Supporting people with dementia and theircarers in health and social care Issued: November 2006 NICE clinical guideline 42guidance.nice.org.uk/cg42 NICE has accredited the process used by the Centre for Clinical Practice at NICE to produceguidelines. Accreditation is valid for 5 years from September 2009 and applies to guidelines producedsince April 2007 using the processes described in NICE's 'The guidelines manual' (2007, updated2009). More information on accreditation can be viewed at www.nice.org.uk/accreditation
Possematolab.med.nyu.eduNek4 Regulates Entry into Replicative
Senescence and the Response to DNA
Damage in Human Fibroblasts
Christine L. Nguyen, Richard Possemato, Erica L.
Bauerlein, Anyong Xie, Ralph Scully and William C. Hahn
Mol. Cell. Biol. 2012, 32(19):3963. DOI:
Published Ahead of Print 30 July 2012.
Updated information and services can be found at: These include: This article cites 59 articles, 24 of which can be accessed freeat: Receive: RSS Feeds, eTOCs, free email alerts (when newarticles cite this article), Nek4 Regulates Entry into Replicative Senescence and the Response to
DNA Damage in Human Fibroblasts
Christine L. Nguyen,a,b Richard Possemato,a Erica L. Bauerlein,a Anyong Xie,c Ralph Scully,c and William C. Hahna,b
Department of Medical Oncology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USAa; Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USAb; and Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts, USAc When explanted into culture, normal human cells exhibit a finite number of cell divisions before entering a proliferative arrest termed
replicative senescence. To identify genes essential for entry into replicative senescence, we performed an RNA interference (RNAi)-
based loss-of-function screen and found that suppression of the Never in Mitosis Gene A (NIMA)-related protein kinase gene NEK4
disrupted timely entry into senescence. NEK4 suppression extended the number of population doublings required to reach replicative
senescence in several human fibroblast strains and resulted in decreased transcription of the cyclin-dependent kinase inhibitor p21.
NEK4-suppressed cells displayed impaired cell cycle arrest in response to double-stranded DNA damage, and mass spectrometric anal-
ysis of Nek4 immune complexes identified a complex containing DNA-dependent protein kinase catalytic subunit [DNA-PK(cs)],
Ku70, and Ku80. NEK4 suppression causes defects in the recruitment of DNA-PK(cs) to DNA upon induction of double-stranded DNA
damage, resulting in reduced p53 activation and H2AX phosphorylation. Together, these observations implicate Nek4 as a novel regu-
lator of replicative senescence and the response to double-stranded DNA damage.
When explanted into culture, human cells derived from nor- mortalization Certain cell types that are solely
mal tissues exhibit a finite number of cell divisions dependent upon p53 inactivation to bypass replicative senescence After extended passage in culture, human cells begin to divide are capable of being immortalized by hTERT overexpression, more slowly and eventually enter an irreversible proliferative ar- leading to the hypothesis that telomere erosion triggers replicative rest termed replicative senescence Senescent cells exhibit a senescence enforced by p53 However, complicating this anal- characteristic large, flattened morphology and, although metabol- ysis, some human cell types, such as keratinocytes, also appear to ically active, have permanently exited the cell cycle. Since senes- require inactivation of the RB pathway, together with loss of p53 cence prevents cells from exceeding a defined replicative limit, function and constitutive hTERT expression, to achieve immor- replicative senescence has been implicated in aging, as well as in tality Other types of cellular stress, including accumulated tumor suppression. In support of this hypothesis, several studies DNA and organelle damage, oxidative stress, and the stresses of have identified senescent cells in premalignant lesions cell culture itself, have also been implicated in triggering RB path- and genetic mutations commonly found in human cancers, way-dependent senescence Therefore, although the p53 and such as loss of the TP53 and RB1 tumor suppressors or constitu- RB pathways clearly play significant roles, the molecular events tive expression of telomerase, have also been shown to interfere that ultimately induce and enforce replicative senescence remain with replicative senescence Oncogenic stress, DNA damage, replication fork stalling, acute To identify genes required for entry into replicative senescence, telomere uncapping, and other types of cellular stress can induce we performed a loss-of-function genetic screen using a pooled an acute senescence response with hallmarks similar to those seen version of the RNAi Consortium (TRC) short hairpin RNA in cells that have entered replicative senescence Sev- (shRNA) library and identified the Never in Mitosis Gene A eral lines of evidence implicate a common set of pathways in the (NIMA)-related protein kinase gene NEK4 as a regulator of repli- regulation of acute and replicative senescence, although the extent cative senescence.
