The Role of RAR and RXR Activation in Retinoid-Induced Tissue Factor Suppression T Tenno1, J Botling2,FO¨ Berg2, S Jossan1, K Nilsson2 and a Siegbahn1

Home , Retinoic acid, Retinoic acid receptor, Retinoid receptor, Retinoid X receptor

Leukemia (2000) 14, 1105–1111  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu The role of RAR and RXR activation in retinoid-induced tissue factor suppression T Tenno1, J Botling2,FO¨ berg2, S Jossan1, K Nilsson2 and A Siegbahn1

Departments of 1Medical Sciences and 2Genetics and Pathology, Uppsala University, S-751 85 Uppsala, Sweden

Excessive expression of tissue factor (TF) is a common finding cells.9–11 RA therapy is also widely used in treatment of APL in leukaemic cells and may contribute to thrombotic compli- patients, giving a remission rate over 90%.12 Nevertheless, cations in patients. Retinoic acid has been shown to induce differentiation and reduce TF expression in acute promyelo- there is a limited knowledge about the mechanisms underly- cytic leukaemia (APL) cells in vitro, and to induce remission in ing the processes of RA-induced TF suppression in APL and APL patients. Treatment of the APL cell line NB4 with the spe- other myeloid leukaemic cells. It is known that RA acts cific retinoic acid receptor-α (RARα) agonists Ro40–6055 or through nuclear steroid receptors: retinoic acid receptors TTNPB resulted in down-regulation of TF expression and in (RARα, β, γ) and retinoid X receptors (RXRα, β, γ). RA treat- β γ induction of differentiation. The activation of RAR , RAR or ment of various cells induces formation of RAR-RXR transcrip- retinoid X receptor (RXR) did not suppress the constitutive TF expression in NB4 cells. Moreover, the RARα antagonist Ro41- tional complexes on the RA response element (RARE) in pro- 5253 blocked the retinoid-induced down-regulation of TF. In moters of RA-regulated genes. Although RXR has been contrast, in the monoblastic U-937 cell line only a partial sup- considered as a silent transcriptional partner,13 the activation pression of TF antigen expression and activity was observed of both RXR and RAR is essential for full transcriptional by treatment with the RAR agonist TTNPB or the RXR agonist activity and induction of differentiation in myeloid cells.14,15 SR11237 alone. However, the combination of TTNPB and The abnormal PML/RARα gene in APL cells results in defective SR11237 resulted in a pronounced down-regulation of TF RA signalling and is responsible for the leukaemic phenotype expression and induction of differentiation in U-937 cells. We 16 show for the first time that the activation of both subunits of of these cells. It was shown that in NB4 cells the activation the RARα-RXR transcriptional complex is needed for TF sup- of RARα or RARβ is sufficient to induce TF down-regu- pression in U-937 cells, whereas in NB4 cells RARα activation lation.17,18 However, the abnormal retinoic acid signalling by alone is sufficient. Thus, distinct molecular mechanisms for TF the PML/RARα nuclear oncoprotein in NB4 cells does not suppression seem to be operating in leukaemic cell lines of allow us to expand these data on TF regulation in other different origin. Leukemia (2000) 14, 1105–1111. Keywords: tissue factor; RAR; RXR; differentiation myeloid and leukaemic cells. In this report, we have used RA nuclear-receptor-specific ligands to reveal the mechanisms of retinoid-induced TF suppression in the APL cell line NB4 and in the monoblastic cell Introduction line U-937. Although RARα activation can be shown to be sufficient for TF suppression in NB4 cells, we show that in a Tissue factor is the main initiator of the blood coagulation cas- cell line with a normal RARα gene the full suppression of TF cades. Expression of this protein is restricted to subendothelial requires activation of both RARα and RXR. Moreover, TF sup- tissues, and the peripheral blood cells and endothelial cells of pression in both of the cell lines was found to be coherent healthy individuals are negative for TF.1 Monocytes, however, with induction of general myeloid differentiation markers, are the only blood cells that can be induced to express this