The objective of this work is to investigate the effect of nitric oxide induced response modification on the cytotoxicity of peroxynitrite and the anti-cancer drug doxorubicin. The data generated from these studies will provide insight into the effect of response modification on DNA transcription, translation of specific anti-oxidant enzymes and DNA repair. The significance work relates to both chronic inflammation and the inflammatory response to solid tumors. In both cases, there is unregulated/inappropriate proliferation of cells. Both are also examples of microenvironments in which there are likely to be areas where the concentration of reactive oxygen and nitrogen species is sub-lethal. In such cases, the effect of these reactive species results in the modification of surrounding cells in such a way which renders them subsequently resistant to otherwise lethal agents such as peroxynitrite and doxorubicin. Specifically, the applicant will response modified A549 cells with a sub-lethal concentration of nitric oxide and then challenge these cells with lethal concentrations of either doxorubicin or peroxynitrite. The applicant will measure the amount and type of DNA damage induced both agents; the alterations in the activation of the nuclear transcription factor NF-kB; and the alteration in the mRNA level of selected anti-oxidant enzymes under the regulation of NF-kB. Alkaline elution will be used to assess the amount of DNA damage; confocal microscopy will be used to assess the type of DNA damage as well as the nuclear translocation of the p65 sub-unit of NF-kB; an NF-kB regulated luciferase reporter gene will be used to measure transcription factor activation; and reverse-transcriptase polymerase chain reaction will be used to quantitate the amount of mRNA for metallothionein, catalase, manganese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase and gamma glutamyl synthetase.
