Skip to main content
NCBI home page
Cell Cycle logoLink to Cell Cycle
. 2013 Sep 24;12(22):3537–3546. doi: 10.4161/cc.26592

microRNA-141 regulates BMI1 expression and induces senescence in human diploid fibroblasts

Manjari Dimri 1, Jeremy D Carroll 1, Joon-Ho Cho 1, Goberdhan P Dimri 1,*
PMCID: PMC3906339  PMID: 24091627

Abstract

Polycomb group protein BMI1 is an important regulator of senescence, aging, and cancer. On one hand, it is overexpressed in cancer cells and is required for self-renewal of stem cells. On the other hand, it is downregulated during senescence and aging. MicroRNAs have emerged as major regulators of almost every gene associated with cancer, aging, and related pathologies. At present, very little is known about the miRNAs that regulate the expression of BMI1. Here, we report that miR-141 posttranscriptionally downregulates BMI1 expression in human diploid fibroblasts (HDFs) via a miR-141 targeting sequence in the 3′ untranslated region of BMI1 mRNA. We also show that overexpression of miR-141 induces premature senescence in HDFs via targeting of BMI1 in normal but not in exogenous BMI1-overexpressing HDFs. Induction of premature senescence in HDFs was accompanied by upregulation of p16INK4a, an important downstream target of BMI1 and a major regulator of senescence. Our results suggest that miR-141-based therapies could be developed to treat pathologies where BMI1 is deregulated.

Keywords: microRNA, senescence, BMI1, miR-141, polycomb group proteins, aging

Introduction

Polycomb group (PcG) proteins, first discovered in Drosophila are evolutionarily conserved gene silencers that determine cell fate decisions and body pattern during development.1 These proteins are often dysregulated in cancer cells.2,3 In particular, BMI1 and EZH2 are known to be overexpressed in a number of human cancers including breast and prostate cancers.4-6 In addition to its role in cancer, BMI1 is known to be required for self-renewal of neural, hematopoietic, intestinal, and mammary stem cells.7-12 Recent studies of breast and prostate cancer stem cells have also established the requirement for BMI1 in their proliferation and self-renewal.13,14 BMI1 can replace c-Myc in promoting the conversion of human fibroblasts into induced pluripotent stem cells (iPSC),15 and the knockdown of BMI1 inhibits conversion of human fibroblasts into iPSC.16 BMI1 also plays an important role in senescence and aging.17 It has been shown that the overexpression of BMI1 results in repression of tumor suppressor p16INK4a, which has emerged as a major regulator of aging and age-associated pathologies.18 Overexpression of BMI1 results in bypass of senescence in most cells, and immortalization of certain epithelial cell types.19,20 Importantly, the deficiency of Bmi1 in mouse results in tissue atrophy, developmental and neurological abnormalities, type II diabetes, and accelerated aging.21-23 BMI1 can also regulate the expression of several genes other than p16INK4a that are relevant to aging and cancer.24-26 Additionally, it was shown that p16INK4a-independent BMI1 targets include genes involved in transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) and endoplasmic reticulum (ER) stress pathways27 and inhibitors of WNT pathway such as DKK1.28

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNA molecules of 19–24 nucleotides in length, which regulate gene expression posttranscriptionally.29,30 MiRNAs regulate a variety of genes and can function as an oncogene (oncomiR) or a tumor suppressor.31 MiRNAs are also known to regulate senescence and aging32,33 and various other pathologies.34-36 An elegant study showed that miR-200c regulates breast cancer stem cell phenotype by targeting PcG protein BMI1.14 Among other miRNAs, miR-15a and miR-16 were shown to control BMI1 expression in ovarian cancer cells,37 and miR-218, which is downregulated in colon cancer cells, was shown to target BMI1.38 Most of these studies relate to regulation of BMI1 expression in cancer cells. At present, it is not known which of these miRNAs or any novel miRNA could regulate BMI1 function during senescence. Here we studied the role of hsa-miR-141 (herein referred to as miR-141) in regulation of BMI1 and its functional role in senescence. We report that miR-141 regulates BMI1 expression, and that its overexpression induces senescence via pRb-p16 pathway in human diploid fibroblasts (HDFs).

Results

MiR-141 induces senescence in normal human diploid fibroblasts (HDFs)

The miR-200 cluster, which includes miR-141 and miR-200c, 2 different miRNAs, acts as a tumor suppressor by inhibiting epithelial-to-mesenchymal transition (EMT) during cancer progression.39-41 As most tumor suppressors are potent inducers of senescence, we determined whether miR-141 can induce senescence in normal HDFs. MRC5 cells were lentivirally transduced to stably overexpress miR-141. Control cells infected with the lentiviral vector pEZX-MR03 alone were also generated. Cells were monitored for the GFP reporter of the vector and stably selected in 1.0 μg/ml puromycin. The overexpression of miR141 was confirmed by qRT-PCR (Fig. 1A). Next, we determined the relative rate of cell proliferation. The results indicated that compared with control, cells overexpressing miR-141 had dramatically reduced cell proliferation (Fig. 1B). Since the retroviral construct does not include miR-200c, the reduced proliferation of cells is likely to be due to the miR-141 overexpression only.

Figure 1.

Figure 1.