which might imply that TF suppression and cell differentiation protein.2 In septic conditions TF expression in monocytes is are regulated concomitantly. known to play a major role in development of disseminated intravascular coagulation (DIC).3 Moreover, pathological expression of TF on leukaemic cells seems to be an important Materials and methods finding for thrombotic complications.4 Acute promyelocytic leukaemia (APL) is the most common myeloid leukaemia Cells associated with thrombotic complications, ultimately leading to DIC.5 APL is a subtype of acute myelogenous leukaemia The NB4 cells, established from a human acute promyelocytic with the characteristic chromosome translocation that disrupts leukaemia (M3)19 (kindly provided by Inge Olsson, University the retinoic acid receptor (RAR)-α gene on chromosome 17 of Lund, Lund, Sweden), and the human monoblastic cell line and fuses it to the promyelocytic leukaemia (PML) gene at U-937 (subline U-937-1), derived from a patient with histo- chromosome 15, thus resulting in the PML/RARα fusion pro- cytic lymphoma20,21 were maintained in RPMI 1640 medium tein. Beyond pathological expression, TF is also expressed on supplemented with 10% foetal bovine serum (Sigma, St Louis, the surface of bone marrow derived myeloid progenitor cells.6 MO, USA), 1% of L-glutamine, 100 U/ml of penicillin and The role of the protein during myeloid maturation is however 50 µg/ml of streptomycin (Biochrom, Berlin, Germany). The still poorly understood. Thus, understanding the mechanisms cells were routinely screened for mycoplasma infection at reg- of the specific regulation of TF in different cells of myeloid ular intervals. The cells at a concentration of 2 × 105/ml were origin is of great importance for interventions aiming at allevi- induced with different retinoids: all-trans retinoic acid (ATRA; ating the thrombotic complications. Sigma), TTNPB, SR11237, Ro40-6055, Ro48-2249, Ro44- Retinoic acid induces differentiation and growth arrest in 4753 and Ro41-5253 (Table 1). Retinoids were dissolved in myeloid precursor cells and in leukaemic cells.7,8 This differ- ethanol to the 1 mM concentration and further dissolved in entiation process has been shown to be accompanied by sup- the culture medium. The final concentration of ethanol in the pression of TF expression in various myeloid leukaemia culture media was less than 0.1%, which was found to have no effect on the TF expression in these cells. During the period Correspondence: A Siegbahn; Fax: 46 18 55 25 62 of incubation we did not observe any changes in the cell Received 16 November 1999; accepted 31 January 2000 viability (.90%). Cell culture medium was checked for endo- The role of RAR and RXR in tissue factor suppression T Tenno et al 1106 Table 1 The specificities of the retinoids used in the study primers and probe. The PCR reaction was carried out in ABI PRISM 7700 Sequence Detection System (Perkin Elmer Ligand Receptor specificities Ref. Applied Biosystems) in a two-step protocol: 10 min at 95°C (denaturation of DNA and activation of AmpliTaq Gold) and ATRA RARα/β/γ agonist 26 50 cycles amplification by 15 s at 95°C and 1 min at 60°C. α β γ TTNPB RAR / / agonist 39 The real-time detection of amplification signal information Ro40–6055 (Am580) RARα agonist 26 Ro48–2249 RARβ agonist 26 was captured by the ABI PRISM 7700 and the appearance Ro44–4753 RARγ agonist 26 of the amplification signal, threshold cycle number (CT), was Ro41–5253 RARα antagonist 40 registered for each sample. The differences in TF mRNA α β γ SR11237 (BMS649) RXR / / agonist 24, 37 expression were determined by the comparative CT method as described in ABI PRISM 7700 User Bulletin No. 2. Briefly, β ∆ the difference in TF and -actin CT values ( CT) was determ- ∆ ined and calibrated to the CT value of the uninduced sample ∆∆ = ∆ − ∆ toxin contamination with limulus amebocyte lysate assay ( CT CTX CT0). The relative TF mRNA expression (Chromogenix AB, Mo¨lndal, Sweden), and endotoxin concen- related to uninduced sample is determined by evaluating the tration was found less than 5 pg/ml at working concentrations expression: 2−∆∆CT. of the reagents.

