Oxidative DNA damage causes premature senescence in mouse embryonic fibroblasts deficient for Krüppel-like factor 4.
Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress-induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4-deficient MEFs. With SA-β-Gal staining, we show that Klf4(-/-) MEFs enter senescence earlier than Klf4(+/+) MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against γ-H2AX indicates that the increased level of DNA damage in Klf4(-/-) MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4(-/-) MEFs, treatment with NAC, reduces the accumulation of DNA damage. Our RT-PCR result demonstrates that Klf4(-/-) MEFs have decreased expression of the antioxidant gene, Gsta4. The downregulation of the Gsta4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress-induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes.
Department of Biology, Colgate University, Hamilton, New York.
Changchang
C
Liu
Department of Biology, Colgate University, Hamilton, New York.|Memorial Sloan-Kettering Cancer Center, Prostate Cancer Research Program, New York, New York.
Stephen
S
La Rosa
Department of Biology, Colgate University, Hamilton, New York.
Engda G
EG
Hagos
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D016922:Cellular Senescence; D004249:DNA Damage; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology*; D017353:Gene Deletion; D042822:Genomic Instability; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53
Krüppel-like factor 4; cellular senescence and reactive oxygen species; genomic instability