All of these events lead to Bax translocation to the mitochondria, cytochrome-c release, caspase activation and apoptosis

All of these events lead to Bax translocation to the mitochondria, cytochrome-c release, caspase activation and apoptosis. implies that randomly formed platinum adducts block transcription. Cisplatin treated DRG neurons exhibit mitochondrial vacuolization and degradationin vitroandin vivo.Taken together, this data suggests that direct mtDNA damage may provide a novel, distinct mechanism for cisplatin-induced neurotoxicity individual from the established nDNA damage pathway. Keywords:Chemotherapy: Neuropathy, DRG Neurons, Cisplatin, Mitochondria: DNA and DNA damage == Introduction == Platinum-based chemotherapy (e.g. cisplatin, carboplatin and oxaliplatin) is used to treat various types of cancers including prostate, testicular, ovarian, and colon cancers. The dose limiting side effect of these drugs is usually peripheral neuropathy, which reduces the quality of life for many long-term malignancy survivors. Cisplatin-induced peripheral neuropathy occurs in about 30% of patients with 20% being forced to discontinue treatment. Patients with cisplatin-induced peripheral neuropathy experience sensory loss, often accompanied by pain, starting in the distal extremities. Although cisplatin treatment is usually customarily discontinued once neuropathic symptoms begin, worsening of the symptoms may continue for weeks or months. This phenomenon, commonly known as coasting, is observed exclusively in platinum-based chemotherapy and suggests that there is ongoing neuronal damage after discontinuing cisplatin. Cisplatin-induced nuclear DNA (nDNA) damage in dorsal root ganglion (DRG) neurons has been thoroughly investigated. Cisplatin covalently binds to nuclear DNA (nDNA) inducing DNA damage and apoptosis. Cisplatin-induced DNA damage up-regulates p53 and induces cell cycle changes in DRG neurons. Cell cycle changes include up-regulation of cyclin D1, activation of CDK4/6 PFI-2 complexes and phosphorylation of Rb. All of PFI-2 these events lead to Bax translocation to the mitochondria, cytochrome-c release, caspase activation and apoptosis. In culture, DRG neuron survival is dependent on maintenance levels of nerve growth factor (NGF). Increasing the concentration of NGF ten fold prevents both Bax translocation to the mitochondria and cell cycle changes observed in cisplatin treated DRG neurons. Previous studies have shown cisplatin binds to mitochondrial DNA in liver and B50 cells. Mitochondrial DNA is usually a circular ~16,300 bp piece of DNA that encodes for 13 proteins essential for the various subunits of the electron transport chain including complex I (NADH dehydrogenase 1, 2, 3, 4, 4L, 5 and 6), complex III (cytochrome b), complex IV PFI-2 (cytochrome c oxidase 1, 2 and 3) and ATP synthase 6 frpHE and 8. Mitochondrial DNA also encodes for 22 tRNA and 2 rRNA that are important for synthesis of these proteins. Both replication and transcription are unidirectional from one start point. The effects of cisplatin on DRG mitochondrial DNA (mtDNA) have not been investigated. Given that mtDNA differs from nDNA in several ways, it follows that cisplatin effects on mtDNA likely differ as well, and that PFI-2 these effects could contribute to cisplatin-induced neurotoxicity. Previous studies in neuronal systems have resolved total platinum-DNA adduct formation without making a variation between nDNA and mtDNA. It has been shown that DRG neurons build up high levels of cisplatin-DNA adductsin vitroandin vivo. In nDNA, cisplatin-DNA adducts are removed and DNA is usually repaired by nucleotide excision repair (NER). Mitochondria do not have NER mechanisms for mtDNA. We demonstrate that cisplatin forms adducts with mtDNA in DRG neurons, and that this alters mtDNA replication and transcription. The resultant cisplatin-induced mitochondrial dysfunction may lead to a novel mechanism of cisplatin neurotoxicity. Our studies were designed to quantitatively assess mtDNA binding and the producing effect on mitochondrial transcription and replication. We have also assessed the effects of cisplatin on mitochondrial morphology and cellular viability. == Materials and Methods == == Cell culture model == DRG from embryonic day 15 Sprague-Dawley rat were dissociated and treated with 20M 2,5 Fluoro-2-deoxyuridine (Sigma, St. Louis, MO) and 20M uridine (Sigma, St. Louis, MO) for 35 days to eliminate supporting cells via established methods. Cultures were managed in EMEM made up of 15% calf bovine serum (Hyclone, Logan, UT), 7mg/ml glucose (Sigma, St. Louis, MO) 1.2mM l-glutamine (Invitrogen, Carlsbad, CA) and 10 ng/ml NGF (Bioproducts for Sciences, Indianapolis, IN). For prolonged cisplatin exposure experiments, we utilized a DRG neuron culture system using increased NGF concentrations (100ng/ml) and the pan caspase inhibitor, zVAD-FMK (75M) (Sigma, St. Louis, MO), to inhibit the apoptotic effects of cisplatin (2g/ml) (Novaplus, Bedford, OH). == Survival Experiments == DRG neurons were cultured into poly-l-lysine (Sigma, St. Louis, MO) coated dishes etched with 1mm grids (Nunc, Rochester, NY). Dishes were marked to ensure counting of the same area with images taken at time 0 prior to treatment. Cells were treated over a PFI-2 period of 144 hours. Images were acquired every 48 hours of the same area taken at time 0 and live cells counted as previously explained. == Long mtDNA PCR assay == The PCR assay.