Reciprocal amplification of caspase-3 activity by nuclear export of a putative human RNA-modifying protein, PUS10 during TRAIL-induced apoptosis

Pus10 is a pseudouridine synthase present in Archaea and Eukarya, but not in Bacteria and yeast. It has been suggested that the human PUS10 (DOBI) gene is needed during TRAIL-induced apoptosis. We analyzed the role of PUS10 in TRAIL-induced apoptosis by immunofluorescence, immunoblotting and several indicators of apoptosis. We examined several TRAIL-sensitive cell lines and we also examined some resistant cell lines after treatment with cycloheximide. PUS10 is mainly present in the nucleus. Early during apoptosis, PUS10 translocates to mitochondria via CRM1-mediated export with the concurrent release of cytochrome c and SMAC. Caspase-3 is required for PUS10 translocation, which reciprocally amplifies the activity of caspase-3 through the intrinsic/mitochondrial pathway. This suggests that in addition to cytoplasmic factors, nuclear factors also have a direct role in the major apoptosis pathways. However, p53 is not involved in TRAIL-induced PUS10 movement. The caspase-3-mediated movement of PUS10 and the release of mitochondrial contents enhancing caspase-3 activity creates a feedback amplification loop for caspase-3 action. Therefore, any defect in the movement or interactions of PUS10 would reduce the TRAIL sensitivity of tumor cells.

(E) HuP10 is present in the nucleus but not in the cytoplasm. IB analysis of nuclear (NE) and cytoplasmic (CE) fractions of PC3 cells using anti-HuP10 antibody. Note: the unknown protein observed in (D) is present in both nucleus and cytoplasm.
(F) The anti-HuP10 antibody recognizes recombinant His-tagged HuP10. IB analysis of cell lysates of un-induced and induced E. coli cells containing recombinant HuP10 and Ni-affinity purified recombinant HuP10 using anti-HuP10 antibody.
(G) HuP10 present in human cells is larger than recombinant HuP10 (~60 kDa) purified from E. coli cells. IB analysis of cell lysate of PC3 cells and purified recombinant HuP10 using anti-HuP10 antibody. Positions of two size markers are indicated on the side.
(I) Determination of the specificity of anti-HuP10 for IF studies. Anti-HuP10 incubated with recombinant HuP10 was used for an immunofluorescence study similar to the one in Figure 1A.
Untreated antibody was used as control. Mito Tracker and DAPI staining are also as in Fig.1A. The MTT assay showed a significant reduction in cell viability when HeLa (B) and LNCaP (E) cells were exposed to CHX plus TRAIL together. Values are mean ± SE (n=3). In (B) ***=p<0.001 vs untreated control and TRAIL alone, and # =p0.001 vs CHX alone. In (E) ***=p<0.001 vs untreated control, and # =p0.001 vs TRAIL and CHX alone. PC3 and MDA-MB-231 cells were treated with different concentrations of LMB for 12 and 5 h, respectively. Cell viability was then determined by MTT assays in three independent experiments with three replicates each. ** and *** are p<0.01 and p<0.001, respectively, vs untreated (0 ng/ml).

Supplementary Figure S6. Effects of caspase inhibitors on TRAIL-induced HuP10 translocation and apoptosis.
(A) IF analyses of PC3 cells growing on coverslips at 80% confluency treated simultaneously with inhibitors of two initiator caspases (-8 and -9, -8 and -10, or -9 and -10), 40 µM each for 3 h followed by 12 h TRAIL (0.5 µg/ml, plus inhibitors) treatment. Staining as in Fig. 1A. These inhibitor treatments do not block the transfer of HuP10 to the cytoplasm (arrows). Bars = 10 µm. (A) The MTT assay shows significant difference in MCF7 cell viability after 12 h simultaneous exposure to TRAIL (0.5 µg/ml) and CHX (0.5 µM) when compared to untreated (control) or treatment with TRAIL or CHX alone. Values are presented as mean ± SE (n=3). ***=p <0.001 individually vs control or TRAIL or CHX treatment.

(B) IB analysis of the lysates of the MCF7 cells treated individually with TRAIL and CHX alone
or combined for 6 h and 12 h using anti-PARP antibody to determine PARP cleavage. ß-actin was used as a loading control. See also Fig. S4H.
(C) Nuclear (NE) and cytoplasmic (CE) fractions of untreated and TRAIL+CHX-treated (12 h) MCF7 cells were analyzed by IB using anti-HuP10 antibody. Nuclear and cytoplasmic markers were lamin A and tubulin, respectively. ß actin was used as a loading control.