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[CANCER RESEARCH 59, 3803–3811, August 1, 1999]

Induction of a Functional Vitamin D Receptor in all-trans-Retinoic Acid-inducedMonocytic Differentiation of M2-type Leukemic Blast Cells1

Rossella Manfredini, Francesca Trevisan, Alexis Grande, Enrico Tagliafico, Monica Montanari, Roberto Lemoli,Giuseppe Visani, Sante Tura, Stefano Ferrari, and Sergio Ferrari2

Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, Universita di Modena e di Reggio Emilia, 41100 Modena [R. M., F. T., A. G., E. T., M. M., St. F., Se. F.], andIstituto di Ematologia L&A Seragnoli, 40138 Bologna [R. L., G. V., S. T.], Italy

ABSTRACT

Different types of acute myeloid leukemia blast cells were induced todifferentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D 3

(VD). M0/M1 leukemic cells are not sensitive to differentiating agents,whereas M3 leukemic cells are induced to undergo granulocytic differen-tiation after ATRA treatment but are not sensitive to VD. M2 leukemicblast cells behave differently because they undergo monocytic differenti-ation with both the differentiation inducers. To gain some insight into thematuration of M2-type leukemic cells, we studied the molecular mecha-nisms underlying monocytic differentiation induced by ATRA and VD inspontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloidleukemia M2-type cell line). Our results indicate that ATRA as well as VDefficiently increases the nuclear abundance of VD receptor (VDR) andpromotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoidX receptor, binds to a DR3-type vitamin D-responsive element, and acti-vates the transcription of a vitamin D-responsive element-regulated re-porter gene. Consistent with these findings, VD-responsive genes areinduced by ATRA treatment of Kasumi-1 cells, suggesting that the geneticprogram underlying monocytic differentiation is activated. The molecularmechanism by which ATRA increases the nuclear abundance of a func-tional VDR is still unknown, but our data clearly indicate that the M2leukemic cell context is only permissive of monocytic differentiation.

INTRODUCTION

Myelopoiesis of acute leukemias is characterized by an alteredbalance between quiescent, cycling, and differentiating cells. AML3

blast cells are, in fact, mainly arrested in the G1 phase of the cell cycleand are unable to progress spontaneously toward terminal differenti-ation (1, 2). The growth advantage of leukemic blast cells is thereforeachieved mainly through a prolonged survival time, probably causedby a maturation arrest and an inefficient activation of the apoptoticprogram (3, 4). A unique and specific genetic disorder has beendiscovered in APL in which the t(15;17) translocation involves thegenetic loci of RARa and the promyelocytic leukemia gene (5–8).Patients affected by this type of leukemia can undergo completeremission when treated with ATRA, which induces terminal granu-locytic differentiationin vitro andin vivo (9, 10). It is thus evident that

the availability of compounds capable of inducing APL blast cells todifferentiate and therefore undergo apoptosis provides an opportunityto modify the course of the malignancy.

In a high percentage of the other AML types, genetic abnormalitiesare extremely heterogeneous and are not specific (11). For example,only 20% of M2-AML is characterized by t (8;21) (12), and a verylow percentage of cases carry t(6;9) (13). Moreover, very few cases ofAML are characterized by t(3;21) (14). In principle, genetic abnor-malities might contribute to the maturation arrest, but no clear corre-lation can be made between the level of the differentiation block andthe genetic aberrations, with the exception of the M3-AML (15).

Furthermore, despite the fact that the vast majority of acute leuke-mia blast cells express several growth factor receptors (16, 17), theyare poorly sensitive to the differentiation activity of the correspondingcytokinesin vitro and in vivo; consequently, the therapeutic use ofhematopoietic growth factors in AML is controversial (18). Interest-ingly, cytokine antagonists can block leukemic myeloid cell prolifer-ation due to an autocrine mechanism (19).

Despite the poor evidence that exists on the physiological role ofATRA and VD in normal hematopoiesis, these inducers could besuitable to force the differentiation block in AML blast cells (20, 21).

It is well known that VD and ATRA exert their action by bindingto specific nuclear receptors with high affinity. These nuclear recep-tors regulate gene expression in target cells by binding to specificDNA-responsive elements (VDRE and RARE) via heterodimerizationwith RXR (22). It has to be pointed out that our previous studiessuggest that myeloid precursors at all stages of differentiation areequipped with the machinery required to trigger the VD-dependentgenetic program, eventually leading to terminal differentiation. Inother words, they contain VDR protein that is fully active in bindingtheir DNA sites and in promoting transcription (23). The response toVD is therefore likely to depend on steps located downstream of thenuclear receptor/DNA interaction, such as chromatin structure, nu-cleosome organization, and gene-nuclear matrix interaction.

To gain some insight into the differentiation potential of differenttypes of AML blast cells (M0/M1, M2, and M3), we have investigatedthe expression levels and function of RARa, VDR, and RXR nuclearreceptors (22, 24) and the ability of ATRA or VD to induce terminalgranulocytic or monocytic differentiationin vitro (25–27). Our datasuggest that a monocytic differentiation window exists in M2-typeblast cells because ATRA and VD can only trigger monocytic differ-entiation in M2-type Kasumi-1 cells (28) as well as in spontaneousleukemic blast cells.