Open in a new tab

MiR-141 inhibits cell proliferation and induces senescence in HDFs. (A) Relative expression of microRNA in control MRC5 and MRC5 fibroblasts stably overexpressing miR-141 was determined by qRT-PCR as described in the “Materials and Methods”. (B) MiR-141 overexpression inhibits cellular proliferation. Equal number of cells were plated and harvested as indicated. Each experiment was done in triplicates, and proliferation curves were generated by plotting the number of cells against the number of days. Error bars represent ± SD *, P < 0.05. (C) Induction of cellular senescence by miR-141 overexpression was determined by SA-β-gal staining and EdU co-staining as described in the “Materials and Methods”. The number of senescent (SA-β-Gal positive), proliferating (EdU positive), and total number of cells in each culture were counted in multiple fields to determine the percentage of senescent and proliferating cells and was plotted as shown. Each experiment was done in triplicates. Error bars represent ± SD *, P < 0.05 (D) MiR-141 overexpression induces marker of DNA damage as seen by the ϒH2AX foci formation. The numbers of ϒH2AX foci/cell were quantified from multiple cells/field, and plotted as shown in the graph. Experiment was done in triplicates. Error bars represent ± SD *, P < 0.05 (E) MiR-141 overexpression inhibits proliferation and promotes senescence as determined by the western blot analysis for expression of pRb, p53, p21, and p16 in indicated set of cells. β-actin was used as the loading control. The signal for each protein was quantified using ImageJ software (NIH) and normalized to β-actin. The normalized value (relative expression) is indicated below each blot. *The relative expression values of phospho- pRb (p-pRb) and pRb are indicated as x/x.

The dramatically reduced proliferation of miR-141-overexpressing cells could be due to the induction of cellular senescence. Hence, we determined the induction of the senescent phenotype using SA-β-gal marker.42 Our results indicated that overexpression of miR-141 led to the induction of cellular senescence, as more than 80% of cells expressing miR-141 stained positively for SA-β-gal (Fig. 1C). Induction of senescent phenotype was further confirmed by γH2AX and 5-ethynyl-2'-deoxyuridine (EdU) staining. The EdU staining was optimized using serum induction of quiescent MRC5 cells (Fig. S2A). The γH2AX staining was optimized using Adriamycin treatment of cells, which induces DNA damage characterized by typical formation of γH2AX foci (Fig. S2B). Our results showed that the cells expressing miR-141 but not the control cells had γH2AX foci characteristic of the senescent cells (Fig. 1D). Cells expressing miR-141 also had a much reduced uptake of EdU, a marker of active cell proliferation (Fig. 1C).

Next, we performed western blot analysis of cells overexpressing miRNA-141 to determine the expression of p16-pRb and p53-p21 pathways, which regulate senescence. The results revealed that compared with vector control cells, MRC5-miR-141 had significantly increased levels of the growth-inhibitory proteins p16INK4a, p21, p53, and decreased levels of the phosphorylated (inactivated form) pRb (Fig. 1E). Based on staining for senescence markers (SA-β-Gal and γH2AX) and western blot analyses, we conclude that miR-141 is a potent inducer of cellular senescence in HDFs. We also determined whether miR-200c also induces senescence in HDFs. MRC5 cells were infected with a lentiviral vector that expresses only miR-200c. The cells were selected and studied for senescent phenotype. Our data indicated that miR-200c is also able to induce senescence in HDFs (Fig. S1).

MiR-141 regulates expression of PcG protein BMI1 and corresponding H2AK119Ub activity in normal HDFs

We have previously shown that BMI1 overexpression bypasses senescence,20 and that its knockdown leads to the induction of cellular senescence in HDFs via p16-pRb pathway.20,43 We surmised whether miR-141 targets BMI1 expression in HDFs. Hence, we wanted to analyze MRC5 fibroblasts overexpressing miR-141 for the expression of BMI1, EZH2, H2AK119Ub, and H3K27me3. Our results indicated that miR-141 overexpression led to downregulation of BMI1 and BMI1-mediated H2AK119Ub activity (Fig. 2A). Interestingly, miR-141 overexpression also led to downregulation of another PcG protein EZH2 and EZH2-dependent PRC2 activity (H3K27Me3 activity) (Fig. 2A). Collectively, these data indicate that miR-141 overexpression leads to downregulation of BMI1, EZH2, and the corresponding PRC1 and PRC2 activities.

Figure 2.

Figure 2.

Open in a new tab

MiR-141 posttranslationally targets BMI1 and upregulates p16 expression at the mRNA level. (A) miR-141 targets PcG proteins BMI1 and EZH2, and PRC activities as shown by western blot analysis of EZH2 and BMI1, and H3K27Me3 and H2AK119Ub in miR-141 overexpressing and control MRC5 cells. The BMI1 and EZH2 expression was normalized to β-actin while H2AK119Ub and H3K27Me3 levels were normalized to total H2A and H3, respectively. (B) MiR-141 targets BMI1 via 3′UTR sequences. Wild-type and mutant miR-141 target site was cloned in pLS-3′UTR reporter vector and the normalized activity of 3′UTR reporters was determined as described in the “Materials and Methods”. Result shows dose-dependent decrease in the luciferase activity of wild-type but not the mutant 3′UTR reporters. The error bars represent the means ± SD of 3 independent experiments. (C) Relative mRNA levels of BMI1 and p16INK4a were analyzed by qRT-PCR from MRC5-derived control and miR-141 overexpressing cells as indicated. (D) Binding of BMI1 and H3K27Me3 to p16INK4a promoter was determined using a ChIP assay in the indicated set of MRC5 stable cell lines. Error bars represent ± SD *P < 0.05.

