title: Mutant AF-2 domain of PML-RARalpha in retinoic acid-resistant NB4 cells: differentiation induced by RA is triggered directly through PML-RARalpha and its down-regulation in acute promyelocytic leukemia.
abstract: To study the molecular mechanism of the differentiation induced by retinoic acid (RA) in acute promyelocytic leukemia (APL), we established a new RA-resistant NB4 subline, NB4/RA. The NB4/RA cells were neither differentiated by a single or a combination of RA isoforms, nor by the addition of clotrimazole (P450-inhibitor) or interferon gamma. However, the combination of RA and 8-(4-chlorophenylthio) adenosine cyclic 3',5'-monophosphate (a cAMP analog, 8-CPT-cAMP) induced differentiation. Immunostaining of NB4/RA cells using anti-PML antibody showed a microgranular pattern which was not restored even by the combination of RA and 8-CPT-cAMP, whereas the microgranular pattern in NB4 cells was rapidly restored to the normal speckled pattern by RA. Western blot analysis revealed that RA alone or the combination with 8-CPT-cAMP did not down-regulate PML-RARalpha in NB4/RA cells, which was in contrast to NB4 cells. The PML-RARalpha fusion gene and transcript in NB4/RA cells were conserved as well as the RARalpha gene and transcripts. Sequence analysis of the PML-RARalpha transcript in NB4/RA cells indicated a Pro (CCG) to Leu (CTG) mutation at codon 900 (type L) in AF-2 domain, while the RARalpha transcript had a normal sequence. These data suggest that differentiation of APL by RA is triggered directly through PML-RARalpha, and is associated with its degradation. Furthermore, there might be another mechanism of differentiation which does not require the down-regulation of PML-RARalpha and the restoration of the PML-staining pattern.
authors.affiliation: Department of Infectious Diseases, Nagoya University Hospital, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kitamura
authors.forename: H
authors.initials: H
authors.lastname: Kiyoi
authors.forename: H
authors.initials: H
authors.lastname: Yoshida
authors.forename: H
authors.initials: H
authors.lastname: Saito
authors.forename: R
authors.initials: R
authors.lastname: Ohno
authors.forename: T
authors.initials: T
authors.lastname: Naoe
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics*; D001483:Base Sequence; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015151:Immunoblotting; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D017354:Point Mutation; D018168:Receptors, Retinoic Acid / Q000235:genetics; D000072482:Retinoic Acid Receptor alpha; D017422:Sequence Analysis, DNA; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D015703:Antigens, CD; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D014212:Tretinoin