MATERIALS AND METHODS

Cell Cultures and Differentiation. Blast cell populations were obtainedby leukapheresis from five patients with M0/M1-AML, five patients with M2-AML, and three patients with M3-AML before any pharmacological treatment.All of the blast cells were purified by Ficoll-Hypaque density gradient cen-trifugation to obtain extremely homogeneous populations (.95% blast cells).The phenotype in each case was defined by morphological, cytochemical (29,30), immunological (31), cytogenetic, and molecular criteria, at least for themolecular analysis of t(8;21) (32) and t(15;17) translocations (33). The blast

Received 12/1/98; accepted 5/21/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the Associazione Italiana per la Ricerca sul Cancro and bya grant from the Consiglio Nazionale delle Ricerche (Progetto Finalizzato: ApplicazioniCliniche della Ricerca Oncologica).

2 To whom requests for reprints should be addressed, at Dipartimento di ScienzeBiomediche, Sezione di Chimica Biologica, Via Campi 287, Universita di Modena eReggio Emilia, 41100 Modena, Italy. Phone: 39059-428514; Fax: 39059-428524; E-mail:[email protected].

3 The abbreviations used are: AML, acute myeloid leukemia; RA, retinoic acid;ATRA, all-trans-RA; VD, vitamin D3; VDR, vitamin D receptor; VDRE, vitamin Dresponsive element; APL, acute promyelocytic leukemia; RARa, RA receptora; RARE,RA responsive element; RXR, retinoid X receptor;b2m, b2-microglobulin; NE, nuclearextract; CE, cytoplasmic extract; CAT, chloramphenicol acetyltransferase; TK, thymidinekinase; DP, direct primer; RP, reverse primer; RT-PCR, reverse transcription-PCR;HMSE-1, human monocytic serine esterase 1; hOC, human osteocalcin; IRF-1, interferonregulatory factor-1; mAb, monoclonal antibody; EMSA, electrophoretic mobility shiftassay.

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cell populations were cultured in Iscove’s medium supplemented with 2 mM

L-glutamine and 20% heat-inactivated FCS. Kasumi-1 (M2 AML) and HL-60(M2/M3 AML) cells were cultured in RPMI 1640 supplemented with 15%heat-inactivated FCS and 2 mM L-glutamine. Differentiation was induced inblast cells as well as in Kasumi-1 and HL-60 (34, 35) cells by treatment witheither 1026 M ATRA (Sigma Chemical Co., St. Luis, MO) or 1027 M VD (F.Hoffmann-La Roche, Basel, Switzerland) as described previously (36). Thedifferentiation was monitored by direct immunofluorescence analysis of sur-face markers (37), such as CD14 (38), CD11b (39), and CD45 (40). Eachantibody was incubated directly into the cellular suspension (5–10mg/106

cells) for 20 min at 4°C. The cells were washed twice with PBS and thenanalyzed by cytofluorometric analysis. Morphology was assessed by cytocen-trifugation followed by May Grunwald Giemsa staining.

RNA Analysis. Total cellular RNA was extracted using a modification ofthe guanidinium-cesium chloride centrifugation technique (41) and digestedwith RQ1-DNase (Promega, Madison, WI) to avoid DNA contamination asdescribed previously (42). RT-PCR reactions were carried out using a modi-fication of a previously described technique (42). RT-PCR analysis wasperformed using oligodeoxynucleotide primers specific for RARa (43), RXR(44), VDR (45, 46), CD14 (47), HMSE-1 (48), hOC (49), andb2m, (50). Theoligonucleotides used as DPs or RPs were as follows: (a) RARa-DP (59-CCAGACTGTCTGCCTCCCTT-39) and RARa-RP (59-GTTTCGGTCGTT-TCTCACAGAC-39); (b) RXRa-DP (59-CATTTCCTGCCGCTCGATTTC-39)and RXRa-RP (59-CTGCTGCTGACGGGGTTCAT-39); (c) VDR-DP (59-GGAGACTTTGACCGGAACGTG-39) and VDR-RP (59-GAACTGGCA-GAAGTCGGAGTA-39); (d) VDR primary transcript-DP (59-CAGTGACGT-GACCAAAGGTATGCCTAG-39) and VDR primary transcript-RP (59-GGGAGACGATGCAGATGGCCATGAGCA-39); (e) CD14-DP (59-TCC-AGAGCCTGTCCGGAGCTCAGA-39) and CD14-RP (59-GCGTTCGC-CCAGTCCAGGATTGTCA-39); (f) HMSE-1-DP (59-CCTCCTATGTG-CACCCAAGAT-39) and HMSE-1-RP (59-GCATCCC-ATCAATCACAG-TGC-39); (g) hOC-DP (59-GAGCCCTCACACTCCTCGCCCTATT-39) andhOC-RP (59-GTAGAAGCGCCGATAGGCCTCCTGA-39); and (h) b2m-DP(59-CTCGCGCTACTCTCTCTTTCT-39) andb2m-RP (59-TCCATTCT-TCAGTAAGTCAACT-39. The primers used for the detection of t(8;21) andt(15;17) were as described previously (32, 33). Northern blot analysis tomonitor p21 (51), E3 (52), and IRF-1 (53) expression was performed asdescribed previously (54).

Preparation of NEs and CEs. NEs and CEs were obtained from thedifferent myeloid cells as described previously (23, 55), before and after 12 hof VD treatment.

Western Blotting. Western blotting was performed using the method de-scribed by Burnette (56), with minor modifications (23). The 9A7g rat mono-clonal anti-VDR (ABR; Affinity Bioreagents, Golden, CO) and Rpa(F) rabbitpolyclonal anti-RARa (a generous gift of Prof. P. Chambon, Institute deGenetique et de Biologie Moleculaire et Cellulaire, Illkirch Cedex, France)were used as primary antibody for the detection of the VDR and RARa

proteins, respectively. The detection was carried out by the enhanced chemi-luminescence method (Amersham Life Science, Little Chalfont, United King-dom).

Gel Shift Assay. Oligonucleotides were synthesized with an automatedsolid-phase DNA synthesizer (Model 394; Applied Biosystem, Inc., FosterCity, CA) with the standard phosphoramidite chemistry and purified by PAGE,followed by electroelution as described previously (57). The DR3 oligonu-cleotide 59-AGCTTCAGGTCAAGGAGGTCAGAGAGC-39(58) and its com-plement were synthesized and used as probe in the gel shift assay. Oligonu-cleotide annealing was performed as described previously (58, 59). Thedouble-stranded oligonucleotide was further purified by PAGE, electroeluted,and 59end-labeled (100 ng) as described previously (60). Specific radioactivityranged from 1–33 108 cpm/mg DNA. Gel retardation assays were performedas described previously (60). The following mAbs were used (a) 9A7g (ratmonoclonal anti-VDR; ABR; Affinity Bioreagents); (b) 4RX-1D12 (mousemonoclonal anti-RXRa, -b, and -g; kindly provided by Dr. P. Chambon); and(c) Ab9a (F; mouse monoclonal anti-RARa; kindly provided by Dr. P.Chambon). In all of the gel shift mobility experiments, the VDR specificity ofthe shifted bands was confirmed by the disruption of shift complex whenanti-VDR antibody was added. In several control experiments, VDRE speci-ficity was also confirmed by adding a 100-fold excess of cold VDRE oligo-nucleotides (data not shown). The use of anti-RARa or anti-RXRa mAb has

the ability to detect a supershifted band when these nuclear receptors arecomplexed in a heterodimeric conformation. All antibodies were added to thesamples at 1:20 final dilution. The reaction mixtures were then loaded on a 5%polyacrylamide gel in 0.53 TBE (45 mM Tris, 45 mM boric acid, and 1 mMEDTA), prerun for 1 h at room temperature. Finally the gel was fixed, vacuumdried, and exposed to X-ray films for 2–4 h.

Cell Transfection. The following plasmids were used in the transfectionexperiments: (a) pCMVb (Clontech Laboratories Inc., Palo Alto, CA), ab-galactosidase expression vector; (b) pOsteo/CABP-CAT, which contains theCAT reporter gene under the control of the VDRE of the hOC gene (2511/2480; Ref. 61) and the minimal promoter region of the chicken Calbindin gene(62); and (c) pTK/RAREb2-CAT (kindly provided by Prof. V. Colantuoni,Universita di Napoli “Federico II”, Napoli, Italy), containing the CAT reportergene under the control of the RAREb2 and the minimal promoter region of thehuman TK gene (2104/151). Cells (2.53 107) were transfected by electro-poration with 30mg of pOsteo/CABP-CAT or pTK/RAREb2-CAT plasmidand 3mg of pCMVb plasmid. Electroporation was performed at 250 V and 960mF (Gene Pulser Apparatus; Bio-Rad, Hercules, CA). Transfected cells werecultured in RPMI 1640 containing 15% FCS with or without VD or ATRA for48 h and then harvested forb-galactosidase and CAT assay (63).b-Galacto-sidase activity was used to normalize the amount of cell extract to be assayedfor CAT activity (63).

RESULTS

Differentiation Capacity of Myeloid Blast Cells Arrested atDifferent Levels of Maturation after 5 Days of Treatment withATRA or VD. Fig. 1 shows the results obtained by cytofluorometricanalysis of CD14, CD11b, and CD45 expression in leukemic cellpopulations. The induction of the CD11b marker is associated withgranulocytic differentiation, whereas the simultaneous induction ofCD14 and CD11b is associated with monocytic differentiation. Theexpression of panleukocytic CD45 antigen indicates the percentage ofcell viability. Fig. 1A shows the results obtained in five cases ofM0/M1-AML populations: all cell populations studied express a highlevel of CD45 and very low levels of CD14 and CD11b before andafter treatment with ATRA or VD.

The pattern of surface marker expression in five cases of M2-AMLpopulations before differentiation treatment is characterized by a highlevel of expression of CD45 and a very low expression of CD14 andCD11b (Fig. 1B). After VD or ATRA treatment, a high percentage ofcells express CD14 and CD11b, indicating that monocytic differenti-ation occurs in these cells (39), independent of the inducer used (Fig.1B).

