The copper chaperone Atox1 is involved in the intracellular sequestration of cisplatin

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The role of copper (Cu) in the etiology of cancer is widely recognized and recent findings link the expression of Cu transporters directly to the development of resistance to platinum drugs particularly cisplatin (DDP). The Cu chaperone Atox1 is essential for the efflux of Cu and is known to exchange Cu with the importer Ctr1 and the exporters Atp7a and Atp7b. We studied the uptake, efflux and Pt-DNA adduct formation in fibroblast cell lines from Atox1^-/^- transgenic and wild type (Atox1⁺/⁺) mice following 24 h exposure to 2 µM DDP and performed parallel experiments with ⁶⁴Cu to define the mechanisms by which Atox1 contributes to the intracellular transport of DDP (all values are mean ± SEM). The whole cell accumulation of DDP in the Atox1^-/- cells was reduced to 62% of that in the Atox1^+/+ cells (167.03 ± 18.15 and 103.32 ± 23.41 picomol/mg protein (p = 0.03) in the Atox1⁺/⁺ and Atox1^-/^- cells, respectively). In contrast, following a 24 h exposure to 2 µM ⁶⁴Cu the Cu level was 86% higher in the Atox1^-/- than in the Atxo1^+/+ cells (174.02 ± 3.73 and 324.45 ± 16.00 picomol /mg protein in the Atox1^+/+ and Atox1^-/^- cells, respectively (p = 0.00001)). Efflux of DDP was similar in the 2 cell lines but that of ⁶⁴Cu was more rapid in the Atox1⁺/⁺ cells (p= 0.05). At the end of a 60 min efflux period 44.68 ± 0.91% of the ⁶⁴Cu remained in the Atox1^+/+ cells whereas 82.74 ± 11.50 % persisted in the Atox1^-/- cells The extent of Pt-DNA adduct formation in the Atox1^-/^- cells was only 55% of that in the Atox1^+/+ cells (18.77 ± 5.50 and 34.38 ± 6.72 and picomol/mg DNA, p < 0.0005). While both cell types concentrated the majority of DDP in the nuclear and microsomal fractions, the Atox1⁺/⁺ cells showed significantly higher levels in both compartments than the Atox1^-/^- cells (p < 0.005). Nuclear Pt was 212.20 ± 1.40 picomol/mg protein in the Atox1⁺/⁺ cells and 165.19 ± 21.60 in the Atox1^-/^- cells. Microsomal Pt was 221.78 ± 10.99 picomol/mg protein in the Atox1^+/+ cells and 176.34 ± 8.88 in the Atox1^-/^- cells (p < 0.005). Cytosolic Pt levels were similar in both cell lines being 21.44 ± 0.46 picomol Pt/mg protein in the Atox1⁺/⁺ and 18.54 ± 0.50 in the Atox1^-/^- cells. Whereas loss of Atox1 impairs Cu efflux and increases cellular levels of Cu, loss of Atox1 reduces cellular, nuclear and DNA levels of Pt following exposure to DDP. Thus, Atox1 influences the cellular pharmacology of both Cu and DDP; in the case of Cu loss of Atox1 primarily affects efflux whereas in the case of DDP the primary effect is on influx. Whether Atox1 directly binds DDP, as it does Cu, or whether its effect on DDP cellular pharmacology is indirect remains to be established.