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Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein ␤ Are

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Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein ␤ Are [CANCER RESEARCH 64, 370–377, January 1, 2004] Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein ␤ Are Required for 1,25-Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL60 Cells Yan Ji and George P. Studzinski Department of Pathology and Laboratory Medicine, The University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey ABSTRACT several levels of phosphorylation (11, 12). When hypophosphorylated, pRb can block the cell cycle traverse by binding members of the E2F Derivatives of vitamin D (deltanoids) are well known to have the ability transcription factor family through its pocket domain (13, 14), thus to induce differentiation of a variety of malignant cells, including human preventing E2F proteins from transactivating the genes that encode leukemia cells, but the signaling pathways that lead to such an outcome are unclear. In this study we investigated the role of the retinoblastoma various components of the machinery required for the cell cycle protein (pRb) and the CCAAT/enhancer-binding protein (C/EBP) ␤ in traverse and DNA replication (15–17). However, pRb can also inter- act with transcription factors that regulate differentiation, such as 1,25-dihydroxyvitamin D3 (1,25D3)-induced monocytic differentiation of human leukemia HL60 cells. It was found that in this system, pRb is MyoD (18), HBP1 (19, 20), CCAAT/enhancer-binding protein (C/ up-regulated within 12 h of exposure to the inducer, and the kinetics of its EBP) ␣ (21), and C/EBP␤ (22). Thus, pRb is implicated in myogen- increase parallel the appearance of the early markers of differentiation, esis (18, 23), adipogenesis (24, 25), osteogenesis (26), and hemato- CD14 and monocyte-specific esterase. The increase in pRb expression was poiesis (27). Notably, pRb binding to C/EBP␤ is associated with accompanied by a similar increase in C/EBP␤ protein, and these two 12-O-Tetradecanoylphorbol-13-acetate-induced macrophage differen- proteins coimmunoprecipitated, suggesting formation of a complex. Oli- tiation of myelomonocytic leukemia U937 cells (22). This is in gonucleotides antisense to pRb or C/EBP␤ (but not to C/EBP␣) or con- keeping with the observation that in normal hematopoiesis, C/EBP␤ is taining the C/EBP-binding sequence (“decoys”), all inhibited 1,25D - 3 ␣ ␦ induced differentiation. Inhibition of signaling by vitamin D receptor or expressed in the monocyte lineage, whereas C/EBP and C/EBP are by mitogen-activated protein kinase (MAPK) extracellular signal-regu- predominantly expressed in the granulocyte and eosinophil lineages, respectively (21, 28, 29). lated kinase and c-Jun-NH2-terminal kinase pathways using pharmaco- logical inhibitors ZK159222, PD98059, or SP600125, respectively, inhib- What controls pRb and C/EBP expression in hematopoietic cells is ited pRb and C/EBP␤ expression and differentiation in a coordinate not established. In the case of deltanoid-induced differentiation, it was manner. In contrast, inhibition of the p38MAPK pathway by SB202190 demonstrated that there is an increased expression of pRb before the potentiated differentiation and the up-regulation of pRb and C/EBP␤.We onset of the G1 block (30) and that it is accompanied by an increased suggest that 1,25D3 may signal monocytic differentiation of HL60 cells in activity of three mitogen-activated protein kinase (MAPK) pathways a vitamin D receptor-dependent manner that includes activation of extra- (30–33), but the relationship between these events is not clear. We cellular signal-regulated kinase and c-Jun-NH -terminal kinase MAPK 2 have addressed this issue and further investigated the kinetics of pathways, which then up-regulate pRb and C/EBP␤ expression and in turn initiate the differentiation process. retinoblastoma (RB) gene up-regulation in 1,25-dihydroxyvitamin D3 ␤ (1,25D3)-treated HL60 cells, as well as the participation of C/EBP in INTRODUCTION this form of differentiation. Data presented here show that both pRb ␤ and C/EBP are required for 1,25D3-induced differentiation of HL60 Differentiation therapy is conceptually an elegant approach to the cells, form a complex, and appear to be under the control of vitamin eradication of neoplastic cells from the human body because cytotox- D receptor (VDR) and MAPK-transduced signals. icity is avoided, whereas normal mature cells are unaffected by the differentiation agents. Clear-cut successes have already been MATERIALS AND METHODS achieved, such as the use of retinoids for the treatment of acute Chemicals and Antibodies. 