Granulocytic differentiation is observed when leukemic blast cellsof the M3 type are treated with ATRA; in fact, only CD11b is clearlyinduced in all populations studied (Fig. 1C). In contrast, VD treatmenthas no effect on these cells.

Morphological analysis is consistent with the pattern of surfacemarker expression in all types of AML cells (data not shown).

VDR, RARa, and RXR Expression in M0/M1, M2, and M3-AML Blast Cells. To clarify whether the different sensitivity toATRA or VD could depend on the differential expression of VDR,RARa, and RXR nuclear receptors, we performed RT-PCR analysisin all types of AML blast cells. Fig. 2A shows that RARa, RXRa, andVDR mRNAs are present in the vast majority of spontaneous leuke-mic cell populations studied, although with a different abundance. Theonly exceptions are a M0/M1 population expressing a low level ofRARa mRNA and no VDR mRNA (Lane 4) and one M3-AMLpopulation (Lane 12) in which VDR mRNA is not detectable.

RT-PCR products ofb2m mRNA used to monitor the amount ofthe RNA in each sample are also reported in Fig. 2A, Lanes 1–13.

Nuclear receptor expression is detectable in Kasumi-1 (M2-typeAML) and HL-60 (M2/M3-type AML) cell lines before and after 12 hof ATRA or VD treatment (Fig. 2B).

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MONOCYTIC DIFFERENTIATION OF AML-M2 BLAST CELLS



Furthermore, t(8;21) can be detected by RT-PCR in only one of fivecases of M2-AML (Fig. 2A, C. C.), whereas all M3-AMLs weret(15;17) positive (data not shown).

VDR Protein Expression in M0/M1, M2, and M3-AML BlastCells. Western blot analysis was performed to detect VDR protein inCEs (data not shown) and NEs obtained from M0/M1 (Fig. 3A), M2(Fig. 3B), and M3 (Fig. 3C) AML blast cells. The results show that noVDR can be detected before VD treatment in either the cytoplasmic

(data not shown) or the nuclear compartment (Fig. 3,A—C,Lane 1).On the contrary, VDR is clearly present in NEs of all types of AMLblast cells after 12 h of VD treatment (Fig. 3A—C, Lane 3). Surpris-ingly, 12 h of ATRA treatment causes the appearance of VDR only inM2 blast cell NEs (Fig. 3B, Lane 2), and not in the other types of blastcells studied. The same VDR expression pattern is observed in theother four cases of this type of AML (data not shown).

Fig. 1. Expression of CD45, CD14, and CD11b in M0/M1, M2, and M3-AML cellpopulations. The histograms show the levels of expression, as evaluated by flow cyto-metric analysis, of CD45 (hatched bars), CD14 (M), and CD11b (f) surface markers inAML M0/M1 (five blast cell populations;A), AML M2 (five blast cell populations;B),and AML M3 (three blast cell populations;C) cells treated with ATRA or VD for 5 daysor left untreated. The values are expressed in percentages. The reported results are theaverage of three different sets of experiments. Variability in each experiment was less than10%.

Fig. 2. Detection by RT-PCR of RARa, VDR, and RXR mRNAs in AML-M0/M1, M2,and M3 blast cell populations as well as in HL-60 and Kasumi-1 cells. PanelA: Lanes 1–5, fivecases of AML-M0/M1;Lanes 6–10,five cases of AML-M2;Lanes 11–13, three cases ofAML-M3; Lane 14, negative control performed without the cDNA template. PanelB: Lane 1,proliferating HL-60 cells;Lane 2,HL-60 cells treated for 12 h with ATRA;Lane 3, HL-60cells treated for 12 h with VD;Lane 4,proliferating Kasumi-1 cells;Lane 5, Kasumi-1 cellstreated for 12 h with ATRA;Lane 6, Kasumi-1 cells treated for 12 h with VD;Lane 7, negativecontrol performed without the cDNA template. RT-PCR products ofb2m mRNA are shownat the bottom of both panels. The different genes studied and the size of the amplifiedfragments are labeled on theleft andright sideof each panel, respectively.

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RARa protein is clearly detected in NEs obtained from untreatedM2-AML blast cells (Fig. 3D, Lane 1), and it is not modulated after12 h of ATRA or VD treatment (Fig. 3D, Lanes 2and3). This patternof RARa protein expression was also observed in NEs obtained fromthe other types of AML blast cells (data not shown).

Differentiation Effects of ATRA and VD in Kasumi-1 andHL-60 Cells. To better characterize ATRA-induced monocytic dif-ferentiation of M2 blast cells, we have compared the differentiationcapacity of Kasumi-1 cells derived from the peripheral blood of apatient with M2-AML (28) with the well-known differentiation ca-pacity of HL-60 cells (M2/M3-AML) after 5 days of treatment withATRA or VD. Morphological analysis performed on differentiatedcells shows that only monocytic differentiation can be induced inKasumi-1 cells with both inducers, whereas HL-60 cells differentiateto monocytes when treated with VD and differentiate to granulocytesafter ATRA treatment (data not shown).