MiR-141 posttranscriptionally regulates BMI1 expression

Next, we explored the mechanism of BMI1 downregulation by miR-141. We determined whether miR-141 transcriptionally or posttanscriptionally regulates BMI1 expression. We performed BMI1 promoter reporter assays using a BMI1 promoter region cloned in pGL3 vector.43 Our data indicated that miR141 did not affect BMI1 promoter activity, therefore miR-141 does not transcriptionally downregulate BMI1 (Fig. S3A). As miRNAs most often regulate their target genes posttranscriptionally, we examined the possibility of postranscriptional regulation of BMI1 by miR-141. To test this possibility, we examined 3′untranslated (UTR) of BMI1 by miRANDA, an miRNA prediction software. The BMI1 3′UTR showed a putative miR-141 site, although with a lower mirSVR score (−0.1125), but high homology in the seed sequence (6 bp homology) (Fig. 2B). We generated wild-type and mutant 3′UTR reporters in pLightSwitch_3′UTR reporter vector as described in the “Materials and Methods”. 293T cells were transiently transfected with plasmid expressing luciferase 3′UTR reporters with wild-type (pLS-miR-141WT) or mutant miR-141 targeting sequences (pLS-miR-141Mut), and luciferase activity of the different reporters was determined. The results showed a dose-dependent decrease in the activity of the wild-type but not the mutant reporters (Fig. 2B).

MiR-141 mediated BMI1 downregulation leads to upregulation of p16INK4a

As p16INK4a is a major regulator of senescence and is also a downstream transcriptional target of BMI1, we examined the expression of p16INK4a by qRT-PCR and the binding of BMI1 to p16INK4a promoter by ChIP analysis. We also found the downregulation of BMI1 by miR-141 correlated well with upregulation of p16INK4a mRNA as determined by qRT-PCR analysis (Fig. 2C). Next we determined whether BMI1 binding to p16INK4a promoter is quantitatively decreased in cells expressing exogenous miR141. Results showed a 50% decrease in the BMI1 binding to p16INK4a promoter sequences in the miR-141 overexpressing cells (Fig. 2D). Similar to the binding of BMI1, H3K27me3 binding was also reduced in miR-141 overexpressing cells (Fig. 2D).

MiR-141 inhibitor upregulates BMI1 and downregulates expression of senescence-related proteins

We further confirmed the BMI1-repression by miR-141 by stably expressing a miR-141 inhibitor in MRC5 cells. We generated cells stably expressing miR-141 inhibitor and confirmed downregulation of miR-141 using qRT-PCR (Fig. 3A). Next, we analyzed control and miR-141 inhibitor-expressing cells for BMI1 and regulators of senescence, and senescent phenotype. Western blot analysis of cells expressing miR-141 showed that miR-141 inhibition led to upregulation of BMI1 and EZH2 and downregulation of p53, p21 and p16 (Fig. 3B). We also performed staining of cells for SA-β-Gal, EdU, and γH2AX (Fig. 3C and D). Since these cells were early passage and had very few senescent but high numbers of proliferating cells, the decrease in the number of senescent cells was not evident. Nonetheless, there was a modest increase in number of EdU-positive cells (Fig. 3C) and a modest decrease in number of γH2AX foci/cell (Fig. 3D). We also overexpressed miR-141 in control and miR-141 inhibitor-expressing cells and examined the induction of senescence using SA-β-Gal staining. The results indicated that miR-141 inhibitor can abrogate senescence induction by miR-141 (Fig. S4).

Figure 3.

Figure 3.

Open in a new tab

Exogenous expression of miR-141 inhibitor upregulates BMI1 and downregulates senescence-related proteins. (A) The relative expression of miR-141 in control and miR-141 inhibitor expressing cells was determined by qRT-PCR analysis. (B). Control and miR-141 inhibitor expressing MRC5 cells were analyzed for the expression of BMI1, EZH2 and indicated senescence-related proteins by western blot analysis. The normalized relative expression of each protein was determined as described in Figure 1. (C) and (D) Control and miR-141 inhibitor expressing cells were studied for senescence using SA-β-Gal/EdU staining (C), and γH2AX foci formation (D). Error bars represent ± SD *, P < 0.05.

Exogenous BMI1 expression rescues miR-141 mediated repression of BMI1 and induction of senescence

If the induction of senescence by miR-141 in HDFs solely results due to the targeting of BMI1 via its target site in the 3′UTR of BMI1, then the exogenous expression of BMI1 cDNA lacking 3′UTR should overcome senescence induced by miR-141. To test this possibility, we stably overexpressed miR-141 in vector control (MRC5-B0) and MRC5 cells expressing an exogenous BMI1 (MRC5-BMI1). The resulting set of cells was analyzed for cell proliferation and induction of senescence markers (Figs. 4 and 5). Our results showed that overexpression of miR-141 led to minimal downregulation of BMI1 in MRC5-BMI1 cells, which is likely due to the reduction in the levels of the endogenous BMI1 (Fig. 4B). To further confirm that the exogenous BMI1 is not affected by miR-141, we transiently transfected HA-tagged BMI1 (HA-BMI1) together with miR-141 expressing plasmid into 293T cells. After 48 h, we determined the effect of miR-141 on exogenous BMI1 using western blot analysis using anti-HA (exogenous BMI1) and anti-BMI1 (exogenous and endogenous BMI1) antibodies. The result suggested that the exogenous BMI1 (lacking 3′UTR) is not affected by miR-141 (Fig. S3B).

Figure 4.

Figure 4.

Open in a new tab

MiR-141 induced senescence is mediated via its regulation of BMI1. (A) Relative expression levels of miR-141 in MRC5 fibroblasts stably overexpressing either control (B0) or BMI1. (B) Western blot analysis of BMI1, EZH2, Rb, p53, p21, p16 in indicated set of cells was performed as described in the “Materials and Methods”. β-actin was used as a loading control. The relative expression of each protein was quantified as described in Figure 1. (C) BMI1 overexpression rescues the proliferation defect in miR-141-overexpressing fibroblasts.

