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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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Overexpression of Klf4 suppresses centrosome amplification in Klf4−/−MEFs. Centrosome staining was conducted with an antibody against γ-tubulin and detected with Alexa Fluor 488-conjugated antibody. Hoechst stain (blue) was used to visualize the nuclei. (A) A representative image of centrosome staining of Klf4+/+ and Klf4−/− MEFs. The inset shows a Klf4+/+ cell with 2 centrosomes and a Klf4−/− cell with abnormal number of centrosomes (≥ 3). Shown is a typical result of 4 independent experiments. (B) histogram showing quantification in percentages of cells with ≥3 centrosomes in Klf4+/+, Klf4−/−, and GFP or Klf4-GFP transfected Klf4-/ -MEFs. One hundred cells were counted per cell type per experiments. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells. (C) A graph showing the percentage of green Klf4−/− cells (positive for GFP-control or Klf4-GFP) with ≥3 centrosomes. One hundred green cells were counted per cell type per experiments. N = 5; * p < 0.05 compared to Klf4−/− cells transfected with GFP alone.
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Figure 2: Overexpression of Klf4 suppresses centrosome amplification in Klf4−/−MEFs. Centrosome staining was conducted with an antibody against γ-tubulin and detected with Alexa Fluor 488-conjugated antibody. Hoechst stain (blue) was used to visualize the nuclei. (A) A representative image of centrosome staining of Klf4+/+ and Klf4−/− MEFs. The inset shows a Klf4+/+ cell with 2 centrosomes and a Klf4−/− cell with abnormal number of centrosomes (≥ 3). Shown is a typical result of 4 independent experiments. (B) histogram showing quantification in percentages of cells with ≥3 centrosomes in Klf4+/+, Klf4−/−, and GFP or Klf4-GFP transfected Klf4-/ -MEFs. One hundred cells were counted per cell type per experiments. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells. (C) A graph showing the percentage of green Klf4−/− cells (positive for GFP-control or Klf4-GFP) with ≥3 centrosomes. One hundred green cells were counted per cell type per experiments. N = 5; * p < 0.05 compared to Klf4−/− cells transfected with GFP alone.

Mentions: We previously showed that Klf4 plays a role in regulating centrosome duplication in MEFs - whereas 2-3% of Klf4+/+ MEFs exhibited centrosome amplification, defined as the presence of 3 or more centrosomes per cell, approximately 25% of the Klf4−/−MEFs had centrosome amplification, [18]. To determine if re-expressing Klf4 corrects the numerical centrosome abnormality in Klf4−/−MEFs, we performed immunofluorescent staining of centrosomes with an antibody against γ-tubulin. An example of the results of such staining in Klf4+/+ and Klf4−/− is shown in Figure 2A, which demonstrates the normal distribution of 2 centrosomes per cell in Klf4+/+MEFs but up to 8 centrosomes in a Klf4−/−MEF. We quantified the total number of cells with 3 or more centrosomes in Klf4+/+, GFP-control-transfected Klf4−/−, and Klf4-GFP-transfected Klf4−/−MEFs. As shown in Figure 2B, there was a significant increase in cells with 3 or more centrosomes in GFP-transfected Klf4−/− as compared to Klf4+/+ cells (2-3% and 17%, respectively). Overexpression of Klf4 in Klf4−/−MEFs significantly reduced the number of cells with 3 or more centrosomes to an average of 7%. To further demonstrate a direct link between Klf4 levels and the extent of centrosome number correction, we overexpressed Klf4-GFP in Klf4−/−MEFs and counted only the cells that were positive for GFP and have ≥3 centrosomes. As shown in Figure 2C, overexpression of Klf4-GFP in Klf4−/−MEFs resulted in a significant decrease in the percentage of cells with ≥3 centrosomes compared to GFP-control-transfected Klf4−/−MEFs (12% and 32%, respectively).