1,25D was a gift from Dr. Milan Uskokovic promyelocytic leukemia (1–3). Derivatives of vitamin D, dubbed 3 (Bio Xell, Inc., Nutley, NJ). The antibodies used to detect pRb (IF-8, mouse “deltanoids” in analogy to the retinoids (4), also hold promise for use polyclonal antibody), VDR (C-20), C/EBP␣ (14AA, rabbit polyclonal anti- as components of differentiation regimens, and several are currently body), C/EBP␤ (C19, rabbit polyclonal antibody), Crk-L (C-20, rabbit poly- used in clinical trials, e.g., EB 1089 in patients with advanced breast clonal antibody), antimouse IgG-horseradish peroxidase, and antirabbit IgG- or colorectal cancer (5), and this deltanoid has already been approved horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa for the treatment of advanced hepatocarcinoma (6). However, pro- Cruz, CA). Mitogen-activated protein/ERK kinase/extracellular signal-regu- gress in generation of even more effective deltanoids should be lated kinase inhibitor PD98059 was purchased from Cell Signaling Technol- accelerated by a better understanding of the mechanisms by which ogy (Beverly, MA). The p38 kinase inhibitor SB202190 was purchased from these compounds signal differentiation. Calbiochem-Novabiochem Corp. (San Diego, CA). c-Jun-NH2-terminal kinase The retinoblastoma protein (pRb), a product of a prominent cancer inhibitor SP600125 was a generous gift from Signal Research Division, Cel- gene (7–10), has a less well appreciated or understood role in normal gene Corp. (Warren, NJ), and ZK159222 was a gift from Schering AG (Berlin, Germany). cell differentiation. It is a ubiquitously expressed protein that exists at Cell Culture. HL60-G cells (34), a subclone of human promyelocytic leukemia cells (35), were grown in suspension culture at 37°C in a closed Received 9/25/03; revised 10/24/03; accepted 10/31/03. atmosphere in RPMI 1640 (Mediatech, Washington, D.C.) with 10% heat- Grant support: New Jersey Commission for Cancer Research and Grant RO1- inactivated, defined iron-supplemented bovine calf serum (Hyclone, Logan, CA44722 (to G. P. Studzinski) from the National Cancer Institute, NIH. The costs of publication of this article were defrayed in part by the payment of page UT) and 1% L-glutamine. The cell number and viability were determined by charges. This article must therefore be hereby marked advertisement in accordance with hemocytometer counts and trypan blue (0.4%) exclusion. For all experiments, 18 U.S.C. Section 1734 solely to indicate this fact. the cells were suspended at 2.5 ϫ 105 cells/ml fresh medium containing the Requests for reprints: George P. Studzinski, Department of Pathology and Labora- desired concentrations of 1,25D . Cells in the control groups received an equal tory Medicine, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, 3 New Jersey 07013. Phone: (973) 972-5869, Fax: (973) 972-7293; E-Mail: studzins@ volume of ethanol in which 1,25D3 was dissolved. Each experiment was umdnj.edu. repeated at least three times. 370 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. RETINOBLASTOMA PROTEIN IN DIFFERENTIATION Determination of Markers of Differentiation. These methods were de- a final concentration of 5 ␮M in the culture medium for 24 h before treatment ϫ 6 scribed previously (36). Briefly, aliquots of 1 10 cells were washed twice with 1,25D3. with 1ϫ PBS and then incubated with 0.5 ␮l of MY4-RD1 and 0.5 ␮l of -FITC Decoy Oligonucleotide Inhibition Assay. A decoy oligonucleotide inhi- (Coulter, Miami, FL) at room temperature for 45 min to analyze the expression bition assay was used to study the role of various transcription factors (37). of surface cell markers CD14 and CD11b, respectively. The cells were then Decoy and mutant decoy oligonucleotides were phosphorothioated oligonu- suspended in 0.5 ml of 1ϫ PBS and analyzed using an Epics Profile II cleotides, synthesized by the Molecular Resource Facility of the New Jersey instrument (Coulter Electronics, Hialeah, FL). The thresholds for side scatter Medical School. The decoy C/EBP oligonucleotides sequences were as fol- and forward scatter were set using ␥-1 FITC/␥-2A phycoerythrin as subclass lows: 5Ј-tgcaGATTGCGCAATCtgca-3Ј and its complement; and SC C/EBP control. CD11b FITC/CD14 PE-positive sample was used to adjust color (5Ј-tgcaGAGACTAGTCTCtgca-3Ј) and its complement. HL60-G cells were compensation, and 104 cells were analyzed in each sample. preincubated with the decoy at a final concentration of 5 ␮M of the oligonu- For assessment of differentiation by monocyte-specific esterase (MSE), also cleotides in culture medium for 24 h before treatment with 1,25D3. known as nonspecific esterase, smears were made by resuspending 2 ϫ 106 Coimmunoprecipitation. Seize Primary Mammalian Immunoprecipitation cells in 100 ␮lof1ϫ PBS and spreading them on slides. The air-dried smears Kit (Pierce Biotechnology, Rockford, IL) was used. Immunoprecipitations were fixed for 30 s in formalin (25%) acetone (45%) mixture buffer and then were performed according to the manufacturer’s recommended procedures. First, 50 ␮g of anti-C/EBP␤ or anti-RB antibody were coupled to 100 ␮lof washed with distilled H2O and stained for 45 min at room temperature with the following solution: 67 mM sodium phosphate buffer (pH 7.6; 8.9 ml); hexazo- AminoLink Plus coupling gel in a spin column and incubated overnight at 4°C. ␮ tized pararosaniline (0.6 ml); 10 mg of ␣-naphthyl acetate; and 0.5 ml of Then, 200 l of whole cell protein extract were incubated at 4°C with antibody ethylene glycol monomethyl ether.
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