Figure 5.

Figure 5.

Open in a new tab

Exogenous BMI1 rescues senescence-inducing function of miR-141. The effect of miR-141 overexpression was determined in control (B0) and BMI1-overexpressing MRC5 cells. (A) Abrogation of cellular senescence induced by miR-141 (seen by reduction in number of SA-β-Gal positive cells) and restoration of proliferation (increased EdU-positive cells) was studied as described in “Materials and Methods”. The total number of cells were counted to determine the percentage of senescent (SA-β-gal positive) and proliferating cells (EdU positive) and plotted as a graph. (B) The number of ϒH2AX foci/cell were quantified using ImageJ and plotted as shown in the graph. The experiment was done in triplicates. Error bars represent ± SD *, P < 0.05.

Importantly, there was no significant induction of p16INK4a by miR-141 in MRC5-BMI1 cells, suggesting that the exogenous BMI1 can rescue the effect of endogenous BMI1 downregulation and consequent upregulation of p16INK4a (Fig. 4B). We also examined cells for proliferation and induction of senescence using SA-β-Gal marker and γH2AX foci formation assays (Figs. 4C and 5A and B). Our results indicated that miR-141 cannot inhibit proliferation of cells or induce senescence in MRC5 cells that are made to overexpress an exogenous BMI1 (Figs. 4C and 5A and B).

Collectively, our results strongly suggest that miR-141 induce senescence via targeting of BMI1 in a posttranscriptional manner.

MiR-141 is upregulated in senescent cells

To determine the physiological significance of our findings, we determined whether miR-141 is upregulated by senescence-inducing signals. We analyzed expression of miR-141 in proliferating (early passage) and senescent (late passage) MRC5 cells (Fig. 6A). We also analyzed MRC5 cells that were treated with either hydrogen peroxide (H2O2) or sodium butyrate (Fig. 6B), which are known to induce premature or stress-induced senescence in HDFs.24,44 Our data indicate that compared with proliferating MRC5 cells, miR-141 is upregulated in MRC5 cells that are undergoing replicative or premature senescence (Fig. 6).

Figure 6.

Figure 6.

Open in a new tab

MiR-141 is upregulated in senescent cells. (A) Expression of miR-141 was studied by qRT-PCR in early passage (proliferating) and late passage (senescent) cells. (B) MRC5 cells were either mock treated or treated with premature senescence-inducing reagents H2O2 (100 µM for 4 h followed by 24 h growth in normal medium without H2O2) and sodium butyrate (NaB) (4 mM for 24 h followed by 24 h growth in normal medium without NaB), and expression of miR-141 was determined by qRT-PCR analysis. In each case induction of senescence was confirmed by staining for SA-β-Gal and EdU markers. Error bars represent ± SD *, P < 0.05.

Discussion

MiR-200c/141 cluster functions as a potential tumor suppressor. The cluster includes 2 different miRNAs: miR-200c and miR-141. The expression of miR-141 and miR-200c inhibits EMT and metastasis via targeting of ZEB1, SIP1, and ZEB2.40,41,45 The ZEB/miR-200c/141 feedback loop has been suggested to be a major control of cellular plasticity in the development and cancer.39-41 The miR-200c/141 cluster is known to be silenced via DNA methylation in invasive breast cancer cells,46,47 and miR-200c was shown to inhibit breast cancer stem cell phenotype.14 While these studies point toward the tumor suppressor role of miR-200c and miR-141, paradoxically, these miRNAs are also reported to function as oncogenes in certain cells,48,49 suggesting a complex context-dependent role of miR-200c and miR-141. Many tumor suppressor genes, such as p53, pRb, p16INK4a, p57, p27, and p21, are potent inducers of cellular senescence, which by itself is a natural tumor suppressor mechanism.50 Importantly, miR-200c was reported to target PcG protein BMI1, a potential oncoprotein, which is a negative regulator of p16INK4a and can bypass senescence when exogenously expressed in human and rodent cells.20,22 Moreover, miR-200c was recently shown to induce apoptosis and senescence in endothelial cells via inhibition of ZEB1, a known target of miR-200c.51 While the function of miR-200c is well studied, relatively little is known about the functional role of miR-141, particularly its role in cellular senescence is not known. Here we studied the role of miR-141 in HDFs, which has been long studied for the cellular senescence since the days of Hayflick.50 Our studies show that miR-141 induces a robust senescent phenotype in HDFs, suggesting that miR-141 acts as a tumor suppressor with respect to senescence.

MiR-141 and miR-200c can target several important genes that are involved in a variety of cellular processes such as growth, proliferation, senescence, and apoptosis. The known targets of miR-200 family include important regulators of EMT such as ZEB1 and ZEB2 (SIP1).40,41,45 A large number of genes can be targeted by these miRNAs. For example, miRANDA analysis of both miR-200c and miR-141 shows that each of these miRNAs can target several genes to some extent. Interestingly, although miR-200c and miR-141 targeting sites have different seed sequences, each of them can also target the same gene perhaps via different targeting sites. For example, both miR-200c and miR-141 can target EMT regulators ZEB1 and ZEB2.40,41,45 The studies reported here show that another important common target of miR-141 and miR-200c is the PcG protein BMI1. Although the target sequence of miR-200c in 3′UTR of BMI1 shares 7 bp homology with the miR-200c seed sequence,14,52 the miR-141 target site shares only 6 bp homology with its seed sequence in 3′UTR of BMI1. The target sequences with only 6 bp homology in miRNA seed sequences can also be targeted efficiently by the respective miRNAs.53 For example, miR-24 can target several proliferation-regulatory genes by targeting 3′UTR sequences that may not necessarily share 8 bp homology in miR-24 seed sequences.54 Indeed, we show that miR-141 can efficiently target BMI1 via its target sequence in the 3′UTR of BMI1 that shares only 6 bp homology with the seed sequences of the miR-141. Our data show that miR-141 posttranscriptionally targets BMI1 and induces a robust senescent phenotype. More importantly, our data also show that the expression of an exogenous BMI1 gene that lacks the 3′UTR can overcome the senescence-inducing activities of miR-141 in HDFs.

