Novel mutation in the PML/RARalpha chimeric gene exhibits dramatically decreased ligand-binding activity and confers acquired resistance to retinoic acid in acute promyelocytic leukemia.
OBJECTIVE
All-trans retinoic acid (RA) resistance in acute promyelocytic leukemia (APL) has been a serious clinical problem in differentiation-inducing therapy. However, the mechanisms underlying acquired RA resistance in APL patients are not well understood.


METHODS
We recently established a spontaneous RA-resistant APL cell line (UF-1) from a patient and used this cell line as an excellent in vitro model for RA-resistant clinical situations. We investigated the structural and functional abnormalities of chimeric PML/RARalpha gene in UF-1 cells and preserved materials from the original patient.


RESULTS
A novel point mutation was detected in the ligand-binding (E) domain of the RARalpha portion of the PML/RARalpha gene in UF-1 cells. This mutation resulted in amino acid substitution of Arg611 (CGG) for Trp611 (TGG) in the short-form PML/RARalpha protein, which corresponded to Arg276 in wild-type RARalpha. Importantly, the same mutation was also detected in the preserved materials from the original patient. COS-1 cells were transiently transfected with cDNA encoding wild-type and mutant PML/RARalpha constructed by site-directed mutagenesis and performed RA-binding assay. Interestingly, RA-binding activity was dramatically decreased in the mutant PML/RARalpha compared with that of the wild-type chimeric protein, suggesting that this single amino acid substitution is critical for RA binding.


CONCLUSIONS
These results strongly suggest that a novel point mutation in the ligand-binding domain of the RARalpha portion (Arg611) of the chimeric PML/RARalpha gene decreased sensitivity to all-trans RA. We conclude that acquisition of the PML/RARalpha mutation is one possible mechanism for development of RA resistance in patients with APL in vivo.
Takayama
N
N
Division of Hematology, Keio University School of Medicine, Tokyo, Japan.
Kizaki
M
M
Hida
T
T
Kinjo
K
K
Ikeda
Y
Y
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000235:genetics*; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D014212:Tretinoin / Q000378:metabolism / Q000627:therapeutic use; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D008024:Ligands; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D014212:Tretinoin