of overlapping pathways is not clear. The expression of the simianvirus 40 (SV40) oncoprotein large T antigen (LT) permits human MATERIALS AND METHODS
cells to bypass replicative senescence LT binds and inacti- Cell culture and lentiviral and retroviral constructs. Culture conditions
vates p53 and RB, resulting in a loss of cell cycle checkpoints re- and retroviral or lentiviral infections were as described during the time in quired to enforce the senescent state. However, in some cell types,senescence is dependent solely upon the tumor suppressor p53,and inactivation of p53 alone is sufficient to bypass replicative Received 2 April 2012 Returned for modification 4 May 2012 senescence or to extend the time to senescence Others have Accepted 18 July 2012 identified cell lines in which p53 inactivation results in an exten- Published ahead of print 30 July 2012 sion of the time to replicative senescence, but this second prolif- Address correspondence to William C. Hahn, email@example.com.
erative barrier is now dependent upon the retinoblastoma protein C.L.N. and R.P. contributed equally to this article.
Supplemental material for this article may be found at In addition, overexpression of the telomerase catalytic subunit Copyright 2012, American Society for Microbiology. All Rights Reserved.
human telomerase reverse transcriptase (hTERT) in presenescent human cells leads to telomere length stability and facilitates im- October 2012 Volume 32 Number 19 Molecular and Cellular Biology Nguyen et al.
which the construction of the original TRC shRNA library was being Analysis of telomere length and telomerase activity. Telomere re-
developed shRNAs in the TRC library were created by ligating striction fragment (TRF) Southern blotting was performed as described oligonucleotides constructed on solid surfaces and then sequencing the previously and the mean TRF length was measured as described products. Therefore, the order in which shRNAs were produced did not previously using 0.5% agarose gels. The program Image J was used correspond to particular classes of genes at that time. Human foreskin to quantify the signal intensity of fibroblasts (HFF) were isolated from human neonatal foreskin by dispase- the images shown. Telomerase activity was measured by a telomere repeat mediated separation of the dermis from the epidermis. BJ fibroblasts were amplification protocol as described previously cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) Proliferation, BrdU incorporation, and SA ␤-Gal assays. Prolifera-
(Gibco) containing 20% Medium 199 (Gibco), 15% fetal bovine serum tion assays were performed in triplicate using a Coulter Particle Counter (Sigma), glutamine, and penicillin/streptomycin (Gibco). 293T cells were every 5 to 10 days for rapidly dividing cultures and every 10 to 20 days for cultured in high-glucose DMEM (Gibco) containing 10% fetal bovine presenescent cultures. Population doublings were defined as log (cells serum (Sigma), glutamine, and penicillin/streptomycin (Gibco). The counted/cells plated). P values were determined by Student's paired two- shRNA sequences used in the previously described lentiviral vector sided t test. The doubling time was defined as days/cumulative PD over 13 pLKO.1-puro are listed in Table S2 in the supplemental material. Infec- to 17 days. Bromodeoxyuridine (BrdU) incorporation was measured by tions were performed at the minimal titer required to achieve ⬃100% indirect immunofluorescence of cells that had been cultured with 30 infection efficiency to ensure that simultaneously infected cultures had g/ml BrdU for the indicated amount of time using a fluorescein isothio- undergone the same number of population doublings (PD), unless oth- cyanate (FITC)-conjugated anti-BrdU antibody (BU1/75 [ICR1]; Ab- erwise specified. Validation experiments were performed using indepen- cam). At least 100 cells were counted in each triplicate experiment. Senes- dently derived and propagated BJ fibroblasts expressing the individual cence-associated ␤-galactosidase (SA ␤-Gal) staining was performed as shRNAs as described below. The Nek4 cDNA was cloned from 293T cell described previously Briefly, cells were fixed with 0.2% glutaralde- total cDNA by PCR and ligated into pBabe retroviral vectors engineered to hyde for 5 min at room temperature. After washing with PBS, the cells form Flag fusions, and the constructs were sequence verified.
