MOLECULAR CARCINOGENESIS 54:889–899 (2015)
Oxidative DNA Damage Causes Premature Senescence in Mouse Embryonic Fibroblasts Deficient for Krüppel-Like Factor 4 Changchang Liu,1 Stephen La Rosa,1, 2 and Engda G. Hagos1* 1 2
Department of Biology, Colgate University, Hamilton, New York Memorial Sloan-Kettering Cancer Center, Prostate Cancer Research Program, New York, New York
Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress-induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4-deficient MEFs. With SA-b-Gal staining, we show that Klf4/ MEFs enter senescence earlier than Klf4þ/þ MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against g-H2AX indicates that the increased level of DNA damage in Klf4/ MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4/ MEFs, treatment with NAC, reduces the accumulation of DNA damage. Our RT-PCR result demonstrates that Klf4/ MEFs have decreased expression of the antioxidant gene, Gsta4. The downregulation of the Gsta4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress-induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes. © 2014 Wiley Periodicals, Inc. Key words: Krüppel-like factor 4; cellular senescence and reactive oxygen species; genomic instability
INTRODUCTION ¨ ppel-like factor 4 (KLF4) belongs to the Kru ¨ ppelKru like factor family of zinc-finger-containing transcription factors and has been shown to regulate various cellular processes including development, proliferation, inflammation, apoptosis, and differentiation [1– 6]. KLF4 also plays a role in tumorigenesis and reprogramming of somatic cells to induced pluripotent stem cells [7–10]. In many human cancers KLF4 is regarded as a tumor suppressor. For example, the level of KLF4 is downregulated in certain cancers such as gastric, colorectal, esophageal, bladder, lung, and pancreatic cancer [11–16]. Interestingly, in contrast to its role as a tumor suppressor in many cancer types, KLF4 has been found to act as a tumor promoter in certain tumorous contexts as it is upregulated in primary breast ductal carcinoma and oral squamous cell carcinoma [17,18]. In cell cultures, KLF4 expression is associated with conditions that initiate cell-cycle arrest such as contact inhibition and serum deprivation [4,19]. Cells that are arrested in growth are rich in KLF4, as KLF4 functions to inhibit cell cycle progression by upregulating cyclin-dependent kinase (CDK) inhibitors such as p21 [20]. Previous reports have shown that KLF4 was transcriptionally activated by p53 ß 2014 WILEY PERIODICALS, INC.
following DNA damage, and as a result, cells were arrested at both the G1-S and G2-M boundaries [21]. Furthermore, we previously showed that KLF4 plays a role in maintaining genomic stability in
Abbreviations: Klf4, Krüppel-like factor 4; ROS, reactive oxygen species; MEFs, mouse embryonic fibroblasts; CDK, Cyclin-dependent kinase; SOD, superoxide dismutase; CAT, catalase; GST, glutathione Stransferase; DOX, Doxorubicin; PFTa, Pifithrin alpha; Gsta4, glutathione S-transferase-alpha 4; DMEM, Dulbecco's modified Eagle's medium; NAC, N-acetyl-cysteine; SA-b-gal, Senescence associated-bgalactosidase; PBS, phosphate buffered saline; RT-qPCR, reverse transcriptase- quantitative polymerase chain reaction. Conflict of interest: none. Authors' contributions: C.L. carried out the all the experimental studies, participated in the design of the study and drafted the manuscript. S.L.R. participated in ROS staining and Flow Cytometry. EH conceived of the study, participated in its design and coordination, carried out cell transfections, microscope imaging, and Western blots, and, drafted the manuscript. All authors read and approved the final manuscript. Grant sponsor: Picker Research Fellowship from Colgate University Research Council *Correspondence to: Department of Biology, Colgate University, Hamilton, NY 13346-1398. Received 21 November 2013; Revised 9 March 2014; Accepted 27 March 2014 DOI 10.1002/mc.22161 Published online 30 April 2014 in Wiley Online Library (wileyonlinelibrary.com).
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mouse embryonic fibroblasts (MEFs). Specifically, Klf4/ MEFs show evidence of genomic instability, including aneuploidy, chromosome aberrations, increased DNA double strand breaks (DSBs), centrosome amplification, and anchorage-independent growth [22]. Notably, re-expression of KLF4 in Klf4/ MEFs corrects the genomic instability in these cells [23]. Cellular senescence is a mechanism used by cells to limit the growth of cells at high risk for tumorigenesis [24]. Cells with high levels of DNA damage can trigger cellular senescence and irreversibly lose the ability to undergo cell proliferation. This response prevents genomic instability and is achieved mainly by activation of senescence-associated genes such as p53 and p21 [24,25]. The pathways controlled by p53 to induce senescence can be triggered by reactive oxygen species (ROS), which can result in oxidative DNA damage [26]. ROS are products of cellular metabolic processes and can introduce DNA damage by modifying bases, cross-linking DNA and proteins, and causing DNA strand breakage [27]. Thus the cellular oxidative stress associated with increased ROS is a main cause of senescence in MEFs maintained under standard tissue culture conditions, as MEFs cultured in reduced levels of oxygen failed to senesce [26–28]. Previous studies have shown that cells regulate the levels of ROS and prevent dangerous accumulation of ROS by expressing antioxidants that chemically reduce ROS to non-harmful by-products [29]. For example, cellular antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) are responsible for reducing ROS and maintaining an appropriate homeostatic level of ROS [26]. When there is an elevated level of ROS present in cells, proper antioxidant defenses must be expressed to prevent or rescue cells from adverse cell fates. It has been shown that exposing cells with high oxidative stress levels to antioxidants allows cells to either repair or arrest in order to restore proper organelle and cell function [30,31]. Despite growing evidence that KLF4 maintains genomic stability as exemplified above, the mechanisms by which KLF4 exerts its functions are not well established. Since KLF4 has been shown to be a crucial mediator of p53 in the DNA damage response [21], and we have reported that MEFs null for Klf4 are genetically unstable [22,23], we sought to determine whether KLF4 plays a role in maintaining genomic stability by preventing DNA damage-induced oxidative stress in MEFs null for Klf4. Results presented in this report provide insights into the potential mechanism by which KLF4 plays a role in suppressing DNA damage accumulation as well as in oxidative stress responses, and implicate KLF4 in genome maintenance and protection against aberrant ROS production. Molecular Carcinogenesis
RESULTS /
Klf4
MEFs Enter Senescence Earlier than Klf4þ/þ MEFs
We first examined the growth characteristics of MEFs derived from Klf4þ/þ and Klf4/ embryos by calculating the relative cell number of primary cells of each genotype starting at passage 3. As shown in Figure 1A, pre-senescent Klf4/ MEFs proliferated at a slower rate compared to Klf4þ/þ MEFs. In addition, Klf4/ MEFs ceased proliferating at passage 6 whereas Klf4þ/þ MEFs continued to proliferate until passage 15 (Figure 1A). We next determined at what passage the MEFs enter senescence by assaying for the senescence marker, SA-b-Gal, beginning at passage 3. Klf4/ MEFs started to exhibit the typical flattened and enlarged senescence phenotypic morphology [32] at passage 4 and started to show SA-b-Gal activity (Figure 1B). The percentage of MEFs with SA-b-Gal staining was quantified and analyzed with Student’s t-test. The percentage of Klf4/ MEFs with SA-b-Gal staining increased with passage number. Significantly more Klf4/ MEFs assayed positive for SA-Gal and entered senescence at passage 6, as compared to Klf4þ/þ MEFs from the same passage (Figure 1B,C). Consistent with the relative cell number curve, Klf4þ/þ MEFs started to show positive SA-b-Gal at about passage 14 and expressed high levels of SA-bGal at passage 16, a much later time point compared to Klf4/ MEFs (Figure 1D). The Accumulation of p21 Leads MEFs to Enter Senescence Previous studies have shown that cell cycle regulators such as p21 are involved in the induction of premature senescence [33–35]. To characterize the molecular nature of the premature senescence in Klf4/ MEFs, we examined the levels of p21 and its upstream transcription factor, p53, in Klf4þ/þ and Klf4/ MEFs. We observed that Klf4/ MEFs exhibited higher levels of p21 and p53 compared to Klf4þ/þ MEFs from the same passage (Figure 2A,B). Furthermore, both Klf4þ/þ and Klf4/ MEFs expressed high levels of p21 and p53 at the passage where a high percentage of cells showed positive SA-bGal staining (Figure 2B,C). To investigate whether MEFs experience earlier senescence due to p21 accumulation, we overexpressed p21 by transient transfection in pre-senescence Klf4þ/þ MEFs at passage 8. As shown in Figure 2D, cells transfected with either GFP-control or p21 showed significantly higher levels of protein for GFP or p21, respectively, as compared to mock transfected cells. Next we assayed the transfected Klf4þ/þ MEFs with SA-b-Gal staining 5 d posttransfection for the presence of senescent cells. We found that significantly more MEFs transfected with p21 entered senescence as compared to Klf4þ/þ MEFs transfected with either mock or GFP (Figure 2E,F). This suggests that increased p21 levels are sufficient to induce senescence, consistent with previously
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Figure 1. Klf4/ MEFs enter cellular senescence earlier than Klf4þ/þ MEFs. (A) Relative cell number for Klf4þ/þ and Klf4/ MEFs. Klf4þ/þ and Klf4/ MEFs were passed following the 3T3 protocol. Relative cell number were calculated by counting cell numbers at each passage for Klf4þ/þ and Klf4/ MEFs. The arrows indicate that cells arrest at passage 6 for Klf4/ and passage 15 for Klf4þ/þ. Shown is the representative growth curve of three independent
experiments. (B) Klf4þ/þ and Klf4/ MEFs were stained with SA-bGal to detect senescence from passage 3 to 6 (Klf4/) or from passage 6 to 16 (Klf4þ/þ). Representative images were shown from the indicated passages. (C, D) Quantification of the senescent Klf4þ/þ and Klf4/ MEFs with SA-b-Gal stain. N ¼ 3 (P < 0.05 at p5, p6, and p7 for Klf4/ compared to Klf4þ/þ). Error bars represent standard error.
