Study of mutations arising in vivo and in vitro in a new murine tumor model system: The role of reactive oxygen and nitrogen species.
Creators: name: Sandhu, Jagdeep K.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1998
Subjects: subject: Biology, Molecular.

description: A 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.)