were stained with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyra- Immunoblotting, immunoprecipitation (IP), and reverse transcrip-
noside) overnight at 37°C. The proportion of blue cells was determined by tion (RT)-PCR. Immunoblotting was performed on whole-cell extracts
counting at least 100 cells from each triplicate well at the indicated time isolated in a buffer containing 1.25% NP-40, 1.25% SDS, 12.5 mM NaPO point or 4 PD after selection.
(pH 7.2), 2 mM EDTA or in a buffer containing 20 mM Tris (pH 7.5), 150 Colony formation assays. Cells were trypsinized and seeded at 500
cells per well of a six-well plate in triplicate. Twenty-four hours after mM NaCl, 0.5% NP-40, and 1 mM EDTA and sonicated before separation seeding, the cells were treated with the indicated doses of drug or gamma by SDS-PAGE and incubation with antibodies against ␤-actin– horserad- irradiation. For drug treatment, cells were treated for 1 h before changing ish peroxidase (HRP) (Santa Cruz), p53 (Ab-6; Calbiochem), H-Ras to fresh medium. After approximately 8 days, the cells were fixed with (F235; Santa Cruz), DNA-dependent protein kinase catalytic subunit methanol and stained with Giemsa stain (Sigma). Colonies containing [DNA-PK(cs)] (Ab-4; NeoMarkers), p21 (C-19; Santa Cruz), SV40 LT more than 50 cells were scored.
(sc-148; Santa Cruz), Ku70 (H-308; Santa Cruz), Ku80 (B-1; Santa Cruz), Cell fractionation, extraction, and flow cytometry. Cell fractionation
hemagglutinin (HA) (clone 12CA5; Roche), Flag (M2; Sigma) or cyclin- was performed as previously described Cells were trypsinized, dependent kinase 2 (CDK2) (D-12; Santa Cruz), ataxia-telangiectasia mu- washed with PBS, and resuspended in buffer A (10 mM HEPES [pH 7.9], tated (ATM) (NB100-104; Novus), pSer1981 ATM (10H11.E12; Rock- 10 mM KCl, 1.5 mM MgCl , 10 mM NaF, 340 mM sucrose, 10% glycerol, land), and pRb (G3-245; BD Biosciences). The Nek4 polyclonal antibody 1 mM dithiothreitol [DTT], 0.1% Triton X-100, and protease inhibitors was generated by immunization of a rabbit with the peptide N-CSEPSLSRQ [Roche]). Following low-speed centrifugation at 1,300 ⫻ g, the superna- RRQKQQEQ-C, corresponding to amino acids 528 to 543 of the Nek4 tant was collected as the cytoplasmic fraction. The pellet was resuspended protein sequence, followed by affinity purification of harvested antiserum in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, and protease inhibitors [Roche]). Following low-speed centrifugation at 1,700 ⫻ g, the Lysates for immunoprecipitation were prepared in a buffer containing supernatant was collected as the soluble nuclear fraction. The pellet was 20 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, and 1 mM EDTA, then resuspended in SDS-PAGE sample loading buffer and sonicated, followed by sonication, and immunoprecipitation was performed using representing the insoluble chromatin fraction.
