Figure 1.
Arsenic-induced degradation of mutant p53 protein is inhibited by MG132, an inhibitor of 26S proteasome.
Western blots were prepared with extracts from HaCaT (A) and MIA PaCa-2 (B) cells, which were untreated or pretreated with 4 µM MG132 for 2 h, and then untreated or treated with ATO for 6 h.
Figure 2.
Arsenic trioxide induces expression of Pirh2 E3 ligase.
(A) The level of Pirh2 transcript is increased by ATO. RT-PCR analysis was performed with total RNA isolated from MIA PaCa-2 and HaCaT cells untreated or treated with 7.5 µM ATO for 2–6 h. Actin mRNA was amplified as a loading control. (B) Western blots were prepared with extracts from MIA PaCa-2 and HaCaT cells untreated or treated as in (A), and then probed with antibodies against Pirh2 and actin, respectively.
Figure 3.
Ectopic expression of Pirh2 promotes arsenic-induced degradation of mutant p53 protein.
(A–B) Western blots were prepared with extracts from HaCaT (A) and MIA PaCa-2 (B) cells, which were transfected with pcDNA3 or pcDNA3-FLAG-Pirh2 for 24 h, and then treated with 5 or 7.5 µM ATO for 6 h. The blots were then probed with antibodies against FLAG tag, p53, and actin, respectively. (C) Schematic presentation of the Pirh2 protein along with locations of the Zn Finger and RING domains, two substitution mutations C145S and C148S in the RING domain (Pirh2-DN), and deletion of the RING domain in Pirh2 (Pirh2-ΔRING). (D–E) Ectopic expression of Pirh2-DN or Pirh2-ΔRING has little if any effect on the level of mutant p53. Western blots were prepared with extracts from HaCaT (D) and MIA PaCa-2 (E) cells, which were transfected with pcDNA3, pcDNA3-FLAG-Pirh2-DN, pcDNA3-FLAG-Pirh2-ΔRING, or pcDNA3-FLAG-Pirh2 for 24 h. The blots were then probed with antibodies against FLAG tagged Pirh2, p53, and actin, respectively.
Figure 4.
Knockdown of Pirh2 inhibits arsenic-induced degradation of mutant p53 protein.
Western blots were prepared with extracts from HaCaT (A) and MIA PaCa-2 (B) cells, which were transfected with scrambled siRNA #1 (Scr-1) (lanes 1–2), Scr-2 (lanes 5–6), siRNA against Pirh2-1 (siPirh2-1) (lanes 3–4), or siPirh2-2 (lanes 7–8), and then treated with ATO for 6 h. The blots were then probed with antibodies against Pirh2, p53, and actin, respectively.
Figure 5.
Pirh2 physically associates with mutant p53 protein for polyubiquitination.
(A) HaCaT and MIA PaCa-2 cells were treated with 5 or 7.5 µM ATO for 6 h. Cell extracts from HaCaT (left) and MIA PaCa-2 (right) cells were immunoprecipitated with anti-p53 or control IgG. The immunocomplexes were then used to detect mutant p53 and Pirh2 along with whole-cell lysates as input control. (B) The experiment was performed as described in (A), except that anti-Pirh2 antibody was used in immunoprecipitation. (C–E) In vitro synthesized 35S-labeled wild-type p53 (C), R175H (D), and R273H (E) were mixed with GST, GST-tagged Pirh2, Pirh2-DN, or Pirh2-ΔRING. The complexes were then mixed with a buffer containing E1, E2 (UbcH5b), and Ub and then incubated at 30°C for 2 h. Ubiquitinated p53 was analyzed by SDS-PAGE and detected by autoradiography. (F) A model of Pirh2-mediated degradation of mutant p53 induced by ATO.
Figure 6.
Arsenic trioxide cooperates with HSP90 or HDAC inhibitor to decrease mutant p53 expression and tumor cell proliferation.
(A–B) Western blots were prepared with extracts from HaCaT (A) and MIA PaCa-2 (B) cells, which were untreated or treated with 1 µM 17AAG or 2 µM SAHA for 12 h. The blots were then probed with antibodies against Pirh2 and actin, respectively. (C–D) Western blots were prepared with extracts from HaCaT (C) and MIA PaCa-2 (D) cells, which were untreated or treated with 7.5 µM ATO, 1 µM 17AAG or 2 µM SAHA, alone or in combination for 12 h. The blots were then probed with antibodies against p53 and actin, respectively. (E–F) HaCaT (E) and MIA PaCa-2 (F) cells were treated as in (C–D) for 24 h. Surviving cells from both control and treated groups were counted and presented as Mean ± SD from three separate experiments. *, p<0.05.