Analysis of TF mRNA Flow cytometry

Total RNA was isolated from 2 × 106 cells with TRIZOL The surface antigen expression was analysed by immunofluo- reagent (Life Technologies, St Louis, MO, USA). RNA concen- rescence with a flow cytometer (EPICS XL, Coulter Electronics, tration was determined at 260 nm with a spectrophotometer Hialeah, FL, USA). For indirect immunofluorescence experi- (SPECTRAmax 250, Molecular Devices, Sunnyvale, CA, USA), ment the cells were washed twice with PBS + 0.1% BSA, incu- and the integrity of 28S and 18S ribosomal RNA was checked bated for 30 min on ice with the primary antibody, washed in ethidium bromide-stained agarose gel. Total RNA (1 µg) twice, incubated for 30 min on ice with fluorescein-isothiocy- was subjected to a reverse transcription (RT) reaction for anate (FITC)-labelled rabbit anti-mouse (Dakopatts, Glostrup, 90 min at 37°Cin20µl volume containing 200 U MMLV RT Denmark) and finally washed twice. The primary antibodies enzyme, 0.5 mM dNTP mixture, 10 mM DTT, 0.25 µg were monoclonal antibodies to TF (TF9-9C3; American Diag- × oligo(dT)12–18 primer and 1 first strand buffer (50 mM Tris- nostica, Greenwich, CT, USA) and Leu M5 (CD11c) HCI, 75 mM KCI, 3 mM MgCl2) (Life Technologies). (Camfolio, Becton Dickinson, Mountain View, CA, USA). The The relative amount of reverse transcribed TF mRNA was expression of CD11b and CD49f was determined by FITC- quantitated with the TaqMan real-time polymerase chain reac- labelled monoclonal antibodies BEAR1 and GoH3, respect- tion (PCR) assay. The method is based on 59–39 nuclease ively (Immunotech, Marseilles, France). The anti-Aspergillus activity of the Taq polymerase, in which the enzyme degrades niger glucose oxidase monoclonal IgG1 (DAK-G01; the dual-labelled fluorogenic target-specific TaqMan probe, Dakopatts) was used as a negative control. Mean channel resulting in an accumulation of fluorescent emission as the fluorescence intensity and percentage of positive cells were reaction proceeds.22 Table 2 shows the sequences of PCR pri- determined for each sample. mers and fluorescent TaqMan probes for TF and the house- keeping gene β-actin. PCR reaction mixture of 25 µl contained the TaqMan buffer A (50 mM KCl, 10 mM EDTA, 10 mM Procoagulant activity Tris-HCl pH 8.3, 60 nM passive reference ROX), 3 mM MgCl2, dNTP mixture (0.2 mM of each), 1.25 U AmpliTaq Gold (Perkin Elmer, Foster City, CA, USA), 300 nM forward and The procoagulant activity of TF was determined in a pre- reverse primers, 200 mM probe and 1 µl of RT reaction pro- viously described two-stage amidolytic assay.23 Briefly, ali- duct. For each sample two parallel reactions were performed – quots with 3 × 105 cells were washed twice with PBS and one with TF primers and probe, and the other with β-actin placed in the wells of a 96-well microtitre plate. The reaction mixture contained 0.6 mM of the chromogenic substrate, S- 2222 (Chromogenix AB), 10 µM of ionomycin (Calbiochem, Table 2 Primers and probes used for TaqMan real-time PCR La Jolla, CA, USA) and the coagulation factor concentrate Prothromplex-T TIM 4 (Immuno, Vienna, Austria) at a final 9 R Q TF TaqMan probe 5 -T TG GAG CTG TGG CAT TTG concentration of 1 U/ml of FVII and 1.2 U/ml of FX. The reac- TGG TCA TCA TCC-39a 9 9 tion was started by adding of 2 mM final concentration of TF forward primer 5 -GCC AGG AGA AAG GGG AAT-3 ° TF reverse primer 59-CAG TGC AAT ATA GCA TTT GCA CaCl2 and the solution was incubated for 30 min at 37 C. The GTA GC-39 change in absorbance at 405 nm was determined with a β-Actin TaqMan probe 59-ARTG CCC TQCC CCC ATG CCA spectrophotometer (SPECTRAmax 250, Molecular Devices). TCC TGC GT-39a β-Actin forward primer 59-TCA CCC ACA CTG TGC CCA TCT ACG A-39 β-Actin reverse primer 59-CAG CGG AAC CGC TCA TTG Statistical analysis CCA ATG G-39

aThe indexes stand for the labelling-sites for (R) the reporter fluor- Statistical analysis was performed by Student’s t-tests for escent dye FAM (6-carboxyfluorescein) and (Q) the quencher flu- dependent samples and P values less than 0.05 were con- orescent dye TAMRA (6-carboxytetramethylrhodamine). sidered statistically significant.

