Characterization of the Retinoid Binding Properties of the Major Fusion Products Present in Acute Promyelocytic Leukemia Cells

Abstract

AbstractThe bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor α (PML/RARα) 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 RARα, 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/RARα products. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RARα product has been found associated with a poorer prognosis than bcr1-PML/RARα. In the present study we have investigated the structural and functional properties of the bcr3-PML/RARα in comparison to the previously characterized bcr1-PML/RARα. 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/RARα APL patients and from bcr3-PML/RARα 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/RARα receptor than to bcr1-PML/RARα or RARα (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/RARα product but not in the bcr1-PML/RARα product (Kd = 0.77 nmol/L). By competition binding experiments we showed that 9-cis-RA binds with higher specificity to the bcr3-PML/RARα isoform than to the bcr1-PML/RARα or RARα. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RARα product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the βRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RARα products can be measured.