Flag M2-conjugated agarose beads (Sigma). Immune complexes were For extraction of cells prior to flow cytometric analysis, the cell pellets eluted with the Flag peptide as recommended by the manufacturer were resuspended in 500 l of extraction buffer containing 0.5% Triton (Sigma). For immunoprecipitations of Nek4 complexes, Nek4 (2631C1a; X-100, 0.2 g/ml EDTA, 1% bovine serum albumin (BSA) in PBS. After Santa Cruz) antibody was conjugated to Dynabeads Protein G (Invitro- 10 min on ice, samples were fixed with 3 ml methanol at ⫺20°C. The cells gen). Immunoprecipitates were incubated for ⬎4 hours at 4°C before were stained with antibodies specific for DNA-PK(cs) (18-2; Abcam) or being washed three times with lysis buffer. Immune complexes were ␥H2AX (JBW301; Millipore), followed by a fluorescent secondary anti- eluted with SDS-PAGE sample loading buffer and boiled before separa- body for analysis via flow cytometry. Analysis was performed on a BD tion by SDS-PAGE.
The following primer pairs were used for quantitative RT-PCR (qRT- Immunofluorescence assay. Immunofluorescence assays were per-
PCR): Nek4 (5=-GGAGCTATGGAGAGGTGACG-3=; 5=-CACAGAAGC formed as previously described Briefly, cells were plated onto cover- CCATGACAATG-3=), p53 (5=-CCGCAGTCAGATCCTAGCG-3=; 5=-AA slips, fixed with either 4% formaldehyde or methanol, permeabilized with 1% Triton X-100, and blocked with 10% serum and 0.1% cold-water fish GGAGGAC-3=; 5=-CAGCCGGCGTTTGGAGTGGTAGAA-3=), p16 (5=- skin gelatin (Sigma) in PBS plus 1% BSA, 0.02% saponin, and 0.05% sodium azide. The primary antibody used in this study was ␥H2AX G-3=), HDM2 (5=-GGATCCTTTGCAAGCGCCAC-3=; 5=-TCAAAGGA (JBW301; Millipore), followed by secondary mouse antibody-Alexa Fluor CAGGGACCTGCG-3=), and GAPDH (glyceraldehyde-3-phosphate 488 (Invitrogen). Nuclei were counterstained with Hoechst 33258 dye.
Comet assay. Comet assays were performed using the CometSlide kit
TTCCCGTTCTCAG-3=). qRT-PCR was performed in triplicate using (Trevigen) according to the manufacturer's protocol. Briefly, cells were SYBR green master mix (Applied Biosystems; ABI 7300). Threshold cycle treated with the indicated amount of drug, trypsinized, and resuspended (C ) values were determined using ABI Prism software, which includes in the provided melted agarose mixture. The mixture was pipetted onto automatic background correction and threshold selection.
slides and allowed to harden, after which the slides were submerged in Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR FIG 1 Pooled shRNA screen to identify genes affecting senescence. (A) Flow chart of the experimental design used in the study. BJ cells were infected with
individual pools of shRNAs and passaged when nearly confluent until control cultures had entered replicative senescence. The graph shows the cumulative
population doublings of the pools during the course of the experiment. DNA was isolated from these six pools at the final time point, and shRNAs were amplified
by PCR, cloned, and sequenced to determine the identities of individual shRNAs in each pool. gDNA, genomic DNA. (B) Suppression of CHEK2 expression. One
control shRNA (shGFP no. 3) and four shRNAs targeting CHEK2 (sh1 to sh4) were introduced into BJ cells at PD 45, and knockdown was measured by qRT-PCR.