Figure 2. Early senescence in Klf4/ MEFs is associated with p21 induction. Western blot analysis of p21 in Klf4þ/þ (A) and Klf4/ (B) MEFs at early passages (3, 6, and 7). (C) Western blot analysis of p21 levels in Klf4þ/þ MEFs at late passages (10,14, and 16). (D) Western blot analysis of either GFP or p21 in pre-senescent Klf4þ/þ MEFs transfected with either p21 or GFP at passage 8. (E) SA-b-Gal analysis of transfected MEFs with mock, GFP or p21. SA-b-Gal activity was assayed 3 d after transfection. (F) The percentage of MEFs described in (E) positive for SA-b-Gal activity was quantified. Error bars represent standard error. N ¼ 3; P < 0.05.
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published data that p21 is required for cells to enter senescence [33–35]. Klf4/ MEFs Enter Premature Senescence in a p53 Dependent Manner The tumor suppressor gene, p53, has been shown to play an important role in regulating cell cycle checkpoints through activation of p21 and other target genes [21]. Since overexpression of p21 leads pre-senescent MEFs to enter senescence (Figure 2E), we hypothesized that manipulating of p53 using drugs that interfere with its function will affect the levels of p21 and thus the entry of senescence in Klf4deficient cells. To determine whether the early cellular senescence shown is caused by p53 upregulation, we induced p53 in pre-senescent passage 3 Klf4/ MEFs with Nutlin-3, a MDM2 antagonist that induces senescence in wild type MEFs by activating p53 [36,37]. Western blot confirmed that Nutlin-3 treatment induced p53 activity and its downstream target p21 in Klf4/ MEFs as compared with DMSOtreated control Klf4/ MEFs (Figure 3A). In addition, as shown in Figure 3B,C, treatment with 5 mM Nutlin3 induced senescence morphology and increased SAb-Gal staining in Klf4/ MEFs. Our data strongly suggest that p53 induction is capable of driving premature senescence in Klf4/ MEFs. We have previously shown that pre-senescent Klf4/ MEFs exhibited higher levels of DNA damage
Figure 3. p53 induction results in premature senescence in Klf4/ MEFs. (A) Klf4/ MEFs of passage 3 were treated with 10 mM Nutlin-3 or DMSO as a control for 24 hr. Proteins were then extracted and subjected to western blot analysis of p53, p21 and b-actin. (B) Klf4/ MEFs from passage 3 were treated with DMSO or 5 mM of Nutlin-3 for 1 wk followed by 1-wk incubation in DMEM. The cells were stained with SA-b-Gal to detect senescent cells. (C) A quantification of MEFs in (B) is shown N ¼ 4; P < 0.05. Error bars represent standard error. (D) Klf4þ/þ
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as compared to Klf4þ/þ [22], To investigate whether the early cellular senescence associated with p53 upregulation in Klf4/ MEFs is due to DNA damage, we next treated the pre-senescent passage 8 Klf4þ/þ MEFs with either Doxorubicin (DOX), to induce DNA damage or Pifithrin alpha (PFTa), a p53 transactivation inhibitor [38,39]. As shown by western blot in Figure 3D, 0.1 mM DOX alone induced g-H2AX, a DNA damage marker, and activated the p53 pathway. On the other hand, inhibition of p53 with PFTa in DOXtreated MEFs induced DNA damage, but was unable to activate the expression of p53 and p21 (Figure 3D). Furthermore, whereas 45% MEFs showed SA-b-Gal staining with DOX treatment, PFTa significantly reduced the percentage of senescent cells to less than 20% (Figure 3E,F). Thus, inhibition of p53 by PFTa blocked p53-mediated induction of p21 and significantly prevented DNA damage-inducedsenescence. Increased Oxidative DNA Damage in Klf4/ MEFs Could Be Rescued by NAC Treatment Cellular senescence can be induced by DNA damage [40]. We recently showed that KLF4 is involved in DNA damage repair [22,25]. To investigate whether Klf4/ MEFs enter premature senescence due to DNA damage, we performed immunostaining for the presence of g-H2AX foci, a DNA damage marker, in Klf4þ/þ and Klf4/ MEFs,
MEFs were treated with DMSO, 0.1 mM Doxorubicin (Dox), or 0.1 mM Doxorubicin plus 30 mM Pifithrin a (PFTa) for 24 hr. Protein extracts were analyzed by western blot analysis for p53, p21, and b-actin and gH2AX. (E) Klf4þ/þ MEFs from early passage 8 were treated with DMSO, 0.1 mM Doxorubicin, or 0.1 mM Doxorubicin plus 30 mM PFTa for 1 wk. MEFs were then stained with SA-b-Gal to detect senescence. (F) A quantification of cells in (E) is shown. Error bars represent standard error. N ¼ 3; P < 0.05, P < 0.001.
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Figure 4. DNA damage in Klf4/ MEFs rescued by NAC treatment. (A) Immunostaining was performed for g-H2AX (green) on Klf4þ/þ and Klf4/, and NAC-treated Klf4/ pre-senescence MEFs. DAPI (blue) was used to stain the nuclei. (B) The percentage of cells with more than five g-H2AX foci from each cell line was quantified. N ¼ 3.
with or without NAC [41]. As shown in Figure 4A and B, over 50% Klf4/ MEFs contained more than five g-H2AX foci compared to only 10% Klf4þ/þ MEFs. Klf4/ MEFs treated with 10 mM NAC, a ROS scavenger, showed reduced g-H2AX foci as compared to untreated Klf4/ MEFs.
whether KLF4 regulates Gsta4 expression using RTPCR and qPCR. Consistent with the microarray data, we found that the mRNA levels of Gsta4 in Klf4/ MEFs were significantly downregulated compared to those in wild type MEFs (Figure 5C,D).
Klf4/ MEFs Experience Higher Levels of Oxidative Stress
DISCUSSION
ROS can react with DNA and introduce breaks to the DNA strands [42]. Because increasing ROS levels are linked to cellular senescence [43–47], we next examined whether Klf4/ MEFs experience higher levels of ROS compared to Klf4þ/þ MEFs. To visualize ROS in MEFs, we treated Klf4þ/þ and Klf4/ pre-senescent MEFs with 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCF-DA), which is oxidized to the fluorescent DCF by ROS and trapped within cells [39]. The cells were then stained with DAPI and visualized under a confocal fluorescence microscope. MEFs lacking Klf4 exhibited higher ROS levels compared to the Klf4þ/þ MEFs, as more Klf4/ MEFs exhibited positive ROS staining (Figure 5A). To quantify the levels of ROS in Klf4þ/þ and Klf4/ MEFs, we stained the cells with H2DCF-DA and performed FACS analysis on Klf4þ/þ, Klf4/, and Klf4/ MEFs with NAC. Klf4/ MEFs exhibited significantly higher levels of ROS compared to the wild type (Figure 5B). Moreover, in both DCF staining and FACS analysis, the ROS direct scavenger, NAC, reduced the levels of ROS significantly in Klf4/ MEFs (Figure 5A,B). To determine why there is more ROS in Klf4/ MEFs, we studied the levels of antioxidant genes in Klf4þ/þ and Klf4/ MEFs. Glutathione S-Transferase alpha 4 (Gsta4) encodes an enzyme that catalyzes the degradation of the products of oxidative metabolism [48]. Our prior microarray studies found that Gsta4 levels decreased by ninefold in Klf4/ MEFs as compared to Klf4þ/þ MEFs [49]. Here we determined
A hallmark of cancer, genomic instability can manifest as chromosomal aberrations, microsatellite instability, DSBs, and frequent base-pair mutations [50,51]. Genomic instability can arise when mutations occur in genes responsible for DNA damage repair, DNA replication, or segregation during mitosis [52]. Studies have shown that genomic instability is related to the initiation of tumorigenesis and increased rate of tumor growth and cancer development [52,53]. In many human cancers, KLF4 functions as a tumor suppressor [11–16]. KLF4 can exert its anti-carcinogenesis function by mediating the p53 activation of p21 and initiating cell cycle checkpoints [21,54]. In addition to its anti-proliferative effect, KLF4 has been shown to be involved in maintaining genomic integrity [22,23]. We have previously demonstrated that MEFs lacking KLF4 exhibit genomic instability as evidenced by the presence of aneuploidy, more DNA damage, and chromosomal aberrations [22]. Moreover, we recently reported that restoration of KLF4 to Klf4/ MEFs reduces the extent of DNA damage and aneuploidy [23]. In light of the previous studies which demonstrated the role of KLF4 in maintaining genomic stability, we investigated whether Klf4/ MEFs experience early cellular senescence due to DNA damage. In the current study we report that Klf4/ MEFs enter senescence prematurely in a p53-dependent manner (Figures 1 and 2). Moreover, we find that Klf4/ MEFs
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Figure 5. Klf4/ MEFs exhibit higher levels of ROS and reduced levels of antioxidant. (A) Klf4þ/þ MEFs, Klf4/ MEFs and Klf4/ MEFs treated with 10 mM NAC for 2 d were stained with 10 mM CM-H2DCFDA to detect ROS. DAPI was used to visualize the nuclei. (B) Klf4þ/þ MEFs and Klf4/ MEFs from late passage were grown with or without 10 mM NAC for 2 d. MEFs were then treated with 10 mM CM-H2DCFDA before FACS analysis. Data shown is representative of three
independent experiments. N ¼ 3; P < 0.05, P < 0.001. (C) Semiquantitative RT-PCR was performed to assess the level of Gsta4 in Klf4þ/þ and Klf4/ MEFs at passage 3. b-Actin was used as a loading control. (D) RT-qPCR was performed to assess the level of Gsta4 normalized to b-Actin. Shown is the fold reduction in Klf4/ MEFs compared to Klf4þ/þ MEFs (error bars represent standard deviation of three independent experiments, P < 0.05).
