Regulation of copper transporter 2 expression by copper and cisplatin in human ovarian carcinoma cells

Abstract

Down-regulation of copper transporter 1 (CTR1) reduces uptake and sensitivity, whereas down-regulation of CTR2 enhances both. Cisplatin (DDP) triggers the rapid degradation of CTR1 and thus limits its own accumulation. We sought to determine the effect of DDP and copper on the expression of CTR2. Changes in CTR1 and CTR2 mRNA and protein levels in human ovarian carcinoma 2008 cells and ATOX1(+/+) and ATOX1(-/-) mouse embryo fibroblasts in response to exposure to DDP and copper were measured by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and deconvolution microscopy. DDP triggered rapid degradation of CTR1 in 2008 human ovarian cancer cells. However, it increased the expression of CTR2 mRNA and protein levels. Expression of CTR2 was heavily modulated by changes in intracellular copper concentration; copper depletion produced rapid disappearance of CTR2, whereas excess copper increased the level of CTR2 protein. This increase was associated with an increase in CTR2 mRNA and prolongation of the CTR2 half-life. Consistent with prior observations that short hairpin RNA interference-mediated knockdown of CTR2 enhanced DDP uptake and tumor cell kill, reduction of CTR2 by copper starvation also enhanced DDP uptake and cytotoxicity. Comparison of the ability of copper and DDP to modulate the expression of CTR1 in ATOX1(+/+) and ATOX1(-/-) indicated that ATOX1 participates in the regulation of CTR2 expression. Unlike CTR1, the expression of CTR2 is increased rather than decreased by DDP. Therefore, these two copper transporters have opposite effects on DDP sensitivity. CTR2 expression is regulated by copper availability via the copper-dependent regulator ATOX1.

Fig. 1.

Measurement of CTR2 in 2008 cells after 1-h pretreatment with either 200 μM CuSO₄, 100 μM BCS, 30 μM DDP, or drug-free media. A, relative mRNA levels measured by qRT-PCR. B, Western blot and densitometric analysis of CTR2 protein levels. C, representative deconvolution micrographs of 2008 cells stained for CTR2 (red).

Fig. 2.

Time course of changes produced in CTR2 protein levels by a 1-h exposure to either 200 μM CuSO₄ (A), 100 μM BCS (B), or 4.5 μM DDP (C).

Fig. 3.

Measurement of CTR2 half-life by Western blot. A, blot showing lysates extracted from cells at 0, 5, 10, 20, 30, or 45 min after the start of exposure to 100 μg/ml cyclohexamide. Cells were pretreated with either drug-free media, 200 μM CuSO₄, or 30 μM DDP for 1 h before cyclohexamide exposure; ♦, control; □, DDP pretreatment; ▴, copper pretreatment. B, logarithmic representation of the percentage of remaining CTR2 protein as a function of time after the start of cyclohexamide treatment. C, deconvolution microscopy of 2008 cells after exposure to drug-free control media or 50 nM bortezomib in the presence or absence of 100 μM BCS.

Fig. 4.

A, uptake of DDP after 1 h pretreatment with either 200 μM CuSO₄, 100 μM BCS, or drug-free media. Data shown are whole-cell nanograms of platinum accumulation per microgram of sulfur. B, DDP concentration-survival curves for 2008 cells with (◊) and without (■) pretreatment with 200 μM CuSO₄ for 1 h. C, DDP concentration-survival curves for 2008 cells with (◊) and without (■) pretreatment with 100 μM BCS for 1 h.

Fig. 5.

A, deconvolution microscopic images of 2008 cells after 1 h of pretreatment with either 200 μM CuSO₄, 100 μM BCS, or drug-free media. Red, CTR2; green, NPC proteins. B, Western blot analysis of CTR2, tubulin, and lamin B in the whole cell, cytosolic, and nuclear fractions of 2008 cells. C, Western blot analysis of CTR2 in the cytosolic and nuclear fractions of 2008 cells after 1-h pretreatment with either 200 μM CuSO₄, 100 μM BCS, or drug-free media. Loading controls were anti-tubulin (cytosol) and anti-laminin B1 (nucleus).

Fig. 6.

A, deconvolution microscopic images of ATOX1(+/+) and ATOX1(−/−) cells after 1 h pretreatment with either 200 μM CuSO₄, 100 μM BCS, or drug-free media. CTR2 is shown in red. B, Western blot analysis of CTR2 in ATOX1(+/+) and ATOX1(−/−) cells after a 1-h pretreatment with either 200 μM CuSO₄, 100 μM BCS, or drug-free media. C, relative CTR2 mRNA levels measured by qRT-PCR of ATOX1(+/+) (black) and ATOX1(−/−) cells (gray). D, model of CTR2 regulation by ATOX1.