TY - JOUR
T1 - Characterization of the retinoid binding properties of the major fusion products present in acute promyelocytic leukemia cells
AU - Benedetti, L
AU - Levin, A. A
AU - Scicchitano, Bianca Maria
AU - Grignani, F
AU - Allenby, G
AU - Diverio, D
AU - Lo Coco, F
AU - Avvisati, G
AU - Ruthardt, M
AU - Adamo, S
AU - Pelicci, P. G
AU - Nervi, C.
PY - 1997
Y1 - 1997
N2 - 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
AB - 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
KW - Animals
KW - Antineoplastic Agents
KW - Binding, Competitive
KW - COS Cells
KW - Chromosomes, Human, Pair 15
KW - Chromosomes, Human, Pair 17
KW - DNA-Binding Proteins
KW - Gene Expression Regulation, Leukemic
KW - Humans
KW - Kruppel-Like Transcription Factors
KW - Leukemia, Promyelocytic, Acute
KW - Neoplasm Proteins
KW - Oncogene Proteins, Fusion
KW - Prognosis
KW - Protein Binding
KW - Recombinant Fusion Proteins
KW - Remission Induction
KW - Structure-Activity Relationship
KW - Transcription Factors
KW - Transcription, Genetic
KW - Transfection
KW - Translocation, Genetic
KW - Tretinoin
KW - Animals
KW - Antineoplastic Agents
KW - Binding, Competitive
KW - COS Cells
KW - Chromosomes, Human, Pair 15
KW - Chromosomes, Human, Pair 17
KW - DNA-Binding Proteins
KW - Gene Expression Regulation, Leukemic
KW - Humans
KW - Kruppel-Like Transcription Factors
KW - Leukemia, Promyelocytic, Acute
KW - Neoplasm Proteins
KW - Oncogene Proteins, Fusion
KW - Prognosis
KW - Protein Binding
KW - Recombinant Fusion Proteins
KW - Remission Induction
KW - Structure-Activity Relationship
KW - Transcription Factors
KW - Transcription, Genetic
KW - Transfection
KW - Translocation, Genetic
KW - Tretinoin
UR - http://hdl.handle.net/10807/97038
M3 - Article
SN - 0006-4971
VL - 90
SP - 1175
EP - 1185
JO - Blood
JF - Blood
ER -