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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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Related in: MedlinePlus

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

Mentions: We then determined if re-expression of Klf4 corrects the DNA damage observed in Klf4−/−MEFs by transfecting GFP-control or Klf4-GFP into Klf4−/− cells. Figure 4A shows an example of GFP-positive green cells with γ-H2AX and 53BP1 staining in GFP- or Klf4-GFP-transfected Klf4−/−MEFs. As shown in Figure 4B, at baseline, transfection of Klf4−/− cells with Klf4-GFP significantly reduced the number of green cells with γ-H2AX foci as compared to GFP-transfected cells. At 1 and 4 h after irradiation, the number of cells with γ-H2AX foci increased in Klf4-GFP-transfected Klf4−/−MEFs and then returned to a level below that of GFP-transfected MEFs at 24 h post-irradiation. A similar trend is noted for 53BP1 except that Klf4-GFP-transfectedMEFs had lower percentage of cells with ≥5 53BP1 foci relative to GFP-transfected cells at all time-points (Figure 4C). These data suggest that re-expression of Klf4 in Klf4−/−MEFs results in a more efficient repair of DNA damage compared to control Klf4−/− cells.


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−/−MEFs transfected with GFP or Klf4-GFP. (A) Immunostaining was conducted for γ-H2AX and 53BP1 in Klf4−/−MEFs transfected with GFP or Klf4-GFP.hoechst stain (blue) was used to visualize nuclei. Shown is a representative result of three independent experiments for the non-irradiated cells. (B) Histogram showing quantification of cells with ≥ 5 γ-H2AX foci in Klf4−/−MEFs cells transfected with GFP or Klf4-GFP, with or without γ-irradiation. (C) Histogram showing quantification of cells with ≥ 5 53BP1 foci in Klf4−/−MEFs cells transfected with GFP or Klf4-GFP, with or without γ-irradiation. For (B) and (C), foci were counted for non-irradiated cells, and at 1, 4 and 24 h post irradiation. One hundred green cells were counted per cell type per experiment. N = 5; * p < 0.05, ** p < 0.01 compared to GFP-transfected Klf4−/− cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3750599&req=5

Figure 4: Immunostaining for γ-H2AX and 53BP1 in Klf4−/−MEFs transfected with GFP or Klf4-GFP. (A) Immunostaining was conducted for γ-H2AX and 53BP1 in Klf4−/−MEFs transfected with GFP or Klf4-GFP.hoechst stain (blue) was used to visualize nuclei. Shown is a representative result of three independent experiments for the non-irradiated cells. (B) Histogram showing quantification of cells with ≥ 5 γ-H2AX foci in Klf4−/−MEFs cells transfected with GFP or Klf4-GFP, with or without γ-irradiation. (C) Histogram showing quantification of cells with ≥ 5 53BP1 foci in Klf4−/−MEFs cells transfected with GFP or Klf4-GFP, with or without γ-irradiation. For (B) and (C), foci were counted for non-irradiated cells, and at 1, 4 and 24 h post irradiation. One hundred green cells were counted per cell type per experiment. N = 5; * p < 0.05, ** p < 0.01 compared to GFP-transfected Klf4−/− cells.
Mentions: We then determined if re-expression of Klf4 corrects the DNA damage observed in Klf4−/−MEFs by transfecting GFP-control or Klf4-GFP into Klf4−/− cells. Figure 4A shows an example of GFP-positive green cells with γ-H2AX and 53BP1 staining in GFP- or Klf4-GFP-transfected Klf4−/−MEFs. As shown in Figure 4B, at baseline, transfection of Klf4−/− cells with Klf4-GFP significantly reduced the number of green cells with γ-H2AX foci as compared to GFP-transfected cells. At 1 and 4 h after irradiation, the number of cells with γ-H2AX foci increased in Klf4-GFP-transfected Klf4−/−MEFs and then returned to a level below that of GFP-transfected MEFs at 24 h post-irradiation. A similar trend is noted for 53BP1 except that Klf4-GFP-transfectedMEFs had lower percentage of cells with ≥5 53BP1 foci relative to GFP-transfected cells at all time-points (Figure 4C). These data suggest that re-expression of Klf4 in Klf4−/−MEFs results in a more efficient repair of DNA damage compared to control Klf4−/− cells.

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