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Characterization of the Retinoid Binding Properties of the Major Fusion Products Present in Acute Promyelocytic Leukemia Cells
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.
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.
Blood, Vol. 90 No. 3 (August 1), 1997:
pp. 1175-1185
© 1997 by The American Society of Hematology.
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