Abstract
unstable as evidenced by the presence of high level of DNA damage, centrosome amplification, and aneuploidy. AIM: To investigate role and possible mechanisms of Klf4 in preserving genetic stability. METHODS: To determine the ability of Klf4 restoration in correcting the observed genetic instability in MEFS null for Klf4 (Klf4-/-), we overexpressed Klf4 in Klf4-/MEFs and compared the results to wild type (Klf4+/+) and non-transfected Klf4-/MEFs. Cell cycle profiles and proliferation curve was performed to determine growth characteristics. Quantification of chromosome number and immunohistochemical staining of centrosomes were performed to assess chromosome integrity. To determine the role of Klf4 in correcting DNA damage, we overexpressed or not Klf4 in Klf4-/MEFs followed by γ-irradiation or not in parallel with Klf4+/+ and Klf4-/MEFs. Using immunohistochemistry, γH2AX and p53BP1 foci were counted at 0, 1h, 4h and 24h after γ-irradiation. The levels of p53, p21, cyclin E, Cdk2 and γH2AX in Klf4+/+ and Klf4-/MEFs, with and without Klf4 overexpression, were determined by Western blotting. RESULTS: Overexpression of Klf4 in Klf4-/MEFs suppressed cell proliferation compared to untransfected Klf4+/+ and untransfected Klf4-/MEFs. Centrosome staining showed a 10-fold decrease in the percent of cells with ≥3 centrosomes after overexpressing Klf4 in Klf4-/MEFs compared to untransfected Klf4-/MEFs. A higher percentage of Klf4-/MEFs cells were detected with increased and sustained ≥5 γH2AX and p53BP1 foci following γ-irradiation compared to irradiated Klf4+/+ MEFs. Overexpression of Klf4 in Klf4-/MEFs reduced the percentage of cells with ≥5 γH2AX foci to a level similar to that in Klf4+/+ MEFs, while the percent of cells with ≥5 p53BP1 foci were reduced to the same level as irradiated Klf4+/+ but at a faster rate. Karyotype analysis showed that Klf4-transfected Klf4-/MEFs had a 25% decrease in number of cells exhibiting aneuploidy in contrast to untransfected Klf4-/MEFs. Western blot analysis showed that p53, cyclin E, Cdk2 and γH2AX levels were reduced and p21 was elevated following Klf4 overexpression in Klf4-/MEFs. CONCLUSION: Results of this study demonstrate that overexpression of Klf4 in MEFs null for the Klf4 alleles display evidence of improved genetic stability as illustrated by reduced DNA damage, aneuploidy, and centrosome amplification. These results indicate that KLF4 plays a crucial role in preserving genetic stability.
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