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 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.
Benedetti
L
L
Department of Histology and Medical Embryology, University La Sapienza, Rome, Italy.
Levin
A A
AA
Scicchitano
B M
BM
Grignani
F
F
Allenby
G
G
Diverio
D
D
Lo Coco
F
F
Avvisati
G
G
Ruthardt
M
M
Adamo
S
S
Pelicci
P G
PG
Nervi
C
C
D000077556:Alitretinoin; D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D001667:Binding, Competitive; D019556:COS Cells; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics / Q000648:ultrastructure; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000145:classification / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000145:classification / Q000378:metabolism*; D011379:Prognosis; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012074:Remission Induction; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014178:Translocation, Genetic; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; C092037:ZBTB16 protein, human; D000077556:Alitretinoin; D014212:Tretinoin