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Blood, Vol. 90 No. 3 (August 1), 1997:
pp. 1175-1185
Characterization of the Retinoid Binding Properties of the Major Fusion Products Present in Acute Promyelocytic Leukemia Cells
By
Laura Benedetti,
Arthur A. Levin,
Bianca M. Scicchitano,
Francesco Grignani,
Gary Allenby,
Daniela Diverio,
Francesco Lo Coco,
Giuseppe Avvisati,
Martin Ruthardt,
Sergio Adamo,
Pier Giuseppe Pelicci, and
Clara Nervi
From the Department of Histology and Medical Embryology and the Department of Cellular Biotechnology and Hematology, University "La Sapienza," Rome, Italy; the Department of Toxicology and Pathology, Hoffmann-La Roche, Nutley, NJ; the Department of Medicine, University of Perugia, Perugia, Italy; and the Department of Experimental Oncology, European Institute of Oncology, Milano, Italy.
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ABSTRACT |
The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor  (PML/RAR) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are present in different lengths of PML (amino acids 1-552 and 1-394, respectively), contain most of the functional domains of PML and RAR, bind all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcription factors. T-RA is an effective inducer of clinical remission only in patients carrying the t(15; 17) and expressing the PML/RAR products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR product has been found associated with a poorer prognosis than bcr1-PML/RAR. In the present study we have investigated the structural and functional properties of the bcr3-PML/RAR in comparison to the previously characterized bcr1-PML/RAR. In particular, we have measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to both of these isoforms. T-RA binding analysis of nuclear and cytosolic extracts prepared from bcr3-PML/RAR APL patients and from bcr3-PML/RAR COS-1 transfected cells indicates that this protein is present only as high-molecular-weight nuclear complexes. Using saturation binding assays and Scatchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR receptor than to bcr1-PML/RAR or RAR (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). Moreover, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 and 0.075 nmol/L) were detectable in the bcr3-PML/RAR product but not in the bcr1-PML/RAR product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RAR isoform than to the bcr1-PML/RAR or RAR. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the  RARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR products can be measured.
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INTRODUCTION |
THE CHARACTERISTIC balanced translocation t(15q+; 17q-) of acute promyelocytic leukemia (APL) fuses a portion of the promyelocytic leukemia (PML) gene on chromosome 15 to the second coding exon of the nuclear retinoic acid receptor (RAR) on chromosome 17. As a result of this translocation a PML/RAR chimeric gene is formed which is transcriptionally active in all cases of APL.1,2 The chimeric transcript encodes a PML/RAR fusion protein that retains both the PML "ring finger" and dimerization domains and the RAR DNA-binding and RA-binding domains.3,4 The breakpoints on chromosome 15 are clustered at three different sites in the PML locus: in intron 6 (breakpoint cluster region 1; bcr1), exon 6 (bcr2), and intron 3 (bcr3), leading to three different fusion proteins.5-8 The most frequent products are the bcr1- (also known as "long" or type "B") and bcr3- (or "short" or type "A") PML/RAR,4,5 which present different lengths of PML (amino acids 1-552 and 1-394, respectively). In rare APL cases, the RAR gene fuses with the PLZF (promyelocytic leukemia zinc finger) gene on chromosome 119,10 or with the nucleophosmin gene on 5,11 suggesting that aberrant RAR gene expression may be associated with leukemogenesis.
APL has become the first model of differentiation therapy targeted to a defined genetic defect.3,4,12 All-trans-retinoic acid (t-RA) induces disease remission of APL patients by triggering terminal differentiation of malignant cells.3,4,12 However, there is diversity of t-RA sensitivity in APL cases: t-RA induces cell differentiation and clinical remission in patients with the t(15; 17) translocation,4,12 but APL patients and cultured cells from patients with the t(11; 17) fail to differentiate in response to t-RA. In the single t(5; 17) APL case a reduced response to t-RA therapy compared to PML/RAR cases10 was found. In addition, within APLs with the t(15; 17), evaluation of patients in remission after treatment with t-RA alone or with t-RA followed by chemotherapy showed that the expression of the bcr3-PML/RAR isoform is significantly associated with a poorer prognosis compared to those with the bcr1-PML/RAR isoform.12-14
The mechanisms underlying the heterogeneous t-RA response in APL are unclear. Retinoids activate two classes of nuclear receptor proteins of the steroid and thyroid hormone superfamily, the RARs (, , and  ), and the retinoid X receptors (RXRs , , and ), for gene transcriptional activation.15,16 T-RA binds only to RARs, whereas 9-cis-retinoic acid (9-cis-RA) is a bifunctional ligand capable of binding to both RARs and RXRs.17-19 Whether 9-cis-RA has a different spectrum of biologic activity from t-RA is presently unknown.
