Effects of neutrophils and reactive nitrogen oxide species in tumors as determined by genetic manipulation of the Mutatect mouse tumor model.

摘要

Most human tumors exhibit accumulation of mutations throughout their progression. In addition, many human tumors are infiltrated with inflammatory cells, such as neutrophils and macrophages. These cells can generate potentially mutagenic species, including nitric oxide-derived species. We hypothesize that these species contribute to the accumulation of mutations in tumors. Our lab previously developed the ‘Mutatect’ mouse tumor model to study this hypothesis. Mutatect fibrosarcoma cells can form solid tumors in C57BL/6 mice when injected subcutaneously. These tumors are variably infiltrated with neutrophils. The neutrophils contain inducible nitric oxide synthase, and tumor cells contain nitrotyrosine, an indicator of nitric oxide-derived damage. Neutrophil number correlates with mutation arising in vivo in Mutatect cells at the hypoxanthine phosphoribosyltransferase (hprt) locus, a marker of genetic damage. Dietary vitamin E significantly lowers the hprt mutations. I genetically engineered Mutatect cells to produce interleukin-8, a neutrophil attractant. Cells expressing high levels of interleukin-8 formed small tumors, whereas cells expressing lower levels produced large tumors. A biochemical assay was developed to quantify neutrophil content in tumors. Interleukin-8-expressing tumors had a significantly higher neutrophil content and hprt mutations than non-expressing tumors. Interleukin-8 gene instability was observed in high interleukin-8-expressing tumors. Dietary vitamin E dramatically inhibited both hprt mutations and interleukin-8 instability. It also affected neutrophil distribution in tumors. High level of protein nitrotyrosine was seen in tumors with high neutrophil content (and mutations). Using proteomics tools, a significant amount of nitrotyrosine could be identified in histones, the major nuclear proteins associated with DNA. Mechanism of nitric oxide-derived mutagenicity was also examined. Previous studies implicate the role of intracellular S-nitrosoglutathione, which was recently found to be detoxified by formaldehyde dehydrogenase. I developed several cell-lines with down-regulated formaldehyde dehydrogenase levels using RNA interference and antisense techniques. A metabolic cycle, involving formaldehyde dehydrogenase, capable of regulating protein S-nitrosation was identified. This thesis has contributed significantly towards understanding the role of inflammatory cells and the nitric oxide-derived factors in solid tumors. It has also allowed us to understand the mechanism(s) of S-nitrosation and tyrosine nitration in proteins, both of which may have physiological and pathological relevance.