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

Bianca Maria Scicchitano, L Benedetti, A. A Levin, F Grignani, G Allenby, D Diverio, F Lo Coco, G Avvisati, M Ruthardt, S Adamo, P. G Pelicci, C. Nervi

Risultato della ricerca: Contributo in rivistaArticolo in rivista

34 Citazioni (Scopus)

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
Lingua originaleEnglish
pagine (da-a)1175-1185
Numero di pagine11
RivistaBlood
Volume90
Stato di pubblicazionePubblicato - 1997

Keywords

  • Animals
  • Antineoplastic Agents
  • Binding, Competitive
  • COS Cells
  • Chromosomes, Human, Pair 15
  • Chromosomes, Human, Pair 17
  • DNA-Binding Proteins
  • Gene Expression Regulation, Leukemic
  • Humans
  • Kruppel-Like Transcription Factors
  • Leukemia, Promyelocytic, Acute
  • Neoplasm Proteins
  • Oncogene Proteins, Fusion
  • Prognosis
  • Protein Binding
  • Recombinant Fusion Proteins
  • Remission Induction
  • Structure-Activity Relationship
  • Transcription Factors
  • Transcription, Genetic
  • Transfection
  • Translocation, Genetic
  • Tretinoin

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