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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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Determination of micronuclei (MN) formation in MEFs. Micronuclei (MN) analysis was conducted in cytokinesis-arrested cells prepared from Klf4+/+ and from Klf4−/−MEFs transfected with GFP or Klf4-GFP. (A)Klf4+/+ and Klf4−/−MEFs transfected with GFP or Klf4-GFP were treated with cytochalasin-B (CCB) and Hoechst stain (blue) was used to visualize nuclei. Shown are representative images of cells containing MN. (B) Histogram showing quantification of percent of binucleated cells with MN 24h post CCB treatment. At least 200 green cells were counted for each genotype per experiment. N = 3; *** p < 0.001 compared to GFP-transfected Klf4−/− cells.
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Figure 6: Determination of micronuclei (MN) formation in MEFs. Micronuclei (MN) analysis was conducted in cytokinesis-arrested cells prepared from Klf4+/+ and from Klf4−/−MEFs transfected with GFP or Klf4-GFP. (A)Klf4+/+ and Klf4−/−MEFs transfected with GFP or Klf4-GFP were treated with cytochalasin-B (CCB) and Hoechst stain (blue) was used to visualize nuclei. Shown are representative images of cells containing MN. (B) Histogram showing quantification of percent of binucleated cells with MN 24h post CCB treatment. At least 200 green cells were counted for each genotype per experiment. N = 3; *** p < 0.001 compared to GFP-transfected Klf4−/− cells.

Mentions: Micronuclei are formed due to DSBs and represent a mechanism by which errors in chromosome segregation and DNA breaks are eliminated from the nucleus of the cell [24]. Given the increased proliferation rate, DNA damage and aneuploidy observed in Klf4−/− in comparison with Klf4+/+MEFs, we further assessed the frequency of micronuclei formation which can originate from DNA breaks, chromosome fragments or lagging chromosomes during aberrant cell division in Klf4−/−MEFs. We used cytochalasin-B, an inhibitor of cytokinesis that allows easily distinguishing between mononucleated non-dividing cells and binucleated dividing cells. To examine the effect of Klf4 on the frequency of binucleated cells containing micronuclei in Klf4−/−MEFs, we overexpressed GFP-control or Klf4-GFP in Klf4−/−MEFs and treated cells with cytochalasin-B. At 24 h following cytochalasin-B addition, we stained cells with Hoechst and analyzed only the green, binucleated cells and quantified the frequency of cells with micronuclei. An example of binucleated cells containing micronuclei staining in Klf4+/+ and binucleated green Klf4−/− cells is shown in Figure 6A. As shown in Figure 6B, approximately 4% of Klf4+/+ cells have binucleated cells with micronuclei, while 60-70% of GFP-control-transfected Klf4−/−MEFs have binucleated cells with micronuclei. Importantly, re-expression of Klf4-GFP in Klf4−/−MEFs reduced the levels of binucleated cells with micronuclei to 20-30% (Figure 6B).


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)

Determination of micronuclei (MN) formation in MEFs. Micronuclei (MN) analysis was conducted in cytokinesis-arrested cells prepared from Klf4+/+ and from Klf4−/−MEFs transfected with GFP or Klf4-GFP. (A)Klf4+/+ and Klf4−/−MEFs transfected with GFP or Klf4-GFP were treated with cytochalasin-B (CCB) and Hoechst stain (blue) was used to visualize nuclei. Shown are representative images of cells containing MN. (B) Histogram showing quantification of percent of binucleated cells with MN 24h post CCB treatment. At least 200 green cells were counted for each genotype per experiment. N = 3; *** p < 0.001 compared to GFP-transfected Klf4−/− cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Determination of micronuclei (MN) formation in MEFs. Micronuclei (MN) analysis was conducted in cytokinesis-arrested cells prepared from Klf4+/+ and from Klf4−/−MEFs transfected with GFP or Klf4-GFP. (A)Klf4+/+ and Klf4−/−MEFs transfected with GFP or Klf4-GFP were treated with cytochalasin-B (CCB) and Hoechst stain (blue) was used to visualize nuclei. Shown are representative images of cells containing MN. (B) Histogram showing quantification of percent of binucleated cells with MN 24h post CCB treatment. At least 200 green cells were counted for each genotype per experiment. N = 3; *** p < 0.001 compared to GFP-transfected Klf4−/− cells.
Mentions: Micronuclei are formed due to DSBs and represent a mechanism by which errors in chromosome segregation and DNA breaks are eliminated from the nucleus of the cell [24]. Given the increased proliferation rate, DNA damage and aneuploidy observed in Klf4−/− in comparison with Klf4+/+MEFs, we further assessed the frequency of micronuclei formation which can originate from DNA breaks, chromosome fragments or lagging chromosomes during aberrant cell division in Klf4−/−MEFs. We used cytochalasin-B, an inhibitor of cytokinesis that allows easily distinguishing between mononucleated non-dividing cells and binucleated dividing cells. To examine the effect of Klf4 on the frequency of binucleated cells containing micronuclei in Klf4−/−MEFs, we overexpressed GFP-control or Klf4-GFP in Klf4−/−MEFs and treated cells with cytochalasin-B. At 24 h following cytochalasin-B addition, we stained cells with Hoechst and analyzed only the green, binucleated cells and quantified the frequency of cells with micronuclei. An example of binucleated cells containing micronuclei staining in Klf4+/+ and binucleated green Klf4−/− cells is shown in Figure 6A. As shown in Figure 6B, approximately 4% of Klf4+/+ cells have binucleated cells with micronuclei, while 60-70% of GFP-control-transfected Klf4−/−MEFs have binucleated cells with micronuclei. Importantly, re-expression of Klf4-GFP in Klf4−/−MEFs reduced the levels of binucleated cells with micronuclei to 20-30% (Figure 6B).

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