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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

Determination of ploidy in Klf4+/+ and Klf4−/−MEFs. Karyotype analysis was conducted in metaphase chromosome spreads prepared from Klf4+/+ and from Klf4−/−MEFs transfected or not with GFP or Klf4-GFP. (A) Shown is a typical result of three independent experiments for Klf4+/+ and Klf4−/−MEFs. (B-E) Histograms showing quantification of chromosome number in Klf4+/+ and in Klf4−/−MEFs untransfected or transfected with GFP or Klf4-GFP. Spreads from 100 cells were examined per genotype.
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Figure 5: Determination of ploidy in Klf4+/+ and Klf4−/−MEFs. Karyotype analysis was conducted in metaphase chromosome spreads prepared from Klf4+/+ and from Klf4−/−MEFs transfected or not with GFP or Klf4-GFP. (A) Shown is a typical result of three independent experiments for Klf4+/+ and Klf4−/−MEFs. (B-E) Histograms showing quantification of chromosome number in Klf4+/+ and in Klf4−/−MEFs untransfected or transfected with GFP or Klf4-GFP. Spreads from 100 cells were examined per genotype.

Mentions: We recently reported that Klf4−/−MEFs exhibited genetic instability manifested by the presence of aneuploidy [18]. To determine whether re-expression of Klf4 corrects this abnormality, we transfected Klf4−/−MEFs with Klf4-expressing plasmids and performed cytogenetic analysis. An example of metaphase chromosome spreads in Klf4+/+ and Klf4−/− is shown in Figure 5A. As seen in Figure 5B, analysis of metaphase chromosome spreads demonstrated that while Klf4+/+MEFs showed a distribution of chromosome numbers between the 35–44 and 75–84 (approximating haploid and diploid, respectively), Klf4−/−MEFs consistently had higher numbers of chromosomes with many cells displaying greater than 95 chromosomes per cell, indicating the presence of aneuploidy. Overexpression of Klf4 in Klf4−/−MEFs, however, resulted in a reduction in the number of cells exhibiting aneuploidy as compared to untransfected Klf4−/− or GFP-control-transfected Klf4−/−MEFs.


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 ploidy in Klf4+/+ and Klf4−/−MEFs. Karyotype analysis was conducted in metaphase chromosome spreads prepared from Klf4+/+ and from Klf4−/−MEFs transfected or not with GFP or Klf4-GFP. (A) Shown is a typical result of three independent experiments for Klf4+/+ and Klf4−/−MEFs. (B-E) Histograms showing quantification of chromosome number in Klf4+/+ and in Klf4−/−MEFs untransfected or transfected with GFP or Klf4-GFP. Spreads from 100 cells were examined per genotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Determination of ploidy in Klf4+/+ and Klf4−/−MEFs. Karyotype analysis was conducted in metaphase chromosome spreads prepared from Klf4+/+ and from Klf4−/−MEFs transfected or not with GFP or Klf4-GFP. (A) Shown is a typical result of three independent experiments for Klf4+/+ and Klf4−/−MEFs. (B-E) Histograms showing quantification of chromosome number in Klf4+/+ and in Klf4−/−MEFs untransfected or transfected with GFP or Klf4-GFP. Spreads from 100 cells were examined per genotype.
Mentions: We recently reported that Klf4−/−MEFs exhibited genetic instability manifested by the presence of aneuploidy [18]. To determine whether re-expression of Klf4 corrects this abnormality, we transfected Klf4−/−MEFs with Klf4-expressing plasmids and performed cytogenetic analysis. An example of metaphase chromosome spreads in Klf4+/+ and Klf4−/− is shown in Figure 5A. As seen in Figure 5B, analysis of metaphase chromosome spreads demonstrated that while Klf4+/+MEFs showed a distribution of chromosome numbers between the 35–44 and 75–84 (approximating haploid and diploid, respectively), Klf4−/−MEFs consistently had higher numbers of chromosomes with many cells displaying greater than 95 chromosomes per cell, indicating the presence of aneuploidy. Overexpression of Klf4 in Klf4−/−MEFs, however, resulted in a reduction in the number of cells exhibiting aneuploidy as compared to untransfected Klf4−/− or GFP-control-transfected Klf4−/−MEFs.

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