| Summary |
Our goal was to determine whether markers of oxidative stress induced DNA damage such as oxidatively induced non-DSB clustered DNA lesions (OCDLs) can be used as general precancerous or cancer biomarkers. Secondly, in order to gain mechanistic insights on the in vivo processing of clusters, we assessed the radioprotective effects of hypothermia with respect to OCDL accumulation and repair. By inducing reticulum sarcoma in mice, we compared the number of OCDLs in tissue DNA samples from a certain organ of the mouse bearing the tumor, e.g. duodenum tissue, with another duodenum DNA sample that came from a normal mouse. The cancer cells used for inducing tumor in the mice were M5076. These are transplantable murine reticulum sarcoma originating in the ovary of C57B1/6 mice and are highly invasive and metastatic. When subcutaneously transplanted into mice, a solid tumor arises before it metastasizes. This told us whether when an organism is carrying a tumor in a specific organ, if all tissues (even distant to the tumor) are under oxidative stress indicative of DNA damage by bystander effect. In the second part we used lung carcinoma cells injected into SCID mice. Once again, we assessed the DNA damage in the different tissue samples from the injected mice and compared it with normal mice. A group of SCID mice were fed with antioxidants, to see if inhibition of ROS/RNS can decrease OCDL accumulation. The tumor was also injected in a group of B6 normal mice to see the effect of the presence of a fully functioning immune system. In the third part, we tested the effect of hypothermia on cellular OCDL repair capacity after treatment with ionizing radiation. Peripheral blood lymphocytes from three patients were treated with 2 Gy IR ex vivo, and subsequently incubated at either 37°C or 13°C for up to 24 hours. The repair kinetics of OCDLs at hypothermic and normothermic environments was compared. We used repair enzymes as probes to compare the level of OCDLs. These enzymes, APEl, OGGI, and NTHl (E.coli), have functional activity in vitro i.e. isolated DNA carrying clustered lesions. Once a lesion in a cluster is detected, they excise the damaged base, cleave the DNA strand and create a single stranded break in each strand which in turn leads to a double strand break. We measured these additional breaks as clusters using standard neutral agarose gel electrophoresis and number average length analysis (NALA). A variety of factors accounts for the importance of this study. Early detection of a growing tumor is a significant aspect of its successful treatment. The proposed investigation has the potential to provide a gateway into an efficient means of early tumor detection. Future experiments can build on our study, by looking for the differences in particular gene expression in the different tissues that may cause a varying response to oxidative stress. |