exhibit high levels of DNA damage (Figure 3). Increased DNA damage in pre-senescent Klf4/ MEFs correlates with increased ROS compared to Klf4þ/þ MEFs, and as a result, Klf4/ MEFs display elevated levels of cell cycle inhibitor, p21 which induces premature senescence (Figures 2, 4, and 5). Our results are consistent with reports that cells deficient in genes involved in genome maintenance, such as Brca1, Ku86, and Atm, enter senescence much earlier compared to their wild type counterparts [44,45,55,56]. We previously showed that post-senescent MEFs lacking Klf4 exhibit higher levels of DNA damage [22,23]. Consistent with previous findings, we show here that pre-senescent MEFs lacking Klf4 also display higher levels of DNA damage (Figure 4A,B). Thus, we hypothesize that Klf4/ MEFs experience premature senescence as a result of accumulated DNA damage. In order to test this hypothesis, we treated the Klf4/ cells with NAC, which has been shown recently to prevent mutagenesis and carcinogenesis by ameliorating genotoxic effects [57]. We observed that NAC rescued Klf4/ MEFs from extensive DNA damage (Figure 4A,B). In accord with previous findings that DNA damage induces cellular senescence as a protective mechanism against tumorigenesis, our data suggests that Klf4/ MEFs experience premature senescence as a response to the extensive damage in their DNA.
Expression of p21, a CDK inhibitor, has been shown to be upregulated by the p53 tumor suppressor gene in response to DNA damage [58]. To determine whether p21 was involved in the premature senescence in Klf4null cells, we performed Western blot analysis using p21 antibody. As shown in Figure 2, Klf4/ MEFs exhibit significantly higher levels of p21 compared to Klf4þ/þ MEFs of the same passage, and Klf4þ/þ cells later show comparable levels of p21 as they enter senescence. Moreover, overexpression of p21 in early pre-senescence passage Klf4þ/þ MEFs leads to premature senescence (Figure 2). Together, these data implicate p21 in the premature senescence observed in Klf4/ MEFs. Furthermore, we also observed a significant increase of p53, which transcriptionally regulates p21 (Figure 2). Previous studies in cardiomyocytes and wild type MEFs showed that p53 induces senescence [39,40]. To determine if the activation of p21 led to senescence downstream of p53, we used drugs that affect p53 expression. As shown in Figure 3, upregulation of p53 with Nutlin-3 induces premature senescence in Klf4/ MEFs, whereas inhibition of p53 with PFTa delays cellular senescence. Our data strongly suggest that the premature senescence of Klf4/ MEFs is associated with p53 activation. KLF4 inhibits cell proliferation by functioning as a cell cycle checkpoint protein to activate transcription of the CDK inhibitor, p21 [1,20]. Interestingly, we
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PREMATURE SENESCENCE IN Klf4-NULL MEFs
observed that the activation of p21 by p53 in presenescent MEFs is independent of KLF4 function (Figure 2). This suggests that p53 activation of p21 in MEFs entering senescence does not require KLF4. This KLF4-independent p21 induction supports a previous report [59] that HCT116 cells that have lost KLF4 show an increase of p21 in response to p53 upregulation. Taken together, we conclude that p53 transactivation of p21 does not require KLF4 in presenescent MEFs, and the exact molecular mechanism for KLF4-independent p21 transcription awaits further investigation. In post-senescent Klf4/ MEFs, we previously reported that despite high levels of p53, the levels of p21 were greatly reduced [22]. Interestingly, in the current study we observe a modest increase of p53 but high levels of p21 in primary Klf4/ MEFs entering senescence compared to Klf4þ/þ MEFs (Figure 2). The increased level of p53 in Klf4/ is most likely due to DNA damage [22]. Moreover, restoration of KLF4 to the MEFs reduces the levels of p53 and corrects DNA damage [23]. We thus propose that persistent DNA damage in the Klf4/ MEFs activates DNA damage response and results in increased p53 levels compared to that in Klf4þ/þ MEFs. Furthermore, we observed that fewer than 60% of pre-senescent Klf4/ MEFs contained over 5 g-H2AX foci (Figure 4). On the other hand, in the postsenescence MEFs, 80–90% of Klf4/ MEFs contained over five g-H2AX foci [22,23]. The greater extent of DNA damage in the immortalized MEFs corresponds to a higher increase of p53, and the milder DNA damage in pre-senescence MEFs correlates with a modest increase of p53 [22], (Figure 2). Recently, published studies demonstrated that p53 determines the fate of a cell poised to undergo either cell cycle arrest or apoptosis [59]. Low dosage of DNA damage induces a modest increase of p53, which activates KLF4 and in turn, p21, resulting in cell cycle arrest [59]. On the other hand, extensive DNA damage induces high levels of p53, which suppresses KLF4 but activates BAX, leading to apoptosis [59]. In light of the role of p53 in determining DNA damage response outcomes, we propose that in the pre-senescent MEFs, the absence of Klf4 causes mild DNA damage, which leads to a modest increase of p53 (Figure 2). As a result, the activation of p53 induces p21 and premature senescence (Figure 3). In post-senescence MEFs, extensive DNA damage results in p53 upregulation, which inhibits p21 and subjects the cells to apoptosis [22]. Moreover, most recently we have shown that restoring KLF4 in Klf4 null MEFs reduced DNA damage, p53 levels, and apoptosis [23]. The upregulation of p53 in Klf4/ MEFs is consistent with previous literature, which found that KLF4 represses p53 activity [60]. More recently, we reported that restoration of KLF4 to Klf4/ MEFs decreases the level of apoptosis [23]. The decreased level of apoptosis is likely due to the decrease in p53 levels, Molecular Carcinogenesis
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thus providing additional evidence of KLF4 repression of p53. The finding that KLF4 represses p53 also supports studies that reported an anti-apoptotic effect of KLF4, as KLF4 is likely to rescue cells from programmed cell death to cell arrest by decreasing the levels of p53 [60]. Thus, in KLF4-depleted cells the levels of p53 are restored and cause p53-dependent apoptosis. These findings imply that the cytostatic activity of KLF4 is inhibited by p53 during extensive DNA damage, so that the severely damage cells undergo programmed cell death. Most recently, we have shown that overexpressing KLF4 in Klf4 null MEFs corrects DNA damage [23]. To investigate whether the source of DNA damage in the Klf4/ MEFs is induced by oxidative stress, we treated MEFs null for Klf4 with NAC. It has been shown that NAC reduces genomic toxicity through a wide range of mechanisms including inhibiting mutations during DNA repair, blocking mutagens, and acting as a direct scavenger of ROS [57]. ROS are highly reactive products of the electron transport chain with important physiological effects in oxygen detection, acquired immune response, and skeletal muscle activity; however, unregulated ROS levels can cause genomic instability and carcinogenesis [61]. Here we showed that Klf4/ MEFs exhibited higher levels of ROS compared to Klf4þ/þ MEFs (Figure 5A,B). Addition of NAC reduced the levels of ROS in Klf4/ MEFs (Figure 5A,B). This result provides novel evidence that KLF4 regulates cellular ROS levels. Next, we investigated whether KLF4 regulates ROS levels through antioxidant genes. Gsta4 belongs to the GST family that neutralizes hazardous endogenous oxidative compounds by catalyzing the conjugate addition of reduced glutathione [61]. Moreover, overexpression of Gsta4 reduced the levels of lipid peroxidation in the presence of DNA damage and protected cells from oxidative stress [61]. We have shown with microarray data that MEFs lacking Klf4 exhibit decreased expression of antioxidant genes [49]. In the current study, our semi quantitative RT-PCR and quantitative PCR results suggest that KLF4 reduces the levels of ROS by sustaining the expression of Gsta4 (Figure 5). However, whether or not KLF4 influences Gsta4 expression through direct regulation remains unknown. In conclusion, the present study demonstrates that Klf4/ MEFs have decreased antioxidant gene expression and increased ROS levels compared to Klf4þ/ þ MEFs. High levels of ROS cause persistent DNA damage in Klf4/ MEFs. As a part of the DNA damage response, p53 and p21 are induced. Upregulation of the cytostatic cell cycle regulators leads to premature senescence in Klf4/ MEFs (Figure 6). These findings provide insights into the potential mechanism by which KLF4 plays a role in DNA damage and oxidative stress responses, and implicate KLF4 in maintaining genomic stability and protection against aberrant ROS production in MEFs.