The error bars represent standard deviations (SD) from triplicate experiments. (C) Proliferation of cells upon suppression of CHEK2. Cells expressing two
shRNAs that suppress CHEK2 (red data points, corresponding to the red bars in panel B) were grown in serial passages, together with cells expressing an shRNA
targeting p53 (green data points) and two control cell lines expressing shGFP (blue data points). On the indicated days, the number of PD achieved by each culture
lysis solution and then run in a standard electrophoresis chamber, incu- lentiviral vector, we propagated each of the pools for several weeks bated with SYBR green, and visualized via epifluorescence. The percent- to identify cells that had bypassed replicative senescence. Most of age of DNA in the tail was analyzed with Comet Assay IV software (Per- these pools displayed significant proliferation past that of control cells, suggesting that each shRNA pool harbored shRNAs that gavethe desired phenotype After this period of extended proliferation, DNA was isolated from the pooled cultures, and the Loss-of-function screen to identify senescence regulators. To
shRNAs present within the extended-proliferation populations identify genes that regulate the entry of human cells into replica- were identified by PCR followed by Sanger sequencing. We iden- tive senescence, we performed a pooled-format loss-of-functionscreen in late-passage BJ human foreskin fibroblasts us- tified 17 different genes by this method, 10 of which were identi- ing a portion of the TRC shRNA library composed of 5 pools fied multiple times In addition, we included two genes containing a total of 13,799 shRNA constructs targeting 2,939 identified only once for further evaluation based upon prior re- genes (see Table S1 in the supplemental material). We selected BJ ports implicating these genes as tumor suppressors (FBXW7 and fibroblasts for this screen, since prior work had shown that sup- pression of p53 extends the life span of these cells and that consti- For these genes, we obtained 3 to 6 additional shRNAs from tutive expression of hTERT alone suffices to immortalize the cells the TRC collection and introduced the shRNAs individually into Infection of 1 ⫻ 105 cells was performed at a multiplicity independently propagated presenescent BJ cells to determine of infection of 0.5 to ensure that the majority of the cells in the whether the shRNAs suppressed their target genes, as well as culture were infected with a single lentivirus-delivered shRNA.
whether they permitted BJ cells to bypass senescence. Although After selection with puromycin to purify cells that integrated a the shRNAs identified in the screen suppressed the expression of October 2012 Volume 32 Number 19 Nguyen et al.
TABLE 1 Identities of cloned shRNAs
precursors, as determined by quantitative RT-PCR (data notshown).
No. of shRNAsb Long-term culture of BJ cells in which NEK4 expression is sup- pressed (BJ shN4) revealed an extension of the time to replicative Zinc finger protein 193 senescence of 10 to 20 PD compared to BJ shGFP cells EH domain binding protein 1 NIMA-related kinase 4 The upper limit of this range attained by BJ shN4 no. 2 cells is Cyclic AMP (cAMP)-dependent protein similar to the extension of proliferative capacity seen upon expres- kinase catalytic subunit sion of SV40 LT, although we note that BJ shN4 no. 2 cells entered Checkpoint kinase 2 senescence at PD 90 while cells expressing LT entered crisis at PD WD repeat domain 79 90 and data not shown). These observations demonstrate Ras family member that suppression of NEK4 extended the proliferative limit of BJ Transcription factor 20 fibroblasts, similar to that induced by elimination of known rep- Runt-related transcription factor 3 licative-senescence checkpoints. To confirm that NEK4 plays a Cholinergic receptor, muscarinic 3 similar role in other human fibroblast cell lines, we introduced the Cell division cycle 2 NEK4-specific or control GFP-specific shRNAs into indepen- Casein kinase 1 epsilon Multiple C2 domains, transmembrane 1 dently isolated HFF, as well as IMR90 and WI-38 fibroblasts, and found that NEK4 expression was suppressed and that Fbox and WD40 domain protein 7 suppression of NEK4 resulted in an extension of the time to senes- Kruppel-like factor 11 cence by 5 to 17 PD Thus, suppression of NEK4 delays Ras family member the initiation or enforcement of replicative senescence in human a Boldface, eliminated from follow-up due to low frequency of identification or previous experience with the genes or shRNAs.