This observation was confirmed by flow cytometric analysis ofsurface marker expression (Fig. 4). In fact, a clear induction of CD11band CD14 expression (peak 1andpeak 2, respectively) is observed inKasumi-1 cells after treatment with ATRA (Fig. 4B) or with VD (Fig.4C), even if a bimodal distribution of CD14/CD11b expression isobserved, due to the presence of negative cells. Despite this observa-tion, the majority of Kasumi-1 cells differentiate upon ATRA and VDtreatment. HL-60 cells express only CD11b after ATRA treatment(Fig. 4E) and coexpress CD14 and CD11b after VD treatment (Fig.4F). A high expression level of CD45 is evident in both cell linesbefore and after treatment with the differentiation inducers (Fig.4A—F, peak 3).

VDR and RARa Expression in Kasumi-1 and HL-60 Cellsbefore and after 12 h of ATRA or VD Treatment. Western blotanalysis reveals immunoreactive RARa bands in the NEs of both celllines before and after treatment for 12 h with ATRA or VD (Fig. 5A,Lanes 1–6). Thus, ATRA treatment is not crucial for RARa detection.The apparent molecular weight of this nuclear receptor is not homo-geneous in the two different cell types. Because RARa is a phospho-protein, the size variability ranging fromMr 54,000 toMr 58,000 isprobably due to different levels of phosphorylation (64). Two mainRARa bands are detected in proliferating and ATRA-treated Ka-sumi-1 cells (Fig. 5A, Lanes 1and2), and only one band ofMr 58,000

Fig. 3. Western blot analysis of VDR expression in AML-M0/M1, M2, and M3 blastcell populations. The presence of VDR protein was assayed in NEs obtained from AML-M0/M1 (A), M2 (B), and M3 (C) blast cell populations treated for 12 h with ATRA or VDor left untreated.Lane 1, NEs of untreated cells;Lane 2, NEs of ATRA-treated cells;Lane3, NEs of VD-treated cells.D, RARa expression in NEs obtained from AML-M2 blastcell populations treated for 12 h with ATRA or VD or left untreated. The estimatedmolecular mass of VDR and RARa is indicated on theright sideof each panel.

Fig. 4. Flow cytometric analysis of CD45, CD14,and CD11b expression in Kasumi-1 and HL-60 cellstreated with ATRA or VD for 5 days or left un-treated.Peak 1 corresponds to the expression ofCD11b, peak 2 corresponds to the expression ofCD14, andpeak 3corresponds to the expression ofCD45. The expression of the markers listed abovehas been evaluated in proliferating Kasumi-1 cells(A) and in Kasumi-1 cells treated with ATRA (B) orVD (C), in proliferating HL-60 cells (D), and inHL-60 cells treated with ATRA (E) or VD (F).

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is detected in VD-treated Kasumi-1 cells (Fig. 5A, Lane 3), whereasin HL-60 cells, only aMr 54,000 band is evident with both inducers(Fig. 5A, Lanes 4–6).

VD treatment is crucial for VDR detection because NEs obtainedfrom untreated Kasumi-1 and HL-60 cells do not show the receptorband (Fig. 5B, Lanes 1and4). VDR is clearly observed in the NEs ofboth cell lines after VD treatment (Fig. 5B, Lanes 3and 6). InKasumi-1 cells, a clear VDR band is evident in the NEs after ATRAtreatment (Fig. 5B, Lane 2). This result is superimposable to thatobtained in spontaneous M2-AML blast cells (Fig. 3B).

On the contrary, ATRA-treated HL-60 cells, in keeping with theresults described for M3 spontaneous blast cells (Fig. 3C), do notshow any increase of VDR abundance in the nuclear compartment(Fig. 5B,Lane 5).

To clarify whether the VDR induction in Kasumi-1 cells coulddepend on transcriptional regulation by ATRA, we performed RT-PCR analysis on the VDR primary transcript in Kasumi-1 and HL-60cells before and after the differentiation induction. In fact, it has beenrecently described that a functional RARE is present in intron 1C ofthe VDR gene (46).

Our results clearly indicate that in both cellular contexts, VDRprimary transcript is detectable after ATRA treatment, suggesting thatVDR protein expression in Kasumi-1 cells is not due to transcriptionalregulation (data not shown).

DNA Binding Activity of VDR in Kasumi-1 and HL-60 Cells.Because Kasumi-1 cells are induced to monocytic differentiation bothwith ATRA and VD, we investigated the ability of VDR, which ispresent in the NEs after ATRA treatment, to bind a DR3-type VDREin EMSA. As shown in Fig. 6A, Lanes 2and5, in untreated Kasumi-1cells, a major complex is detected, even if the band intensity is lowerthan that in VD- or ATRA-treated cells (Fig. 6B, Lanes 8and11; Fig.6C, Lanes 14and17, respectively). This complex certainly containsRXRa because a clear supershifted band is detected with the anti-RXR mAb (Fig. 6A,Lanes 4and7). Very little or no RARa and VDRare present in the complex because it is not affected by anti-RARa oranti-VDR mAb (Fig. 6A, compareLanes 2and3 andLanes 5and6).

