Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation

Abstract

Chronic oxidative stress may play a critical role in the pathogenesis of many human cancers. Here, we report that mouse embryonic stem (ES) cells deficient in DNA mismatch repair responded abnormally when exposed to low levels of ionizing radiation, a stress known to generate oxidative DNA damage. ES cells derived from mice carrying either one or two disrupted Msh2 alleles displayed an increased survival following protracted exposures to low-level ionizing radiation as compared with wild-type ES cells. The increases in survival exhibited by ES cells deficient in DNA mismatch repair appeared to have resulted from a failure to efficiently execute cell death (apoptosis) in response to radiation exposure. For each of the ES cell types, prolonged low-level radiation treatment generated oxidative genome damage that manifested as an accumulation of oxidized bases in genomic DNA. However, ES cells from Msh2(+/-) and Msh2(-/-) mice accumulated more oxidized bases as a consequence of low-level radiation exposure than ES cells from Msh2(+/+) mice. The propensity for normal cells with mismatch repair enzyme deficiencies, including cells heterozygous for inactivating mismatch repair enzyme gene mutations, to survive promutagenic genome insults accompanying oxidative stresses may contribute to the increased cancer risk characteristic of the hereditary nonpolyposis colorectal cancer syndrome.

Figure 1

Mouse ES cells carrying either one or two disrupted Msh2 genes appear tolerant to lethal effects of low-level radiation exposure. Displayed are clonogenic survival curves obtained from analysis of wt mouse ES cells (Msh2^+/+) and ES cells carrying disrupted Msh2 genes (Msh2^+/− and Msh2^−/−) after (A) prolonged (24, 48, and 72 h) exposure to low-level ionizing radiation (0.004 Gy/min) or after (B) treatment with acute dose ionizing radiation (1.0 Gy/min). Each symbol represents the mean of multiple, triplicate experiments; the SEM is shown by using error bars. In some cases, the error bars are smaller than the symbol.

Figure 2

Mouse ES cells containing defective Msh2 genes fail to efficiently execute cell death (apoptosis) in response to low-level radiation treatment. Apoptosis induction was assessed by using flow cytometry to monitor the appearance of cells containing fragmented DNA (see Materials and Methods) for wt ES cells (Msh2^+/+) and for ES cells carrying disrupted Msh2 genes (Msh2^+/− and Msh2^−/−) after exposure to low-level ionizing radiation for 24 h. Mean values ± SEM are displayed.

Figure 3

DNA from mouse ES cells containing disrupted Msh2 genes accumulates more oxidative base damage as a consequence of low-level radiation exposure than wt ES cells. (A) Increases in oxidized dGua (8OHdGua) in DNA from different ES cell lines treated with low-level radiation, assessed by using HPLC-ECD (see Materials and Methods), are displayed as a ratio of the number of detected 8OHdGua per 10⁶ dGua in irradiated vs. unirradiated cell cultures. (B) Levels of the oxidized bases 8-hydroxyadenine (8OHAde) and thymine glycol (TG) as well as levels of 5-methylcytosine (5-MeCyt) in DNA from ES cells with and without defective mismatch repair (i.e., Msh2^+/+ and Msh2^−/−, respectively) were determined before and after low-level irradiation by GC/MS-SIM (see Materials and Methods). For both HPLC-ECD and GC/MS-SIM, DNA from ES cells was collected and pooled following at least five independent exposures before being subjected to analysis for oxidized bases.