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independent experiments, each done in triplicate, were performed. Analysis of Senescence-Associated-b-Galactosidase Activity The expression of galactosidase as a marker for senescent MEFs was determined using a senescenceassociated-b-galactosidase (SA-b-Gal) activity assay as described in Ref. [33]. The senescence-associated-bgalactosidase staining kit was purchased from Cell Signaling (Danvers, MA), and the staining was performed following manufacturer’s protocol. Briefly, cells treated with X-gal were incubated at 378C overnight without CO2. Twenty-four hours later cells were washed twice with phosphate buffered saline (PBS). Cells then were mounted with 70% glycerol and images were captured using an Olympus IX51 microscope. Results are from three independent experiments each done in duplicate and are presented as the percentage of MEFs positive for SA-b-Gal staining. Figure 6. A working model for how Klf4 deficiency causes ROS accumulation that leads to premature senescence. Lack of Klf4 leads to the downregulation of antioxidant genes such as Gsta4 and causes the accumulation of ROS. Elevated oxidative stress causes increased DNA damage. As a result, the p53 gene, a tumor suppressor induces the expression of cell cycle inhibitor p21. Prolonged upregulation of p21 leads to cellular senescence.
MATERIALS AND METHODS Cell Culture, Growth Curve Analysis, and Drug Treatment Mice heterozygous for the Klf4 alleles (Klf4þ/) on a C57BL/6 background were crossbred. MEFs that are wild type (Klf4þ/þ) or null (Klf4/) for the Klf4 alleles were derived from Day 13.5 embryos. MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin-streptomycin at 378C and 5% CO2. Following 3T3 protocol, pre-senescent cells were passed every 3 d until they reached senescence. Cell numbers were counted at each passage and the relative cell number was calculated. To overexpress p21 and GFP that served as a control in MEFs, cells were transiently transfected with 4 mg plasmid DNA (per well in a 6well plate) using Lipofectamine LTX reagent (Life Technologies, Grand Island, NY) according to manufacturer’s instructions. To determine the expression of p53, MEFs were treated with indicated drugs 24 hr after plating. Nutlin-3, Pifithrin-a and DOX were dissolved in dimethyl sulfoxide (DMSO) to 10 mM (Nutlin-3, Pifthrin-a) or 100 mM (Doxorubicin) stock concentration and added to the cells at 5, 30, and 0.1 mM respectively. N-acetyl-cysteine (NAC) was purchased from Sigma–Aldrich (St. Louis, MO). To rescue DNA damage, NAC was dissolved in PBS to 1M stock concentration. Cells were incubated for 2 d with 10 mM NAC at 378C. For all experiments, three Molecular Carcinogenesis
Immunoblotting Protein extraction and western blot analysis were performed as previously described [22]. Briefly, the membranes were incubated with the following primary antibodies to: p21 (BD Pharmingen, Franklin Lakes, NJ), p53 (Abcam, Cambridge, MA), g- H2AX (Cell Signaling), and b-Actin (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The antibody-antigen complex was visualized by an Immun-StarTM HRP Chemiluminescence Kit and ChemiDocTM XRSþ System (Bio-Rad Laboratories, Hercules, CA). Anti-rabbit secondary antibody and anti-mouse secondary antibody were purchased from Cell Signaling and Abcam, respectively. Representative western blot image of three independent experiments were shown. Immunostaining MEFs grown on glass coverslips were washed briefly with PBS. They were then fixed with 16% methanolfree formaldehyde for 5 min at room temperature followed by three washings with PBS before blocking in solution [3% bovine serum albumin (BSA), 0.2% Triton X-100 in PBS] for 1 hr at room temperature. Rabbit anti-g-H2AX primary antibody (Cell Signaling) diluted in blocking solution was incubated at room temperature for an hour and detected with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology) for 1 hr at room temperature. Cells were then washed once and counterstained with DAPI (Life Technologies) for 5 min at room temperature in the dark. Finally cells were washed three times, mounted in Prolong Antifade kit (Molecular Probes by Life Technologies), and visualized with a Zeiss 710 confocal laser scanning microscope (Carl
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Zeiss, Thornwood, NY). Two independent experiments, each done with duplicates, were performed. Reactive Oxygen Species Detection MEFs grown on cover slips with or without 10 mM NAC were washed with PBS. Cells were then incubated with freshly prepared 10 mM CM-H2DCF-DA, a membrane permeable fluorescent dye which is converted to DCF upon interaction with ROS (Molecular Probes by Life Technologies) (Ex: 495 nm, Em: 520 nm) at 378C for 40 min with a 10 min recovery in DMEM in the dark. Cells were washed three times with PBS and counterstained with DAPI for 5 min at room temperature in the dark. Finally cells were washed three times with PBS and mounted in Prolong Antifade kit and imaged with a Zeiss 710 confocal laser scanning microscope. Three independent experiments, each done with duplicates, were performed. Flow Cytometry Quantification of ROS levels was performed as follows. 105 cells/well were plated in 6-well plates with or without 10 mM NAC and cultured at 378C in 5% CO2. After 2 d, cells were rinsed in PBS, trypsinized and re-suspended in DMEM with 10% FBS. Pelleted cells were washed again and then incubated in 10 mM CM-H2DCF-DA, Ex: 495 nm, Em: 520 nm, at 378C for 40 min with a 10 min recovery in DMEM in the dark. Pelleted live cells were re-suspended in PBS and analyzed with a BD Accuri C6 flow cytometer with the FL1 laser (515–545 nm). Data are presented as the mean percentage of three independent experiments. Semi-Quantitative RT-PCR and RT-qPCR Total RNA from cultured Klf4þ/þ and Klf4/ MEFs was isolated at passage 3 using RNeasy1 Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RNA was subjected to DNase I treatment in order to remove any contaminating genomic DNA. cDNA was prepared from 1 mg of RNA and amplified with OneStep RT-PCR Kit (Qiagen). PCR products were analyzed by ethidium bromide staining on a 2% agarose gel. RNA was subjected for RT-qPCR analysis using Express One-Step SYBR GreenER Universal (Life Technologies) following manufacturer’s protocol. The expression of Gsta4 was normalized to the expression level of b-Actin. Gene expression level in Klf4/ MEFs was compared to that in Klf4þ/þ MEFs, which was set to 1. Data shown represents three independent experiments, each performed in triplicates. PCR reactions were performed using the following primers purchased from Integrated DNA Technologies (Coralville, IA). Gsta4F: 50 TCA AAC TCC ACT CCA GCC G 30 ; Gsta4R: 50 CTC GAG TGC CTG GAG ACA A 30 ; b-ActinF: 50 ATG GAG GGG AAT ACA GCC C 30 ; b-ActinR: 50 TTC TTT GCA GCT CCT TCG TT 30 . Molecular Carcinogenesis
Statistical Analysis The Student’s t-test was used to test for significant differences between treatments. ACKNOWLEDGMENTS This work was supported in part by a Picker Research Fellowship from Colgate University Research Council. We acknowledge the lab of Dr. Vincent W. Yang (Stony Brook Medical School, NY) for providing GFP and p21 constructs. REFERENCES 1. Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem 1996;271: 20009–20017. 2. Garrett-Sinha LA, Eberspaecher H, Seldin MF, de Crombrugghe B. A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in certain mesenchymal cells. J Biol Chem 1996;271:31384– 31390. 3. McConnell BB, Ghaleb AM, Nandan MO, Yang VW. 2007; The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology. Bioessays 29:549– 557. 4. Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Kruppel-like factors 4 and 5: The yin and yang regulators of cellular proliferation. Cell Res 2005;15:92–96. 5. Dang DT, Bachman KE, Mahatan CS, Dang LH, Giardiello FM, Yang VW. Decreased expression of the gut-enriched Kruppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients. FEBS Lett 2000;476:203– 207. 6. Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S, Jain MK. Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages. J Biol Chem 2005;280:38247– 38258. 7. Ghaleb AM, Aggarwal G, Bialkowska AB, Nandan MO, Yang VW. Notch inhibits expression of the Kruppel-like factor 4 tumor suppressor in the intestinal epithelium. Mol Cancer Res 2008;6:1920–1927. 8. Kanai M, Wei D, Li Q, et al. Loss of Kruppel-like factor 4 expression contributes to Sp1 overexpression and human gastric cancer development and progression. Clin Cancer Res 2006;12:6395–6402. 9. Xu J, Lu B, Xu F, et al. Dynamic down-regulation of Kruppel-like factor 4 in colorectal adenoma-carcinoma sequence. J Cancer Res Clin Oncol 2008;134:891–898. 10. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676. 11. Wei D, Gong W, Kanai M, et al. Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res 2005;65: 2746–2754. 12. Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE, Yang VW. Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene 2004;23:395–402. 13. Wang N, Liu ZH, Ding F, Wang XQ, Zhou CN, Wu M. Down-regulation of gut-enriched Kruppel-like factor expression in esophageal cancer. World J Gastroenterol 2002;8:966– 970. 14. Ohnishi S, Ohnami S, Laub F, et al. Downregulation and growth inhibitory effect of epithelial-type Kruppel-like
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Oxidative DNA Damage Causes Premature Senescence in Mouse Embryonic Fibroblasts Deficient for Krüppel-Like Factor 4 Changchang Liu,1 Stephen La Rosa,1, 2 and Engda G. Hagos1* 1 2
Department of Biology, Colgate University, Hamilton, New York Memorial Sloan-Kettering Cancer Center, Prostate Cancer Research Program, New York, New York
Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor with tumor suppressor activity in various cancer types. Cells that sustain double strand breaks (DSBs) in their DNA due to high levels of reactive oxygen species (ROS) can develop genomic instability, which can result in cancer formation. One protective response to increased levels of ROS is the induction of cellular senescence. Recently, we found that mouse embryonic fibroblasts (MEFs) null for Klf4 are genetically unstable, as evidenced by the presence of DNA DSBs. However, it is yet unknown whether KLF4 is involved in regulating oxidative stress-induced DNA damage. Therefore, we sought to determine the mechanisms by which ROS induce genomic instability in Klf4-deficient MEFs. With SA-b-Gal staining, we show that Klf4/ MEFs enter senescence earlier than Klf4þ/þ MEFs, and western blot shows accumulation of p21 and p53 with increasing passages. In addition, immunostaining against g-H2AX indicates that the increased level of DNA damage in Klf4/ MEFs positively correlates with ROS accumulation. Consistent with ROS as a source of DSB in Klf4/ MEFs, treatment with NAC, reduces the accumulation of DNA damage. Our RT-PCR result demonstrates that Klf4/ MEFs have decreased expression of the antioxidant gene, Gsta4. The downregulation of the Gsta4 correlates with significant levels of ROS accumulation, as shown by DCFDA and FACS analysis, and thus the oxidative stress-induced premature senescence. Together these findings suggest a mechanism by which KLF4 protects against DNA damage and oxidative stress at least in part through the regulation of Gsta4 and likely related genes. © 2014 Wiley Periodicals, Inc. Key words: Krüppel-like factor 4; cellular senescence and reactive oxygen species; genomic instability
INTRODUCTION ¨ ppel-like factor 4 (KLF4) belongs to the Kru ¨ ppelKru like factor family of zinc-finger-containing transcription factors and has been shown to regulate various cellular processes including development, proliferation, inflammation, apoptosis, and differentiation [1– 6]. KLF4 also plays a role in tumorigenesis and reprogramming of somatic cells to induced pluripotent stem cells [7–10]. In many human cancers KLF4 is regarded as a tumor suppressor. For example, the level of KLF4 is downregulated in certain cancers such as gastric, colorectal, esophageal, bladder, lung, and pancreatic cancer [11–16]. Interestingly, in contrast to its role as a tumor suppressor in many cancer types, KLF4 has been found to act as a tumor promoter in certain tumorous contexts as it is upregulated in primary breast ductal carcinoma and oral squamous cell carcinoma [17,18]. In cell cultures, KLF4 expression is associated with conditions that initiate cell-cycle arrest such as contact inhibition and serum deprivation [4,19]. Cells that are arrested in growth are rich in KLF4, as KLF4 functions to inhibit cell cycle progression by upregulating cyclin-dependent kinase (CDK) inhibitors such as p21 [20]. Previous reports have shown that KLF4 was transcriptionally activated by p53 ß 2014 WILEY PERIODICALS, INC.
