Tumor Growth Induces Oxidative Clustered DNA Lesions Damage in Distant Mouse Tissue in vivo

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Thomas Kryston (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Our goal was to ascertain if DNA damage induced by oxidative stress is capable of being exploited as a precancerous or cancer biomarker. The specific DNA damage that we assessed was oxidatively induced non-DSB clustered DNA lesions (OCDLs). We further wanted to investigate if OCDL formation could be decreased by reducing reactive oxygen species (ROS) and oxidative stress. To accomplish this, we utilized the superoxide dismutase antioxidant, Tempol. Finally, we wanted to assess the role of specific cytokines and their part of inducing OCDLs in tissues both proximal and distal to a tumor mass.  For our first experiment, lung carcinoma cells were injected into nude mice in collaboration with the National Cancer Institute. We evaluated the DNA damage in various tissue samples, distal and proximal to the tumor, and compared the level the damage to control B6 mice with normal immune system. To conclude if reducing ROS effects OCDL accumulation, a cohort of NUDE mice were fed the antioxidant Tempol. To establish the effects of a fully functioning immune system on OCDL formation, tumor cells were also injected into a cohort of normal B6 mice.  The second experiment was performed to evaluate the specific roles that cytokines and inflammation play in inducing OCDL damage in tissues from tumor bearing mice. A cytokine analysis was previously performed by Redon et al. which indicated in the presence of Sarcoma, monocyte chemoattractant protein-1 (MCP-1) was up regulated. To implement the specific role of MCP-1 in mediating the "bystander effect", our experiment entailed MCP-1 being knocked out for a group of mice and assessing the amount of OCDL damage in various tissues.   We utilized repair enzymes as probes to measure the level of OCDLs. These repair enzymes, human APE1, human OGG1, and E. coli Endo III, have functional activity in vivo. Once detection of a lesion in a cluster occurs, excision of the damaged base and of the DNA strand will transpire. Each DNA strand will now display a single stranded break which, if within 1-10 base pairs, results into a double stranded break. These additional breaks are measured as clustered lesions and assessed via neutral agarose gel electrophoresis and calculated with number average length analysis (NALA).  Earlier prognosis and detection of a growing tumor is a significant aspect of successful treatment. From these experiments, we hope to establish OCDLs as precancerous or cancer biomarkers in the case of high oxidative stress. It is hopeful one day clinical biopsies can be performed to screen for this specific damage and indicate early complications and tumor growth.   Future experiments can be projected based on our study. Different tumors can be utilized to assess OCDL damage. An inclusion of wider variety of tissues will further establish the complex DNA damage induced by the bystander/distal effect with ROS and inflammation as mediators of this damage.  

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Thesis

Date: 2010

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