Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein ␤ Are

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[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 interact 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 pharmacological 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

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. The MSE-positive cells were determined coupling gel overnight. Next, immunoprecipitated proteins were eluted from ␤ by counting 500 cells in each group under a light microscope. the column and run on SDS-PAGE gel (7% for pRb and 10% for C/EBP ), ␤ Western Blotting. Western blotting was performed using nuclear extracts. transferred to a membrane, and probed with anti-pRb or anti-C/EBP . The Twenty ␮g of protein samples were separated on SDS-PAGE (7% for pRb and protein bands were visualized with a chemiluminescence assay system (Am- 10% for all of the other proteins) gels and transferred to nitrocellulose mem- ersham Pharmacia Biotech. Inc). branes (Amersham Pharmacia, Little Chalfont, United Kingdom). The mem- Statistical Methods. All experiments were repeated a minimum of three times. Significance of differences between mean values was assessed by branes were blocked with 5% milk in Tris-buffered saline/0.1% Tween 20 for ANOVA analysis followed by the Bonferroni post-test. All computations 1 h, subsequently blotted with primary antibodies, and then blotted with were performed using an IBM personal computer using Microsoft EXCEL ϩ horseradish peroxidase-conjugated secondary antibody for 1 h. The protein ANALYSE-IT Program. bands were visualized with a chemiluminescence assay system (Amersham). The membranes were stripped according to the manufacturer’s protocol (Am- ersham) and reprobed with Crk-L. The absorbance of each band was quanti- RESULTS tated using an Image QuaNT program (Molecular Dynamics, Sunnyvale, CA). Reverse Transcription-PCR. Total RNA was extracted using Trizol rea- The Kinetics of Up-Regulation of RB Gene Expression Parallel gent. GeneAmp RNA PCR Core Kits (Perkin-Elmer, Boston, MA) were used. the Expression of Early Markers of Monocytic Differentiation. It Ј The primers were as follows: (a) RB upstream primer (5 -TACCTAGCT- was observed previously that levels of the pRb are increased in HL60 CAAGGGTTAAT-3Ј) and RB downstream primer (5Ј-TAGCCATATGCA- cells treated with concentrations of 1,25D that are insufficient to CATGAATG-3Ј); (b) C/EPB␤ upstream primer (5Ј-GTTCTTGACGTTCT- 3 cause cell cycle arrest but that induce markers of differentiation (30). TCGGCCG-3Ј) and C/EPB␤ downstream primer (5Ј-TGGACAAGCACAGC- GACGAGT-3Ј), and (c) ␤-actin upstream primer (5Ј-TGACGGGGTCAC- This showed that in addition to its well known role in controlling the CCACACTGTGCCCAGCTA-3Ј) and ␤-actin downstream primer (5Ј-CTA- cell cycle transitions (15, 38), pRb is likely to function in cellular GAAGCATTTGCCGGTGGACGATGGAGGG-3Ј). Reverse transcription- events associated with monocytic differentiation, which precede the PCR was performed according to the manufacturer’s recommended procedure. cell cycle blocks induced by 1,25D3. However, the relationship of pRb For reverse transcription, samples were incubated in a Perkin-Elmer GeneAmp up-regulation to the early differentiation events of 1,25D3-treated cells PCR System 9600 at 42°C for 15 min and then incubated at 99°C for 5 min and was not evident