The use of platinum compounds is increasing in the treatment of cancer. Cisplatin, carboplatin and oxaliplatin produce a dose-related and dose-limiting sensory neuropathy. We have demonstrated that cisplatin binds to neuronal DNA, activates DMA-damage recognition pathways, initiates aberrant cell cycle entry, and induces apoptosis in vitro and in vivo. We now propose to test the hypothesis that platinum compounds produce neuronal injury by a common mechanism that involves separate nuclear (n-DNA) and mitochondrial DNA (mt-DNA) binding followed by synergistic activation of parallel death pathways. A new approach to measuring mt-DNA platination has been developed using inhibition of the polymerase chain reaction. We will determine whether the number of platinum adducts in mt-DNA of DRG neurons is sufficient to prevent replication and transcription of the mitochondrial genome. Function of respiratory chain components will be measured. Inability to repair Pt-DNA lesions in mt-DNA would result in attrition of mitochondria and chronic neuronal death explaining the clinical phenomenon of """"""""coasting"""""""" or progression of neuropathy after drug cessation. We will use DRG neurons from Bax and cyclin D1 knockout mice to determine whether platinum-induced inhibition of mitochondrial function is sufficient to cause neuronal death. We will use the mitochondrial DNA synthesis inhibitor dideoxycytidine (ddC) to determine whether inhibition of mitochondrial DNA replication is independently sufficient to cause cell death. The mitochondrial genome will be protected by selectively increasing mitochondrial glutathione. The modifier and catalytic subunits ofglutamate cysteine ligase (GCL) andglutathione synthetase (GS) willbe targeted to mitochondria in an adeno-viral vector to reduce formation of mt-DNA adducts. Both nerve growth factor (NGF) and pigment epithelium derived growth factor (PEDF) have been demonstrated to partially protect DRG from cisplatin-induced apoptosis. We will determine whether a combination of therapeutic strategies that block n-DNA induced apoptosis and protect mt-DNA promote long-term survival of cisplatin treated DRG in vitro. If the combination strategy is effective,we will test it in an animal model by developing methods to provide long-term delivery of growth factors and viral targeting of GCL and GS to DRG in vivo. The goal is to develop a mechanism-based therapy that will prevent the major dose-limiting side effect of the platinum compounds.
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