Page 117 - 2019_01-Haematologica-web

2-Bromopalmitate promotes APL differentiation
ABCD
EFGH
Figure 4. 2BP stabilizes RARα protein and enhances transcriptional activity of RARα. (A-D) NB4(A-B) and HL60(C-D) cells were incubated with 5 mM of 2BP for indi- cated hours. The mRNA (B and D) and protein (A and C) of RARα were detected by quantitative real-time PCR and western blots. β-actin was detected as the loading control. P value between the line-pointed group is shown. (E) NB4 and HL60 cells were incubated with 5 mM of 2BP for indicated hours and protein of RXRα was detected by western blot with β-actin as the loading control. (F-G) ATRA-sensitive NB4 and HL60 (F) or ATRA-resistant NB4-MR2, NB4-LR1, and NB4-LR2 cells (G) were treated with 5 mM 2BP in the presence or absence of ATRA for 48 hours and RARα protein was detected by western blot. The protein bands on the gels were quantified by densitometry from three independent experiments and shown in the bottom panel (A,C,E,F and G). Scanning was performed at optimal exposure time where band intensity was proportional to the concentration of protein present. Gel photographic images were stored as GRAYSCALE pictures in TIF format and were processed using ImageJ Software. P value between the line-pointed group is shown. (H) NB4 cells were treated with 5 mM 2BP and/or 10-8M ATRA for 48 hours, and the expression of indicated genes was detected by quantitative real-time PCR with specific primers. ** and *** indicated P value between 2BP plus ATRA and ATRA was < 0.01 and < 0.001, respectively. Each experiment was repeated three times.
tion (Figure 3A and Online Supplementary Figure S5). In con- trast, ATRA combined with 2BP could induce granulocytic differentiation of NB4-MR2 (Figure 3A-B), NB4-LR1 and NB4-LR2 (Online Supplementary Figure S5) cells as evi- denced by mature granulocytic morphologic features and increased CD11b and CD11c expression, indicating that 2BP could overcome ATRA resistance of promyelocytes.
In vivo, ATRA-resistant leukemic blasts from transgenic mice expressing human MRP8-PML-RARα mutant were intravenously transplanted into sublethally irradiated iso- genic FVB/N recipients to generate ATRA-resistant leukemic mice.33 Twenty-six days later, all vehicle-treated leukemic mice died abruptly (Figure 3C). ATRA-treated leukemic mice died in 27 days, verifying that the ATRA resistance was lethal for leukemic mice. In contrast, administration of 2BP along with ATRA overcame the resistance and extended the survival of leukemic mice to 36 days (Figure 3C). It was discovered that 2BP with ATRA facilitated the remission of swollen spleen on ATRA-resistant leukemic mice (Figure 3D). Consistent with this, more morphologically differentiated cells were observed in PB and BM in the ATRA/2BP-treated group (Figure 3E). These data indicated that 2BP presents a potential to overcome ATRA resistance.
2BP accumulates RARα protein and enhances ATRA-dependent transcriptional activity of RARα
As previously documented, administration of ATRA induced a progressive degradation of wild-type RARα as well as the PML-RARα chimeric protein.14,15 To elucidate the mechanism of the positive effect of 2BP on APL cell differentiation, RARα and PML-RARα fusion protein in NB4 cells under 2BP treatment were detected. Treatment of 2BP for 48 hours significantly increased RARα protein level (Figure 4A), but not its mRNA level in NB4 (Figure 4B) and HL60 cells (Figure 4C-D). In contrast, RXRα, the binding partner of RARα protein in transactivating target genes as heterodimers, was not affected by 2BP (Figure 4E). Furthermore, we detected the effects of 2BP on RARα protein in the presence of ATRA, which was documented to induce degradation of RARα.34 When NB4 and HL60 cells were cotreated with ATRA and 2BP, the protein level of RARα was increased compared to ATRA-treated group (Figure 4F). Moreover, accumulated RARα protein induced by 2BP in the absence (MR2 and LR1) or presence of ATRA (MR2 and LR2) could also be seen in ATRA resist- ance NB4-MR2, NB4-LR1 or NB4-LR2 cells (Figure 4G). In addition, using immunofluorescent staining with anti- PML antibody, we observed that ATRA restored the PML-
haematologica | 2019; 104(1)
107