following DNA damage, and as a result, cells were arrested at both the G1-S and G2-M boundaries [21]. Furthermore, we previously showed that KLF4 plays a role in maintaining genomic stability in
Abbreviations: Klf4, Krüppel-like factor 4; ROS, reactive oxygen species; MEFs, mouse embryonic fibroblasts; CDK, Cyclin-dependent kinase; SOD, superoxide dismutase; CAT, catalase; GST, glutathione Stransferase; DOX, Doxorubicin; PFTa, Pifithrin alpha; Gsta4, glutathione S-transferase-alpha 4; DMEM, Dulbecco's modified Eagle's medium; NAC, N-acetyl-cysteine; SA-b-gal, Senescence associated-bgalactosidase; PBS, phosphate buffered saline; RT-qPCR, reverse transcriptase- quantitative polymerase chain reaction. Conflict of interest: none. Authors' contributions: C.L. carried out the all the experimental studies, participated in the design of the study and drafted the manuscript. S.L.R. participated in ROS staining and Flow Cytometry. EH conceived of the study, participated in its design and coordination, carried out cell transfections, microscope imaging, and Western blots, and, drafted the manuscript. All authors read and approved the final manuscript. Grant sponsor: Picker Research Fellowship from Colgate University Research Council *Correspondence to: Department of Biology, Colgate University, Hamilton, NY 13346-1398. Received 21 November 2013; Revised 9 March 2014; Accepted 27 March 2014 DOI 10.1002/mc.22161 Published online 30 April 2014 in Wiley Online Library (wileyonlinelibrary.com).
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mouse embryonic fibroblasts (MEFs). Specifically, Klf4/ MEFs show evidence of genomic instability, including aneuploidy, chromosome aberrations, increased DNA double strand breaks (DSBs), centrosome amplification, and anchorage-independent growth [22]. Notably, re-expression of KLF4 in Klf4/ MEFs corrects the genomic instability in these cells [23]. Cellular senescence is a mechanism used by cells to limit the growth of cells at high risk for tumorigenesis [24]. Cells with high levels of DNA damage can trigger cellular senescence and irreversibly lose the ability to undergo cell proliferation. This response prevents genomic instability and is achieved mainly by activation of senescence-associated genes such as p53 and p21 [24,25]. The pathways controlled by p53 to induce senescence can be triggered by reactive oxygen species (ROS), which can result in oxidative DNA damage [26]. ROS are products of cellular metabolic processes and can introduce DNA damage by modifying bases, cross-linking DNA and proteins, and causing DNA strand breakage [27]. Thus the cellular oxidative stress associated with increased ROS is a main cause of senescence in MEFs maintained under standard tissue culture conditions, as MEFs cultured in reduced levels of oxygen failed to senesce [26–28]. Previous studies have shown that cells regulate the levels of ROS and prevent dangerous accumulation of ROS by expressing antioxidants that chemically reduce ROS to non-harmful by-products [29]. For example, cellular antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) are responsible for reducing ROS and maintaining an appropriate homeostatic level of ROS [26]. When there is an elevated level of ROS present in cells, proper antioxidant defenses must be expressed to prevent or rescue cells from adverse cell fates. It has been shown that exposing cells with high oxidative stress levels to antioxidants allows cells to either repair or arrest in order to restore proper organelle and cell function [30,31]. Despite growing evidence that KLF4 maintains genomic stability as exemplified above, the mechanisms by which KLF4 exerts its functions are not well established. Since KLF4 has been shown to be a crucial mediator of p53 in the DNA damage response [21], and we have reported that MEFs null for Klf4 are genetically unstable [22,23], we sought to determine whether KLF4 plays a role in maintaining genomic stability by preventing DNA damage-induced oxidative stress in MEFs null for Klf4. Results presented in this report provide insights into the potential mechanism by which KLF4 plays a role in suppressing DNA damage accumulation as well as in oxidative stress responses, and implicate KLF4 in genome maintenance and protection against aberrant ROS production. Molecular Carcinogenesis
RESULTS /
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MEFs Enter Senescence Earlier than Klf4þ/þ MEFs
We first examined the growth characteristics of MEFs derived from Klf4þ/þ and Klf4/ embryos by calculating the relative cell number of primary cells of each genotype starting at passage 3. As shown in Figure 1A, pre-senescent Klf4/ MEFs proliferated at a slower rate compared to Klf4þ/þ MEFs. In addition, Klf4/ MEFs ceased proliferating at passage 6 whereas Klf4þ/þ MEFs continued to proliferate until passage 15 (Figure 1A). We next determined at what passage the MEFs enter senescence by assaying for the senescence marker, SA-b-Gal, beginning at passage 3. Klf4/ MEFs started to exhibit the typical flattened and enlarged senescence phenotypic morphology [32] at passage 4 and started to show SA-b-Gal activity (Figure 1B). The percentage of MEFs with SA-b-Gal staining was quantified and analyzed with Student’s t-test. The percentage of Klf4/ MEFs with SA-b-Gal staining increased with passage number. Significantly more Klf4/ MEFs assayed positive for SA-Gal and entered senescence at passage 6, as compared to Klf4þ/þ MEFs from the same passage (Figure 1B,C). Consistent with the relative cell number curve, Klf4þ/þ MEFs started to show positive SA-b-Gal at about passage 14 and expressed high levels of SA-bGal at passage 16, a much later time point compared to Klf4/ MEFs (Figure 1D). The Accumulation of p21 Leads MEFs to Enter Senescence Previous studies have shown that cell cycle regulators such as p21 are involved in the induction of premature senescence [33–35]. To characterize the molecular nature of the premature senescence in Klf4/ MEFs, we examined the levels of p21 and its upstream transcription factor, p53, in Klf4þ/þ and Klf4/ MEFs. We observed that Klf4/ MEFs exhibited higher levels of p21 and p53 compared to Klf4þ/þ MEFs from the same passage (Figure 2A,B). Furthermore, both Klf4þ/þ and Klf4/ MEFs expressed high levels of p21 and p53 at the passage where a high percentage of cells showed positive SA-bGal staining (Figure 2B,C). To investigate whether MEFs experience earlier senescence due to p21 accumulation, we overexpressed p21 by transient transfection in pre-senescence Klf4þ/þ MEFs at passage 8. As shown in Figure 2D, cells transfected with either GFP-control or p21 showed significantly higher levels of protein for GFP or p21, respectively, as compared to mock transfected cells. Next we assayed the transfected Klf4þ/þ MEFs with SA-b-Gal staining 5 d posttransfection for the presence of senescent cells. We found that significantly more MEFs transfected with p21 entered senescence as compared to Klf4þ/þ MEFs transfected with either mock or GFP (Figure 2E,F). This suggests that increased p21 levels are sufficient to induce senescence, consistent with previously
PREMATURE SENESCENCE IN Klf4-NULL MEFs
Figure 1. Klf4/ MEFs enter cellular senescence earlier than Klf4þ/þ MEFs. (A) Relative cell number for Klf4þ/þ and Klf4/ MEFs. Klf4þ/þ and Klf4/ MEFs were passed following the 3T3 protocol. Relative cell number were calculated by counting cell numbers at each passage for Klf4þ/þ and Klf4/ MEFs. The arrows indicate that cells arrest at passage 6 for Klf4/ and passage 15 for Klf4þ/þ. Shown is the representative growth curve of three independent
experiments. (B) Klf4þ/þ and Klf4/ MEFs were stained with SA-bGal to detect senescence from passage 3 to 6 (Klf4/) or from passage 6 to 16 (Klf4þ/þ). Representative images were shown from the indicated passages. (C, D) Quantification of the senescent Klf4þ/þ and Klf4/ MEFs with SA-b-Gal stain. N ¼ 3 (P < 0.05 at p5, p6, and p7 for Klf4/ compared to Klf4þ/þ). Error bars represent standard error.
