Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells.

Benedetti, Laura, Levin, Arthur A., Scicchitano, Bianca M., Grignani, Francesco, Allenby, Gary, Diverio, Daniela, Lo Coco, Francesco, Avvisati, Giuseppe, Ruthardt, Martin ORCID: https://orcid.org/0000-0003-1021-3811, Adamo, Sergio, Pelicci, Pier Giuseppe and Nervi, Clara 1997. Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells. Blood 90 (3) , pp. 1175-1185. 10.1182/blood.V90.3.1175

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Abstract

The bcr1- and bcr3- promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR alpha) 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 alpha, 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 alpha products. 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 have investigated the structural and functional properties of the bcr3-PML/RAR alpha in comparison to the previously characterized bcr1-PML/RAR alpha. 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 alpha APL patients and from bcr3-PML/RAR alpha 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 alpha receptor than to bcr1-PML/RAR alpha or RAR alpha (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 alpha product but not in the bcr1-PML/RAR alpha 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 alpha isoform than to the bcr1-PML/RAR alpha 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 betaRARE, in transiently transfected COS-1 cells. These results provide evidence indicating that preferential retinoid binding to the different PML/RAR alpha products can be measured.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine
Publisher:	American Society of Hematology
ISSN:	0006-4971
Date of Acceptance:	7 March 1997
Last Modified:	01 Mar 2024 14:45
URI:	https://orca.cardiff.ac.uk/id/eprint/166042

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