Roles of Atox1 and p53 in the trafficking of copper-64 to tumor cell nuclei: implications for cancer therapy

Abstract

Owing to its cytotoxicity, free copper is chelated by protein side chains and does not exist in vivo. Several chaperones transport copper to various cell compartments, but none have been identified that traffic copper to the nucleus. Copper-64 decays by β (+) and β (-) emission, allowing positron emission tomography and targeted radionuclide therapy for cancer. Because the delivery of (64)Cu to the cell nucleus may enhance the therapeutic effect of copper radiopharmaceuticals, elucidation of the pathway(s) involved in transporting copper to the tumor cell nucleus is important for optimizing treatment. We identified Atox1 as one of the proteins that binds copper in the nucleus. Mouse embryonic fibroblast cells, positive and negative for Atox1, were used to determine the role of Atox1 in (64)Cu transport to the nucleus. Mouse embryonic fibroblast Atox1(+/+) cells accumulated more (64)Cu in the nucleus than did Atox1(-/-) cells. HCT 116 colorectal cancer cells expressing p53 (+/+) and not expressing p53 (-/-) were used to evaluate the role of this tumor suppressor protein in (64)Cu transport. In cells treated with cisplatin, the uptake of (64)Cu in the nucleus of HCT 116 p53(+/+) cells was greater than that in HCT 116 p53(-/-) cells. Atox1 expression increased in HCT 116 p53(+/+) and p53(-/-) cells treated with cisplatin; however, Atox1 localized to the nuclei of p53(+/+) cells more than in the p53(-/-) cells. The data presented here indicate that Atox1 is involved in copper transport to the nucleus, and cisplatin affects nuclear transport of (64)Cu in HCT 116 cells by upregulating the expression and the nuclear localization of Atox1.

Figure 1

Internalization (a) and nuclear localization (b) of [⁶⁴Cu]copper acetate in MEF cell lines positive (Δ) and negative ■ for Atox1 (n=3). Data are presented as %ID/mg and %ID/Nuclei. (*) p<0.05, (**) p<0.01. The data are representative of three experiments.

Figure 2

Atox1 level in HCT116 p53+/+ and p53-/- cell lines. Western blot analysis of lysates prepared from p53+/+ and p53-/- cell lines (a). Ratio of Atox1 to β-actin in p53+/+ and p53-/-cell lines (b). (*) p<0.05 (n=3). These results are representative of three independent experiments.

Figure 3

Effect of Atox1 knock-down on nuclear localization of [⁶⁴Cu]copper acetate in HCT116 p53+/+ and p53-/- cells. Nuclear localization of [⁶⁴Cu]copper acetate in HCT116 p53+/+ and p53-/- cell lines before and after Atox1 knock-down (n=3) (a). Western blot showing Atox1 knock-down in HCT116 p53+/+ and p53-/- cells (b). (*) p<0.05 (n=3).

Figure 4

Effect of p53 knock-down on Atox1 expression in MEF Atox1+/+ cells. (a) Confocal images of MEF Atox1+/+ cells after KD of p53 using p53-siRNA. Cells were seeded on glass coverslips, treated with 20 pmol p53-siRNA for 48 h and fixed with 4% PFA. p53 (green) was detected with Mouse Anti-Human p53 antibody and Atox1 (red) was detected with Goat Anti-mouse HAH1 antibody. (b) Western blot showing p53 knock-down in MEF Atox1+/+ cells. (c) Nuclear localization of [⁶⁴Cu]copper acetate in MEF Atox1+/+ and Atox1-/- cell lines before and after p53 knock-down (n=3). These results are representative of two independent experiments.

Figure 5

Internalization (a) and nuclear localization (b) of [⁶⁴Cu]copper acetate in HCT116 cell lines positive and negative for p53 (n=3) in %ID/mg. Cells were pre-treated with cisPt for 24 h and [⁶⁴Cu]copper acetate incubation time is 2 h. (*) p<0.05, (***) p<0.001. This experiment is representative of two experiments.

Figure 6

Effect of cisPt on Atox1 expression in HCT116 p53+/+ and p53-/- cell lines. Confocal images of HCT116 p53+/+ (a) and p53-/- (d) cells treated and non treated with cisPt; cells were seeded on glass coverslips, treated with 40 μM cisPt for 24h h then fixed with 4% PFA; p53 (green) was detected with Mouse Anti-Human p53 antibody and Atox1 (red) was detected with Mouse Anti-Human Atox1 antibody. Quantification of the fluorescence of Atox1 in HCT116 p53+/+ (b) and p53-/- (e) cells treated and non treated with cisPt. Western blot of Atox1 in HCT116 p53+/+ (c) and p53-/- (f) cells treated and non treated with cisPt. (*) p<0.05, (***) p<0.001.

Figure 7

Nuclear localization of Atox1 in HCT116 p53+/+ and p53-/- cells, treated and non-treated with cisPt. Confocal images of Atox1 (red) and nuclear staining (blue) in HCT116 p53+/+ (a, upper left panel) and p53-/- (a, upper right panel) cells; Images show the colocalized pixels from the total Z-stack between Atox1 and DAPI staining in HCT116 p53+/+ (a, lower left panel) and p53-/- (a, lower right panel) cells treated and non treated with cisPt. (b) Pixel density analysis of the Atox1 and DAPI colocalized images. (c) Western blot showing Atox1 levels in HCT116 p53+/+ and p53-/- nuclei before and after 24 h treatment with cisPt.