We showed that BRCA1 regulates intracellular ROS levels in several breast carcinoma cell lines and in a non-tumor human mammary epithelial cell line, based on carboxy-DCF fluorescence measurements by flow cytometry and confocal microscopy. Carboxy-DCF is O−
, and other oxygen species, and so provides an estimate of the total burden of ROS. Although the changes in ROS levels due to BRCA1 were not always large in magnitude, they were easily detectable. Such changes may be physiologically significant, particularly if they are prolonged, since ROS contribute to so many normal and pathologic processes [5
]. A breast cancer-associated mutation (T300G, or C61G) abrogated the ability of BRCA1 to reduce ROS levels; and expression of the T300G mutant protein actually increased ROS levels, raising the possibility that the mutant protein functions as a dominant negative.
BRCA1 also reduced the levels of protein nitration and H2
-induced 8-oxoguanine lesions in DNA, two measures of oxidative damage to cellular macromolecules. The BRCA1-mediated reduction in protein nitrotyrosine levels was observed in both carcinoma and non-tumor cell lines. This reduction may be due to a BRCA1-mediated reduction in superoxide ion levels, since peroxynitrite, the reactive intermediate involved in protein nitration, is formed by the reaction of superoxide with nitric oxide [12
]. Thus, BRCA1 may protect the integrity of cellular macromolecules. In this regard, we have shown that BRCA1 can stimulate a low grade endoplasmic reticulum stress response, a cytoprotective response to proteins damaged by oxidative and other causes [13
Our findings are consistent with our previous study showing that BRCA1 can stimulate antioxidant gene expression and protect cells against oxidative stress [7
]. They are also consistent with an in vivo study of suggesting that BRCA1-deficient mice are more prone to oxidant-induced lethality [14
]. While the precise mechanism(s) by which BRCA1 reduces ROS levels is not yet clear, our findings suggest that loss of the endogenous BRCA1 protein, which is commonly observed in sporadic breast cancers [2
], is sufficient to cause increased ROS levels in carcinoma cells, and thus might contribute to development of the breast cancer phenotype.
REF1 is a multi-functional protein that acts an apurinic/apyrimidinic endonuclease in the base excision pathway of DNA repair and a regulator of stress-induced transcription factors. Although the mechanisms are not entirely clear, REF1 expression is induced by oxidative stress; and REF1 protects cells against ROS-induced cell killing [15
]. Here, we showed that REF1 knockdown increases ROS levels in a manner that is partly reversed by BRCA1 over-expression and vice versa
. These findings suggest that with regard to regulation of ROS levels, BRCA1 and REF1 do not work in a simple series model with one down-stream of the other.
An interesting finding of this study is that cell transfection with plasmid expression vectors or siRNA can itself induce oxidative stress, a factor that needs to be taken into account in studies in which over- or under-expression models are utilized in studies of ROS production.