With respect to retinoid binding properties, we and others6,20 have previously reported that the bcr1-PML/RAR isoform binds retinoids with the same affinity and specificity as the wild-type RAR receptor and is present predominantly in high-molecular-weight nuclear complexes.20
In the present investigation we have characterized the structural and functional properties of the bcr3-PML/RAR and PLZF/RAR in comparison to those of the bcr1-PML/RAR product. We show that in cells from bcr3- PML/RAR APL patients and bcr3-PML/RAR or PLZF/RAR COS-1-transfected cells, these fusion proteins are present in high-molecular-weight nuclear complexes. Moreover, our results provide evidence for preferential retinoid binding to the different PML/RAR products. In fact, we found that 9-cis-RA binds with higher affinity and specificity to the bcr3-PML/RAR isoform than to the bcr1-PML/RAR or RAR. This effect was also associated with a differential activation of retinoic acid responsive elements in COS-1 cells transfected with bcr3-PML/RAR and either TRE (thyroid response element) or RARE (RAR response element). Taken together, these results indicate that preferential retinoid binding to different gene fusion products can be measured and suggest the possibility of specific differentiation therapy in APL patients.
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MATERIALS AND METHODS |
9-cis-[10-3H]retinoic acid (24 Ci/mmol) and unlabeled retinoids used in this study were synthesized by the Department of Medicinal Chemistry, Hoffmann-La Roche, Nutley, NJ. All-trans-[3H]-retinoic acid (50.7 Ci/mmol) was purchased from DuPont/NEN (Boston, MA). COS-1 cells were obtained from American Type Culture Collection (ATCC; Rockville, MD). Rabbit anti-RAR polyclonal serum RP(F )21 was kindly provided by Prof P. Chambon (Strasbourg, France).
Patients samples and processing.
Fresh leukemic cells were obtained from peripheral blood (PB) and bone marrow (BM) aspirates of newly diagnosed, informed APL patients presenting an initial percentage of blasts that was more than 80%. Patients were classified as M3 or M3-variant according to the French-American-British (FAB) classification.22 Characterization of the PML/RAR isoform (bcr1-bcr2-bcr3) was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay as previously described.23 Leukemic cells were isolated by Ficoll-Hypaque density gradients (Pharmacia, Uppsala, Sweden).
RNA preparation and Northern blot analysis.
Total RNA was prepared by the guanidinium isothiocyanate-CsCl procedure24 from Ficoll-Hypaque-isolated APL blasts washed twice with calcium-magnesium free phosphate solution. Total RNA (10 µg/sample) was electrophoresed through a 1.2% denaturing agarose-formaldeyde gel. After electrophoresis, RNA was transferred to Nytran (Schleicher & Schuell, Hayward, CA) by capillary blotting and then cross-linked by UV irradiation. The cDNA probes were radiolabeled with [-32P]dCTP (3,000 Ci/mmol; DuPont-NEN) via random priming using the kit and protocols supplied by Bethesda Research Laboratories. The cDNA probes for human RAR, , and 25,26 were obtained from Prof P. Chambon. The cDNA probes for human RXR27 and for mouse RXR and 28 were obtained from Dr R.M. Evans (The Salk Institute, San Diego, CA). The cDNA probe for chicken glyceraldehyde-3-dehydrogenase (GAPDH)29 was used as control probe to standardize the level of gene expression.
Cell culture, transfection, and retinoic acid binding assays.
COS-1 cells were transiently transfected by electroporation as previously described19 with pSG5 expression vectors containing cDNAs for human RAR, bcr1-, and bcr3-PML/RAR,30 or with a pMT2 expression vector24 containing a cDNA encoding the human PLZF/RAR.9 The PLZF/RAR cDNA was reconstructed from wild-type RAR and PLZF cDNAs by PCR and conventional cloning strategies (M. Ruthardt and P.G. Pelicci, manuscript submitted). After transfection (48 to 72 hours), cells (3 to 7 × 107) were removed by trypsinization and collected by centrifugation. Nuclear and cytosolic extracts were prepared from COS transfected cells and from fresh blasts from APL patients as previously described.19,31,32 Extracts were incubated with 10 nmol/L [3H]-t-RA for 18 hours at 4°C. [3H]-t-RA binding was analyzed by high-performance liquid chromatography (HPLC) performed at 4°C using a size exclusion column Superose 6 HR 10/30 (Pharmacia). The eluent was PTG buffer containing 0.4 mol/L KCl and the flow rate was 0.4 mL/min.20
Apparent molecular weights (MW) were calculated on the basis of the elution times of a series of marker proteins used to calibrate the system, such as: blue dextran, MW 2,000,000; thyroglobulin, MW 669,000; apoferritin, MW 443,000; -amylase MW 200,000; alcohol dehydrogenase, MW 150,000; bovine albumin, MW 66,000; ovalbumin, MW 45,000; carbonic anhydrase, MW 29,000; and lactalbumin, MW 14,200.