Leukemia The role of RAR and RXR in tissue factor suppression T Tenno et al 1107 Results tor subtype-specific ligands in the absence or presence of RXR agonist. The expression of TF antigen, determined by flow RAR activation induces suppression of TF in APL cytometric analysis, was significantly (P , 0.005) inhibited by − (NB4) cells the RARα agonist Ro40–6055 (10 8 M) (Figure 2a).26 No changes in TF expression were found after treatment of NB4 Retinoid-induced differentiation in myeloid cells is known to cells with the same concentrations of RARβ or RARγ agonists, require the activation of the RAR-RXR nuclear receptor com- Ro48–2249 or Ro44–4753, respectively. Similar to the experi- plex.14 Therefore, we first studied if the activation of the RAR- ments with the RAR pan-agonist TTNPB (Figure 1), the acti- RXR complex is also a requirement for retinoid-induced sup- vation of the RAR transcriptional partner RXR by SR11237 did pression of TF mRNA in the APL cell line NB4. The NB4 cells not affect TF expression in NB4 APL cells (Figure 2a). were treated with the RXR-selective synthetic agonist SR11237 The essential role of RARα for TF regulation in NB4 cells (0.1 µM), the RAR-selective agonist TTNPB (0.01 µM) or with was confirmed by blocking of the RARα by the synthetic the combination of the ligands.24 To achieve maximum speci- antagonist Ro41–5253. Treatment of NB4 cells with various − − ficity we used the lowest effective ligand concentrations. We concentrations (from 10 6 M to 10 8 M) of ATRA induced have found that ATRA-induced TF suppression in NB4 cells efficient suppression of TF expression (Figure 2b). Thirty is an early event, followed by the other differentiation related minutes preincubation of the cells with Ro41–5253 resulted 11 − changes, and thus retinoid-induced TF changes were meas- in almost complete inhibition of the effects induced by 10 8 M ured after 24 h. As shown in Figure 1a the activation of RARs ATRA (P , 0.05). The antagonist, however, failed to block TF with the selective ligand TTNPB resulted in almost complete down-regulation at higher ATRA concentrations (Figure 2b). (95%, P , 0.005) down-regulation of TF mRNA levels, These results indicate that in the NB4 APL cell line, possessing whereas induction by combination of RAR and RXR agonists the abnormal PML/RARα fusion protein, the retinoid induced did not give any further suppression. The RXR agonist TF suppression is dependent on activation of the RARα alone. SR11237 alone induced only partial suppression of TF mRNA. Consistent with the results obtained from TF mRNA measurements we found a similar suppression of TF antigen Activation of both RARα and RXR are essential for TF levels in NB4 cells (Figure 1b). After 24-h treatment of NB4 suppression in U-937 cells cells with TTNPB the expression of TF antigen was inhibited by 75% (P , 0.005). No marked effect on TF antigen To investigate if activation of RARα is a general requirement expression was found when NB4 cells were activated by RXR for retinoid-induced TF suppression in myeloid cells, we stud- ligand SR11237 alone (Figure 1b). Moreover, SR11237 did not ied the monoblastic U-937 cell line that constitutively − enhance the effect of TTNPB on TF expression (Figure 1b). To expresses TF.27 The cells were treated with TTNPB (10 8 M) − confirm the regulation of TF expression by the RAR agonist, and SR11237 (10 7 M) as described for NB4 cells. Figure 3 we also evaluated the functional properties of expressed TF shows that incubation of the U-937 cells with TTNPB or by measuring procoagulant activity in a two-stage amidolytic SR11237 alone did not affect the TF mRNA levels, but a slight assay.23 RAR ligand TTNPB induced 40% (P , 0.05) sup- reduction of TF antigen and TF procoagulant activity was pression of procoagulant activity, while the activation of RXR recorded after 48 h. To achieve a complete suppression of TF had no effect (Figure 1c). The incomplete suppression of PCA expression the combination of RAR and RXR agonists was by retinoids in NB4 cells is in agreement with earlier pub- necessary. The combination of TTNPB and SR11237 resulted lished data,11,17,18 and the residual activity may result from the in TF mRNA, TF antigen and TF PCA down-regulated by 81%, expression of cancer procoagulant with coagulation factor X 80% and 78%, respectively (P , 0.01). activating capacity in NB4 cells.25 A moderate effect on TF antigen expression by RARα ligand Ro40–6055 was observed in U-937 cells after 48 h (Figure 4a). In accordance with results described above the combi- Activation of RARα in NB4 cells suppresses TF nation of RXR and RARα agonists induced a more pronounced expression (74%, P , 0.05) down-regulation of TF. Thus, the activation of the normal RARα-RXR transcriptional complex is essential To determine the RAR subtype responsible for TF down-regu- for retinoid induced TF suppression in these cells. To confirm lation in NB4 cells, we first induced the cells with RAR recep- the role of RARα in regulation of TF expression in myeloid