After 12 h of treatment of Kasumi-1 cells with ATRA or VD, VDRis clearly detected in the major complex (Fig. 6B, Lane 8;Fig. 6C,Lane 14) because a consistent decrease in intensity is caused by theVDR mAb (Fig. 6B, Lane 12;Fig. 6C, Lane 15). No supershift of themajor complex is evident in Kasumi-1 NEs after VD or ATRAtreatment, when they are matched with RARa mAb (Fig. 6B, compareLanes 8and 9; Fig. 6C, compareLanes 17and 18). After VD andATRA treatment of Kasumi-1 cells, the NEs showed an increasedabundance of RXR because an increased intensity of the supershiftedband is detected with the RXR mAb (Fig. 6B, Lanes 10and13; Fig.6C, Lanes 16and19).

These data clearly show that in ATRA- or VD-treated Kasumi-1cells, VDR can heterodimerize with RXR and efficiently bind to theDR3-responsive element. In HL-60 cells, on the contrary, a VDR-containing bandshift complex is observed only after VD treatment(Fig. 6E, compareLanes 30and31) and not upon ATRA treatment(Fig. 6F, compareLanes 36and37).

Our results indicate that VDR-VDRE complex formation is greatlyenhanced by VD or ATRA treatment in Kasumi-1 cells, even if wecannot exclude that a ligand-independent complex might be present inuntreated cells (Fig. 6A, compareLanes 5and6).Fig. 5. Western blot analysis of RARa and VDR in Kasumi-1 and HL-60 cells.A,

RARa protein expression in NEs from Kasumi-1 and HL-60 cells.B, VDR proteinexpression in NEs from Kasumi-1 and HL-60 cells.Lane 1, proliferating Kasumi-1 cells;Lane 2, Kasumi-1 cells treated for 12 h with ATRA;Lane 3, Kasumi-1 cells treated for12 h with VD;Lane 4, proliferating HL-60 cells;Lane 5, HL-60 cells treated for 12 h withATRA; Lane 6, HL-60 cells treated for 12 h with VD. Estimated molecular masses ofRARa and VDR are indicated on theright sideof each panel.

Fig. 6. EMSA of VDR after binding to a DR3-type DNA element in NEs of Kasumi-1and HL-60 cells. NEs from untreated, VD-treated (12 h), or ATRA-treated (12 h)Kasumi-1 cells (A, B, andC, respectively) and from untreated, VD-treated, and ATRA-treated HL-60 cells (D, E, and F, respectively) were assayed for specific binding to a32P-labeled DR3-type DNA element. To assess the identity of nuclear receptors partici-pating in the shifted complexes, antibodies to RARa, RXR, and VDR were used. Thearrow indicates the position of shifted complexes containing VDR and RXR.

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Ability of Endogenous VDR to Activate Transcription of aVDRE-dependent Promoter in Kasumi-1 and HL-60 Cells.Toexamine the transactivation capacity of endogenous VDR and RARa,we transiently transfected Kasumi-1 and HL-60 cells with the reporterconstructs pOsteo/CaBP-CAT and pRAREb2/TK-CAT and thentreated them for 48 h with ATRA or VD. As shown in Fig. 7, bothATRA treatment and VD treatment of Kasumi-1 cells cause transcrip-tional activation of the pOsteo/CaBP-CAT reporter gene (Fig. 7A,Lanes 4and6), whereas ATRA treatment but not VD treatment causesthe transcriptional activation of pTK/RAREb2-CAT (Fig. 7B, Lanes10 and12).

This observation provides further evidence that in Kasumi-1 cells,ATRA specifically activates transcription through a VDRE just asefficiently as VD. The effect of ATRA on VDRE-dependent tran-scription is restricted to M2-type blast cells because no transcriptionalactivation of pOsteo/CaBP-CAT is evident after ATRA treatment ofHL-60 cells (Fig. 7C, Lane 16); in these cells, reporter activity isobserved only after VD treatment (Fig. 7C, Lane 18).

The pTK/RAREb2-CAT construct is transactivated quite consis-tently in HL-60 cells after ATRA treatment, but not after VD treat-ment (Fig. 7D, Lanes 24and22, respectively).

These results suggest that the pOsteo/CaBP-CAT activation ob-served in Kasumi-1 cells after ATRA treatment is due to a VDRinduction.

Even if several reports indicate that a VD-independent transactiva-tion may occur mainly in kidney and intestine epithelial cells (65–67),pOsteo/CaBP-CAT transactivation observed in Kasumi-1 cells after

ATRA treatment seems to be more dependent on increased VDRnuclear abundance than on ligand-independent activation, as alsodemonstrated by VDR protein expression and EMSA experiments.

Expression of ATRA and VD Primary Response Genes inKasumi-1 and HL-60 Cells. To monitor the ability of ATRA andVD to activate the genetic program underlying monocytic differenti-ation in the M2 cellular context, we have investigated the expressionof VD-responsive genes (Fig. 8) such as HMSE-1, CD14, and hOC byRT-PCR and the expression of genes controlled by a functionalVDRE, such as p21waf-1 (51), or RARE, such as E3 (52), IRF-1 (53)and p21waf-1 (68), by Northern blot analysis after different times ofATRA or VD treatment.