For saturation binding studies as well as competitive binding assays, bound RA was separated from free radioactivity using PD10 desalting columns (Pharmacia) as described.19,20 In saturation binding studies, incubations were performed, for 18 hours at 4°C, in the presence of increasing concentrations of [3H]-t-RA or [3H]-9-cis-retinoic acid. Binding in the presence of 200-fold molar excess of unlabeled t-RA or 9-cis-RA was defined as nonspecific binding. Specific binding resulted from total binding minus nonspecific binding. Linear least-square analysis of the Scatchard plot was performed with the aid of the computer program BDATA-EMF.33 A fixed concentration of [3H]-t-RA (10 nmol/L) and increasing concentrations of unlabeled competing ligand (ranging between 0.5 nmol/L and 1µmol/L) were used in incubations for competitive binding assays. The EC50 values (retinoid concentrations that inhibit 50% of the total specific RA binding) were calculated using the nonlinear least squares regression analysis program ALLFIT.34
Sodium dodecyl sulfate (SDS)-gel electrophoresis and Western immunoblotting.
Nuclear extracts were diluted 1:1 with 2× SDS sample buffer (2% SDS, 0.125 mol/L Tris-HCl, pH 6.8, 20% glycerol, 0.02% bromophenol blue, and 10% 2-mercaptoethanol). Proteins were then fractionated by electrophoresis on an 8% SDS polyacrylamyde gel and electroblotted to nitrocellulose membrane (Hybond C Super; Amersham Life Science Inc, Arlington Heights, IL). Proteins that reacted with the anti-RAR RP(F ) antibody21 (used at a 1:500 dilution) were detected using the ECL Western blotting detection kit (Amersham) with the reagents provided according to the manufacturer's instructions.
Transient cotransfection of COS-1 cells and transactivation assays.
COS-1 cells were plated in six-well 35-mm culture dishes at a density of 2.5 × 105 cells/well. Twenty-four hours after plating cells were transiently transfected using the Lipofectamine reagent (GIBCO-BRL, Gaithersburg, MD). Each well received 0.5 µg of the pSG5 expression vectors containing the coding regions for human RAR, bcr1- or bcr3-PML/RAR, 0.5 µg (RARE)3-tk-LUC,35 or TRE2-tk-LUC,36 or the RAR pr-LUC reporter,8,35 and the plasmid encoding -galactosidase (pSV-Gal) (0.3 µg) was cotransfected to monitor transfection efficiency and for normalization of reactions. After 5 hours, transfecting medium was removed and cells were incubated in Dulbecco's modified Eagle's medium containing 5% heat-inactivated, charcoal stripped fetal calf serum. The next day retinoids were added for 16 hours to minimize retinoid isomerisation. Cells were washed twice with calcium-magnesium-free phosphate solution and lysate using a Reporter Lysis Buffer (Promega Corp, Madison, WI). Luciferase activity was measured using a luminometer (Berthold, Wildbad, Germany).37 Reporter gene expression was calculated in arbitrary units, relative to -galactosidase expression. The EC50 values (retinoid concentrations which induced 50% of maximal response) were calculated using the nonlinear least squares regression analysis program ALLFIT.34
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RESULTS |
Retinoic acid binding in bcr3-PML/RAR APL patients.
Previous retinoid binding studies performed in the APL cell line NB4, which express the bcr1-PML/RAR product,6 showed the presence of specific [3H]-t-RA binding corresponding to CRABP, RARs, monomeric form of bcr1-PML/RAR (110,000), and to the high-molecular-weight complexes formed by the interaction of bcr1-PML/RAR with itself or with other nuclear proteins.20,38 We have now investigated the t-RA binding properties of fresh APL blasts cells obtained from patients who expressed the bcr3 isoform of the PML/RAR transcript. Nuclear and cytosolic extracts were labeled with 10 nmol/L [3H]-t-RA in the absence or in the presence of 200-fold mol/L excess of unlabeled t-RA to determine nonspecific binding, and the t-RA binding was analyzed by HPLC size exclusion chromatography. The HPLC profile of nuclear extracts prepared from these cells showed the presence of three main peaks of specific [3H]-t-RA binding eluting at 36, 42, and 47 minutes, corresponding to molecular weights of about 500,000, 50,000, and 18,000, respectively (Fig 1). Similar HPLC profiles were consistently found in blasts isolated either from BM or from PB of bcr3-PML/RAR APL patients. The specific t-RA binding peaks corresponding to molecular weights of 50,000 and 18,000 probably represent binding to RARs and CRABP, respectively. The presence of CRABP in cytosol extracts prepared from these cells was established by the presence of a main peak of specific binding eluting at 47 minutes and corresponding to a molecular weight of 18,000. In addition, in cytosolic extracts, two small peaks of specific t-RA binding activity eluting at a retention time of 28 and 34 minutes, which probably represented contamination of cytosol with nuclear extract, could be detected.
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| Fig 1.