We also noticed that another important PcG protein EZH2 is also downregulated in cells overexpressing miR-141. Coordinated regulation of PcG proteins by certain microRNAs including miR-200c has been previously reported.52 Our data suggesting coordinated regulation of BMI1 and EZH2 by miR-141 is consistent with the published report.52 The 3′UTR of EZH2 does not appear to have target sites for miR-141 according to miRANDA analysis and other miR site prediction software. Although it is possible that EZH2 downregulation is a consequence of senescence induction, we have previously noticed concurrent co-regulation of BMI1 and EZH2.24 As BMI1 upregulates WNT signaling,28 it is possible that WNT signaling connects concurrent co-regulation of BMI1 and EZH2 via c-Myc. As the most important senescence-relevant target of BMI1 is p16INK4a, the induction of senescence by miR-141 is almost certainly due to induction of p16INK4a, and induction of p53 and p21 could possibly be a consequence of senescence induction by miR-141. Finally, our studies also show that miR-141 is upregulated in cells that are undergoing either replicative or premature stress-induced senescence. Thus, it is possible that miR-141 upregulation may at least partially account for the downregulation of BMI1 and upregulation of p16INK4a in senescent cells. Our studies have important implications for cancer and age-related pathologies, as these studies suggest that by modulating the expression of miR-141, BMI1 expression can be repressed or activated, which may then correct its dysregulation in a particular pathological condition. For example, anti-miR-141 could be used to restore BMI1 expression and stem cell function in aged tissues, and miR-141 overexpression could be used to inhibit cancer progression in cases where BMI1 is overexpressed.

Materials and Methods

Cells and cell culture methods

MRC5 strain of HDFs was obtained from the NIA Aging Cell Repository (Coriell Institute for Medical Research). 293T cells were obtained from the American Type Culture Collection (ATCC). The cells were cultured as described previously.42,43

Expression vectors, promoter constructs, transfections, retrovirus, and lentivirus production

Lentiviral vectors pEZX-MR03 expressing either hsa-miR-141 or hsa-miR-200c, and pEZX-AM03 expressing hsa-miR-141 inhibitor and a miRNA scrambled control clone were obtained from the Genecopoeia. Retroviral vector overexpressing wild-type BMI1 (pBabe-BMI1) and method for producing lentiviruses and retroviruses have been described previously.28,55

Antibodies and western blot analyses

Various antibodies were obtained from commercial sources. A rabbit polyclonal antibody (pAb) against pRb, a p53 mouse monoclonal antibody (mAb), a p21 mAb, and a p16INK4a mAb were obtained from Santa Cruz Biotechnology . The BMI1 mAb, a EZH2 mAb, and a β-actin mAb were obtained from Invitrogen, BD Biosciences, and Sigma-Aldrich, respectively. pAbs against H3K27me3 and H2AK119Ub were obtained from Millipore. pAbs against total H3 and H2A were obtained from Cell Signaling Technology. Western blot analyses were done using specific antibodies as described previously24,56.

Proliferation, senescence associated β-galactosidase assay, EdU, and γH2AX staining

The proliferation assays were performed as described.42,43 Senescence was determined using SA-β-Gal marker.42,43 EdU (5′ ethynyl -2’-deoxyuridine, a thymidine analog) and SA-β-Gal co-staining was performed as described.57 Images were taken with a Nikon Eclipse Ti microscope camera under 10× magnification, and stained cells were counted as described.58 Other markers of senescence, such as γH2AX foci formation using immunostaining was done as described.59 Briefly, cells were fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100 for 5 min, and immunostained with γH2AX (S139)-A555 conjugated antibody (BD Pharmingen). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), washed, and mounted with Prolong antifade mounting medium. Images were taken with an Olympus confocal microscope under 60× magnification as described.58 Quantification of H2AX foci was performed using ImageJ (NIH) software.

3′UTR reporter vectors and luciferase assays

For the reporter assays, the potential target site for hsa-miR-141 in the 3′ untranslated region (3′UTR) of BMI1 was identified by miRANDA, an miRNA prediction software (www.microRNA.org). Wild-type and mutant oligonucleotides were designed to amplify about 70 bp surrounding the seed sequences of the miR-141 miRNA target site, which can make the stem-loop structure. The double-strand oligonucleotides were annealed and cloned into XbaI and XhoI site of the pLightSwitch_3UTR Reporter vector (SwitchGear Genomics). These constructs have been referred to as pLS-miR141WT and pLS-miR141Mut. The 3′UTR reporter assays were performed as recommended by the manufacturer (SwitchGear Genomics). For transfection, 293T cells were plated in 6-well plates. For each well, 1 µg of the 3′UTR constructs were transfected with 0–3 µg of the pEZX-MR03-hsa-miR-141 plasmid. pTK-Cluc vector (SwitchGear Genomics) was also transfected as an internal control using FuGENE (Promega) transfection reagent. After 48 h, cells were lysed, and the luciferase assay performed using LightSwitch Dual Assay System (SwitchGear Genomics).