from those studies. Therefore, to determine whether 5°C for 5 min. For PCR, samples were incubated in GeneAmpPCR System the up-regulation of pRb expression can be considered as one of the 9600 as follows: 95°C for 105 s; 35 cycles of 95°C for 15 s and 60°C for 30 s; earliest detectable events during 1,25D3-induced differentiation, we and 72°C for 7 min. The reverse transcription-PCR products were separated in compared the kinetics of this up-regulation and of the appearance of 1.2% agarose gels and stained with ethidium bromide. The intensities of the two early markers of monocytic differentiation, CD14 and MSE, also bands were measured using Image QuaNT program (Molecular Dynamics). known as the nonspecific esterase. Interestingly, the increase in pRb For real-time reverse transcription-PCR, LighterCycler-FastStart DNA Master mRNA closely paralleled the appearance of CD14 and MSE in HL60 SYBR Green I kit (Roche, Indianapolis, IN) was used. Reverse transcription was performed as referred to above, and PCR was performed according to the cells induced to differentiate with either 1 or 10 nM 1,25D3 (Fig. 1, A manufacturer’s recommended procedure. Briefly, 2 ␮l of cDNA samples from and B). Furthermore, there was also a good correlation between reverse transcription reaction were mixed with 2 ␮l of SYBR Green I, 2.4 ␮l increased levels of pRb and these early differentiation markers (Fig. ␮ ␮ of MgCl2 (25 mM), 1 l of upstream and downstream primers (7.5 M) each, 1C), whereas the myeloid marker CD11b, which appears later in ␮ and 11.6 l of PCR-grade distilled H2O in a capillary and placed in the monocytic differentiation, and the RB-related pocket protein p130, LightCycler. The PCR reaction temperature was set as described above. Data which is associated with replicative quiescence (39–41), showed were analyzed using Relative Quantification software from Roche. increases with kinetics that were different from those observed for Antisense (AS) Oligonucleotides. AS oligonucleotides were used to in- CD14, MSE, and pRb (compare Fig. 1, C and D). Thus, the up- hibit the expression of products of the genes of interest. Phosphorothioate regulated expression of pRb is an early event in 1,25D3-induced oligodeoxynucleotides were synthesized by the Molecular Resource Facility of monocytic differentiation of HL60 cells, whereas the p130 pocket the New Jersey Medical School. The sequences of the phosphorothioate- protein and CD11b marker signify a more mature monocytic AS-RB oligonucleotide and C/EBP␣ and C/EBP␤ AS oligodeoxynucleotides ␣ phenotype. that target the translation initiation sites of RB gene, C/EBP gene, and ␤ C/EBP␤ gene, respectively, were as follows: AS-RB (5Ј-GGGGGTTTT- The C/EBP Is Up-Regulated in HL60 Cells by 1,25D3 in GGGcGGCATGAC-3Ј) and scrambled (SC)-RB (5Ј-GTGCGAGTGGCGT- Concert with pRb and Forms a Complex with It. Rb protein is GAGTGCGT-3Ј); AS-C/EBP␣ (5Ј-GAAGTCGGCCGACTCCAT-3Ј) and known to function by binding to and modifying the activity of tran- SC-C/EBP␣ (5Ј-ATGCATGCATGCAGCGCC-3Ј); and AS-C/EBP␤ (5Ј- scription factors, including members of the E2F and the C/EBP CACCAGGCGTTGCATGAA-3Ј) and SC-C/EBP␤ (5Ј-ACTGACTGACTGA- families (18, 22, 25). E2F transcription factors regulate many genes Ј CGACG-3 ). HL60-G cells were preincubated with the AS oligonucleotides at whose products are required for the G1 traverse and DNA replication 371