Size exclusion HPLC analysis of cytosolic (A) and nuclear (B) extracts prepared from fresh blasts from the PB of an APL patient expressing the bcr3-PML/RAR isoform. Extracts (200 µL) were incubated with 10 nmol/L [3H]-t-RA in the absence ( ) or in the presence ( ) of 200-fold mol/L excess of unlabeled t-RA for 18 hours at 4°C. The samples were then fractionated over a Superose 6 HR 10/30 size exclusion column as described in Materials and Methods.
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Retinoic acid binding in bcr3-PML/RAR-COS-1 transfected cells and immunoblot analysis.
To easily characterize the t-RA ligand binding properties of the bcr3-PML/RAR isoform in comparison to the previously reported RAR and bcr1-PML/RAR, COS-1 cells were transiently transfected with the cDNAs for RAR, bcr1-PML/RAR, bcr3-PML/RAR, or expression plasmid (mock). Nuclear extracts prepared from these cells were first examined by immunoblot analysis using the anti-RAR RP(F ) antibody21 to confirm the production of intact receptors. Immunoblot analysis showed the presence of immunoreactive proteins corresponding to the approximate MW calculated from their respective cDNA sequences30,39,40 (RAR 50,000; bcr1-PML/RAR 105,000; bcr3-PML/RAR 90,000) (Fig 2A). As previously described,20 endogenous RAR was not immunodetectable in nuclear extracts from mock-transfected cells (Fig 2A). In pSG5/bcr1-PML/RAR extracts a small degree of degradation was detected, as evidenced by an immunoreactive band showing an MW of about 40,000. We next examined the binding of [3H]-t-RA to these proteins by HPLC size exclusion analysis (Fig 2B). The HPLC profile obtained from nuclear extracts prepared from pSG5/bcr3-PML/RAR transiently transfected COS-1 cells showed the presence of two main peaks of specific t-RA binding activity eluting at 31 and 37 minutes and then corresponding to an MW of more than 1,000,000 and of about 550,000, respectively. These HPLC profiles were very similar to those of nuclear extracts prepared from blasts obtained from bcr3-PML/RAR APL patients. Conversely to RAR and pSG5/bcr1-PML/RAR, a complete absence of the monomeric form of this receptor was found (Fig 2B). In fact, t-RA specific binding activity was not present in fractions corresponding to lower MW proteins. We concluded that the pSG5/bcr3-PML/RAR product is present only as macromolecular complexes that may represent pSG5/bcr3-PML/RAR homodymers and/or high-molecular-weight complexes with other nuclear proteins.
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| Fig 2.
Immunoblot analysis and HPLC analysis of [3H]-t-RA binding in nuclear extracts prepared from mock, pSG5/RAR, pSG5/bcr1-PML/RAR and pSG5/bcr3-PML/RAR transiently transfected COS-1 cells. (A) Immunoblot analysis: Nuclear extracts were subjected to 8% polyacrylamide slab gel electrophoresis followed by immunoblotting using the anti-RAR RP(F ) antibody21 as described in Materials and Methods. (B) Size exclusion HPLC analysis. Nuclear extracts were analyzed as described in the legend of Fig 1. () [3H]-t-RA alone; () [3H]-t-RA plus 200-fold mol/L excess of unlabeled t-RA. Arrows indicate the elution times of marker proteins (in thousands) used to calibrate the Superose 6 HR 10/30 size exclusion column as indicated in Materials and Methods.
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The HPLC profile of cytosolic extracts prepared from COS-1 transfected cells showed the presence of a specific t-RA binding component corresponding to an MW of 18,000 and probably representing the endogenous CRABP and similar peaks of specific binding present in nuclear extracts probably resulting from a lysis of a small percentage of nuclei during the preparation of cytosolic extracts (data not shown).
Saturation binding and Scatchard analysis of t-RA and 9-cis-RA.
Previous studies20 indicated that the bcr1-PML/RAR isoform bound t-RA with similar affinity as RAR (Kd 0.13 nmol/L and 0.09 nmol/L, respectively). To characterize the binding affinities of t-RA to the bcr3-PML/RAR product we first performed saturation binding and Scatchard analysis. Nuclear extracts prepared from pSG5/bcr3-PML/RAR COS-1 transfected cells were labeled for 18 hours with increasing concentrations of [3H]-t-RA. Bound and free radiolabeled were separated with PD10 desalting columns and counted as described in Materials and Methods. A typical binding curve and Scatchard plot for [3H]-t-RA binding to the bcr3-PML/RAR is shown in Fig 3. The binding of t-RA to the bcr3-PML/RAR was specific, saturable, and exhibited high affinity similar to that described for RAR and bcr1-PML/RAR isoform.20 Scatchard analysis yielded a linear plot consisting of the presence of a single class of binding sites (r = -.91). The Kd value obtained was 0.47 ± 0.01 nmol/L. These data indicate that t-RA binds with relative lower affinity (threefold to fivefold) to bcr3-PML/RAR compared with the previously reported Kd values measured for RAR and bcr1-PML/RAR receptors.20
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| Fig 3.
