Study of mutations arising in vivo and in vitro in a new murine tumor model system: The role of reactive oxygen and nitrogen species.

dc.contributor.advisorBirnboim, H. C.,
dc.contributor.authorSandhu, Jagdeep K.
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 59-10, Section: B, page: 5305.
dc.description.abstractA transplantable murine tumor model system has been established in our laboratory to study the contribution of the tumor microenvironment in tumor progression using the hypoxanthine phosphoribosyltransferase (HPRT) gene as a marker of chromosome fragmentation or loss. MN-11 was identified as the most stable clone with a low spontaneous mutant frequency (MF) and 1000 times increased sensitivity than the parental line in detecting mutagenic events induced by $\sp{60}$Co $\gamma$-rays. This cell line can grow in vitro and in vivo as a subcutaneous tumor in syngeneic C57BL/6 mice. This model system allows the ex vivo detection of mutants that may arise in vivo in tumors. Such mutants are resistant to the cytotoxic drug, 6-thioguanine when grown in vitro. The frequency of mutants arising in MN-11 cells grown as subcutaneous tumors was found to be 4-fold higher than that in otherwise identical cells grown in culture. The mutant frequency of individual tumors varied considerably in a non-gaussian manner. These results suggest that the conditions within solid tumors are mutagenic and/or clastogenic. Histochemical analysis of MN-11 tumors showed the presence of granulocytes, macrophages, mast cells and lymphocytes. Biochemical measurements of total nitric oxide synthase (NOS) activity in tumor homogenates demonstrated a positive correlation between NOS activity and MF. The expression of inducible (iNOS) was estimated by Western blot analysis and localized by immunohistochemistry, The synthase was present in some tumors as a 130 kDa band, similar to the human iNOS, but not in others. The iNOS-positive cells included predominantly granulocytes and not tumor cells, lymphocytes or vascular endothelial cells. Granulocytes were found mainly in necrotic areas, less frequently in non-necrotic and at the periphery of the tumor. Positive correlations were observed between the number of infiltrating granulocytes and MF, and between % tumor necrosis and MF. These results are consistent with the notion that endogenously generated nitric oxide (NO$\cdot$) may contribute to an increase in MF in MN-11 tumors. Administration of vitamin E to tumor-bearing mice resulted in a significant decrease in MF by 50%. A series of nitrogen monoxide (NO)-donating drugs that differed in their rate of NO$\cdot$ release was tested to induce mutants both in vitro and in vivo. The results show that NO-donating drugs can induce mutants in MN-11 cells but they varied widely in their mutagenic potential. Glyceryl trinitrate and sodium nitroprusside were mutagenic in MN-11 cells. The mutation can be blocked in vitro by pretreatment with vitamin E. As a positive control, exposure of tumor-bearing mice to ionizing radiation and cisplatin also showed an increase in MF. Administration of glyceryl trinitrate to tumor-bearing mice produced a large increase in MF that could also be blocked by vitamin E. (Abstract shortened by UMI.)
dc.format.extent220 p.
dc.publisherUniversity of Ottawa (Canada)
dc.subject.classificationBiology, Molecular.
dc.titleStudy of mutations arising in vivo and in vitro in a new murine tumor model system: The role of reactive oxygen and nitrogen species.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010

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