Effects of oxidative and alkylating damage on microsatellite instability in nontumorigenic human cells.
Microsatellite instability is a phenomenon that is well characterized in mismatch repair-deficient tumor cell lines, including the potential etiological role of endogenous DNA damage. However, our understanding of microsatellite mutational mechanisms in repair-proficient, nontumorigenic cells is limited. We determined microsatellite mutation frequencies for human lymphoblastoid cells using an episomal DNA shuttle vector in which a (TTCC/AAGG)(9) microsatellite is inserted in-frame within the herpes simplex virus thymidine kinase (HSV-tk) gene. The responses of plasmid-bearing cells to reactive oxygen species or alkylating agents were compared after treatment with hydrogen peroxide (H(2)O(2)) and N-ethyl-N-nitrosourea (ENU). H(2)O(2) treatment induced a statistically significant increase in overall HSV-tk mutation frequency relative to controls, with catalase reducing the effect. H(2)O(2) treatment increased the mutation frequency within the microsatellite and the HSV-tk coding region to a similar extent (five and six-fold, respectively, relative to the control). Mutational specificity analyses demonstrated that the proportion of mutations within the microsatellite is not statistically different among the H(2)O(2), catalase, and PBS treatment groups. In contrast, treatment of cells bearing the microsatellite vector with ENU altered the mutational spectrum, relative to solvent control. ENU induced the expected base substitutions within the HSV-tk coding region, but did not increase the microsatellite mutation frequency. The low level of microsatellite mutagenesis observed after reactive oxygen species (ROS) insult likely reflects the normal repair processes of these nontumorigenic, repair-competent cells. Our ex vivo experiments demonstrate the manner in which repetitive DNA in normal human cells might respond to endogenous mutagens.
Maneval
Mandy L
ML
Jake Gittlen Cancer Research Institute, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
Eckert
Kristin A
KA
D000477:Alkylating Agents / Q000633:toxicity*; D002999:Clone Cells; D004249:DNA Damage; D004121:Dimethyl Sulfoxide / Q000633:toxicity*; D005038:Ethylnitrosourea / Q000633:toxicity; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D018895:Microsatellite Repeats / Q000235:genetics*; D009153:Mutagens / Q000633:toxicity; D018384:Oxidative Stress; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics
D000477:Alkylating Agents; D009153:Mutagens; D006861:Hydrogen Peroxide; D013937:Thymidine Kinase; D005038:Ethylnitrosourea; D004121:Dimethyl Sulfoxide