Figure 1 RAR activation is required for suppression of TF expression in NB4 cells. NB4 cells were untreated or incubated for 24 h with − − TTNPB (10 8 M), SR11237 (10 7 M) or with the combination of the retinoids. The expression of TF mRNA (a), TF antigen as percent of positive cells (b) and TF procoagulant activity (c) were determined as described in Methods. The results are the mean of at least three experiments, ± s.d.

Leukemia The role of RAR and RXR in tissue factor suppression T Tenno et al 1108

Figure 2 TF expression in NB4 cells is dependent on RARα activation. (a) The cells were untreated or treated with RXR agonist SR11237 − − (10 7 M), isotype specific RAR agonists at concentration 10 8 M as indicated or with the combination of RXR and the specific RAR agonist for − 24 h. (b) NB4 cells were untreated or pretreated with specific RARα antagonist Ro41–5253 (10 5 M) for 30 min and subsequently incubated with ATRA for 24 h at the indicated concentrations. Data are presented as the percentage of positive cells as determined by flow immunofluorescence, and the mean of three experiments (± s.d.) is shown.

Figure 3 Activation of both RAR and RXR are required for suppression of TF expression in U-937 cells. U-937 cells were incubated as indicated by Figure 1. The expression of TF mRNA was determined after 24 h (a), whereas TF antigen (b) and TF procoagulant activity (c) expressions were determined after 48 h. The results are the mean of at least three experiments, ± s.d.

Figure 4 TF expression in U-937 cells is dependent on RARα and RXR activation. (a) U-937 cells were treated with RXR agonist SR11237 − − (10 7 M), isotype specific RAR agonists at concentration 10 8 M as indicated or with the combination of RXR and the specific RAR agonist for − 48 h. (b) U-937 cells were untreated or pretreated for 30 min with specific RARα antagonist Ro41–5253 (10 5 M) and subsequently incubated with ATRA for 24 h at the indicated concentrations. The percentage of positive cells as determined by flow immunofluorescence is shown. The results are the mean of three experiments, ± s.d.

