The Toxicity Of Arsenical Active Metabolites And The Contribution Of Removing Arsenic Trioxide To APL Relapse

Objective:Part one:In order to better understand the different effects of the three arsenic compounds, namely arsenic trioxide ?As₂O₃? and its active intermediate metabolites ?i.e., trivalent monomethylarsonous acid ?MMA^?? and dimethylarsinous acid ?DMA^??? onthe induction of apoptosis of acute promyelocytic leukemia ?APL? cells NB4, we have compared the induction of endoplasmic reticulum ?ER? stress in NB4 cells after exposure to the three arsenic species ?i.e., iAs^?, MMA^? and DMA^??.Part two:We futher investigated the effects of three arsenic species on the induction of SUMOlytion in PML-transfected 293T cells. Moreover, we also determined the changes of the solubility and modification of PML or PML-RAR? protein in 293T and HeLa cells by removing iAs^? from PML- or PML-RAR?-transfected cells ?e.g.,293T and HeLa cells?.Methods:Acute promyelocytic leukemia ?APL? NB4 cells,293T cells, HeLa cells, and three arsenic species ?i.e., iAs^?, MMA^?, DMA^??, PMLIV plasmid, PML? plasmid, PML-RARa plasmid were used in the current study. Flow cytometry was applied to measure the apoptosis. Western blot was used to detect the expressions of apoptosis-related ?e.g., cleaved-PARP and cleaved caspase 3? and ER stress-related protein such as PERK, JNK, eIF2?, ATF4, CHOP, ASK1. JNK inhibitor and CHOP short hairpin RNA ?shRNA? were used to evaluate the effect of arsenicals on induction of the ER stress in NB4 cells.Regarding the transfction expereminent,293T or HeLa cells were transfected with Flag-PML ?? or ?? or Flag-PML-RARa. Cells were lysed with RIPA buffer, and then separated intosoluble ?Supernatant? and insoluble fractions ?Pellet?.Western blot was used to detect the changes of the solubility and modification of PML or PML-RARa protein.Results:Part one:Inorganic iAs^? and MMA^? have shown no appreciable effects on induction of ER stress, while DMA^? significantly induced ER stress in NB4 cells at the low dose. Especially, the phosphorylated protein kinase-like endoplasmic reticulum kinase ?PERK? was significantly activated along with induced phosphorylated JNK. More interestingly, inhibition of p-JNK was able to significantly prevente cell death, while CHOP shRNA did not significantly decrease the apoptosis in NB4 cells following exposure to DMA^?, suggesting the JNK signaling pathwaywas predominately involved in induction of ER stress by DMA^?.Part two:Interestingly, among the three arsenic compounds ?e.g., iAs^?, MMA^?, DMA^??, only iAs^? was observed to induce a shift of PML protein from supernatants to insoluble fractions in a concentration-dependent manner, while no changes were observed following exposure to MMA^? and DMA^?. When iAs^? was removed from PML-or PML-RARa-transfected cells, we found the PML or PML-RARa protein could transfer from pellet to soluble faction. Thus, further study needs to be clearly identified.Conclusions:Part one:DMA^? could significantly induce ER stress in NB4 cells in comparison with other two arsenic species iAs^? and MMA^? at the low dose?1?m-M?, and it has been confirmed that DMA^? induced apoptosis in NB4 cells mainly through ASK1-JNK pathway.Part two:Among the three arsenic compounds ?e.g., iAs^?, MMA^?, DMA^??, only iAs^? was able to induce the transfer of the PML or PML-RARa protein from the diffuse nuclear fraction of the nucleoplasm ?RIPA soluble fraction? to the nuclear matrix-associated NBs ?RIPA resistant fraction?. In addition, iAs^? could promote the modification of the PML or PML-RARa proteins. After removing i Asm, the PML or PML-RARa protein could transfer from the pellet to soluble faction.