MOLECULAR AND CELLULAR BIOLOGY, 0270-7306/97/$04.00+0 Aug. 1997, p. 4859–4869 Vol. 17, No. 8 Copyright © 1997, American Society for Microbiology Opposite Effects of the Acute Promyelocytic Leukemia PML-Retinoic Acid Receptor  (RAR) and PLZF-RAR Fusion Proteins on Retinoic Acid Signalling MARTIN RUTHARDT, 1 UGO TESTA, 2 CLARA NERVI, 3 PIER FRANCESCO FERRUCCI, 4 FRANCESCO GRIGNANI, 1 ELENA PUCCETTI, 4 FAUSTO GRIGNANI, 1 CESARE PESCHLE, 2,5 AND PIER GIUSEPPE PELICCI 1,4 * Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, 4 Istituto di Medicina Interna e Scienze Oncologiche, Policlinico Monteluce, Perugia University, 06100 Perugia, 1 Department of Haematology and Oncology, Istituto Superlore di Sanita `, 00161 Rome, 2 and Dipartmento di Istologia ed Embriologia Medica, University of Rome “La Sapienza,” 00161 Rome, 3 Italy, and Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541 5 Received 3 December 1996/Returned for modification 5 February 1997/Accepted 14 April 1997 Fusion proteins involving the retinoic acid receptor  (RAR) and the PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemias (APLs). APLs with the PML-RAR or the PLZF-RAR fusion protein are phenotypically indistinguishable except that they differ in their sensitivity to retinoic acid (RA)-induced differentiation: PML-RAR blasts are sensitive to RA and patients enter disease remission after RA treatment, while patients with PLZF-RAR do not. We here report that (i) like PML-RAR expression, PLZF-RAR expression blocks terminal differentiation of hematopoietic precursor cell lines (U937 and HL-60) in response to different stimuli (vitamin D 3 , transforming growth factor 1, and dimethyl sulfoxide); (ii) PML-RAR, but not PLZF-RAR, increases RA sensitivity of hematopoietic precursor cells and restores RA sensitivity of RA-resistant hematopoietic cells; (iii) PML-RAR and PLZF-RAR have similar RA binding affinities; and (iv) PML-RAR enhances the RA response of RA target genes (those for RAR, RAR, and transglutaminase type II [TGase]) in vivo, while PLZF-RAR expression has either no effect (RAR) or an inhibitory activity (RAR and type II TGase). These data demonstrate that PML-RAR and PLZF-RAR have similar (inhibitory) effects on RA-independent differentiation and opposite (stimulatory or inhibitory) effects on RA-dependent differentiation and that they behave in vivo as RA-dependent enhancers or inhibitors of RA-responsive genes, respectively. Their different activities on the RA signalling pathway might underlie the different responses of PML-RAR and PLZF-RAR APLs to RA treatment. The PLZF-RAR fusion protein contains an approximately 120-amino-acid N-terminal motif (called the POZ domain), which is also found in a variety of zinc finger proteins and a group of poxvirus proteins and which mediates protein-protein interactions. Deletion of the PLZF POZ domain partially abrogated the inhibitory effect of PLZF-RAR on RA-induced differentiation and on RA-mediated type II TGase up-regulation, suggesting that POZ-mediated protein interactions might be responsible for the inhibitory transcriptional activities of PLZF-RAR. Acute promyelocytic leukemia (APL) is cytogenetically characterized by a reciprocal translocation that always involves chromosome 17, with a break within the locus encoding for the retinoic acid receptor  (RAR). Chromosome partners are chromosome 15, with the breakpoint located within the PML locus, or, less frequently, chromosome 11, with the breakpoint within the PLZF locus (15, 51). The hybrid genes so formed encode a PML-RAR or PLZF-RAR fusion protein, which retain equivalent portions of RAR (5, 10, 22, 24, 42, 43). The 15;17 and 11;17 translocations are primary chromosome aberrations and are often the only cytogenetic anomalies in the neoplastic metaphases (37). Experimental evidence for leuke- mogenic potential, however, is available only for PML-RAR. PML-RAR blocks terminal differentiation of hematopoietic precursor cell lines in vitro (14, 16), and, in vivo, mice trans- genic for PML-RAR manifest myeloid differentiative alter- ations with the phenotypic features of promyelocytic leukemia (2a, 18). Despite the fact that PML-RAR and PLZF-RAR APLs are clinically indistinguishable, they differ in one important feature: their response to retinoic acid (RA). PML-RAR APL blasts are highly sensitive to RA, and most patients treated with RA achieve disease remission (9, 20, 33, 50). In contrast, PLZF-RAR-expressing APLs are not sensitive to RA treatment (13, 31). The mechanisms through which PML-RAR blocks differ- entiation are not well understood. The cellular localization of PML-RAR is anomalous with respect to the corresponding wild-type proteins: RAR is nuclear diffuse and PML is local- ized within specific subnuclear structures called PML nuclear bodies, whereas PML-RAR has a distinct nuclear distribution pattern (microspeckled localization) (12, 27, 52). It was initially proposed that PML-RAR exerts a dominant-negative action on wild-type PML and RXR, an RAR cofactor (26, 53, 54), in that expression of PML-RAR provokes PML and RXR de- localization within microspeckles (12, 27, 44, 52). This hypoth- esis, however, has been recently challenged by the demonstra- tion that delocalization of neither PML nor RXR is critical for the action of PML-RAR on differentiation (17). Nothing is known about the biological activity or cellular localization of the PLZF-RAR fusion protein. * Corresponding author. Mailing address: European Institute of On- cology, Department of Experimental Oncology, Via Ripamonti, 435, 20141 Milan, Italy. Phone: 39/2/57489 831. Fax: 39/2/574 89/851. E- mail: pgpelicci@ieo.cilea.it. 4859 on February 20, 2016 by guest http://mcb.asm.org/ Downloaded from