Fig. 8 shows that both ATRA and VD treatment of Kasumi-1 cellscauses a rapid induction of HMSE-1, CD14, and hOC expression,demonstrating VDR involvement in ATRA-dependent monocytic dif-ferentiation of M2-type blast cells. In fact, these VD-responsive genesare not induced in ATRA-treated HL-60 cells. In addition, neither VDtreatment nor ATRA treatment is able to modify the expression of RAprimary responsive genes, such as E3 and IRF-1, that are constitu-tively expressed in Kasumi-1 cells, suggesting that no induction ofgranulocytic differentiation occurs in these cells (data not shown).

The same analysis performed on HL-60 cells shows a rapid induc-tion of E3 and IRF-1 expression after ATRA treatment, as expected.Despite the differential phenotypic effect observed in HL-60 cells

Fig. 7. CAT assays of transfected Kasumi-1 and HL-60 cells. pOSTEO/CaBPCAT (Aand C) or pTK/RAREb2-CAT (B and D) was transfected in Kasumi-1 (A and B) andHL-60 (C andD) cells. Where indicated, cells were treated with ATRA or VD for 48 h.Amounts of cell extracts used were normalized to the same value of theb-galactosidaseactivity.

Fig. 8. Detection by RT-PCR of HMSE-1, hOC, and CD14 mRNAs in Kasumi-1 andHL-60 cells treated with ATRA or VD or left untreated.Lane 1, proliferating Kasumi-1cells;Lane 2, Kasumi-1 cells treated for 12 h with ATRA;Lane 3, Kasumi-1 cells treatedfor 12 h with VD; Lane 4, proliferating HL-60 cells;Lane 5, HL-60 cells treated for 12 hwith ATRA; Lane 6, HL-60 cells treated for 12 h with VD;Lane 7, negative controlperformed without the cDNA template. RT-PCR products ofb2m mRNA are shown in thebottom panel.

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with ATRA or VD treatment, p21waf-1 expression is induced after 1 hby both differentiating agents, suggesting that its expression is notsufficient to induce monocytic differentiation, as suggested recently(51).

The level of the constitutively expressedb2m mRNA was assessedto monitor RNA abundance in all samples (Fig. 8,Lanes 1–6).

DISCUSSION

Differentiation of AML blast cells is blocked at different levels ofmaturation, as demonstrated by the French-American-British classi-fication (29) and the immunological classification (31). Blast cellsmaintain the capacity to divide, but the rate of cell division decreasesprogressively as blast cells undergo a series of differentiation transi-tions. The consequence of this biological behavior is that the vastmajority of leukemic blast cells are arrested in the G1 phase of the cellcycle, and, despite the expression of cyclin-dependent inhibitors,never reach spontaneous terminal differentiation (1, 2, 69). Differen-tiation induction therapy based on treatment with hemopoietic growthfactors such as granulocyte colony-stimulating factor, granulocytemacrophage colony-stimulating factor, or monocyte colony-stimulat-ing factor is inefficient because blast cells are not responsive to thesecytokines, despite the expression of the corresponding receptors (16–18). In vitro experiments indicate that these hematopoietic growthfactors are more efficient in recruiting blast cells to the proliferativepathway than to the differentiative pathway (70).

Efficacy of differentiation therapy based on ATRA has been dem-onstrated only in APL blast cells (9, 10, 71). Because differentiationtherapy with physiological inducers is a highly attractive approach tocontrol leukemic cell growth (72), we have evaluated the capacity ofATRA and VD to induce myeloid differentiation in different types ofAML blast cells.

We found that spontaneous M2-type AML blast cells, independentof the presence of t(8;21), are induced to monocytic differentiationwith either ATRA treatment or VD treatment. Spontaneous M0/M1blast cells are not responsive to these inducers. M3-type AML blastcells, as already described, are responsive only to ATRA, and not toVD.

Our original observation is that VDR protein is detected afterATRA treatment in the nuclear compartment of M2 cells, but not inthe NEs of M0/M1 and M3 cells, suggesting that both ATRA and VDare able to increase the nuclear abundance of VDR in M2-type blastcells.

To gain some insight into the molecular mechanism underlyingmonocytic differentiation induced by ATRA in M2-AML blast cells,we studied VDR and RARa expression and VDR function in twoAML cell lines: (a) Kasumi-1 (M2-type cell line); and (b) HL-60(M2/M3-type cell line). Kasumi-1 cells are representative of sponta-neous M2-AML blast cells because only monocytic differentiation isachieved by ATRA or VD treatment (Fig. 4). HL-60 cells are aclassical model of myeloid maturation because they differentiate togranulocytes or monocytes when treated with ATRA or VD, respec-tively (Fig. 4; Ref. 35).

Our results indicate that nuclear VDR protein is detected in Ka-sumi-1 cells after either ATRA or VD treatment (Fig. 5). Furthermore,VDR is functionally active in this cellular context because it iscapable of binding a DR3 oligonucleotide after heterodimerizationwith RXR (Fig. 6) and activating the transcription of the transfectedpOsteo/CaBP-CAT vector (Fig. 7). These results are obtained inHL-60 cells after VD treatment, but not after ATRA treatment. On theother hand, ATRA can transactivate the pTK/RAREb2-CAT vector(Fig. 7) in HL-60 cells and in Kasumi-1 cells, suggesting that RARais functionally active in both cellular contexts.

