BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells

Abstract

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20-45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC(50)) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC(50) : 30-40 microM) compared with MCF-7 (IC(50) : 60-70 microM) and MDA-MB231 (IC(50) : 90-100 microM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC(50) : 45-50 microM) compared with MCF-7 (IC(50) : 1-5 microM) and MDA-MB231 (IC(50) : 5-10 microM) (P<0.02), as well as to paclitaxel (Tax) (IC(50) : >2 microM for HCC1937, 0.1-0.2 microM for MCF-7 and 0.01-0.02 microM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/(WT)BRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.

Figure 1

Differential sensitivity among BRCA1-defective and BRCA1-competent cells to CDDP. In (A) HCC1937 cells, (B) MCF-7 cells and (C) MDA-MB231 cells, the dose-related activity of CDDP is shown as tumour cell growth. Analysis was performed after 48 h exposure to the drug and the IC₅₀ was calculated by interpolate logarithmic curve. In (D), the value of IC₅₀ determined for each cell line is shown. ° and °°: P<0.005.

Figure 2

Differential sensitivity among BRCA1-defective and BRCA1-competent cells to Dox. In (A) HCC1937 cells, (B) MCF-7 cells and (C) MDA-MB231 cells, the dose-related activity of Dox is shown. Interpolate logarithmic curve and the IC₅₀ of cell growth are shown in each quadrant. In (D), the value of IC₅₀ determined for each cell line is shown. ° and °°: P<0.02.

Figure 3

Differential sensitivity among BRCA1-defective and BRCA1-competent cells to Tax. In (A) HCC1937 cells, (B) MCF-7 cells and (C) MDA-MB231 cells, the dose-related activity of Tax is shown. Interpolate logarithmic curve and the IC₅₀ of cell growth are shown in each quadrant. In (D), the value of IC₅₀ determined for each cell line is shown. ^*: Maximum of concentration used, no IC₅₀ was achieved at this dose of drug. ° and °°: P<0.001.

Figure 4

cDNA transfection reconstitutes BRCA1 expression. Western blot analysis of BRCA1 protein expression in HCC1937, HCC-1937/^WTBRCA1 and MCF-7 cells.

Figure 5

Full-length transfection of BRCA1 in HCC1937 (HCC1937/^WTBRCA1) induces resistance to CDDP and restores sensitivity to Dox and Tax. In (A)–(C), interpolate logarithmic curves and the IC₅₀ of cell growth after 48 h exposure to drugs are shown in each quadrant. In (D)–(F), the values of IC₅₀ determined for both cell lines are shown. °: P<0.02; °°: P<0.05; °°°: P<0.001; ^*: no IC₅₀ was achieved at this drug concentration.

Figure 6

Effects of BRCA1-restored expression on drug-induced apoptotic cell death. In (A) percentages of apoptotic cells in untreated controls and after 48 h exposure to CDDP, Dox and Tax, respectively, for both BRCA1-defective and -competent cells are shown. In (B), flow cytometric profiles of Annexin-V FITC staining in a representative experiment are shown. Percentages of stained cells are reported in each quadrant.