Figure 2. Early senescence in Klf4/ MEFs is associated with p21 induction. Western blot analysis of p21 in Klf4þ/þ (A) and Klf4/ (B) MEFs at early passages (3, 6, and 7). (C) Western blot analysis of p21 levels in Klf4þ/þ MEFs at late passages (10,14, and 16). (D) Western blot analysis of either GFP or p21 in pre-senescent Klf4þ/þ MEFs transfected with either p21 or GFP at passage 8. (E) SA-b-Gal analysis of transfected MEFs with mock, GFP or p21. SA-b-Gal activity was assayed 3 d after transfection. (F) The percentage of MEFs described in (E) positive for SA-b-Gal activity was quantified. Error bars represent standard error. N ¼ 3; P < 0.05.
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published data that p21 is required for cells to enter senescence [33–35]. Klf4/ MEFs Enter Premature Senescence in a p53 Dependent Manner The tumor suppressor gene, p53, has been shown to play an important role in regulating cell cycle checkpoints through activation of p21 and other target genes [21]. Since overexpression of p21 leads pre-senescent MEFs to enter senescence (Figure 2E), we hypothesized that manipulating of p53 using drugs that interfere with its function will affect the levels of p21 and thus the entry of senescence in Klf4deficient cells. To determine whether the early cellular senescence shown is caused by p53 upregulation, we induced p53 in pre-senescent passage 3 Klf4/ MEFs with Nutlin-3, a MDM2 antagonist that induces senescence in wild type MEFs by activating p53 [36,37]. Western blot confirmed that Nutlin-3 treatment induced p53 activity and its downstream target p21 in Klf4/ MEFs as compared with DMSOtreated control Klf4/ MEFs (Figure 3A). In addition, as shown in Figure 3B,C, treatment with 5 mM Nutlin3 induced senescence morphology and increased SAb-Gal staining in Klf4/ MEFs. Our data strongly suggest that p53 induction is capable of driving premature senescence in Klf4/ MEFs. We have previously shown that pre-senescent Klf4/ MEFs exhibited higher levels of DNA damage
Figure 3. p53 induction results in premature senescence in Klf4/ MEFs. (A) Klf4/ MEFs of passage 3 were treated with 10 mM Nutlin-3 or DMSO as a control for 24 hr. Proteins were then extracted and subjected to western blot analysis of p53, p21 and b-actin. (B) Klf4/ MEFs from passage 3 were treated with DMSO or 5 mM of Nutlin-3 for 1 wk followed by 1-wk incubation in DMEM. The cells were stained with SA-b-Gal to detect senescent cells. (C) A quantification of MEFs in (B) is shown N ¼ 4; P < 0.05. Error bars represent standard error. (D) Klf4þ/þ
Molecular Carcinogenesis
as compared to Klf4þ/þ [22], To investigate whether the early cellular senescence associated with p53 upregulation in Klf4/ MEFs is due to DNA damage, we next treated the pre-senescent passage 8 Klf4þ/þ MEFs with either Doxorubicin (DOX), to induce DNA damage or Pifithrin alpha (PFTa), a p53 transactivation inhibitor [38,39]. As shown by western blot in Figure 3D, 0.1 mM DOX alone induced g-H2AX, a DNA damage marker, and activated the p53 pathway. On the other hand, inhibition of p53 with PFTa in DOXtreated MEFs induced DNA damage, but was unable to activate the expression of p53 and p21 (Figure 3D). Furthermore, whereas 45% MEFs showed SA-b-Gal staining with DOX treatment, PFTa significantly reduced the percentage of senescent cells to less than 20% (Figure 3E,F). Thus, inhibition of p53 by PFTa blocked p53-mediated induction of p21 and significantly prevented DNA damage-inducedsenescence. Increased Oxidative DNA Damage in Klf4/ MEFs Could Be Rescued by NAC Treatment Cellular senescence can be induced by DNA damage [40]. We recently showed that KLF4 is involved in DNA damage repair [22,25]. To investigate whether Klf4/ MEFs enter premature senescence due to DNA damage, we performed immunostaining for the presence of g-H2AX foci, a DNA damage marker, in Klf4þ/þ and Klf4/ MEFs,
MEFs were treated with DMSO, 0.1 mM Doxorubicin (Dox), or 0.1 mM Doxorubicin plus 30 mM Pifithrin a (PFTa) for 24 hr. Protein extracts were analyzed by western blot analysis for p53, p21, and b-actin and gH2AX. (E) Klf4þ/þ MEFs from early passage 8 were treated with DMSO, 0.1 mM Doxorubicin, or 0.1 mM Doxorubicin plus 30 mM PFTa for 1 wk. MEFs were then stained with SA-b-Gal to detect senescence. (F) A quantification of cells in (E) is shown. Error bars represent standard error. N ¼ 3; P < 0.05, P < 0.001.
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Figure 4. DNA damage in Klf4/ MEFs rescued by NAC treatment. (A) Immunostaining was performed for g-H2AX (green) on Klf4þ/þ and Klf4/, and NAC-treated Klf4/ pre-senescence MEFs. DAPI (blue) was used to stain the nuclei. (B) The percentage of cells with more than five g-H2AX foci from each cell line was quantified. N ¼ 3.
with or without NAC [41]. As shown in Figure 4A and B, over 50% Klf4/ MEFs contained more than five g-H2AX foci compared to only 10% Klf4þ/þ MEFs. Klf4/ MEFs treated with 10 mM NAC, a ROS scavenger, showed reduced g-H2AX foci as compared to untreated Klf4/ MEFs.
whether KLF4 regulates Gsta4 expression using RTPCR and qPCR. Consistent with the microarray data, we found that the mRNA levels of Gsta4 in Klf4/ MEFs were significantly downregulated compared to those in wild type MEFs (Figure 5C,D).
Klf4/ MEFs Experience Higher Levels of Oxidative Stress
DISCUSSION
ROS can react with DNA and introduce breaks to the DNA strands [42]. Because increasing ROS levels are linked to cellular senescence [43–47], we next examined whether Klf4/ MEFs experience higher levels of ROS compared to Klf4þ/þ MEFs. To visualize ROS in MEFs, we treated Klf4þ/þ and Klf4/ pre-senescent MEFs with 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCF-DA), which is oxidized to the fluorescent DCF by ROS and trapped within cells [39]. The cells were then stained with DAPI and visualized under a confocal fluorescence microscope. MEFs lacking Klf4 exhibited higher ROS levels compared to the Klf4þ/þ MEFs, as more Klf4/ MEFs exhibited positive ROS staining (Figure 5A). To quantify the levels of ROS in Klf4þ/þ and Klf4/ MEFs, we stained the cells with H2DCF-DA and performed FACS analysis on Klf4þ/þ, Klf4/, and Klf4/ MEFs with NAC. Klf4/ MEFs exhibited significantly higher levels of ROS compared to the wild type (Figure 5B). Moreover, in both DCF staining and FACS analysis, the ROS direct scavenger, NAC, reduced the levels of ROS significantly in Klf4/ MEFs (Figure 5A,B). To determine why there is more ROS in Klf4/ MEFs, we studied the levels of antioxidant genes in Klf4þ/þ and Klf4/ MEFs. Glutathione S-Transferase alpha 4 (Gsta4) encodes an enzyme that catalyzes the degradation of the products of oxidative metabolism [48]. Our prior microarray studies found that Gsta4 levels decreased by ninefold in Klf4/ MEFs as compared to Klf4þ/þ MEFs [49]. Here we determined
A hallmark of cancer, genomic instability can manifest as chromosomal aberrations, microsatellite instability, DSBs, and frequent base-pair mutations [50,51]. Genomic instability can arise when mutations occur in genes responsible for DNA damage repair, DNA replication, or segregation during mitosis [52]. Studies have shown that genomic instability is related to the initiation of tumorigenesis and increased rate of tumor growth and cancer development [52,53]. In many human cancers, KLF4 functions as a tumor suppressor [11–16]. KLF4 can exert its anti-carcinogenesis function by mediating the p53 activation of p21 and initiating cell cycle checkpoints [21,54]. In addition to its anti-proliferative effect, KLF4 has been shown to be involved in maintaining genomic integrity [22,23]. We have previously demonstrated that MEFs lacking KLF4 exhibit genomic instability as evidenced by the presence of aneuploidy, more DNA damage, and chromosomal aberrations [22]. Moreover, we recently reported that restoration of KLF4 to Klf4/ MEFs reduces the extent of DNA damage and aneuploidy [23]. In light of the previous studies which demonstrated the role of KLF4 in maintaining genomic stability, we investigated whether Klf4/ MEFs experience early cellular senescence due to DNA damage. In the current study we report that Klf4/ MEFs enter senescence prematurely in a p53-dependent manner (Figures 1 and 2). Moreover, we find that Klf4/ MEFs
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Figure 5. Klf4/ MEFs exhibit higher levels of ROS and reduced levels of antioxidant. (A) Klf4þ/þ MEFs, Klf4/ MEFs and Klf4/ MEFs treated with 10 mM NAC for 2 d were stained with 10 mM CM-H2DCFDA to detect ROS. DAPI was used to visualize the nuclei. (B) Klf4þ/þ MEFs and Klf4/ MEFs from late passage were grown with or without 10 mM NAC for 2 d. MEFs were then treated with 10 mM CM-H2DCFDA before FACS analysis. Data shown is representative of three
independent experiments. N ¼ 3; P < 0.05, P < 0.001. (C) Semiquantitative RT-PCR was performed to assess the level of Gsta4 in Klf4þ/þ and Klf4/ MEFs at passage 3. b-Actin was used as a loading control. (D) RT-qPCR was performed to assess the level of Gsta4 normalized to b-Actin. Shown is the fold reduction in Klf4/ MEFs compared to Klf4þ/þ MEFs (error bars represent standard deviation of three independent experiments, P < 0.05).
