Abstract 2028: Krüppel-like factor 4 null mouse embryonic fibroblasts exhibit DNA repair defects post exposure to gamma-irradiation

Abstract

Abstract BACKGROUND: Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that exerts a potent inhibitory effect on cell cycle progression. KLF4 functions as a tumor suppressor in colorectal cancer and is involved in regulating centrosome duplication. Abnormal amplification of centrosomes has been shown to contribute to genetic instability by increasing the frequency of mitotic defects. AIM: To determine whether KLF4 is involved in maintaining genetic stability during cell division. METHODS: Mice heterozygous for the Klf4 alleles (Klf4+/−) on a C57BL/6 background were crossbred. Mouse embryonic fibroblasts (MEFs) that are wild type (Klf4+/+), heterozygous (Klf4+/−) or null (Klf4−/−) for the Klf4 alleles were derived from day-13.5 embryos. Post-senescence MEFs were derived by passing cells to senescence and maintaining them in culture until they spontaneously regained proliferative. Immuno-detection of histone variant γ-H2AX was performed to assess chromosome integrity. The levels of cell cycle regulators such as Cdk2, Cyclin E, and Cyclin D were determined by Western blotting. The presence of double strand breaks (DSB) was determined by western blot and immunofluorescence for histone variant -H2AX contains nuclear foci. Flow cytometry has been used to determine the level of DNA damage in both wildtype and klf4 null cells following gamma-irradiation. Furthermore, Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. RESULTS: Cells that exhibits DSBs in their DNA can develop genomic instability, which can result in cancer formation. Compared to Klf4+/+ and Klf4−/− MEFs exposed to α-irradiation showed time-dependent increase in levels of histone variant -H2AX that persisted for up to 48 hours after the α-irradiation. On the other hand, Klf4+/+ halt cell cycle progression to allow repair of DNA damage. Western blot analysis showed that Cdk2, Cyclin E, Cyclin D and histone variant -H2AX expression were high in Klf4−/− MEFs. Furthermore, we found many of the down-regulated genes in Klf4-null MEFs encode DNA repair, and antioxidant genes. CONCLUSIONS: Results of this study demonstrate that Klf4 null mouse embryonic fibroblasts exhibit an impaired DNA damage response post gamma irradiation, when compared to wild-type cells. This data strongly suggest that the role for Klf4 in DNA damage repair in mouse embryonic fibroblast cells. These results indicate that KLF4 plays a crucial role in maintaining genetic stability and support the previous findings that KLF4 is a tumor suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2028. doi:1538-7445.AM2012-2028