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)

Overexpression of Klf4 suppresses centrosome amplification in Klf4−/−MEFs. Centrosome staining was conducted with an antibody against γ-tubulin and detected with Alexa Fluor 488-conjugated antibody. Hoechst stain (blue) was used to visualize the nuclei. (A) A representative image of centrosome staining of Klf4+/+ and Klf4−/− MEFs. The inset shows a Klf4+/+ cell with 2 centrosomes and a Klf4−/− cell with abnormal number of centrosomes (≥ 3). Shown is a typical result of 4 independent experiments. (B) histogram showing quantification in percentages of cells with ≥3 centrosomes in Klf4+/+, Klf4−/−, and GFP or Klf4-GFP transfected Klf4-/ -MEFs. One hundred cells were counted per cell type per experiments. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells. (C) A graph showing the percentage of green Klf4−/− cells (positive for GFP-control or Klf4-GFP) with ≥3 centrosomes. One hundred green cells were counted per cell type per experiments. N = 5; * p < 0.05 compared to Klf4−/− cells transfected with GFP alone.
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Figure 2: Overexpression of Klf4 suppresses centrosome amplification in Klf4−/−MEFs. Centrosome staining was conducted with an antibody against γ-tubulin and detected with Alexa Fluor 488-conjugated antibody. Hoechst stain (blue) was used to visualize the nuclei. (A) A representative image of centrosome staining of Klf4+/+ and Klf4−/− MEFs. The inset shows a Klf4+/+ cell with 2 centrosomes and a Klf4−/− cell with abnormal number of centrosomes (≥ 3). Shown is a typical result of 4 independent experiments. (B) histogram showing quantification in percentages of cells with ≥3 centrosomes in Klf4+/+, Klf4−/−, and GFP or Klf4-GFP transfected Klf4-/ -MEFs. One hundred cells were counted per cell type per experiments. N = 5; * p < 0.05, ** p < 0.01 compared to Klf4+/+ cells. (C) A graph showing the percentage of green Klf4−/− cells (positive for GFP-control or Klf4-GFP) with ≥3 centrosomes. One hundred green cells were counted per cell type per experiments. N = 5; * p < 0.05 compared to Klf4−/− cells transfected with GFP alone.
Mentions: We previously showed that Klf4 plays a role in regulating centrosome duplication in MEFs - whereas 2-3% of Klf4+/+ MEFs exhibited centrosome amplification, defined as the presence of 3 or more centrosomes per cell, approximately 25% of the Klf4−/−MEFs had centrosome amplification, [18]. To determine if re-expressing Klf4 corrects the numerical centrosome abnormality in Klf4−/−MEFs, we performed immunofluorescent staining of centrosomes with an antibody against γ-tubulin. An example of the results of such staining in Klf4+/+ and Klf4−/− is shown in Figure 2A, which demonstrates the normal distribution of 2 centrosomes per cell in Klf4+/+MEFs but up to 8 centrosomes in a Klf4−/−MEF. We quantified the total number of cells with 3 or more centrosomes in Klf4+/+, GFP-control-transfected Klf4−/−, and Klf4-GFP-transfected Klf4−/−MEFs. As shown in Figure 2B, there was a significant increase in cells with 3 or more centrosomes in GFP-transfected Klf4−/− as compared to Klf4+/+ cells (2-3% and 17%, respectively). Overexpression of Klf4 in Klf4−/−MEFs significantly reduced the number of cells with 3 or more centrosomes to an average of 7%. To further demonstrate a direct link between Klf4 levels and the extent of centrosome number correction, we overexpressed Klf4-GFP in Klf4−/−MEFs and counted only the cells that were positive for GFP and have ≥3 centrosomes. As shown in Figure 2C, overexpression of Klf4-GFP in Klf4−/−MEFs resulted in a significant decrease in the percentage of cells with ≥3 centrosomes compared to GFP-control-transfected Klf4−/−MEFs (12% and 32%, respectively).

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