Potential role of p53 on metallothionein induction in human epithelial breast cancer cells

Abstract

The expression and induction of metallothionein has been associated with protection against oxidative stress and apoptosis. This study examines the effect of tumour suppressor protein p53 on metallothionein expression following CdCl2 treatment in eight human epithelial breast cancer cell lines differing in p53 and oestrogen-receptor status. Cells were treated with 10 microM CdCl2 for 24 h and metallothionein protein levels were measured by cadmium binding assay. MCF7 cells which are p53-positive (p53+) and oestrogen-receptor-positive showed a large induction in metallothionein synthesis by 10.79+/-1.36-fold. Other breast cancer cell lines which are p53-negative (p53-) and oestrogen-receptor-negative or weakly oestrogen-receptor-positive showed a small induction ranging from 1.40+/-0.10 to 3.65+/-0.30-fold. RT-PCR analysis showed an induction of metallothionein mRNA in MCF7 cells by about 1.61+/-0.08-fold, while in HCC1806 cells (p53-, oestrogen-receptor-negative) by 1.11+/-0.13-fold, and in MDA-MB-231 (p53-, oestrogen-receptor-negative) by 1.25+/-0.06-fold. Metallothionein localisation was determined by immunohistochemical staining. Prior to metal treatment, metallothionein was localised mainly in the cytoplasm of MCF7 and MDA-MB-231 cells. After treatment with 10 microM CdCl2 for 24 h, MCF7 cells showed intense nuclear and cytoplasmic staining for metallothionein, while MDA-MB-231 cells showed staining in the cytoplasm with weak nuclear staining. Apoptosis induced by 10-40 microM CdCl2 at time points between 4 and 48 h was examined with TUNEL assay. In MCF7 cells, apoptosis increased with higher concentrations of CdCl2, it peaked at 6-8 h and appeared again at 48 h for all concentrations of CdCl2 tested. In MDA-MB-231 cells, apoptosis remained at low levels for 10-40 microM CdCl2 at all time points. Studies on cadmium uptake showed similar uptake and accumulation of cadmium at 8 and 24 h in all the cell lines. The data demonstrate that treatment of epithelial breast cancer cells with 10 microM CdCl2 for 24 h caused a greater induction of metallothionein protein and mRNA expression in p53+ and oestrogen-receptor-positive cells as compared to p53- and oestrogen-receptor-negative or weakly oestrogen-receptor-positive cells. This effect may be associated with the occurrence of apoptosis and suggests a role for p53 and oestrogen-receptor on the expression and induction of metallothionein in epithelial cells.

Figure 1

Effects of CdCl₂ on cell viability. MCF7, MDA-MB-231, and HCC1806 cells were treated with various concentrations of CdCl₂ for 12 h. Cell viability was determined with Procheck Cell Viability Assay and expressed as a per cent of control. Results are mean±s.e. of three independent experiments. * Significantly different from control at P<0.05.

Figure 2

Induction of MT expression with CdCl₂. Epithelial breast cancer cells were treated with 10 μM CdCl₂ for 24 h. MT protein levels were determined with ¹⁰⁹Cadmium-haeme assay and expressed as μg MT mg total protein⁻¹. Results are mean±s.e. of three independent experiments. *Ratio of induced MT level/basal MT level is significantly different from MCF7 cells at P<0.05. ^# Basal MT level is significantly different from MCF7 cells at P<0.05.

Figure 3

Immunohistochemical staining for MT with rabbit polyclonal anti-MT serum which cross-reacts with human MT. Control cells: MCF7 (A), and HCC1806 (B). Cells treated with 10 μM CdCl₂ for 24 h: MCF7 (C), and HCC1806 (D). Positive staining is brown in colour. Magnification 400×.

Figure 4

MT-II expression at 23 PCR cycles. RT–PCR was used to analyse total RNA samples derived from three epithelial breast cancer cell lines: HCC1806, MDA-MB-213, and MCF7. PCR products were electrophoresed on agarose gel and stained with ethidium bromide. Control cells are represented by C and cells treated with 10 μM CdCl₂ for 24 h are represented by Cd. Intensities of cDNA bands were analysed by densitometry and MT-II mRNA expression was normalised with β-actin. Changes in MT-II mRNA expression following treatment were calculated as a ratio of treated cells/control cells and indicated in the graph. Results are mean±s.e. of three independent experiments. *Significantly different from control cells at P<0.05.

Figure 5

In situ detection of apoptotic bodies using TUNEL technique. Control cells: MCF7 (A), and MDA-MB-231 (B). Cells treated with 40 μM CdCl₂ for 8 h: MCF7 (C), and MDA-MB-231 (D). Cells treated with 40 μM CdCl₂ for 24 h: MCF7 (E), and MDA-MB-231 (F). TUNEL-positive apoptotic bodies are stained brown. Magnification 400×.

Figure 6

In situ detection of cadmium-induced apoptosis using the TUNEL technique. (A) MCF7 and MDA-MB-231 cells were treated with 40 μM CdCl₂ for different time periods. (B) MCF7 and MDA-MB-231 cells were treated with various concentrations of CdCl₂ for 8 h. Apoptotic bodies were counted in randomly selected fields. Results are mean±s.e. of 10 randomly selected fields. *Significantly different from control at P<0.05.

Figure 7

Uptake of CdCl₂ by different cell lines. MCF7, MDA-MB-231 and MDA-MB-435 cells were treated with 10 μM of ¹⁰⁹CdCl₂ and disintegrations per minute (d.p.m.) were measured and expressed as d.p.m. mg protein⁻¹ at (A) 8 h, and (B) 24 h after treatment. Results are mean±s.e. of three independent experiments.