Fig. 1. Kinetics of the up-regulation of retinoblastoma protein parallel the appearance of the early markers of differentiation. HL60-G cells were treated with 1 (A)or10nM (B) 1,25-dihydroxyvitamin D3. The differentiation marker CD14 was determined at the indicated intervals by flow cytometry, monocyte-specific esterase was determined by cytochemistry, and retinoblastoma mRNA was determined by quantitative real-time reverse transcription-PCR. The protein levels of retinoblastoma protein (C) and p130 (D) were determined by Western blots of extracts of cells treated with 5 nM 1,25-dihydroxyvitamin D3. Note the different time scale in A/B and C/D. Error bars represent the SDs in this and all subsequent figures. Fold increase refers to the levels of retinoblastoma or p130 mRNA.

␤ (15, 17), and their binding to pRb negatively influences their activity. C/EBP Transcription Factor Positively Regulates 1,25D3- In contrast, binding of pRb to members of the C/EBP transcription Induced Differentiation of HL60 Cells. C/EBP transcription factors factor family has been suggested to have a positive effect on their control gene expression by binding to the cognate C/EBP DNA functions, which include activation of genes required for differentia- sequence in the promoter regions of the genes that they regulate (43, tion of several cell types (24, 28, 29, 42). We therefore investigated in 44). We therefore initiated the study of the role of the C/EBP family the 1,25D3-HL60 cell differentiation model to determine whether of proteins in 1,25D3-induced differentiation by incubating the cells there is an interaction between pRb and C/EBP␤, previously implicated in monocytic differentiation (22). Immunoprecipitates of C/EBP␤ showed the presence of pRb, and in a reciprocal experiment, C/EBP␤ was detected (Fig. 2), suggesting formation of a complex, as detected previously in another cell line (22). Additionally, we found ␤ that 1,25D3 increased the expression of C/EBP in a dose-dependent manner, which paralleled the increased expression of pRb in these cells (Fig. 2). Quantitation of the signals on Western blots showed a similar magnitude of the increase, with the maximum being 5-fold higher pRb and 4-fold higher C/EBP␤ protein levels in cells treated Ն with 10 nM 1,25D3 (Fig. 2C). Thus, pRb and RB mRNA expression increased in a time- and dose-dependent manner after exposure to ␤ 1,25D3 and correlated with an increased expression of C/EBP and early markers of differentiation

Up-Regulation of RB Gene Expression Is Necessary for 1,25D3- Induced Differentiation of HL60 Cells. We initially tested the hy- pothesis that pRb enhances the ability of C/EBP␤ to activate the transcription of differentiation-related genes by reducing the expression of pRb by an AS oligonucleotide (AS-RB), followed by a determination of the ability of 1,25D3 to induce differentiation. As illustrated in Fig. 3A and quantitated in Fig. 3B, the AS-RB oligonucleotide effectively blocked the expression of early markers of differentiation in cells exposed to a low concentration (1 nM) of 1,25D3 for 24 h, but an exposure to an oligonucleotide with a SC sequence (SC-RB) did not. Although quantitation of the expression of CD11b was also attempted (Fig. 3A), no significant expression of this late myeloid marker was noted at this time (24 h) of 1,25D3 exposure. A Western analysis of proteins extracted from these cells showed an almost complete inhibition by AS-RB, but not by SC-RB, of the

1,25D3-induced up-regulation of pRb (Fig. 3, C and D). This analysis also demonstrated that the expression of C/EBP␤ and VDR is not affected by AS-RB, indicating that the presence of up-regulated C/EBP␤, or VDR, is not sufficient for the induction of differentiation Fig. 2. Dose-dependent up-regulation of retinoblastoma protein and CCAAT/enhancer- (Fig. 3, C and D). Also, it is evident that the expression of C/EBP␤ binding protein ␤ and formation of a complex. A, Western blots. Crk-L was determined as a loading and transfer control, and the signal was used for subsequent quantitation. B, and VDR is not regulated by pRb because they are unaffected by the Western blots after reciprocal immunoprecipitation. C, quantitation of three similar .P Ͻ 0.05 relative to the control group ,ء .depletion of pRb by the AS-RB. experiments illustrated in A 372

Fig. 3. Antisense retinoblastoma (RB) blocks differentiation induced by 1,25-dihydroxyvitamin D3 (1,25D3) in HL60 cells. A, flow cytometry demonstrating the marked inhibition P Ͻ 0.05 relative ,ء .(of differentiation by antisense-RB, but not by SC-RB. B, quantitation of flow cytometry and cytochemical determination of monocyte-specific esterase (n ϭ 3 ϩ to the 1,25D3 treatment group and to the 1,25D3 SC-RB treatment group. C, Western blots showing that antisense RB blocks up-regulation of retinoblastoma protein but does not Ͻ ء ␤ block up-regulation of CCAAT/enhancer-binding protein or vitamin D receptor by 1,25D3. Lane numbers are as defined in B. D, quantitation of three similar experiments. , P 0.05 ϩ relative to both the 1,25D3 and 1,25D3 SC-RB treatment groups.