exhibit high levels of DNA damage (Figure 3). Increased DNA damage in pre-senescent Klf4/ MEFs correlates with increased ROS compared to Klf4þ/þ MEFs, and as a result, Klf4/ MEFs display elevated levels of cell cycle inhibitor, p21 which induces premature senescence (Figures 2, 4, and 5). Our results are consistent with reports that cells deficient in genes involved in genome maintenance, such as Brca1, Ku86, and Atm, enter senescence much earlier compared to their wild type counterparts [44,45,55,56]. We previously showed that post-senescent MEFs lacking Klf4 exhibit higher levels of DNA damage [22,23]. Consistent with previous findings, we show here that pre-senescent MEFs lacking Klf4 also display higher levels of DNA damage (Figure 4A,B). Thus, we hypothesize that Klf4/ MEFs experience premature senescence as a result of accumulated DNA damage. In order to test this hypothesis, we treated the Klf4/ cells with NAC, which has been shown recently to prevent mutagenesis and carcinogenesis by ameliorating genotoxic effects [57]. We observed that NAC rescued Klf4/ MEFs from extensive DNA damage (Figure 4A,B). In accord with previous findings that DNA damage induces cellular senescence as a protective mechanism against tumorigenesis, our data suggests that Klf4/ MEFs experience premature senescence as a response to the extensive damage in their DNA.
Expression of p21, a CDK inhibitor, has been shown to be upregulated by the p53 tumor suppressor gene in response to DNA damage [58]. To determine whether p21 was involved in the premature senescence in Klf4null cells, we performed Western blot analysis using p21 antibody. As shown in Figure 2, Klf4/ MEFs exhibit significantly higher levels of p21 compared to Klf4þ/þ MEFs of the same passage, and Klf4þ/þ cells later show comparable levels of p21 as they enter senescence. Moreover, overexpression of p21 in early pre-senescence passage Klf4þ/þ MEFs leads to premature senescence (Figure 2). Together, these data implicate p21 in the premature senescence observed in Klf4/ MEFs. Furthermore, we also observed a significant increase of p53, which transcriptionally regulates p21 (Figure 2). Previous studies in cardiomyocytes and wild type MEFs showed that p53 induces senescence [39,40]. To determine if the activation of p21 led to senescence downstream of p53, we used drugs that affect p53 expression. As shown in Figure 3, upregulation of p53 with Nutlin-3 induces premature senescence in Klf4/ MEFs, whereas inhibition of p53 with PFTa delays cellular senescence. Our data strongly suggest that the premature senescence of Klf4/ MEFs is associated with p53 activation. KLF4 inhibits cell proliferation by functioning as a cell cycle checkpoint protein to activate transcription of the CDK inhibitor, p21 [1,20]. Interestingly, we
Molecular Carcinogenesis
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observed that the activation of p21 by p53 in presenescent MEFs is independent of KLF4 function (Figure 2). This suggests that p53 activation of p21 in MEFs entering senescence does not require KLF4. This KLF4-independent p21 induction supports a previous report [59] that HCT116 cells that have lost KLF4 show an increase of p21 in response to p53 upregulation. Taken together, we conclude that p53 transactivation of p21 does not require KLF4 in presenescent MEFs, and the exact molecular mechanism for KLF4-independent p21 transcription awaits further investigation. In post-senescent Klf4/ MEFs, we previously reported that despite high levels of p53, the levels of p21 were greatly reduced [22]. Interestingly, in the current study we observe a modest increase of p53 but high levels of p21 in primary Klf4/ MEFs entering senescence compared to Klf4þ/þ MEFs (Figure 2). The increased level of p53 in Klf4/ is most likely due to DNA damage [22]. Moreover, restoration of KLF4 to the MEFs reduces the levels of p53 and corrects DNA damage [23]. We thus propose that persistent DNA damage in the Klf4/ MEFs activates DNA damage response and results in increased p53 levels compared to that in Klf4þ/þ MEFs. Furthermore, we observed that fewer than 60% of pre-senescent Klf4/ MEFs contained over 5 g-H2AX foci (Figure 4). On the other hand, in the postsenescence MEFs, 80–90% of Klf4/ MEFs contained over five g-H2AX foci [22,23]. The greater extent of DNA damage in the immortalized MEFs corresponds to a higher increase of p53, and the milder DNA damage in pre-senescence MEFs correlates with a modest increase of p53 [22], (Figure 2). Recently, published studies demonstrated that p53 determines the fate of a cell poised to undergo either cell cycle arrest or apoptosis [59]. Low dosage of DNA damage induces a modest increase of p53, which activates KLF4 and in turn, p21, resulting in cell cycle arrest [59]. On the other hand, extensive DNA damage induces high levels of p53, which suppresses KLF4 but activates BAX, leading to apoptosis [59]. In light of the role of p53 in determining DNA damage response outcomes, we propose that in the pre-senescent MEFs, the absence of Klf4 causes mild DNA damage, which leads to a modest increase of p53 (Figure 2). As a result, the activation of p53 induces p21 and premature senescence (Figure 3). In post-senescence MEFs, extensive DNA damage results in p53 upregulation, which inhibits p21 and subjects the cells to apoptosis [22]. Moreover, most recently we have shown that restoring KLF4 in Klf4 null MEFs reduced DNA damage, p53 levels, and apoptosis [23]. The upregulation of p53 in Klf4/ MEFs is consistent with previous literature, which found that KLF4 represses p53 activity [60]. More recently, we reported that restoration of KLF4 to Klf4/ MEFs decreases the level of apoptosis [23]. The decreased level of apoptosis is likely due to the decrease in p53 levels, Molecular Carcinogenesis
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thus providing additional evidence of KLF4 repression of p53. The finding that KLF4 represses p53 also supports studies that reported an anti-apoptotic effect of KLF4, as KLF4 is likely to rescue cells from programmed cell death to cell arrest by decreasing the levels of p53 [60]. Thus, in KLF4-depleted cells the levels of p53 are restored and cause p53-dependent apoptosis. These findings imply that the cytostatic activity of KLF4 is inhibited by p53 during extensive DNA damage, so that the severely damage cells undergo programmed cell death. Most recently, we have shown that overexpressing KLF4 in Klf4 null MEFs corrects DNA damage [23]. To investigate whether the source of DNA damage in the Klf4/ MEFs is induced by oxidative stress, we treated MEFs null for Klf4 with NAC. It has been shown that NAC reduces genomic toxicity through a wide range of mechanisms including inhibiting mutations during DNA repair, blocking mutagens, and acting as a direct scavenger of ROS [57]. ROS are highly reactive products of the electron transport chain with important physiological effects in oxygen detection, acquired immune response, and skeletal muscle activity; however, unregulated ROS levels can cause genomic instability and carcinogenesis [61]. Here we showed that Klf4/ MEFs exhibited higher levels of ROS compared to Klf4þ/þ MEFs (Figure 5A,B). Addition of NAC reduced the levels of ROS in Klf4/ MEFs (Figure 5A,B). This result provides novel evidence that KLF4 regulates cellular ROS levels. Next, we investigated whether KLF4 regulates ROS levels through antioxidant genes. Gsta4 belongs to the GST family that neutralizes hazardous endogenous oxidative compounds by catalyzing the conjugate addition of reduced glutathione [61]. Moreover, overexpression of Gsta4 reduced the levels of lipid peroxidation in the presence of DNA damage and protected cells from oxidative stress [61]. We have shown with microarray data that MEFs lacking Klf4 exhibit decreased expression of antioxidant genes [49]. In the current study, our semi quantitative RT-PCR and quantitative PCR results suggest that KLF4 reduces the levels of ROS by sustaining the expression of Gsta4 (Figure 5). However, whether or not KLF4 influences Gsta4 expression through direct regulation remains unknown. In conclusion, the present study demonstrates that Klf4/ MEFs have decreased antioxidant gene expression and increased ROS levels compared to Klf4þ/ þ MEFs. High levels of ROS cause persistent DNA damage in Klf4/ MEFs. As a part of the DNA damage response, p53 and p21 are induced. Upregulation of the cytostatic cell cycle regulators leads to premature senescence in Klf4/ MEFs (Figure 6). These findings provide insights into the potential mechanism by which KLF4 plays a role in DNA damage and oxidative stress responses, and implicate KLF4 in maintaining genomic stability and protection against aberrant ROS production in MEFs.
