Figure 4. Inhibition of Pin1 by ATRA or other compounds causes PML-RARα degradation and treats APL in cell and mouse models and even human patients.
(a and b) NB4 cells were treated with ATRA, various Pin1 inhibitors, RAR inhibitor or RAR activator for 72 h, followed by IB for PML-RARα and Pin1 (b), or Giemsa staining (b, upper panels) or FACS with CD14 and CD11b (b, lower panels) for detecting APL cell differentiation. Scale bar, 10μM.
(c–e) Sublethally irradiated C57BL/6J mice were transplanted with 1 × 106 APL cells isolated from hCG-PML-RARα transgenic mice and, 5 days later, treated with ATRA-releasing implants, EGCG, Juglone or placebo for 3 weeks, followed by determining APL cell differentiation status with Giemsa staining (upper) or FACS with Gr-1 and Mac-1 (lower) (c), PML-RARα and Pin1 expression in the bone marrow (d), and the size of the spleen in mice (e) (mean ± s.d. of ten mice). Mac-1 is the same to CD11b. Scale bar, 10μM.
(f–h) Bone marrow samples from healthy controls (n=24) or APL patients before (n=19) or after the treatment with ATRA for 3 (n=3) or 10 days (n=3) or APL patients in complete remission (n=17) were immunostained with anti-Pin1 and anti-PML antibodies (f). Relative levels of Pin1 (g) in the nucleus and PML-RARα in the nuclear plasma outside of the PML nuclear body (h) were semi-quantified (mean ± s.d.). Red arrows, PML-RARα/PML diffusely distributed to the entire nucleus in APL cells containing more Pin1; yellow arrows, PML-RARα/PML localized to the PML body in APL cells containing less Pin1. Scale bar, 5μM.
*P < 0.05, **P < 0.01, ***P < 0.001, as determined by Student’s t-test.