Mutagenesis induced by the tumor microenvironment
This is an Open Access Thesis
Tumors are characterized by microenvironmental heterogeneity, with regions of hypoxia, low pH, and nutrient deprivation. It has been proposed that such conditions may be an important factor contributing to cancer genetic instability. Using a chromosomally based lambda phage shuttle vector as a mutation reporter, we show that growth of cells in hypoxia and/or at low pH results in elevated mutation frequencies in two different reporter genes. To elucidate the mechanism of the tumor microenvironment-induced genetic instability, we first measured the production of DNA damage upon hypoxia and re-oxygenation in culture. We found that under such conditions, the intracellular level of a premutagenic lesion, 8-oxo-dG, was increased by 2- to 3-fold, consistent with the frequent G to T transversions observed among the hypoxia-induced mutation spectrum. We next examined the effect of the tumor microenvironment on two important cellular DNA repair pathways, i.e., the nucleotide excision repair (NER) and the mismatch repair (MMR) pathways. Using an assay for repair based on host cell reactivation of UV-damaged plasmid DNA, cells exposed to hypoxia and low pH were found to have a diminished capacity for DNA NER compared to control cells grown under standard conditions. Furthermore, the expression of the MMR gene, Mlh1, was specifically reduced under hypoxia, along with secondary decrease in Pms2 protein. On a functional level, instability of simple dinucleotide repeats, a hallmark of MMR deficiency, was detected in hypoxic cells. Taken together, our work suggests that the tumor microenvironment may lead to conditions that either cause DNA damage or compromise DNA repair processes, and consequently contribute to mutagenesis and genomic instability. This may constitute a fundamental mechanism of tumor progression in vivo.