Marilyn E. Thompson

BRCA1 Expression Restores Radiation Resistance in BRCA1-defective Cancer Cells through Enhancement of Transcription-coupled DNA Repair

The breast cancer predisposition genes,BRCA1 and BRCA2, are responsible for the vast majority of hereditary breast cancer. Although BRCA2functions to help the cell repair double-stranded DNA breaks, the function of BRCA1 remains enigmatic. Here, we develop a human genetic system to study the role of BRCA1 in oxidative DNA damage. We show that human cancer cells containing mutated BRCA1 are hypersensitive to ionizing radiation. This hypersensitivity can be reversed by the expression of forms ofBRCA1 that are not growth suppressing. Reversal of hypersensitivity requires the ring finger of BRCA1, its transactivation domain, and its BRCT domain. Lastly, we show that unlike BRCA2, BRCA1 does not function in the repair of double-stranded DNA breaks. Instead, it functions in transcription-coupled DNA repair (TCR). TCR ability correlated with radioresistance as cells containing BRCA1 showed both increased TCR and radioresistance, whereas cells withoutBRCA1 showed decreased TCR and radiosensitivity. These findings give physiologic significance to the interaction ofBRCA1 with the basal transcription machinery.

Nuclear Export of BRCA1 Occurs During Early S Phase and is Calcium-dependent

Although the breast cancer susceptibility gene 1 (BRCA1) protein is predominantly nuclear, its localization can vary during the cell cycle in response to cellular insults. For example, in S-phase cells, BRCA1 forms subnuclear foci and localizes to the perinuclear region in response to DNA damage. The present study provides evidence that BRCA1 is transiently excluded from the nucleus during the early part of S phase in the absence of DNA damage. The percentage of MCF-7 human breast cancer cells predominantly expressing nonnuclear BRCA1 significantly correlates with the percentage of cells within early S phase. This redistribution of BRCA1 is partially sensitive to leptomycin B, indicating that CRM-1-mediated nuclear export is involved. Similar results were observed with MCF-12A nonmalignant human mammary cells. The abilities of BAPTA-AM, an intracellular calcium chelator, to inhibit the change in BRCA1 localization, and of A23187, a calcium ionophore, and of thapsigargin to mimic nuclear exclusion of BRCA1, provide evidence for the involvement of calcium in this process. The calcium-mediated change in BRCA1 localization occurs in several cell lines, indicating that this effect is not cell line specific. BRCA2 localization is not affected by A23187. Furthermore, inhibition of calcium-calmodulin interaction and calcium-calmodulin dependent protein kinase II attenuates the calcium-mediated change in BRCA1 localization. These data suggest that BRCA1 nuclear export can be cell cycle-regulated by a calcium-dependent mechanism.

BRCA1 accumulates in the nucleus in response to hypoxia and TRAIL and enhances TRAIL‐induced apoptosis in breast cancer cells

A major contributing factor to the development of breast cancer is decreased functional expression of breast cancer susceptibility gene 1, BRCA1. Another key contributor to tumorigenesis is hypoxia. Here we show that hypoxia increased the nuclear localization of BRCA1 in MCF‐7 and MDA‐MB‐468 human breast cancer cell lines without changing its steady‐state expression level. Nuclear accumulation of BRCA1 was not evident in MCF‐12A or HMEC (human mammary epithelial cell) nonmalignant mammary epithelial cells under the same conditions. Hypoxia also increased the cell surface expression of TRAIL on MDA‐MB‐468 cells. Neutralization of TRAIL precluded the hypoxia‐induced accumulation of BRCA1 in the nucleus, whereas exogenously administered TRAIL mimicked the effect. Treatment of MDA‐MB‐468 cells with TRAIL resulted in a dose‐ and time‐dependent increase in apoptosis. Furthermore, TRAIL‐induced apoptosis in HCC1937 cells, which harbor a BRCA1 mutation, increased synergistically when wild‐type BRCA1 was reconstituted in the cells, and downregulation of BRCA1 expression in MDA‐MB‐468 cells reduced the apoptotic response to TRAIL. These data provide a novel link between hypoxia, TRAIL and BRCA1, and suggest that this relationship may be especially relevant to the potential use of TRAIL as a chemotherapeutic agent.