with a great excess of oligonucleotides containing either this DNA with a minimal agonist activity of 1,25D3 but the ability to block element (decoy C/EBP) or its SC sequence version (SC-decoy VDR-transmitted signals (48). As shown in Fig. 6A, ZK159222 had C/EBP). Although the SC decoy C/EBP had a modest negative effect no significant (P Ͻ 0.05) differentiation-inducing activity of its own, on 1,25D3-induced differentiation, the effect of decoy C/EBP was but when combined with 1,25D3, it markedly inhibited differentiation. significantly (P Ͻ 0.05) greater (Fig. 4, A and B). Western blot Western analysis showed that the expression of both pRb and C/EBP␤ analysis of the effects of the decoy on the levels of pRb and VDR was markedly decreased by the VDR antagonist (Fig. 6B). Thus, VDR showed that neither of these proteins was affected (Fig. 4, C and D). action is required for the 1,25D -induced, differentiation-associated, ␤ 3 Thus, inhibition of binding of C/EBP to its cognate DNA site does up-regulation of pRb and C/EBP␤. not affect the expression of pRb or VDR. Three MAPK Pathways Are Upstream of the Expression of Previous reports have indicated that hematopoietic cells express pRb and C/EBP␤. Monocytic differentiation of HL60 cells is also ␣ several members of the C/EBP family, including C/EBP and influenced by the activity of at least three MAPK pathways (30, 31, C/EBP␤ (21, 22). C/EBP␣ is implicated in granulopoiesis (45), 33, 49, 50). We therefore determined the effect of inhibition of whereas C/EBP␤ is implicated in macrophage function (46, 47). each of these pathways on the expression of the two proteins that Experiments in which AS-C/EBP␣ and AS-C/EBP␤ were used are the focus of these studies, pRb and C/EBP␤. The results showed that only AS-C/EBP␤ reduced 1,25D -induced differenti- 3 showed that the inhibition of the extracellular signal-regulated ation, although not to the extent that resulted from the exposure to kinase/MAPK and c-Jun-NH -terminal kinase/MAPK pathways by the decoy C/EBP (Fig. 5). This difference may be due a greater 2 efficiency of the decoy as compared with the AS strategy, but specific pharmacological inhibitors (51, 52), PD98059 and because we did not test all members of the large C/EBP family SP600125, respectively, also reduced the extent of differentiation ␤ (29), it is possible that another C/EBP protein contributes to and the expression of pRb and C/EBP (Fig. 7). In contrast, the monocytic differentiation. However, our findings show that inhibition of the p38/MAPK pathway by its specific inhibitor, C/EBP␤, not C/EBP␣, is the principal member of the C/EBP SB202190 (53), increased both the 1,25D3-induced differentiation and the expression of pRb and C/EBP␤ (Fig. 7). These data transcription factor family that contributes to the 1,25D3-induced differentiation of HL60 cells. demonstrate that the expression of pRb and C/EBP␤ is under Expression of pRb and C/EBP␤ Is Regulated by VDR. Having transcriptional, although probably indirect, control by MAPK path- established that the expression of VDR is not under the control of RB ways, raising the possibility that VDR transmits the signal from or C/EBP␤ genes, we enquired whether the reverse is true. For this MAPK pathways to the pRb⅐C/EBP␤ complex, as schematized in purpose, we used a carboxylic ester antagonist of 1,25D3 (ZK159222) Fig. 8. 373

Fig. 4. A, decoy CCAAT/enhancer-binding protein (C/EBP) binding sequence oligonucleotide blocks 1,25-dihydroxyvitamin D3 (1,25D3)-induced differentiation in HL60 cells as Ͻ ء ϭ shown by flow cytometry. B, quantitation of data shown in A and of cytochemical determination of monocyte-specific esterase activity (n 3). , P 0.05 relative to both the 1,25D3 ϩ treatment group and the 1,25D3 SC decoy C/EBP treatment group. Lane numbers are as defined in B. C, Western blots showing that decoy C/EBP oligonucleotide does not block the up-regulation of retinoblastoma protein or vitamin D receptor induced by 1,25D3. D, quantitation of three blots similar to those shown in C. No significant changes were detected.

