Latha Narayanan

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells

ABSTRACT The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)29 dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

Other transgenic mutation assays: Tissue specificity of spontaneous point mutations in λsupF transgenic mice

Transgenic mice carrying multiple copies of a recoverable λ phage shuttle vector carrying the supF mutation reporter gene (λsupF) were constructed for the purpose of studying mutagenesis in a whole animal. Spontaneous mutations in rescued supF target genes from mouse liver and skin were analyzed. The mutation frequency was similar in both tissues (in the range of 2 × 10−5), but the spectrum of point mutations was distinct, with transitions common in the skin and transversions more prominent in the liver (P = 0.01). These results may help to elucidate pathways of endogenous mutagenesis in vivo, and they illustrate potentially important tissue‐specific differences in genetic instability.

Diminished DNA repair and elevated mutagenesis in mammalian cells exposed to hypoxia and low pH

The tumor microenvironment is characterized by regions of fluctuating and chronic hypoxia, low pH, and nutrient deprivation. It has been proposed that this unique tissue environment itself may constitute a major cause of the genetic instability seen in cancer. To investigate possible mechanisms by which the tumor microenvironment might contribute to genetic instability, we asked whether the conditions found in solid tumors could influence cellular repair of DNA damage. 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 repair compared with control cells grown under standard culture conditions. In addition, cells cultured under hypoxia at pH 6.5 immediately after UV irradiation had elevated levels of induced mutagenesis compared with those maintained in standard growth conditions. Taken together, the results suggest that cellular repair functions may be impaired under the conditions of the tumor microenvironment, causing hypermutability to DNA damage. This alteration in repair capacity may constitute an important mechanism underlying the genetic instability of cancer cells in vivo.