Effects of NEK4 suppression on telomeres and telomerase.
b The DNA obtained from pooled cultures was amplified by PCR and sequenced in Because primary BJ fibroblasts and HFF can be immortalized by bulk. For three of the five experimental pools (italics), a single shRNA was identified by constitutive expression of hTERT, we examined whether NEK4- this bulk-sequencing method. Amplified shRNAs from these pools were also cloned and sequenced, and the number of shRNAs identified for each gene is reported.
suppressed cells display an extended life span due to changes intelomeres or telomerase. To determine if alterations in NEK4 ex-pression affected telomere length or telomerase activity, we ma-nipulated NEK4 expression in BJ and HT-1080 cells, which show their target genes for 7 of these 10 genes (see Fig. S1A in the sup- intermediate telomere length and telomerase activity and there- plemental material), for 2 genes, we reproducibly found that in- fore can be monitored for alterations in telomere length in the troduction of multiple shRNAs led to an extension of the time to setting of constitutive expression of telomerase either by senescence and and introducing shRNAs targeting GFP or NEK4 (shGFP no. 3, shN4 The two genes identified by this approach were the checkpoint no. 1, or shN4 no. 2) or by introducing an expression vector to control kinase gene, CHEK2, and the NIMA-related kinase gene, overexpress NEK4, and confirmed the effects of these manipula- NEK4. Suppression of CHEK2 expression extended the time to tions on Nek4 protein levels As indicated by a telomere senescence by 8 to 10 PD, depending on the suppression efficiency repeat amplification protocol (TRAP) assay, suppression or over- of the shRNA used and Since prior work had impli- expression of NEK4 failed to induce telomerase activity in BJ fi- cated CHEK2 as a gene that regulates entry into senescence broblasts and data not shown) and failed to alter telome- this observation validated our approach. Of note, two shRNAs rase activity in the HT-1080 cells targeting CHEK2 potently suppressed Chek2 expression (97 to Because the TRAP assay is not quantitative, subtle changes in 99%) and also markedly inhibited cell proliferation so that a pro- telomerase activity or telomerase activity-independent telomere liferation curve could not be generated, suggesting that CHEK2 is length effects may not be detectable by the assay. Therefore, to an essential gene in this cell type and determine if alteration of NEK4 expression could affect telomere Suppression of NEK4 expression extends the time to senes-
length in long-term culture, we collected DNA from these BJ and cence. In addition to CHEK2, we identified several shRNAs spe-
HT-1080 cells after extended passage in culture and subjected the cific for NEK4, also known as STK2 Nek4 is a serine/threo- DNA to Southern blotting using telomere restriction fragments nine kinase related to the Aspergillus G2/M cell cycle regulator (TRF). Manipulating NEK4 expression in BJ or HT-1080 cells NIMA. Although more extensive studies have been performed on failed to induce a measurable change in TRF length over 29 to 30 other Nek family kinases, the biological functions of Nek4 are PD after stable expression of the indicated shRNAs (initiated at relatively uncharacterized (reviewed in reference To validate approximately PD 20) and These observations suggest the extension of time to senescence phenotype observed when we that Nek4 does not directly affect telomerase activity or telomere suppressed NEK4 expression, we generated cell lines that stably expressed either of two distinct shRNAs targeting NEK4 (BJ shN4 Suppression of NEK4 leads to an increased proliferation rate
no. 1 and shN4 no. 2) or control shRNAs targeting the green and reduced transcription of p21. Although there was no detect-
fluorescent protein gene (GFP) (BJ shGFP no. 1 and shGFP no. 2) able affect on telomere length or telomerase activity upon sup- in BJ cells at PD 50. Expression of the shRNAs targeting NEK4 pression of NEK4, we observed that cells expressing shRNAs spe- suppressed Nek4 protein expression, as determined by immuno- cific for NEK4 displayed an increased proliferation rate. At late blotting with an anti-human C-terminal Nek4 rabbit polyclonal passage (PD 55 to 60), BJ shN4 cells proliferated at nearly twice the antibody This antibody recognizes two predicted splice rate of BJ shGFP cells (P ⬍ 0.003) To gain insight into variants of Nek4 in the same proportions as their mRNA the increased proliferation rate of BJ shN4 cells, we examined the Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR FIG 2 Suppression of Nek4 increases the time to replicative senescence in human foreskin fibroblasts. (A) Suppression of NEK4 in BJ, IMR-90, and WI-38 cells
and HFF. Two control shRNAs (shGFP no. 1 and no. 2) and two shRNAs targeting NEK4 (shN4 no. 1 and no. 2) were introduced into BJ, IMR-90, or WI-38 cells
or HFF at 10 to 15 PD preceding the expected onset of replicative senescence, and Nek4 suppression was measured by immunoblotting. Actin is shown as a
loading control. Immunoblots to confirm SV40 LT expression in IMR-90 and WI-38 cells are also included. Lanes X, untransfected cells. (B) Proliferation of
NEK4-suppressed fibroblasts. The number of PD achieved in BJ, IMR-90, or WI-38 fibroblasts or HFF was measured at the time points indicated and reported
as the average number in triplicate experiments of cumulative PD for each line. *, P ⬍ 0.05; **, P ⬍ 0.005; ***, P ⬍ 0.0005 compared to control shGFP cells.