ChIP assays

The chromatin-IP (ChIP) assay was performed as described.24,28,56 Briefly, cells were treated with 1% formaldehyde for 20 min at room temperature. The cross-linked chromatin was isolated and sonicated to yield 200–500 bp fragments. Immunoprecipitations (IPs) were performed using a custom made rabbit polyclonal antibody raised against BMI1, H3K27me3 (Millipore), and a control IgG. The BMI1- and IgG- bound chromatin were amplified using p16INK4a promoter-specific primers by quantitative real-time PCR (qPCR) as described.24,28,56

MicroRNA analysis

Total RNA was extracted using the miRNeasy Mini Kit (Qiagen). Inventoried TaqMan® miRNA Assay Kit (ABI) was used to synthesize single-stranded cDNA according to the manufacturer’s protocol. The quantitative real-time PCR (qRT-PCR) was done according to TaqMan® miRNA assays protocol using miR-141-, miR-200c-, or RNU6B-specific inventoried real-time miRNA expression assays in a Step-one plus RT-PCR machine from ABI. The comparative CT (ΔΔCT) method was used to calculate RQ of miRNA expression using RNU6B as the endogenous control. The statistical significance was set at P < 0.05.

Statistical analysis

All experiments were performed at least three times in triplicates for each group. The results are presented as the mean ± SD. Statistical significance was determined using Student t test, and P < 0.05 was considered significant.

Supplementary Material

Additional material
cc-12-3537-s01.pdf (724.7KB, pdf)

Acknowledgments

This work was supported in part by RO1 CA094150 from the National Cancer Institute, NIH (GPD) and by the Undergraduate Research Fellowship, sponsored by the Offices of the Provost and the Vice President for Research, George Washington University (JDC).

10.4161/cc.26592

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Materials

Supplemental materials may be found here:
www.landesbioscience.com/journals/cc/article/26592