Leukemia The role of RAR and RXR in tissue factor suppression T Tenno et al 1109 cells we blocked RARα activity with Ro41–5253 30 min prior expression in myeloid and leukaemic cells.11,29 The RA- to 48 h ATRA treatment in U-937 cells. There was a concen- induced suppression of TF antigen and TF procoagulant tration-dependent suppression of TF antigen expression by activity has been reported to be mediated by activation of ATRA (Figure 4b). At the lower ATRA concentrations the RARα and RARβ in the APL cell line NB4.17,18 However, the − RARα antagonist blocked the effect of retinoic acid. The reported effect of RARβ ligation by Ro48–2249 (10 8 M)30 or − expression of TF was down-regulated by 57% by 10 nM of CD2019 (10 8 M)18 was found to be less potent when com- ATRA, whereas pre-treatment with Ro41–5253 in 1000-fold pared to TF suppression induced by RARα agonist. In our excess resulted in 16% suppression, being significant at P , experiments with NB4 and U-937 cell lines we did not 0.05. observe any effect of the RARβ agonist Ro48-2249 on the expression of TF antigen (Figures 2a and 4a) nor on the expression of some general myeloid differentiation markers TF and myeloid differentiation markers are inversely like CD11b and CD11c (data not shown). The contradictory regulated results concerning the effect of RARβ-activation on TF regulation might result from some receptor cross-reactivity We have earlier suggested that retinoid-induced TF sup- and different agonist potency of the RARβ ligands used in pression in myeloid cells is accompanied by terminal differen- these studies. In NB4 cells the growth inhibition and differentiation.11 Therefore, we also studied retinoid receptor depen- tiation are induced with retinoids having only RARα agonist dent induction of some myeloid-specific differentiation activity.31,32 This refers to the specific involvement of markers, ie CD11b, CD11c and CD49f (Table 3). We found RARα/PML protein in RA-induced effects in APL cells. How- pronounced expression of CD11b and CD11c in NB4 APL ever, RARβ is expressed in U-937 cells.33 Although we find it cells after RAR agonist treatment, while activation of RXR less likely, we cannot completely rule out the possibility of alone had minor effects. The combination of RAR and RXR RARβ involvement in TF suppression. agonists did not significantly enhance differentiation above Retinoic acid-induced effects are predominantly transduced RAR activation alone. In contrast, in U-937 cells the activation through activation of RAR-RXR heterodimeric complexes in of RAR-RXR complex was needed for induction of differen- the RARE of RA-responsive promoter sites.34 The mechanism tiation-related cell-surface receptors (Table 3). by which PML/RARα protein is involved in the leukaemogen- esis is not fully understood. In the APL cells, the PML/RARα protein binds to the RARE and associates with the nuclear Discussion corepressor proteins like SMRT and N-CoR,35 and being abun- dant in these cells might act in a dominant negative manner. In this study we have shown that the mechanisms of retinoid- Treatment of APL cells with ATRA results in dissociation of induced TF down-regulation in myeloid leukaemic cells are SMRT/N-CoR complex and degradation of the PML/RARα pro- specific for the particular cell type. In the acute promyelocytic tein.36 Thus, the ligation of PML/RARα by RARα agonist seems leukaemia-derived cell line NB4, the suppression of TF was to be sufficient to eliminate the leukaemic consequences in observed solely after RARα activation, whereas activation of NB4 cell line, and so induce the cell differentiation and sup- RARα alone was not sufficient for the complete suppression pression of TF induction. However, recently a cross-talk of TF in the monoblastic lymphoma cell line U-937. In these between RXR agonists and protein kinase A has been reported cells the retinoid-induced suppression of TF appeared to be also to induce maturation in NB4 cells.37 This RARα- dependent on the activation of both members of RARα-RXR independent pathway seems to be an alternative for the ATRA transcriptional complex. The successful suppression of TF induced APL differentiation programme, whereas the conse- expression in these two cell lines was found to be consistent quences for TF expression remains to be revealed. Thus, we with induction of maturation as confirmed by increased can not exclude the relevance of RXR in the suppression of expression of myeloid differentiation markers. TF in APL cells. Retinoids have been shown to transduce signals leading to RXR has been suggested to act as a silent transcriptional cytodifferentiation28 and concomitant suppression of TF partner to RAR in the heterodimeric complexes.13 However, several studies have shown that in normal RARα protein expressing myeloid cells the synergistic activation of both Table 3 The induction of CD11b, CD11c and CD49f expression partners of the RAR–RXR complex is required for activation of by SR11237 and TTNPB RARE transcriptional activity and for many cellular responses, eg induction of cellular differentiation, growth arrest and reti- CD11b CD11c CD49f noblastoma protein phosphorylation.14,15,38 Consistent with these results we show that activation of RARα or RXR alone by ± ± NB4 — 0.40 0.05 0.43 0.14 ND synthetic nuclear receptor agonists leads only to a moderate NB4 TTNPB 1.47 ± 0.56 3.21 ± 0.27 ND NB4 SR11237 0.42 ± 0.08 0.56 ± 0.27 ND suppression of TF mRNA, TF antigen and TF procoagulant NB4 SR11237 2.16 ± 0.82 3.60 ± 0.61 ND activity in the monoblastic U-937 cells. Full down-regulation + TTNPB was observed when both RARα and RXR were activated. Thus, U-937 — 1.75 ± 0.68 0.82 ± 0.33 0.26 ± 0.18 the suppression of TF seems to require the activation of U-937 TTNPB 4.22 ± 1.33 1.42 ± 0.74 0.33 ± 0.24 normal transcriptional complexes, and correlates with other ± ± ± U-937 SR11237 2.12 1.08 0.84 0.26 0.27 0.17 differentiation-associated cellular responses like induction of U-937 SR11237 8.43 ± 0.73 3.09 ± 1.91 1.07 ± 0.47 + TTNPB cell surface antigens (CD11b, CD11c and CD49f). The suppression of TF in the APL cell line NB4, which pos- sesses the PML/RARα fusion protein, is dependent on the Cells were treated for 96 h as indicated and analysed for α expression of cell surface antigens by flow cytometry. Data are activation of RAR alone. However, in the monoblastic presented as MFI (± s.d., n > 3). lymphoma cell line U-937 the initiation of processes leading ND, not done. to TF down-regulation are dependent on simultaneous acti-

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