Recent reports indicate that there is a cross-over of the nuclearsignaling pathways of RA and VD, but it is still unclear whether theinterplay between the different nuclear receptors can activate differentsignaling pathways during hematopoietic differentiation (73–75).

The peculiarity of our results is that both ATRA and VD lead to theactivation of monocytic differentiation in M2-type blast cells. Fur-thermore, the described ATRA-induced pOsteo/CaBP-CAT transac-tivation is also obtained with compounds such as 8-bromo-cyclicAMP (67) and okadaic acid (66, 67), which are known to induce aVD-independent, VDR-mediated transactivation of reporter genes(65). This transcription activation is achieved through a modulation ofVDR phosphorylation levels and does not involve a VDR proteinexpression induction (67), which is, conversely, clearly observed inATRA-treated Kasumi-1 cells. Moreover, the formation of spontane-ous VD-independent complexes between endogenous VDR-RXR het-erodimers and genomic DNA can probably be hypothesized only fortissues characterized by high levels of VDR nuclear protein expres-sion, such as intestinal epithelial cells (65). The existence of basalVDR-RXR/DNA interaction complexes is unlikely in hematopoieticcells, in which the detection of significant amounts of nuclear VDRprotein is strictly dependent on VD treatment (23). We can thusconclude that VDR-mediated activation of gene expression induced inKasumi-1 cells by ATRA probably resides on mechanisms that are notstrictly related to VD-independent transcription activation of VDR.

The molecular mechanisms by which ATRA induces a functionallyactive VDR nuclear protein in Kasumi-1 cells and M2 blast cells arestill unclear. Transcription of the VDR gene, which is probably RAREdependent (46), is activated by ATRA in both Kasumi-1 and HL-60cells because a primary VDR transcript is clearly detectable in boththe cellular contexts by RT-PCR, suggesting that the ATRA-inducedVDR expression observed in Kasumi-1 cells is not strictly regulated atthe transcriptional level.

Furthermore, VDR expression studies in spontaneous leukemicblast cells showed a discrepancy between VDR mRNA and proteinlevels. VDR mRNA is, in fact, detected in all types of AML blast cellsbefore VD treatment (Fig. 2A). VDR protein (Fig. 3), on the otherhand, is detected in the nuclear compartment of all myeloid blast cellsonly after VD treatment and after ATRA treatment of M2-type blastcells. The RARa expression pattern is different because both mRNAand protein are detected in untreated AML blast cells, and protein isnot significantly modulated in the NEs after ATRA treatment (Fig.3D). Comparable results were observed in the HL-60 and Kasumi-1leukemic cell lines (Figs. 2 and 5).

However, these expression studies indicate clearly that VDR pro-tein abundance in untreated leukemic cells is very low, and that afterVD treatment, posttranscriptional mechanisms such as protein stabi-lization (76) or, as described in other systems, increased translationefficiency of compartmentalized mRNA (77) take part in increasingthe VDR protein abundance in the nuclear compartment. This VDRposttranscriptional regulation also occurs in M2-type blast cells afterATRA treatment.

Both ATRA treatment and VD treatment of Kasumi-1 cells (Fig. 8)rapidly induce the expression of VD-responsive genes such asHMSE-1, CD14, and hOC. These data clearly demonstrate that VDRis involved in ATRA-dependent monocytic differentiation of M2-typeblast cells. Moreover, these VD-responsive genes are not induced inATRA-treated HL-60 cells. In addition, neither VD treatment norATRA treatment is able to modify the expression of RA primaryresponsive genes, such as E3 and IRF-1, which are constitutivelyexpressed in Kasumi-1 cells, suggesting that no induction of granu-locytic differentiation occurs in M2-type blast cells, even if RARa isexpressed and is functionally active in transactivating the pTK/RARE/b2-CAT construct. The same analysis performed on HL-60 cells

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shows a rapid induction of E3 and IRF-1 expression after few hoursof ATRA treatment, as expected.

Despite the differential phenotypic effect observed in HL-60 cellswith ATRA or VD treatment, p21waf-1 expression is induced after 1 hby both differentiating agents, suggesting that its expression is notsufficient to induce monocytic differentiation, as suggested recently(51). These results are consistent with a recent report showing thatp21waf-1 expression after ATRA treatment is associated with granu-locytic differentiation of HL-60 cells (68).

In conclusion, our data allow us to hypothesize that the M2 cellularcontext can be considered a differentiation window permissive only ofmonocytic differentiation because both VD and ATRA differentiationinducers are capable of activating this differentiation program throughthe induction of a functional nuclear VDR protein.

This observation might open a new therapeutic application ofATRA, even if the response of AML patients (including M2-typeAML) to treatment with this differentiating agent remains to beverified.

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1999;59:3803-3811. Cancer Res Rossella Manfredini, Francesca Trevisan, Alexis Grande, et al. Leukemic Blast Cells -Retinoic Acid-induced Monocytic Differentiation of M2-type

transInduction of a Functional Vitamin D Receptor in all-

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