Footnotes

References

  • 1.Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer. 2006;6:846–56. doi: 10.1038/nrc1991. [DOI] [PubMed] [Google Scholar]
  • 2.Richly H, Aloia L, Di Croce L. Roles of the Polycomb group proteins in stem cells and cancer. Cell Death Dis. 2011;2:e204. doi: 10.1038/cddis.2011.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Valk-Lingbeek ME, Bruggeman SW, van Lohuizen M. Stem cells and cancer; the polycomb connection. Cell. 2004;118:409–18. doi: 10.1016/j.cell.2004.08.005. [DOI] [PubMed] [Google Scholar]
  • 4.Glinsky GV, Berezovska O, Glinskii AB. Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest. 2005;115:1503–21. doi: 10.1172/JCI23412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Guo WJ, Zeng MS, Yadav A, Song LB, Guo BH, Band V, Dimri GP. Mel-18 acts as a tumor suppressor by repressing Bmi-1 expression and down-regulating Akt activity in breast cancer cells. Cancer Res. 2007;67:5083–9. doi: 10.1158/0008-5472.CAN-06-4368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Kim JH, Yoon SY, Jeong SH, Kim SY, Moon SK, Joo JH, Lee Y, Choe IS, Kim JW. Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast. 2004;13:383–8. doi: 10.1016/j.breast.2004.02.010. [DOI] [PubMed] [Google Scholar]
  • 7.Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature. 2003;423:255–60. doi: 10.1038/nature01572. [DOI] [PubMed] [Google Scholar]
  • 8.Leung C, Lingbeek M, Shakhova O, Liu J, Tanger E, Saremaslani P, Van Lohuizen M, Marino S. Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas. Nature. 2004;428:337–41. doi: 10.1038/nature02385. [DOI] [PubMed] [Google Scholar]
  • 9.Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, Suri P, Wicha MS. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006;66:6063–71. doi: 10.1158/0008-5472.CAN-06-0054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R. Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev. 2005;19:1432–7. doi: 10.1101/gad.1299505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature. 2003;425:962–7. doi: 10.1038/nature02060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Sangiorgi E, Capecchi MR. Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet. 2008;40:915–20. doi: 10.1038/ng.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev Biol. 2007;23:675–99. doi: 10.1146/annurev.cellbio.22.010305.104154. [DOI] [PubMed] [Google Scholar]
  • 14.Shimono Y, Zabala M, Cho RW, Lobo N, Dalerba P, Qian D, Diehn M, Liu H, Panula SP, Chiao E, et al. Downregulation of miRNA-200c links breast cancer stem cells with normal stem cells. Cell. 2009;138:592–603. doi: 10.1016/j.cell.2009.07.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Moon JH, Heo JS, Kim JS, Jun EK, Lee JH, Kim A, Kim J, Whang KY, Kang YK, Yeo S, et al. Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1. Cell Res. 2011;21:1305–15. doi: 10.1038/cr.2011.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Onder TT, Kara N, Cherry A, Sinha AU, Zhu N, Bernt KM, Cahan P, Marcarci BO, Unternaehrer J, Gupta PB, et al. Chromatin-modifying enzymes as modulators of reprogramming. Nature. 2012;483:598–602. doi: 10.1038/nature10953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dimri GP. The search for biomarkers of aging: next stop INK4a/ARF locus. Sci Aging Knowledge Environ. 2004;2004:pe40. doi: 10.1126/sageke.2004.44.pe40. [DOI] [PubMed] [Google Scholar]
  • 18.Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, Kirkland JL, van Deursen JM. Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature. 2011;479:232–6. doi: 10.1038/nature10600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, Beekman C, Theilgaard-Mönch K, Minucci S, Porse BT, Marine JC, et al. The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 2007;21:525–30. doi: 10.1101/gad.415507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Itahana K, Zou Y, Itahana Y, Martinez JL, Beausejour C, Jacobs JJ, Van Lohuizen M, Band V, Campisi J, Dimri GP. Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1. Mol Cell Biol. 2003;23:389–401. doi: 10.1128/MCB.23.1.389-401.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Chatoo W, Abdouh M, David J, Champagne MP, Ferreira J, Rodier F, Bernier G. The polycomb group gene Bmi1 regulates antioxidant defenses in neurons by repressing p53 pro-oxidant activity. J Neurosci. 2009;29:529–42. doi: 10.1523/JNEUROSCI.5303-08.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Jacobs JJ, Kieboom K, Marino S, DePinho RA, van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature. 1999;397:164–8. doi: 10.1038/16476. [DOI] [PubMed] [Google Scholar]
  • 23.Tschen SI, Dhawan S, Gurlo T, Bhushan A. Age-dependent decline in beta-cell proliferation restricts the capacity of beta-cell regeneration in mice. Diabetes. 2009;58:1312–20. doi: 10.2337/db08-1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Bommi PV, Dimri M, Sahasrabuddhe AA, Khandekar J, Dimri GP. The polycomb group protein BMI1 is a transcriptional target of HDAC inhibitors. Cell Cycle. 2010;9:2663–73. doi: 10.4161/cc.9.13.12147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Datta S, Hoenerhoff MJ, Bommi P, Sainger R, Guo WJ, Dimri M, Band H, Band V, Green JE, Dimri GP. Bmi-1 cooperates with H-Ras to transform human mammary epithelial cells via dysregulation of multiple growth-regulatory pathways. Cancer Res. 2007;67:10286–95. doi: 10.1158/0008-5472.CAN-07-1636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kim RH, Lieberman MB, Lee R, Shin KH, Mehrazarin S, Oh JE, Park NH, Kang MK. Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-beta signaling. Exp Cell Res. 2010;316:2600–8. doi: 10.1016/j.yexcr.2010.04.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Gargiulo G, Cesaroni M, Serresi M, de Vries N, Hulsman D, Bruggeman SW, Lancini C, van Lohuizen M. In vivo RNAi screen for BMI1 targets identifies TGF-β/BMP-ER stress pathways as key regulators of neural- and malignant glioma-stem cell homeostasis. Cancer Cell. 2013;23:660–76. doi: 10.1016/j.ccr.2013.03.030. [DOI] [PubMed] [Google Scholar]
  • 28.Cho JH, Dimri M, Dimri GP. A positive feedback loop regulates the expression of polycomb group protein BMI1 via WNT signaling pathway. J Biol Chem. 2013;288:3406–18. doi: 10.1074/jbc.M112.422931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97. doi: 10.1016/S0092-8674(04)00045-5. [DOI] [PubMed] [Google Scholar]
  • 30.Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33. doi: 10.1016/j.cell.2009.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Garzon R, Calin GA, Croce CM. MicroRNAs in Cancer. Annu Rev Med. 2009;60:167–79. doi: 10.1146/annurev.med.59.053006.104707. [DOI] [PubMed] [Google Scholar]
  • 32.Grillari J, Grillari-Voglauer R. Novel modulators of senescence, aging, and longevity: Small non-coding RNAs enter the stage. Exp Gerontol. 2010;45:302–11. doi: 10.1016/j.exger.2010.01.007. [DOI] [PubMed] [Google Scholar]
