Molecular Mechanism Underlying The Formation Of PML Nuclear Bodies In APL Cells By Leptomycin B

Objective:(1)To reveal the molecular mechanism of PML nuclear bodies(PML-NBs)formation by nuclear export inhibitor Leptomycin B(LMB)in acute promyelocytic leukemia(APL)cells;(2)To identify what kind of protein involves in formation of PML protein and PML-NBs in APL cells after LMB treatment.Methods:(1)NB4 cells or primary APL blast cells were treated with three different nuclear export inhibitors(e.g.,LMB,KPT-185,TBB)for 12 h,and the the morphological changes and distribution of PML protein and PML-NBs were detected by immunofluorescence microscopy;(2)PML-RAR? protein was overexpressed in HeLa and PML knockout HeLa cells(PML-/-)and then treated with LMB.The morphological changes of PML-NBs were determined by immunofluorescence microscopy;(3)GFP-PML-I~VI and Flag-PML-RAR?were co-transformed into PML-/-HeLa cells,and changes in PML-NBs in cells were determined by immunofluorescence microscopy;(4)GFP-PML-I-?NES(deletion of NES)was co-transfected with PML-RAR? into PML-/-HeLa cells,and the morphological changes of PML-NBs were determined by the same method as mentioned above;(5)Protein expression was detected by western-blot,and the Image J was used to quantify levels of the protein expression and PML-NBs formation.Results:(1)Three nuclear export inhibitors such as LMB,KPT-185 and TBB could induce the restoration of PML-NBs in NB4 cells and APL blast cells,indicating that LMB probably inhibiting some key nuclear shuttle proteins andresults in restoration of PML-NBs in APL cells;(2)the formation of PML-NBs was only detected only in wild type HeLa(i.e.,PML+/+)cells but not in PML-/-HeLa cells after LMB treatment,implying that endogenous PML might be played a critical role for restoration of PML-NBs in APL cells;(3)We identified that PML-I was most important protein to form PML-NBs among the all PML isoforms after treatment of LMB;(4)Additionally,PML-I protein in the nucleus was significantly increased by LMB as compared to the control group,indicating that increasing of PML-1 by LMB promote the restoration of PML-NBs,which is consistent with the data that co-transfection of GFP-PML-I-?NES(deletion of NES)with PML-RAR? into PML-/-HeLa cells.Conclusion: LMB induced restoration or formation of PML-NBs are predominantly dependent on PML-I protein accumulation in nuclear,which increase the amount of PML protein and lead to the formation of PML-NBs in APL cells.