Saturation binding (A) and Scatchard plot analyses (B) for the binding of [3H]-t-RA to bcr3-PML/RAR. Nuclear extracts from pSG5/bcr3-PML/RAR transiently transfected COS-1 cells were incubated with the indicated concentrations of [3H]-t-RA and analysed using PD10 desalting columns as described in Materials and Methods. Nonspecific binding activity () was subtracted from total binding activity () to calculate specific binding activity ( ). Specific t-RA binding activity () was also plotted in the form of a Scatchard plot (B).
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It has been shown that 9-cis-RA binds with high affinity to each of the members of the RXR and RAR subfamilies.17,41 Scatchard analyses of the binding of [3H]-9-cis-RA to RAR performed by different groups yield Kd values of 0.24 to 0.31 nmol/L.17,41 We examined the binding of [3H]-9-cis-RA to the bcr1-PML/RAR and bcr3-PML/RAR present in nuclear extracts prepared from COS-1 cells transfected with their respective cDNAs. The [3H]-9-cis-RA binding to both bcr1-PML/RAR and bcr3-PML/RAR was specific and saturable (Fig 4). However, saturation kinetics and Scatchard analyses of the binding of [3H]-9-cis-RA to the bcr1-PML/RAR yield kd value of 0.77 nmol/L (r = -.97), whereas two kd values of 0.45 nmol/L (r = -.98) and 0.075 nmol/L (r = -.96) could be calculated for the bcr3-PML/RAR. These data indicated that 9-cis-RA binds with a lower affinity to the bcr1-PML/RAR than to the bcr3-PML/RAR. Moreover, two sites of 9-cis-RA binding with different affinities are present in the bcr3-PML/RAR fusion product. However, the analysis of the maximum binding showed that low-affinity sites were involved in the majority of this binding activity.
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| Fig 4.
Saturation kinetics for the binding of [3H]-9-cis-RA to pSG5/bcr1-PML/RAR (A) and pSG5/bcr3-PML/RAR (B) transiently transfected COS-1 cells. Specific t-RA binding activity () is defined as total binding () minus nonspecific binding (). Scatchard plot analysis of the saturation binding for bcr1-PML/RAR (C) and bcr3-PML/RAR (D).
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Competition binding.
To further characterize the ligand binding properties of the bcr3-PML/RAR isoform, and the different binding affinities of t-RA and 9-cis-RA for the two major PML/RAR products, competition binding experiments were performed in COS-transfected cells using different unlabeled retinoids such as t-RA, 9-cis-RA, CH55,20,42 AM580, and TTNPB.15,16 In these studies, nuclear fractions prepared from pSG5/RAR, pSG5/bcr1-PML/RAR, and pSG5/bcr3-PML/RAR COS-1 transfected cells were incubated for 18 hours at 4°C with 10 nmol/L [3H]-t-RA in the presence of varying concentrations of the unlabeled retinoid (Table 1). According with previous observations,20 t-RA exhibited similar ability to compete for [3H]-t-RA binding to either RAR and bcr1-PML/RAR (EC50 4.9 and 4.8 nmol/L, respectively). For the bcr3-PML/RAR isoform, the calculated EC50 value for competition binding by t-RA was 7.5 nmol/L. TTNPB and AM580 showed a slightly different specificity (about 1.5- to 2-fold) for competing to the three receptors, whereas CH55 had similar EC50 values. Conversely, competition binding of unlabeled 9-cis-RA to RAR, bcr1-PML/RAR and bcr3-PML/RAR yield EC50 values of 29.5 ± 5.5 nmol/L, 27.5 ± 4.1nmol/L, and 4.8 ± 1.3 nmol/L, respectively. These results indicated that 9-cis-RA binds with a higher specificity to the bcr3-PML/RAR isoform than to the RAR or the bcr1-PML/RAR.
Functional transactivation assay in cotransfected COS-1 cells.
We next investigated whether the higher affinity and specificity of 9-cis-RA for the bcr3-PML/RAR isoform resulted in a different ability of this protein to transactivate two different retinoic acid-response elements (RARE) such as the RARE and the TRE, compared with RAR and/or bcr1-PML/RAR. The RARE is the direct repeat sequence (DR5) of the RAR2 promoter. The TRE is a palindromic sequence that also mediates thyroid hormone transactivation. In COS-1 cells, when RAR, bcr1-PML/RAR or bcr3-PML/RAR expression vectors were cotransfected with either the (RARE)3-tk-LUC or TRE2-tk-LUC reporters, treatment with t-RA and 9-cis-RA induced luciferase activity in a dose-dependent manner. However, 9-cis-RA was less potent than t-RA in activating these repoter genes (Table 2). The magnitude of the (RARE)3-tk-LUC induction by t-RA was similar for the three receptors (EC50 ranging between 0.4 to 0.9 nmol/L). 9-cis-RA was able to induce (RARE)3-tk-LUC about fourfold more efficiently in RAR expressing cells than in bcr1- or bcr3-PML/RAR-COS-1 transfected cells (Table 2). However, under these conditions 9-cis-RA had very similar EC50 for the two PML/RAR isoforms. The different transactivation activity between t-RA and 9-cis-RA was abolished when the RAR-pr LUC,8,35 a reporter plasmid containing the RAR gene promoter region (-5 kb to +155) fused to the promoterless luciferase gene was used (Table 3). As recently shown,43 it is possible that the mechanism of ligand-dependent transcription is modulated by a number of positive and negative factors that may not be functional when direct repeat sequences are used.
