoter activity, we identified HBP1, as an 10 MPO promoter-binding protein. We demonstrated by trans- The human cell line K562 (erythroleukemic) was obtained from fection experiments that HBP1 enhances MPO promoter the American Type Culture Collection (Rockville, MD, USA). activity. We isolated and cloned the cDNA for the human Polyclonal rabbit antibody against HBP1 was prepared as HBP1 gene (GenBank Accession Number AF019214, October previously described.10 The pBudCE4.1 vector, Zeocin antibiotic, 2, 1997). The corresponding human HBP1 mRNA sequence and DMRIE-C transfection reagent were obtained from Invitrogen contains a single open reading frame, which codes for a protein (Carlsbad, CA, USA). Anti-CD41a (FITC-conjugated) and other composed of 514 amino acids. In addition to the HMG box-like antibodies used in flow cytometric analyses were obtained from sequence, which is the DNA-binding region, the amino-acid BD Biosciences (San Jose, CA, USA). SYBRs Green PCR Master sequence of HBP1 includes two pocket protein-binding Mix was from Applied Biosystems (Foster City, CA, USA). Correspondence: Dr GE Austin, Atlanta VA Medical Center (113), 1670 Clairmont Road, NE, Decatur, GA 30033, USA; Plasmid construction Fax: þ 1 404 235 3007; E-mail: [email protected] Received 7 January 2005; accepted 30 June 2005; published online Wild-type HBP1 cDNA was subcloned into pBudCE4.1 22 September 2005 vector, which has dual promoters, pCMV and pEF1a, and a HBP1 and myeloid cell growth and differentiation CJ Yao et al 1959 Zeocin-resistance gene for Zeocin selection of stable cells. (30 mg total protein) were placed in SDS buffer and applied to Two types of constructs containing HBP1 were established 10% SDS-PAGE gels and subsequently transferred to nitrocellu- as follows: HBP1/CMV (with HBP1 driven by a CMV lose membranes. Membranes were incubated with the desired promoter) was prepared by ligating HBP1 to pBudCE4.1 at the primary antibodies, followed by suitable secondary antibodies. multiple cloning site of the CMV promoter at SalI and BamHI Antigen detection was performed with an ECL plus kit restriction sites. HBP1/EF1a (with HBP1 driven by an EF1a (Amersham Biosciences Corp., Piscataway, NJ, USA) promoter) was prepared by ligating HBP1 to modified pBudCE4.1 (cutting off the whole segment containing the CMV promoter) at the multiple cloning site of the EF1a promoter Cell proliferation assay at NotI and XhoI restriction sites. All of the constructs were verified by sequencing. The control vector employed was Cell proliferation was measured by a BrdU incorporation assay pBudCE4.1/LacZ/CAT (hereafter referred to as ‘CAT vector’). kit from BD Pharmingen (San Diego, CA, USA). Briefly, the cells The linearization sites of these constructs were: NheI for HBP1/ were pulse labeled with BrdU for 30 min and the levels of BrdU CMV, FspI for HBP1/EF1a, and BspHI for CAT vector. All incorporated into cellular DNA were quantified by anti-BrdU constructs were expanded in Escherichia coli DH5a and antibody; total DNA was stained by 7-AAD. The samples were plasmids were purified using a Maxi-prep kit (Qiagen, Valencia, analyzed by BD FACScan and data were evaluated using CA, USA). WinMDI 2.8 research software. This two-color flow cytometric analysis permits the enumeration and characterization of cells that are actively synthesizing DNA (BrdU incorporation) in Cell culture, establishment of stable cell lines and terms of their cell cycle position (ie, G0/1, S, or G2/M phases transfection (defined by 7-AAD staining intensities). For these and other assays, the statistical significance of the differences between K562 cells were maintained in RPMI 1640 (GibcoBRL, K562 cells and stable transfectants was assessed using a two- Gaithersburg, MD, USA) supplemented with 1% L-glutamine, tailed t-test at Po0.05 or Po0.005. 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated fetal calf serum, and grown in humidified 5% CO2 at 371C. Exponentially growing cells were used for all Apoptosis assay experiments. For determination of growth rate, cells were diluted at an initial concentration of 0.2 Â 106 cells/ml in Apoptosis assays were performed using an Annexin V-FITC complete medium and aliquots were removed daily for detection kit I (BD Biosciences Pharmingen). The cells were determination of cell concentration. In experiments addres- analyzed using a FACScan flow cytometer and data were sing induction of differentiation, cells at an initial concentra- evaluated using WinMDI 2.8 research software. The results were tion of 0.2 Â 106 cells/ml were incubated in complete medium expressed as percentage of apoptotic Annexin V-FITC positive with or without 30 mM hemin or 10 ng/ml TPA (Sigma Chemical cells with respect to total cells counted. Co, St Louis, MO, USA). For growth in soft agar, cells were cultured in RPMI-1640 medium containing 0.3% Bectar agar, and 20% fetal calf serum. After 10 days colonies containing May–Grunwald–Giemsa staining, and benzidine more than 50 cells were enumerated using an inverted staining microscope. To establish stable HBP1-overexpressing cell lines, 1.6 Â 107 Approximately 5 Â 104 cells were spun onto a microscope slide K562 cells were transfected by the linearized constructs (32 mg in a Cytospin 3 cytocentrifuge (Shandon, Thermo Electron each) with 48 ml DMRIE-C reagent (Invitrogen).