Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein.
All-trans-retinoic acid (RA) treatment induces morphological remission in acute promyelocytic leukemia (APL) patients carrying the t(15;17) and expressing the PML/RARalpha product by inducing terminal differentiation of the leukemic clone. RA treatment induces downregulation of PML/RARalpha and reorganization of the PML-nuclear bodies. These events have been proposed to be essential for the induction of APL cell differentiation by RA. Here, we show that in the APL-derived NB4 cell line as well as in myeloid precursor U937 cells expressing the PML/RARalpha (U937/PR9) and in blasts from APL patients, the PML/RARalpha fusion protein is cleaved by a caspase 3-like activity induced by RA treatment. In fact, a caspase 3-like activity is detectable in PML/RARalpha expressing cells after RA treatment, and selective caspase inhibitor peptides are able to prevent the RA-induced degradation of the fusion protein in vivo and in vitro. Using recombinant caspases and PML/RARalpha deletion mutants we mapped a caspase 3 cleavage site (Asp 522) within the alpha-helix region of the PML component of the fusion protein. The extent of PML/RARalpha cleavage directly correlates with the ability of RA to restore the normal PML nuclear bodies (NBs) pattern. However, RA-induced differentiation is not prevented by the persistence of the fusion product and occurs in the absence of normally structured PML NBs. These results indicate that PML/RARalpha is directly involved in conferring RA sensitivity of APL cells and that the RA-induced reassembly of PML NBs is the consequence of the disappearance of PML/RARalpha.
Nervi
C
C
Dipartimento di Istologia ed Embriologia Medica and Dipartimento di Biotecnologie Cellulari e Ematologia, University of Rome "La Sapienza," Rome. nervi@axrma.uniromal.it
Ferrara
F F
FF
Fanelli
M
M
Rippo
M R
MR
Tomassini
B
B
Ferrucci
P F
PF
Ruthardt
M
M
Gelmetti
V
V
Gambacorti-Passerini
C
C
Diverio
D
D
Grignani
F
F
Pelicci
P G
PG
Testi
R
R
D000970:Antineoplastic Agents / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases; D002454:Cell Differentiation / Q000187:drug effects; D002467:Cell Nucleus / Q000648:ultrastructure; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D004790:Enzyme Induction; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism* / Q000473:pathology; D009097:Multienzyme Complexes / Q000378:metabolism; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D046988:Proteasome Endopeptidase Complex; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D000970:Antineoplastic Agents; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; C070576:PML protein, human; D014212:Tretinoin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex