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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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Growth characteristics of Klf4-transfected Klf4−/−MEFs in culture. (A) Cell proliferation rates of Klf4+/+ and Klf4−/−MEFs, transfected or not with various plasmids. Cells were initially seeded at 105 cells/plate in 60 mm plates. On day 0, cells were transfected in triplicates with pmKLF4-pEGFP, GFP control, or mock-transfected in parallel. Number of cells was counted (three plates per transfection) every day over a total of 3 days. The growth rate of the MEFs from each transfection was determined at each time point and the values represent the mean number of cells per well. N = 3 (*** = p < 0.001 for Klf4+/+ and Klf4−/− cells transfected with Klf4-GFP compared to mock- or GFP-transfected Klf4+/+ and Klf4−/− cells, respectively). (B) Quantification of apoptotic Klf4+/+ and Klf4−/−MEFs following GFP or Klf4-GFP overexpression. At 24 h post-transfection, immunostaining for both cleaved caspase 3 and GFP was done and number of cells that are positive for both were counted. N = 3 (Difference did not reach significance for Klf4−/− cells transfected with Klf4-GFP compared to GFP-transfected Klf4−/− cells, p = 0.059). (C) Western blot analysis of Klf4, p53, p21, cyclin E, γH2AX, and β-actin in protein extracts at 24 h post transfection of Klf4+/+ and Klf4−/− cells with GFP or Klf4-GFP.
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Figure 1: Growth characteristics of Klf4-transfected Klf4−/−MEFs in culture. (A) Cell proliferation rates of Klf4+/+ and Klf4−/−MEFs, transfected or not with various plasmids. Cells were initially seeded at 105 cells/plate in 60 mm plates. On day 0, cells were transfected in triplicates with pmKLF4-pEGFP, GFP control, or mock-transfected in parallel. Number of cells was counted (three plates per transfection) every day over a total of 3 days. The growth rate of the MEFs from each transfection was determined at each time point and the values represent the mean number of cells per well. N = 3 (*** = p < 0.001 for Klf4+/+ and Klf4−/− cells transfected with Klf4-GFP compared to mock- or GFP-transfected Klf4+/+ and Klf4−/− cells, respectively). (B) Quantification of apoptotic Klf4+/+ and Klf4−/−MEFs following GFP or Klf4-GFP overexpression. At 24 h post-transfection, immunostaining for both cleaved caspase 3 and GFP was done and number of cells that are positive for both were counted. N = 3 (Difference did not reach significance for Klf4−/− cells transfected with Klf4-GFP compared to GFP-transfected Klf4−/− cells, p = 0.059). (C) Western blot analysis of Klf4, p53, p21, cyclin E, γH2AX, and β-actin in protein extracts at 24 h post transfection of Klf4+/+ and Klf4−/− cells with GFP or Klf4-GFP.

Mentions: MEFs deficient for Klf4 are known to have a higher rate of BrdU incorporation and apoptosis relative to wild type cells [18]. We assessed whether re-expression of Klf4 in Klf4−/−MEFs will affect the proliferative capacity and or apoptosis. We first compared the growth rates of Klf4-GFP-transfected Klf4−/− to mock- or GFP-transfected Klf4−/−MEFs and to mock-, GFP-, or Klf4-GFP-transfected Klf4+/+MEFs up to three days after transfection. As shown in Figure 1A, in Klf4-GFP-transfected Klf4−/−MEFs, cell proliferation was significantly reduced compared to mock-transfected or GFP-control-transfected Klf4−/− or Klf4+/+ cells up to three days post-transfection. Proliferation of Klf4-GFP-transfected Klf4+/+MEFs was also significantly reduced compared to mock-transfected or GFP-control-transfected Klf4−/− and Klf4+/+ cells. Recently, we demonstrated that Klf4−/−MEFs have a higher level of apoptosis than Klf4+/+MEFs [18]. We examined whether re-expression of Klf4 in Klf4−/−MEFs has any effect on apoptosis level. We transfected Klf4-GFP or GFP-control in Klf4+/+ and Klf4−/−MEFs, immunostained them for cleaved caspase 3, and counted the number of GFP-positive cells that were positive for cleaved caspase 3. As shown in Figure 1B, overexpression of Klf4 has no apparent effect on the basal level of apoptosis in Klf4+/+MEFs transfected with Klf4-GFP as compared to the control. Although overexpression of Klf4 in Klf4−/−MEFs lowered the apoptosis level compared to GFP-control-transfected Klf4−/− cells, it did not reach statistically significant value (p = 0.059).


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)

Growth characteristics of Klf4-transfected Klf4−/−MEFs in culture. (A) Cell proliferation rates of Klf4+/+ and Klf4−/−MEFs, transfected or not with various plasmids. Cells were initially seeded at 105 cells/plate in 60 mm plates. On day 0, cells were transfected in triplicates with pmKLF4-pEGFP, GFP control, or mock-transfected in parallel. Number of cells was counted (three plates per transfection) every day over a total of 3 days. The growth rate of the MEFs from each transfection was determined at each time point and the values represent the mean number of cells per well. N = 3 (*** = p < 0.001 for Klf4+/+ and Klf4−/− cells transfected with Klf4-GFP compared to mock- or GFP-transfected Klf4+/+ and Klf4−/− cells, respectively). (B) Quantification of apoptotic Klf4+/+ and Klf4−/−MEFs following GFP or Klf4-GFP overexpression. At 24 h post-transfection, immunostaining for both cleaved caspase 3 and GFP was done and number of cells that are positive for both were counted. N = 3 (Difference did not reach significance for Klf4−/− cells transfected with Klf4-GFP compared to GFP-transfected Klf4−/− cells, p = 0.059). (C) Western blot analysis of Klf4, p53, p21, cyclin E, γH2AX, and β-actin in protein extracts at 24 h post transfection of Klf4+/+ and Klf4−/− cells with GFP or Klf4-GFP.
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Figure 1: Growth characteristics of Klf4-transfected Klf4−/−MEFs in culture. (A) Cell proliferation rates of Klf4+/+ and Klf4−/−MEFs, transfected or not with various plasmids. Cells were initially seeded at 105 cells/plate in 60 mm plates. On day 0, cells were transfected in triplicates with pmKLF4-pEGFP, GFP control, or mock-transfected in parallel. Number of cells was counted (three plates per transfection) every day over a total of 3 days. The growth rate of the MEFs from each transfection was determined at each time point and the values represent the mean number of cells per well. N = 3 (*** = p < 0.001 for Klf4+/+ and Klf4−/− cells transfected with Klf4-GFP compared to mock- or GFP-transfected Klf4+/+ and Klf4−/− cells, respectively). (B) Quantification of apoptotic Klf4+/+ and Klf4−/−MEFs following GFP or Klf4-GFP overexpression. At 24 h post-transfection, immunostaining for both cleaved caspase 3 and GFP was done and number of cells that are positive for both were counted. N = 3 (Difference did not reach significance for Klf4−/− cells transfected with Klf4-GFP compared to GFP-transfected Klf4−/− cells, p = 0.059). (C) Western blot analysis of Klf4, p53, p21, cyclin E, γH2AX, and β-actin in protein extracts at 24 h post transfection of Klf4+/+ and Klf4−/− cells with GFP or Klf4-GFP.
Mentions: MEFs deficient for Klf4 are known to have a higher rate of BrdU incorporation and apoptosis relative to wild type cells [18]. We assessed whether re-expression of Klf4 in Klf4−/−MEFs will affect the proliferative capacity and or apoptosis. We first compared the growth rates of Klf4-GFP-transfected Klf4−/− to mock- or GFP-transfected Klf4−/−MEFs and to mock-, GFP-, or Klf4-GFP-transfected Klf4+/+MEFs up to three days after transfection. As shown in Figure 1A, in Klf4-GFP-transfected Klf4−/−MEFs, cell proliferation was significantly reduced compared to mock-transfected or GFP-control-transfected Klf4−/− or Klf4+/+ cells up to three days post-transfection. Proliferation of Klf4-GFP-transfected Klf4+/+MEFs was also significantly reduced compared to mock-transfected or GFP-control-transfected Klf4−/− and Klf4+/+ cells. Recently, we demonstrated that Klf4−/−MEFs have a higher level of apoptosis than Klf4+/+MEFs [18]. We examined whether re-expression of Klf4 in Klf4−/−MEFs has any effect on apoptosis level. We transfected Klf4-GFP or GFP-control in Klf4+/+ and Klf4−/−MEFs, immunostained them for cleaved caspase 3, and counted the number of GFP-positive cells that were positive for cleaved caspase 3. As shown in Figure 1B, overexpression of Klf4 has no apparent effect on the basal level of apoptosis in Klf4+/+MEFs transfected with Klf4-GFP as compared to the control. Although overexpression of Klf4 in Klf4−/−MEFs lowered the apoptosis level compared to GFP-control-transfected Klf4−/− cells, it did not reach statistically significant value (p = 0.059).

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