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Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts.

El-Karim EA, Hagos EG, Ghaleb AM, Yu B, Yang VW - Mol. Cancer (2013)

Bottom Line: Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer.In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT

Background: Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.

Methods: To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs for Klf4 (Klf4(-/-)), we transfected Klf4(-/-)MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4(+/+)) and untransfected or mock-transfected Klf4(-/-)MEFs.

Results: We show that overexpression of Klf4 in Klf4(-/-)MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.

Conclusion: Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

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Immunostaining for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. (A) Immunostaining was conducted for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. Non-irradiated Klf4+/+ and Klf4−/−MEFs were stained with antibodies against γ-H2AX and 53BP1 and hoechst stain (blue) was used to visualize nuclei. Shown is a representative result of three independent experiments. (B) Histogram showing quantification of cells with ≥ 5 γ-H2AX foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. (C ) Histogram showing quantification of cells with ≥ 5 53BP1 foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. For (B) and (C), foci were counted for non-irradiated cells, and at 1, 4 and 24h post irradiation. One hundred cells were counted per cell type per experiment. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells.
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Figure 3: Immunostaining for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. (A) Immunostaining was conducted for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. Non-irradiated Klf4+/+ and Klf4−/−MEFs were stained with antibodies against γ-H2AX and 53BP1 and hoechst stain (blue) was used to visualize nuclei. Shown is a representative result of three independent experiments. (B) Histogram showing quantification of cells with ≥ 5 γ-H2AX foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. (C ) Histogram showing quantification of cells with ≥ 5 53BP1 foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. For (B) and (C), foci were counted for non-irradiated cells, and at 1, 4 and 24h post irradiation. One hundred cells were counted per cell type per experiment. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells.

Mentions: To determine the role of Klf4 in the DNA damage response and repair process, we first evaluated the extent of double strand breaks (DSBs) with and without γ-irradiation in Klf4+/+ and Klf4−/−MEFs using γ-H2AX and 53BP1 as markers of DNA damage. We induced DNA damage with γ-irradiation and counted cells with 5 or more foci for each marker at 0, 1, 4, and 24h post γ-irradiation. An example of γ-H2AX and 53BP1 staining in non-irradiated Klf4+/+ and Klf4−/−MEFs is shown in Figure 3A. Approximately 25% of non-irradiated Klf4+/+MEFs and 90% of Klf4−/−MEFs had ≥5 γ-H2AX foci (Figure 3B). The number of cells with ≥5 γ-H2AX foci was significantly increased at 1 and 4h post-irradiation in Klf4+/+MEFs and then returned to the basal level by 24 h post-irradiation. In contrast, the number of cells with ≥5 γ-H2AX foci remained elevated in Klf4−/−MEFs up to 24 h post-irradiation. A similar trend was noted for cells with ≥5 53BP1 foci in the two MEFs before and after irradiation (Figure 3C). These results suggest that while wild-type MEFs exhibit a normal DNA damage response following γ-irradiation, cells lacking Klf4 have persistent evidence of DNA damage.


Krüppel-like factor 4 regulates genetic stability in mouse embryonic fibroblasts.

El-Karim EA, Hagos EG, Ghaleb AM, Yu B, Yang VW - Mol. Cancer (2013)

Immunostaining for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. (A) Immunostaining was conducted for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. Non-irradiated Klf4+/+ and Klf4−/−MEFs were stained with antibodies against γ-H2AX and 53BP1 and hoechst stain (blue) was used to visualize nuclei. Shown is a representative result of three independent experiments. (B) Histogram showing quantification of cells with ≥ 5 γ-H2AX foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. (C ) Histogram showing quantification of cells with ≥ 5 53BP1 foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. For (B) and (C), foci were counted for non-irradiated cells, and at 1, 4 and 24h post irradiation. One hundred cells were counted per cell type per experiment. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells.
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Figure 3: Immunostaining for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. (A) Immunostaining was conducted for γ-H2AX and 53BP1 in Klf4+/+ and Klf4−/−MEFs. Non-irradiated Klf4+/+ and Klf4−/−MEFs were stained with antibodies against γ-H2AX and 53BP1 and hoechst stain (blue) was used to visualize nuclei. Shown is a representative result of three independent experiments. (B) Histogram showing quantification of cells with ≥ 5 γ-H2AX foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. (C ) Histogram showing quantification of cells with ≥ 5 53BP1 foci in Klf4+/+ and untransfected Klf4−/−MEFs with and without γ-irradiation. For (B) and (C), foci were counted for non-irradiated cells, and at 1, 4 and 24h post irradiation. One hundred cells were counted per cell type per experiment. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells.
Mentions: To determine the role of Klf4 in the DNA damage response and repair process, we first evaluated the extent of double strand breaks (DSBs) with and without γ-irradiation in Klf4+/+ and Klf4−/−MEFs using γ-H2AX and 53BP1 as markers of DNA damage. We induced DNA damage with γ-irradiation and counted cells with 5 or more foci for each marker at 0, 1, 4, and 24h post γ-irradiation. An example of γ-H2AX and 53BP1 staining in non-irradiated Klf4+/+ and Klf4−/−MEFs is shown in Figure 3A. Approximately 25% of non-irradiated Klf4+/+MEFs and 90% of Klf4−/−MEFs had ≥5 γ-H2AX foci (Figure 3B). The number of cells with ≥5 γ-H2AX foci was significantly increased at 1 and 4h post-irradiation in Klf4+/+MEFs and then returned to the basal level by 24 h post-irradiation. In contrast, the number of cells with ≥5 γ-H2AX foci remained elevated in Klf4−/−MEFs up to 24 h post-irradiation. A similar trend was noted for cells with ≥5 53BP1 foci in the two MEFs before and after irradiation (Figure 3C). These results suggest that while wild-type MEFs exhibit a normal DNA damage response following γ-irradiation, cells lacking Klf4 have persistent evidence of DNA damage.

Bottom Line: Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer.In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.

ABSTRACT

Background: Krüppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors and regulates proliferation, differentiation, apoptosis and somatic cell reprogramming. Evidence also suggests that KLF4 is a tumor suppressor in certain cancers including colorectal cancer. We previously showed that KLF4 inhibits cell cycle progression following DNA damage and that mouse embryonic fibroblasts (MEFs) for Klf4 are genetically unstable, as evidenced by increased rates of cell proliferation, and the presence of DNA double strand breaks (DSBs), centrosome amplification, chromosome aberrations and aneuploidy.

Methods: To determine whether re-expression of Klf4 corrects the observed genetic instability in MEFs for Klf4 (Klf4(-/-)), we transfected Klf4(-/-)MEFs with Klf4-expressing plasmids and compared the results to wild type (Klf4(+/+)) and untransfected or mock-transfected Klf4(-/-)MEFs.

Results: We show that overexpression of Klf4 in Klf4(-/-)MEFs reduced cell proliferation rates and the proportion of cells with DSBs, abnormal centrosome numbers, aneuploidy and micronuclei. In addition, Klf4-transfected Klf4(-/-)MEFs exhibited a more robust DNA damage repair response as demonstrated by the greater rate in disappearance of γ-H2AX and 53BP1 foci following γ-irradiation.

Conclusion: Taken together these findings provide evidence that KLF4 plays a crucial role in the maintenance of genetic stability by modulating the DNA damage response and repair processes.

Show MeSH
Related in: MedlinePlus