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Table 3.
Transactivation Properties of All-trans-RA and 9-cis-RA on the Reporter Plasmid Containing the RAR Gene Promoter Region -5 kb to +155 (RAR pr-LUC)
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T-RA was about threefold to fivefold more potent in activating the TRE2-tk-LUC reporter gene in COS-1 cells expressing the bcr1- and bcr3-PML/RAR than in COS-1 cells expressing RAR. In contrast, 9-cis-RA treatment was equally potent in activating this reporter gene in RAR and bcr1-PML/RAR expressing cells. Remarkably, 9-cis-RA was about 10-fold more active in activating the TRE2-tk-LUC in the bcr3-PML/RAR than in the RAR or in the bcr1-PML/RAR COS-1 transfected cells. These results suggested that the higher affinity and specificity of the binding of 9-cis-RA to the bcr3-PML/RAR product resulted in an increased ability of this protein to specifically transactivate the TRE2-tk-LUC, and not the (RARE)3-tk-LUC, in transiently transfected COS-1 cells.
In agreement with this, preliminary results obtained from blast cells directly isolated from two bcr3-PML/RAR patients showed that 9-cis-RA was more potent than t-RA in inducing cell differentiation, as evaluated by morphology, by the nitroblue tetrazolium (NBT) dye reduction assay and by type II transglutaminase activity induction.38 In fact, the calculated EC50 for these effects were estimated to be approximately 1.8 nmol/L for t-RA and 0.1 nmol/L for 9-cis-RA, respectively.
Additional differences between binding specifity and transcriptional activity were also detected for the RAR- and RAR-selective retinoids AM580 and TTNPB. AM580 and TTNPB in fact, resulted to be very effective in transactivating either TRE2-LUC or (RARE)3-tk-LUC in cotransfected COS-1 cells. However, these retinoids were consistently found to be less active in transactivating these RAREs in the bcr3-PML/RAR COS-1 transfected cells (Table 2).
Retinoic acid binding properties of PLZF/RAR.
We then compared the t-RA binding properties of PML/RAR isoforms with that of the PLZF/RAR, the fusion protein generated by the less frequent chromosomal rearrangement t(11; 17) present in APL and associate with refractoriness to t-RA treatment. Nuclear and cytosolic extracts were prepared from COS-1 cells transfected with a pMT2 expression vector containing a cDNA encoding the human PLZF/RAR. PLZF/RAR was able to bind t-RA and t-RA specific binding activity was found mainly associated with the nuclear fraction (Fig 5). Similarly to PML/RAR, PLZF/RAR was found present in high-molecular-weight nuclear complexes eluting with apparent MW of 950,000 and 500,000. Similar HPLC profile was obtained in nuclear extracts prepared from myeloid U937 precursor cells expressing PLZF/RAR (data not shown). Moreover, as in the case of RAR and bcr1-PML/RAR,17,20,31,41 a single site of specific high-affinity binding could be detected for PLZF/RAR by saturation kinetics and Scatchard analyses. The calculated binding affinities were 0.17 nmol/L for t-RA and 2.7 nmol/L for 9-cis-RA (data not shown).
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| Fig 5.
Size exclusion HPLC analysis of [3H]-t-RA binding to nuclear (A) and cytosolic (B) extracts prepared from pMT2/PLZF/RAR transiently transfected COS-1 cells. () [3H]-t-RA alone; () [3H]-t-RA plus 200-fold mol/L excess of unlabeled t-RA. The Superose 6 HR 10/30 size exclusion column was calibrated with the indicated marker proteins (in thousands) as described in Materials and Methods.
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Expression of RARs and RXRs in APL patients.
Retinoid induced differentiation involves activation of multiple RA-dependent signaling pathways, including RARs and RXRs. Therefore, we have analyzed the pattern of expression of retinoid receptors in leukemic blasts expressing bcr1 or bcr3 isoforms of PML/RAR by Northern blotting analysis of RNA samples from 4 bcr1-PML/RAR and 5 bcr3-PML/RAR APL patients (Fig 6). As previously described,1,44,45 three RAR transcripts of 2.1, 3.0, and 4.8 kb were detectable in mRNAs prepared from all the APL patients. 3.1-kb RAR and 5.6-kb RXR transcripts were relatively highly expressed in APL patients whereas RAR and RXR transcripts were undetectable. As expected, samples from bcr1- and bcr3-PML/RAR APL patients expressed the same subtypes of RAR or RXR receptor mRNAs. RXR transcript was about equally expressed in all the samples whereas slightly different levels of expression of RAR, RAR, and RXR transcripts were detectable. However, a correlation between the expression level of RARs and RXRs and the presence of a particular PML/RAR isoform was not found.
