Fig 1.
Clonal expansion of PML-RARA mutant cells during APL progression.
(A) Sequence analyses of the RARA region of PML-RARA in the RA-resistant patient’s cells. (B) Schematic diagram of PML-RARA. The mutated amino acid residues in the patient’s samples are indicated by red arrows. (C, D) Patient’s clinical course and frequency of mutated PML-RARA transcripts in the patient’s samples during APL treatment: at diagnosis ①, after induction therapy ②, at molecular relapse ③, after high-dose cytarabine therapy ④, after allogenic hematopoietic stem cell transplantation ⑤, and at the terminal stage ⑥.
Fig 2.
The deletion mutation abrogates RA-induced APL cell differentiation.
(A) The expression of PML-RARA protein was examined by Western blot analysis with anti-FLAG antibody in established Wt-PML-RARA and mutant- PML-RARA expressing cells. (B) Cell growth assay in established Wt-PML-RARA and mutant-PML-RARA expressing cells. (C, D) Mean fluorescence intensity (MFI) ratios of CD11b to isotype controls in ATRA- (C) and tamibarotene- (D) treated Wt-PML-RARA and mutatnt-PML-RARA expressing cells. The cells were treated with 1 μM of ATRA or tamibarotene for seven days. Error bars represent mean values ± S.D. of at least three independent experiments. (E) Frequencies of NBT reduction of neutrophils. Cells were treated with 1 μM ATRA or tamibarotene for seven days. Error bars represent mean values ± S.D. of at least three independent experiments.
Fig 3.
PML-nuclear body formation is impaired in deletion mutant PML-RARA-expressing cells.
(A) PML/RARA degradation upon RA treatment in Wt-PML-RARA- and mutant-PML-RARA-transduced cells was analyzed by Western blot using anti-FLAG antibody. The cells were treated with 1 μM of ATRA or tamibarotene for 12 hours. (B) Localization of PML and PML-nuclear body formation was evaluated by immunofluorescence staining in Wt-PML-RARA- and mutated- PML-RARA-transduced HL-60 cells. The cells were treated with 1 μM of ATRA or tamibarotene for seven days.
Fig 4.
The deletion mutation alters PML-nuclear body formation by RA in primary APL cells.
Localization of PML and PML-nuclear body formation was evaluated by Immunofluorescence staining performed with the patient’s APL cells. The cells were treated with 1 μM ATRA or tamibarotene for seven days.