BARD1 is a breast cancer suppressor protein first discovered as a binding partner of BRCA1. It can shuttle between different subcellular locations and regulates the cellular DNA damage response and apoptosis. BARD1 is expressed as multiple splice variant isoforms in breast and ovarian cancers. Here we present preliminary findings that test the hypothesis that specific BARD1 isoforms safeguard tumour cells against chemotherapeutic drugs, leading to drug resistance.
We cloned five BARD1 splice variants (beta, ΔRIN, epsilon, omega and phi) as YFP-fusion plasmids, and compared their localisation and ability to regulate apoptosis when expressed in MCF-7 breast cancer cells. Cells were treated for 16 h with the DNA damaging drug cisplatin (2.5 – 30 μM). BARD1 localisation and apoptosis (nuclear condensation and Bax oligomerisation) were analysed by fluorescence microscopy. Apoptosis was also analysed by Annexin V/DAPI binding by FACS.
Compared to YFP alone, wild type BARD1 increased Annexin V/DAPI binding (4-fold). Interestingly, the BARD1 isoforms omega and beta, which lack the nuclear export signal localised exclusively to the nucleus and were the least apoptotic. BARD1 isoforms lacking a nuclear localisation signal (phi, epsilon and ΔRIN) localised primarily in the cytoplasm and induced the most apoptosis (e.g. Bax oligomerisation up to 4-fold and Annexin V/DAPI binding up to 7-fold). Cisplatin-induced apoptosis was potentiated to different degrees by all forms of BARD1 and was dose dependent. At higher doses (20 μM), cisplatin induced a 2.5-fold increase in apoptosis in negative control cells and this was potentiated 4- to 5-fold by wild type BARD1, ΔRIN, omega and beta variants.
The preliminary findings suggest that low-dose cisplatin resistance conferred to MCF-7 cells by the omega and beta variants may be overcome by increasing drug dose, while lower doses are sufficient to sensitise cells in the presence of wild type BARD1 or the ΔRIN variant.