Several lines of evidence suggest that accumulation of DNA damage coupled with defects in DNA repair play an important role in breast cancer. Our own previous work has shown that nucleotide excision repair is defective in the Li-Fraumeni syndrome,a heritable cancer prone syndrome associated with increased susceptibility to breast cancer. Several groups have shown that defects in the removal of UV- and X-irradiation induced DNA damage are present in newly diagnosed sporadic breast cancer patients and in their healthy first-degree female relatives. Other investigators have shown that levels of oxidative DNA damage (e.g. 8-oxo-7,8-dihydroguanine) are increased in human breast tumors and in surrounding normal tissue and that the breast cancer susceptibility gene, BRCA1, is required for transcription coupled repair of oxidative damage in mutant rodents. These data suggest that both the nucleotide excision and base excision repair pathways may be involved in breast cancer development. It remains to be established whether DNA repair mechanisms are defective in breast cancer cells and which specific repair pathways are of primary importance. We hypothesize that one of the critical steps in mammary cinogenesis is the loss of the normal response to DNA damage and that specific defects in nucleotide excision repair (NER) or base excision repair (BER) may be critical in development and progression of the malignant phenotype. We have examined transcription coupled nucleotide excision repair in normal human mammary epithelial cells (HMEC) AG11134 and the breast cancer cell lines MCF7 and MDA-MB-468. Transcription coupled repair of cyclobutane pyrimidine dimers examined using the gene specific repair assay (Bohr assay) is reduced in both tumor cell lines when compared to normal human mammary epithelial cells and normal skin fibroblasts. These results suggest that these breast cancer cell lines are defective in nucleotide excision repair; however, the mechanism and genetic changes that account for this repair phenotype are unclear. We examined mRNA and protein expression levels of nucleotide excision repair related genes (ERCC1, XPA, XPB, XPF, XPC and RPA) before and after UV irradiation and found that there is an alteration in the expression of the ERCC1 gene at the protein level possibly pinpointing a specific portion of the nucleotide excision repair pathway. On going work is examining mechanistic explanations is focused on alterations in repair related gene function by transfecting a vector containing the ERCC1 gene back into the breast cancer cell lines and evaluating whether this complements the defects in post UV and Mitomycin-C cellular survival and repair defects found. Because of the body of literature that supports the notion that accumulation of endogenous oxidative damage plays a role in breast carcinogenesis we have begun to examine the possible role of BER in breast cancer development by analyzing the capacity of nuclear extracts from these cell lines to recognize and remove the oxidative DNA adducts 8-oxo-7,8-dihydroguanine (8oxodG) and thymine glycol (TG). Thus far nuclear extracts from MCF-7 and MDA-MB-468 cells are proficient in the incision of TG and 8-oxo-dG. This does not eliminate defective BER as a contributing factor in breast tumorigenesis. Recently it has been shown that mitochondrial DNA mutations in colorectal cell lines likely result from unrepaired oxidative damage. This work indirectly suggests that DNA repair in mitochondria of these cancer cells may be compromised. It is possible that this could also be the case for breast cancer cells. Therefore, our future plans for this part of the project will include examination of mitochondrial DNA repair of oxidative lesions.
