DNA mismatch repair at an oncogenic hotspot correlated with phase of the cell cycle and environmentally relevant concentrations of the Arctic pollutant p, p'-DDE

Abstract

Part I: Mismatch repair in G₁ synchronized mammalian cells. Deficiencies in DNA mismatch repair have been found in hereditary nonpolyposis colon cancer (HNPCC), as well as in sporadic cancers, illustrating the importance of this single repair system in maintaining genomic integrity. In bacteria, this repair system functions primarily, after DNA replication, in the correction of polymerase base insertion errors and in mammalian cells it was also assumed that the mismatch repair system functioned within a similar timeframe. However, DNA mismatches occur ubiquitously and their repair before DNA replication is of paramount importance for faithful genome copying. We investigated the activity of the mismatch repair system, in G₁ synchronized NIH 3T3 cells, in the repair of four mismatches at an oncogenic hotspot in the H-ras gene. Our results clearly show that the mismatch repair system is active and accurate during the pre-replicative G₁ phase of the mammalian cell cycle.

Part II: Effects of p, p'-DDE on cell toxicity and DNA mismatch repair ability. Umbilical cord blood, from Inupiat infants in Barrow Alaska, was examined for the presence of several environmental contaminants. All 24 blood samples analyzed contained measurable levels of p, p'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) with an average concentration of 0.33 ug/1. We examined whether this low concentration of p, p'-DDE had detectable effects on NIH 3T3 (mouse embryonic) and WS1 (human fetal) cells in culture. Initial experiments indicated that exposure to p, p'-DDE resulted in a decrease in the cell number of both cell types. Subsequent analysis revealed that this decrease was due to cell death in NIH 3T3 cells and to cell cycle arrest in WS1 cells. We also examined the effect of p, p'-DDE on the ability of both cell types to repair mismatches at an oncogenic hotspot in H-ras. Preliminary results indicate that p, p'-DDE does not have a discernable effect on the ability of either cell type to correctly repair the G:T mismatch. However, p, p'-DDE exposure results in an increased rate of correct repair of the G:A mismatch by both cell types. Overall, this study demonstrates that p, p'-DDE, at concentrations relevant to the Alaskan environment has significantn but different effects on two immature cell types in culture.