  • 33.Lafferty-Whyte K, Cairney CJ, Jamieson NB, Oien KA, Keith WN. Pathway analysis of senescence-associated miRNA targets reveals common processes to different senescence induction mechanisms. Biochim Biophys Acta. 2009;1792:341–52. doi: 10.1016/j.bbadis.2009.02.003. [DOI] [PubMed] [Google Scholar]
  • 34.O’Connell RM, Rao DS, Baltimore D. microRNA regulation of inflammatory responses. Annu Rev Immunol. 2012;30:295–312. doi: 10.1146/annurev-immunol-020711-075013. [DOI] [PubMed] [Google Scholar]
  • 35.Jamaluddin MS, Weakley SM, Zhang L, Kougias P, Lin PH, Yao Q, Chen C. miRNAs: roles and clinical applications in vascular disease. Expert Rev Mol Diagn. 2011;11:79–89. doi: 10.1586/erm.10.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Santos-Rebouças CB, Pimentel MM. MicroRNAs: macro challenges on understanding human biological functions and neurological diseases. Curr Mol Med. 2010;10:692–704. doi: 10.2174/156652410793384169. [DOI] [PubMed] [Google Scholar]
  • 37.Bhattacharya R, Nicoloso M, Arvizo R, Wang E, Cortez A, Rossi S, Calin GA, Mukherjee P. MiR-15a and MiR-16 control Bmi-1 expression in ovarian cancer. Cancer Res. 2009;69:9090–5. doi: 10.1158/0008-5472.CAN-09-2552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.He X, Dong Y, Wu CW, Zhao Z, Ng SS, Chan FK, Sung JJ, Yu J. MicroRNA-218 inhibits cell cycle progression and promotes apoptosis in colon cancer by downregulating BMI1 polycomb ring finger oncogene. Mol Med. 2012;18:1491–8. doi: 10.2119/molmed.2012.00304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Brabletz S, Bajdak K, Meidhof S, Burk U, Niedermann G, Firat E, Wellner U, Dimmler A, Faller G, Schubert J, et al. The ZEB1/miR-200 feedback loop controls Notch signalling in cancer cells. EMBO J. 2011;30:770–82. doi: 10.1038/emboj.2010.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Brabletz S, Brabletz T. The ZEB/miR-200 feedback loop--a motor of cellular plasticity in development and cancer? EMBO Rep. 2010;11:670–7. doi: 10.1038/embor.2010.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol. 2008;10:593–601. doi: 10.1038/ncb1722. [DOI] [PubMed] [Google Scholar]
  • 42.Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92:9363–7. doi: 10.1073/pnas.92.20.9363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Guo WJ, Datta S, Band V, Dimri GP. Mel-18, a polycomb group protein, regulates cell proliferation and senescence via transcriptional repression of Bmi-1 and c-Myc oncoproteins. Mol Biol Cell. 2007;18:536–46. doi: 10.1091/mbc.E06-05-0447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Li SK, Smith DK, Leung WY, Cheung AM, Lam EW, Dimri GP, Yao KM. FoxM1c counteracts oxidative stress-induced senescence and stimulates Bmi-1 expression. J Biol Chem. 2008;283:16545–53. doi: 10.1074/jbc.M709604200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 2008;22:894–907. doi: 10.1101/gad.1640608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Castilla MA, Díaz-Martín J, Sarrió D, Romero-Pérez L, López-García MA, Vieites B, Biscuola M, Ramiro-Fuentes S, Isacke CM, Palacios J. MicroRNA-200 family modulation in distinct breast cancer phenotypes. PLoS One. 2012;7:e47709. doi: 10.1371/journal.pone.0047709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Neves R, Scheel C, Weinhold S, Honisch E, Iwaniuk KM, Trompeter HI, Niederacher D, Wernet P, Santourlidis S, Uhrberg M. Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells. BMC Res Notes. 2010;3:219. doi: 10.1186/1756-0500-3-219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Chen L, Li Y, Fu Y, Peng J, Mo MH, Stamatakos M, Teal CB, Brem RF, Stojadinovic A, Grinkemeyer M, et al. Role of deregulated microRNAs in breast cancer progression using FFPE tissue. PLoS One. 2013;8:e54213. doi: 10.1371/journal.pone.0054213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Mateescu B, Batista L, Cardon M, Gruosso T, de Feraudy Y, Mariani O, Nicolas A, Meyniel JP, Cottu P, Sastre-Garau X, et al. miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response. Nat Med. 2011;17:1627–35. doi: 10.1038/nm.2512. [DOI] [PubMed] [Google Scholar]
  • 50.Dimri GP. What has senescence got to do with cancer? Cancer Cell. 2005;7:505–12. doi: 10.1016/j.ccr.2005.05.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Magenta A, Cencioni C, Fasanaro P, Zaccagnini G, Greco S, Sarra-Ferraris G, Antonini A, Martelli F, Capogrossi MC. miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition. Cell Death Differ. 2011;18:1628–39. doi: 10.1038/cdd.2011.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Cao Q, Mani RS, Ateeq B, Dhanasekaran SM, Asangani IA, Prensner JR, Kim JH, Brenner JC, Jing X, Cao X, et al. Coordinated regulation of polycomb group complexes through microRNAs in cancer. Cancer Cell. 2011;20:187–99. doi: 10.1016/j.ccr.2011.06.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Ellwanger DC, Büttner FA, Mewes HW, Stümpflen V. The sufficient minimal set of miRNA seed types. Bioinformatics. 2011;27:1346–50. doi: 10.1093/bioinformatics/btr149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Lal A, Navarro F, Maher CA, Maliszewski LE, Yan N, O’Day E, Chowdhury D, Dykxhoorn DM, Tsai P, Hofmann O, et al. miR-24 Inhibits cell proliferation by targeting E2F2, MYC, and other cell-cycle genes via binding to “seedless” 3’UTR microRNA recognition elements. Mol Cell. 2009;35:610–25. doi: 10.1016/j.molcel.2009.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Dimri GP, Itahana K, Acosta M, Campisi J. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14(ARF) tumor suppressor. Mol Cell Biol. 2000;20:273–85. doi: 10.1128/MCB.20.1.273-285.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Sahasrabuddhe AA, Dimri M, Bommi PV, Dimri GP. βTrCP regulates BMI1 protein turnover via ubiquitination and degradation. Cell Cycle. 2011;10:1322–30. doi: 10.4161/cc.10.8.15372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Itahana K, Itahana Y, Dimri GP. Colorimetric detection of senescence-associated β galactosidase. Methods Mol Biol. 2013;965:143–56. doi: 10.1007/978-1-62703-239-1_8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Dimri M, Naramura M, Duan L, Chen J, Ortega-Cava C, Chen G, Goswami R, Fernandes N, Gao Q, Dimri GP, et al. Modeling breast cancer-associated c-Src and EGFR overexpression in human MECs: c-Src and EGFR cooperatively promote aberrant three-dimensional acinar structure and invasive behavior. Cancer Res. 2007;67:4164–72. doi: 10.1158/0008-5472.CAN-06-2580. [DOI] [PubMed] [Google Scholar]
  • 59.Nelson G, von Zglinicki T. Monitoring DNA damage during cell senescence. Methods Mol Biol. 2013;965:197–213. doi: 10.1007/978-1-62703-239-1_13. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional material
cc-12-3537-s01.pdf (724.7KB, pdf)

Articles from Cell Cycle are provided here courtesy of Taylor & Francis

RESOURCES