Part II. Mitochondrial mutational status of high nitric oxide adapted cell line BT-20 (BT-20-HNO) as it relates to human primary breast tumors.

Mitochondria combine hydrogen and oxygen to produce heat and adenosine triphosphate (ATP). As a toxic by-product of oxidative phosphorylation (OXPHOS), mitochondria generate reactive oxygen species (ROS). These free radicals may cause damage to mitochondrial DNA (mtDNA) and other molecules in the cell. Nitric oxide (NO) plays an important role in the biology of human cancers, including breast cancer; however, it is still unclear how NO might affect the mitochondrial genome. The aim of the current study is to determine the role of mtDNA in the breast oncogenic process. Using DNA sequencing, we studied one breast cancer cell line as a model system to investigate the effects of oxidative stress. The BT-20 cell line was fully adapted to increasing concentrations of the NO donor DETA-NONOate and is referred to as BT-20-HNO, a high NO (HNO) cell line. The HNO cell line is biologically different from the "parent" cell line from which it originated. Moreover, we investigated 71 breast cancer biopsies and the corresponding noncancerous breast tissues. The free radical NO was able to generate somatic mtDNA mutations in the BT-20-HNO cell line that were missing in the BT-20 parent cell line. We identified two somatic mutations, A4767G and G13481A, which changed the amino acid residues. Another two point mutations were identified in the mtDNA initiation replication site at nucleotide 57 and at the 'hot spot' cytidine-rich D300-310 segment. Furthermore, the NO regulated the mtDNA copy number and selected different mtDNA populations by clonal expansion. Interestingly, we identified eight somatic mutations in the coding regions of mtDNAs of eight breast cancer patients (8/71, 11.2 %). All of these somatic mutations changed amino acid residues in the highly conserved regions of mtDNA which potentially leads to mitochondrial dysfunctions. The other two somatic mtDNA mutations in the displacement loop (D-loop) region [303:315 C(7-8)TC(6) and nucleotide 57] were distributed among 14 patients (14/71, 19.7 %). Importantly, of these 14 patients, six had mutations in the p53 gene. These results validate the BT-20 parent/HNO cell line model system as a means to study ROS damage in mtDNA, as it parallels the results found in a subset of the patient population.