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independent experiments, each done in triplicate, were performed. Analysis of Senescence-Associated-b-Galactosidase Activity The expression of galactosidase as a marker for senescent MEFs was determined using a senescenceassociated-b-galactosidase (SA-b-Gal) activity assay as described in Ref. [33]. The senescence-associated-bgalactosidase staining kit was purchased from Cell Signaling (Danvers, MA), and the staining was performed following manufacturer’s protocol. Briefly, cells treated with X-gal were incubated at 378C overnight without CO2. Twenty-four hours later cells were washed twice with phosphate buffered saline (PBS). Cells then were mounted with 70% glycerol and images were captured using an Olympus IX51 microscope. Results are from three independent experiments each done in duplicate and are presented as the percentage of MEFs positive for SA-b-Gal staining. Figure 6. A working model for how Klf4 deficiency causes ROS accumulation that leads to premature senescence. Lack of Klf4 leads to the downregulation of antioxidant genes such as Gsta4 and causes the accumulation of ROS. Elevated oxidative stress causes increased DNA damage. As a result, the p53 gene, a tumor suppressor induces the expression of cell cycle inhibitor p21. Prolonged upregulation of p21 leads to cellular senescence.
MATERIALS AND METHODS Cell Culture, Growth Curve Analysis, and Drug Treatment Mice heterozygous for the Klf4 alleles (Klf4þ/) on a C57BL/6 background were crossbred. MEFs that are wild type (Klf4þ/þ) or null (Klf4/) for the Klf4 alleles were derived from Day 13.5 embryos. MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin-streptomycin at 378C and 5% CO2. Following 3T3 protocol, pre-senescent cells were passed every 3 d until they reached senescence. Cell numbers were counted at each passage and the relative cell number was calculated. To overexpress p21 and GFP that served as a control in MEFs, cells were transiently transfected with 4 mg plasmid DNA (per well in a 6well plate) using Lipofectamine LTX reagent (Life Technologies, Grand Island, NY) according to manufacturer’s instructions. To determine the expression of p53, MEFs were treated with indicated drugs 24 hr after plating. Nutlin-3, Pifithrin-a and DOX were dissolved in dimethyl sulfoxide (DMSO) to 10 mM (Nutlin-3, Pifthrin-a) or 100 mM (Doxorubicin) stock concentration and added to the cells at 5, 30, and 0.1 mM respectively. N-acetyl-cysteine (NAC) was purchased from Sigma–Aldrich (St. Louis, MO). To rescue DNA damage, NAC was dissolved in PBS to 1M stock concentration. Cells were incubated for 2 d with 10 mM NAC at 378C. For all experiments, three Molecular Carcinogenesis
Immunoblotting Protein extraction and western blot analysis were performed as previously described [22]. Briefly, the membranes were incubated with the following primary antibodies to: p21 (BD Pharmingen, Franklin Lakes, NJ), p53 (Abcam, Cambridge, MA), g- H2AX (Cell Signaling), and b-Actin (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The antibody-antigen complex was visualized by an Immun-StarTM HRP Chemiluminescence Kit and ChemiDocTM XRSþ System (Bio-Rad Laboratories, Hercules, CA). Anti-rabbit secondary antibody and anti-mouse secondary antibody were purchased from Cell Signaling and Abcam, respectively. Representative western blot image of three independent experiments were shown. Immunostaining MEFs grown on glass coverslips were washed briefly with PBS. They were then fixed with 16% methanolfree formaldehyde for 5 min at room temperature followed by three washings with PBS before blocking in solution [3% bovine serum albumin (BSA), 0.2% Triton X-100 in PBS] for 1 hr at room temperature. Rabbit anti-g-H2AX primary antibody (Cell Signaling) diluted in blocking solution was incubated at room temperature for an hour and detected with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Santa Cruz Biotechnology) for 1 hr at room temperature. Cells were then washed once and counterstained with DAPI (Life Technologies) for 5 min at room temperature in the dark. Finally cells were washed three times, mounted in Prolong Antifade kit (Molecular Probes by Life Technologies), and visualized with a Zeiss 710 confocal laser scanning microscope (Carl
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Zeiss, Thornwood, NY). Two independent experiments, each done with duplicates, were performed. Reactive Oxygen Species Detection MEFs grown on cover slips with or without 10 mM NAC were washed with PBS. Cells were then incubated with freshly prepared 10 mM CM-H2DCF-DA, a membrane permeable fluorescent dye which is converted to DCF upon interaction with ROS (Molecular Probes by Life Technologies) (Ex: 495 nm, Em: 520 nm) at 378C for 40 min with a 10 min recovery in DMEM in the dark. Cells were washed three times with PBS and counterstained with DAPI for 5 min at room temperature in the dark. Finally cells were washed three times with PBS and mounted in Prolong Antifade kit and imaged with a Zeiss 710 confocal laser scanning microscope. Three independent experiments, each done with duplicates, were performed. Flow Cytometry Quantification of ROS levels was performed as follows. 105 cells/well were plated in 6-well plates with or without 10 mM NAC and cultured at 378C in 5% CO2. After 2 d, cells were rinsed in PBS, trypsinized and re-suspended in DMEM with 10% FBS. Pelleted cells were washed again and then incubated in 10 mM CM-H2DCF-DA, Ex: 495 nm, Em: 520 nm, at 378C for 40 min with a 10 min recovery in DMEM in the dark. Pelleted live cells were re-suspended in PBS and analyzed with a BD Accuri C6 flow cytometer with the FL1 laser (515–545 nm). Data are presented as the mean percentage of three independent experiments. Semi-Quantitative RT-PCR and RT-qPCR Total RNA from cultured Klf4þ/þ and Klf4/ MEFs was isolated at passage 3 using RNeasy1 Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. RNA was subjected to DNase I treatment in order to remove any contaminating genomic DNA. cDNA was prepared from 1 mg of RNA and amplified with OneStep RT-PCR Kit (Qiagen). PCR products were analyzed by ethidium bromide staining on a 2% agarose gel. RNA was subjected for RT-qPCR analysis using Express One-Step SYBR GreenER Universal (Life Technologies) following manufacturer’s protocol. The expression of Gsta4 was normalized to the expression level of b-Actin. Gene expression level in Klf4/ MEFs was compared to that in Klf4þ/þ MEFs, which was set to 1. Data shown represents three independent experiments, each performed in triplicates. PCR reactions were performed using the following primers purchased from Integrated DNA Technologies (Coralville, IA). Gsta4F: 50 TCA AAC TCC ACT CCA GCC G 30 ; Gsta4R: 50 CTC GAG TGC CTG GAG ACA A 30 ; b-ActinF: 50 ATG GAG GGG AAT ACA GCC C 30 ; b-ActinR: 50 TTC TTT GCA GCT CCT TCG TT 30 . Molecular Carcinogenesis
Statistical Analysis The Student’s t-test was used to test for significant differences between treatments. ACKNOWLEDGMENTS This work was supported in part by a Picker Research Fellowship from Colgate University Research Council. We acknowledge the lab of Dr. Vincent W. Yang (Stony Brook Medical School, NY) for providing GFP and p21 constructs. REFERENCES 1. Shields JM, Christy RJ, Yang VW. Identification and characterization of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J Biol Chem 1996;271: 20009–20017. 2. Garrett-Sinha LA, Eberspaecher H, Seldin MF, de Crombrugghe B. A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in certain mesenchymal cells. J Biol Chem 1996;271:31384– 31390. 3. McConnell BB, Ghaleb AM, Nandan MO, Yang VW. 2007; The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology. Bioessays 29:549– 557. 4. Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Kruppel-like factors 4 and 5: The yin and yang regulators of cellular proliferation. Cell Res 2005;15:92–96. 5. Dang DT, Bachman KE, Mahatan CS, Dang LH, Giardiello FM, Yang VW. Decreased expression of the gut-enriched Kruppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients. FEBS Lett 2000;476:203– 207. 6. Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S, Jain MK. Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages. J Biol Chem 2005;280:38247– 38258. 7. Ghaleb AM, Aggarwal G, Bialkowska AB, Nandan MO, Yang VW. Notch inhibits expression of the Kruppel-like factor 4 tumor suppressor in the intestinal epithelium. Mol Cancer Res 2008;6:1920–1927. 8. Kanai M, Wei D, Li Q, et al. Loss of Kruppel-like factor 4 expression contributes to Sp1 overexpression and human gastric cancer development and progression. Clin Cancer Res 2006;12:6395–6402. 9. Xu J, Lu B, Xu F, et al. Dynamic down-regulation of Kruppel-like factor 4 in colorectal adenoma-carcinoma sequence. J Cancer Res Clin Oncol 2008;134:891–898. 10. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676. 11. Wei D, Gong W, Kanai M, et al. Drastic down-regulation of Kruppel-like factor 4 expression is critical in human gastric cancer development and progression. Cancer Res 2005;65: 2746–2754. 12. Zhao W, Hisamuddin IM, Nandan MO, Babbin BA, Lamb NE, Yang VW. Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene 2004;23:395–402. 13. Wang N, Liu ZH, Ding F, Wang XQ, Zhou CN, Wu M. Down-regulation of gut-enriched Kruppel-like factor expression in esophageal cancer. World J Gastroenterol 2002;8:966– 970. 14. Ohnishi S, Ohnami S, Laub F, et al. Downregulation and growth inhibitory effect of epithelial-type Kruppel-like
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