DISCUSSION The importance of pRb as a regulator of cell cycle progression achieved, at least in part, by its binding to E2F transcription factors is so well appreciated that it tends to overshadow the fact that pRb has many other potential protein partners (18, 22). These proteins include members of the C/EBP transcription factor family, and in this study ⅐ ␤ we focused on the participation the pRb C/EBP complex in 1,25D3- induced monocytic differentiation of human leukemia HL60 cells. It is evident that in this system the up-regulation of pRb and C/EBP␤ expression occurs early in the differentiation process and is required for differentiation and that, although independent of each other, pRb and C/EBP␤ are both under the control of VDR and MAPK pathways. These findings allow the formulation of a hypothetical “road map” that can explain, in part, the routing of signals through which 1,25D3 and other deltanoids induce monocytic differentiation. Phenotypic differentiation of deltanoid-induced HL60 cells is

Fig. 6. The 1,25-dihydroxyvitamin D3 (1,25D3) antagonist ZK159222 inhibits 1,25D3- induced differentiation as well as expression of retinoblastoma protein and CCAAT/ enhancer-binding protein ␤, but not CCAAT/enhancer-binding protein ␣. HL60 cells were treated for 24 h with 1,25D3 (10 nM), alone or in combination with ZK159222 (100 nM). Ͻ ء , P 0.05 compared with treatment with 1,25D3.

known to precede alterations in the cell cycle traverse (33, 54–57). In these cells an increase in early markers of the changing phenotype, Fig. 5. Antisense CCAAT/enhancer-binding protein (C/EBP) ␤ oligonucleotide, but such as CD14, the lipopolysaccharide surface receptor characteristic ␣ not antisense C/EBP oligonucleotide, blocks 1,25-dihydroxyvitamin D3 (1,25D3)- Ͻ of the macrophage (58, 59), or MSE, a cytoplasmic hydrolytic enzyme ء ϭ induced differentiation of HL60 cells (n 3). , P 0.05 relative to the 1,25D3 treatment ϩ ␤ group and the 1,25D3 SC-C/EBP treatment group. present in phagocytic cells (58, 60, 61), can be detected in a propor- 374

Fig. 7. The mitogen-activated protein/ERK kinase 1/2 inhibitor PD98059 and the c-Jun-NH2- terminal kinase inhibitor SP600125 reduce 1,25- dihydroxyvitamin D3 (1,25D3)-induced differentiation (A), as well as retinoblastoma protein and CCAAT/enhancer-binding protein ␤ and mRNA expression (B and D), whereas the p38MAP inhibitor SB202190 has an opposite effect. C and E show quantitation of these experiments. HL60 cells were treated for 48 h with 5 nM 1,25D3, with or without the inhibitors, all at 10 ␮M concentration. P Ͻ 0.05 compared with treatment with ,ء ;n ϭ 3 the same concentration of 1,25D3.

tion of cells as early as 8 h after the addition of a deltanoid (Fig. 1). In a previous study we showed that the ratio of highly phosphoryl- In contrast, changes in the cell cycle traverse are detectable approx- ated to hypophosphorylated pRb varied with the strength of the imately 30–40 h later (62). Because the up-regulation of pRb closely differentiation signal: pRb was highly phosphorylated when the dif- approximates the appearance of the early markers of differentiation, ferentiation signal was weak, and no cell cycle effects could be noted this suggests a role in the differentiation process, independent of its (30). Nonetheless, the weak signal elicited an increase in total pRb cell cycle controls, and this was confirmed by the inhibition of and expression of early differentiation markers, suggesting that the differentiation when pRb expression is down-regulated after the ad- phosphorylation status of pRb is more critical for cell cycle control dition of an AS oligonucleotide to pRb (Fig. 3). than for differentiation. Whether phosphorylation-induced changes in

Fig. 8. A partly hypothetical schematic of the pathways suggested to induce differentiation of HL60 cells by deltanoids. The initial signal pro- vided by 1,25-dihydroxyvitamin D3 is represented as activation (shown by the asterisk) and stabiliza- tion of the vitamin D receptor protein, which then heterodimerizes with an isoform of retinoid X receptor and transactivates genes containing its cognate DNA response element, e.g., p21Cip1. One result is the sensitization of the mitogen-activated protein kinase pathways to the ambient stimuli, which include growth- and stress-inducing factors (66). Subsequently, activation of the activator protein-1 transcription factor may increase transcription of vitamin D receptor (67) and aid the transcription of retinoblastoma and CCAAT/enhancer-binding protein ␤ genes. This multistep process results in an up-regulation of genes respon- sible for the monocytic phenotype.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein β Are Required for 1,25-Dihydroxyvitamin D3 -Induced Monocytic Differentiation of HL60 Cells

Yan Ji and George P. Studzinski

Cancer Res 2004;64:370-377.

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