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| Fig 6.
Expression of RAR's and RXR's mRNAs in fresh cells from APL patients. Total RNA (10 µg) prepared from Ficoll-Hypaque-isolated APL blasts was fractionated, transferred to Nytran membrane, and hybridized to [32P]-labeled cDNA probes for RAR-, -, -, RXR-, -, -. Hybridization to GAPDH was used as a control.
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DISCUSSION |
The combination of t-RA and conventional cytotoxic agents has been found to provide a more effective overall therapy for APL than conventional chemotherapy or retinoid therapy alone.3,4,12,46 Therapies including t-RA induced terminal differentiation of the leukemic promyelocytes, which are then replaced by normal hematopoiesis.3,4 However, the precise molecular mechanism of RA-induced differentiation in APL blasts has yet to be elucidated. It has been shown that the ligand binding domains of RARs and of the other members of the nuclear receptor superfamily also includes homodimerizing and heterodimerizating interfaces, ligand-dependent transcriptional activation function 2 (AF-2), and in some cases, hormone reversible transcriptional repression functions.15,43 All PML/RAR isoforms retain most of the RAR functional domains.3,4 In addition, APL blasts containing bcr1- and bcr3-PML/RAR also express transcripts encoding for RAR, RAR, RXR, and RXR. Moreover, specific retinoid binding probably corresponding to RARs and to CRABPs was detectable in these cells. These data indicate that in APL cells multiple retinoid signaling pathways are probably involved in RA-induced differentiation.
T-RA is an effective inducer of clinical remission only in patients carrying the t(15; 17) and expressing the PML/RAR product.3,4,12,47 APL patients defined as M3 by FAB criteria,22 but lacking the t(15; 17) or presenting t(11; 17), do not respond to t-RA-based treatments.10,47 Recent clinical studies indicate a different prognosis in APL patients treated with t-RA alone or with t-RA followed by chemotherapy on the basis of the fusion product present.13,14,48 In APL, the bcr3-PML/RAR isoform has been associated with the more aggressive microgranular APL variant, to CD2 expression, to higher leukocyte counts, and to poorer prognosis.12-14,49 However, in patients expressing the two major isoforms of PML/RAR treated with combined t-RA and chemotherapy no statistical differences in overall survival and complete remission duration was found.50 These findings stress the importance of a complete analysis of the molecular and clinical characteristics of APL patients to define t-RA sensitivity and response to differentiation therapy.
In this study we investigated the molecular mechanism of t-RA response in APL by analyzing functional properties such as retinoid binding and transcriptional activation of bcr1- and bcr3-PML/RAR, the two predominant fusion proteins present in APL.
In particular, we measured the binding properties of the two endogenous ligands t-RA and 9-cis-RA to bcr1- and bcr3-PML/RAR isoforms. By Scatchard analysis we showed that t-RA binds to the bcr3-PML/RAR isoform with slightly less affinity (Kd = 0.4 nmol/L) than to bcr1 PML/RAR or RAR (Kd = 0.13 nmol/L or 0.09 nmol/L, respectively). A relatively lower t-RA binding specificity for the bcr3-PML/RAR could also be measured in competitive binding analysis. Moreover, these experiments showed that 9-cis-RA is able to bind with about eightfold higher specificity to bcr3-PML/RAR than to RAR or bcr1-PML/RAR (EC50 values of 4.8 nmol/L, 33.5 nmol/L, and 37.5 nmol/L, respectively). This finding was also supported by saturation kinetics studies which indicated that [3H]-9-cis-RA binds with a lower affinity to the bcr1-PML/RAR than to the bcr3-PML/RAR. Two sites of 9-cis-RA binding with different affinities were detectable in the bcr3-PML/RAR fusion product. In fact, for bcr1-PML/RAR a Kd value of 0.77 nmol/L was measured, whereas two Kd values of 0.45 nmol/L and 0.075 nmol/L could be calculated for bcr3-PML/RAR.
