CHARACTERIZATION OF THE RETINOID-BINDING PROPERTIES OF THE MAJOR FUSION PRODUCTS PRESENT IN ACUTE PROMYELOCYTIC LEUKEMIA-CELLS

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alph a (PML/RAR alpha) are the two major fusion proteins expressed in acute promyelocytic leukemia (APL) patients. These proteins, which are pres ent in different lengths of PML (amino acids 1-552 and 1-394, respecti vely), contain most of the functional domains of PML and RAR alpha, bi nd all-trans-retinoic acid (t-RA), and act as t-RA-dependent transcrip tion factors, T-RA is an effective inducer of clinical remission only in patients carrying the t(15;17) and expressing the PML/RAR alpha pro ducts. However, in APL patients achieving complete remission with t-RA therapy the bcr3-PML/RAR alpha product has been found associated with a poorer prognosis than bcr1-PML/RAR alpha. In the present study we h ave investigated the structural and functional properties of the bcr3- PML/RAR alpha in comparison to the previously characterized bcr1-PML/R AR alpha. In particular, we have measured the binding properties of th e 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 alpha APL patients and from bcr3-PML/RAR alpha COS-1 tran sfected cells indicates that this protein is present only as high-mole cular-weight nuclear complexes. Using saturation binding assays and Sc atchard analyses we found that t-RA binds with slightly less affinity to the bcr3-PML/RAR alpha receptor than to bcr1-PML/RAR alpha or RAR a lpha (Kd = 0.4 nmol/L, 0.13 nmol/L or 0.09 nmol/L, respectively). More over, two different high-affinity 9-cis-RA binding sites (Kd = 0.45 an d 0.075 nmol/L) were detectable in the bcr3-PML/RAR alpha product but not in the bcr1-PML/RAR alpha product (Kd = 0.77 nmol/L). By competiti on binding experiments we showed that 9-cis-RA binds with higher speci ficity to the bcr3-PML/RAR alpha isoform than to the bcr1-PML/RAR alph a or RAR alpha. Consistent with these data, the binding of 9-cis-RA to the bcr3-PML/RAR alpha product resulted in increased transcriptional activation of the RA-responsive element (RARE) TRE, but not of the bet a RARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the differen t PML/RAR alpha products can be measured. (C) 1997 by The American Soc iety of Hematology.