Both PML/RAR isoforms retain the RING domain of PML which is probably involved in protein-protein interactions and regions involved in DNA binding.49 However, the bcr3-PML/RAR product lacks the C-terminal proline/serine-rich region of PML.7 It has been shown that human RAR contains overlapping but distinct binding pockets for t-RA and 9-cis-RA in a region which corresponds to the ligand-dependent transcriptional activation function 2 (AF-2 ) domain.51 This region in RAR is predicted to form an amphipatic -helix that is well conserved among nuclear receptors.52 Using alanine scanning mutagenesis, specific amino acids that constitute the high-affinity binding of 9-cis-RA have been defined within this region.53
Our hypothesis of functional differences between the two isoforms is also supported by the fact that different protein complexes are probably formed by the two PML/RAR fusion products. At variance to RAR,20,31 both PML/RAR isoforms were found in high molecular nuclear complexes by HPLC size exclusion analysis of nuclear extracts prepared from bcr1-PML/RAR and bcr3-PML/RAR transiently transfected COS-1 cells. However, the bcr3-PML/RAR product was present only in protein-protein complexes as shown by the absence of any RA-specific binding corresponding to the monomeric form of this protein either in COS-1 transfected cells as well as in APL patients nuclear extracts. In similar experimental conditions, RA-specific binding activity corresponding to the monomeric form of the bcr1-PML/RAR product was always measurable in NB4 and in COS-1 transfected cells.20,38 In addition, identical HPLC profiles were obtained if nuclear extracts from mock, RAR, bcr1-, and bcr3-PML/RAR COS-1 transfected cells were labeled with [3H]-9cis-RA (data not shown).
It has been shown that both PML/RAR and PLZF/RAR fusion proteins contain identical portions of RAR and retain the ability to act as RA-dependent transcription factors, to bind to RA-response elements, and to antagonize the function of the wild-type RAR.5-9,54 Therefore, it appears that they both have the potential to directly influence the RAR-dependent endogenous pathway that control terminal myeloid differentiation. The introduction of PLZF/RAR cDNA into COS-1 cells by transient transfection resulted in high levels of nuclear t-RA specific binding activity. The affinity of t-RA binding to PLZF/RAR was similar to that of PML/RAR. However, APL patients with the t(11; 17) expressing the PLZF/RAR are refractory to t-RA-induced differentiation9,10,54 and the expression of PLZF/RAR in hematopoietic precursor cells blocks terminal differentiation (M. Ruthardt and P.G. Pelicci, manuscript submitted). Therefore, it seems that despite PML/RAR and PLZF/RAR having similar retinoid-binding properties they have different effects in vivo on the RA-signaling pathway. The biochemical consequences of PML or PLZF sequences on the in vivo activity of the two RAR fusion proteins might differ and underlie their different biologic activities. However, the presence of both PML/RAR and PLZF/RAR in similar high-molecular-weight nuclear complexes suggests the involvement of these complexes in the block of promyelocytic differentiation.
Previous reports indicate only minor differences between the two PML/RAR isoforms in the activation of various RAREs in a variety of target cells.5-8 In this study we extended these previous findings, most of which had been obtained using a single t-RA concentration, by analyzing the t-RA and 9-cis-RA dose-dependent activation of different RAREs in RAR, bcr1-PML/RAR and bcr3-PML/RAR COS-1 transfected cells. We found that bcr1-PML/RAR and bcr3-PML/RAR overexpressed in COS-1 cells had similar alterations in activating (RARE)3-tk-LUC and RAR pr-LUC when compared with wild-type RAR. However, the higher affinity and specificity of 9-cis-RA binding to the bcr3-PML/RAR product resulted in an increased ability (about 10-fold) of this fusion product to specifically transactivate the TRE2-tk-LUC. It should be noted that the TRE is usually present in few RA target genes and is less responsive to RA than the DR-5 motif, thus far the strongest and the most abundant class of RARE present in the promoter of the RAR and in the promoters of several RA-responsive genes.43,55 In agreement with these results, we found the 9-cis-RA to be more potent than t-RA in the induction of differentiation of blast cells from two bcr3-PML/RAR APL patients in culture (data not shown).
In conclusion, our results indicate that distinct functional properties are associated with the two major PML/RAR isoforms. This in turn may provide a rationale for targeting differential retinoid therapy at the specific molecular lesion of APL patients.
| |
NOTE ADDED IN PROOF |
The manuscript by M. Ruthardt and P.G. Pelicci listed as submitted is now in press (Ruthardt M, Testa U, Nervi C, Ferrucci PF, Grignani F, Puccetti E, Grignani F, Peschle C, Pelicci PG: Mol Cell Biol, 1997).
| |
FOOTNOTES |
Submitted August 7, 1996;
accepted March 7, 1997.
Supported by grants from the European Economic Community (Biomed and Biotech), from the Associazione Italiana per la Ricerca sul Cancro (AIRC), from Ministero Università e Ricerca Scientifica e Tecnologica (MURST), and from CNR special project A.C.R.O., CT 95.00.454.39.
Address reprint requests to Clara Nervi, MD, PhD, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via A. Scarpa 14, 00161 Rome, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hearly marked
``advertisment'' in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr. P. Chambon for providing RAR antibody and RARs cDNA and Dr R.M. Evans for providing RXRs cDNA. The RAR pr-luc was kindly provided by Dr A. Dejean.
| |
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Abstract
Introduction
Abstract