steady-state protein levels of various cell cycle regulators. The lev- levels Suppression of NEK4 by shN4 no. 1 resulted in a els of p53, cyclin E, cyclin D1, CDK2, and p27KIP1 (p27) appear decrease in p21 mRNA levels smaller than that observed upon similar in BJ shGFP and BJ shN4 cells As determined by expression of shN4 no. 2 (0.81- and 0.27-fold, respectively), but qRT-PCR, the mRNA levels of p16 also appear similar in BJ shGFP we note that the levels of p21 mRNA and protein were still reduced and BJ shN4 cells (data not shown).
compared to those in BJ shGFP cells and To confirm However, we found that the levels of cyclin-dependent kinase the observed decrease in p21 transcription, we utilized a firefly inhibitor 1A, p21CIP1 (referred to below as p21), were reduced in luciferase construct in which luciferase expression is driven by the BJ shN4 cells compared to BJ shGFP cells To determine p21 promoter Similar to what we observed using qRT-PCR, whether the decrease in p21 levels was due to decreased transcrip- transient cotransfection of the luciferase construct, together with tion rather than a posttranscriptional alteration, we performed shN4 no. 1 and no. 2, led to decreased expression of luciferase quantitative RT-PCR and found that BJ shN4 cells exhibit de- (0.58- and 0.38-fold, respectively) compared to cotransfection creased transcription of p21, as evidenced by reduced p21 mRNA with shGFP no. 1 (P ⬍ 0.4) left), indicating that suppres- October 2012 Volume 32 Number 19 Nguyen et al.
FIG 3 Effects of NEK4 suppression on telomerase and the telomere. (A) Nek4 overexpression and suppression in HT-1080 cells. HT-1080 cells were infected with
either a control vector, a Nek4 overexpression construct, a control shRNA (shGFP no. 3), or each of two NEK4-specific shRNAs (shN4 no. 1 and shN4 no. 2), as
indicated. Nek4 levels were measured by immunoblotting. ␤-Actin is shown as a loading control. (B) Telomerase activity in the presence of Nek4 overexpression
or suppression. The telomere repeat amplification protocol was utilized to measure telomerase activity from 2 g of lysates from HT-1080 cells, shown in panel
A, or BJ cells, as indicated. An internal control (IC) for PCR inhibition is shown (bottom), as is a negative control for heat-inactivated lysate (Boil). (C) Telomere
length Southern blot of HT-1080 cells. HT-1080 cells, shown in panel A, were cultured for 5, 14, or 29 PD postselection. DNAs collected from these cultures were
subjected to TRF Southern blotting, and the intensity of hybridization is shown. Size markers in kb are shown on the left. (D) Telomere length Southern blot of
BJ cells. BJ cells expressing control or NEK4-specific shRNAs were cultured for 10, 20, or 30 PD postselection, and the DNA collected was subjected to TRF
Southern blotting as in panel C.
sion of NEK4 decreases transcription from the p21 promoter.
that the BJ shN4 cells undergo NEK4, however, remains Overexpression of NEK4 did not alter luciferase expression, while suppressed in late-passage and senescing BJ shN4 cells overexpression of p53 led to dramatic upregulation of luciferase Thus, our observations indicate that the extended life span of BJ expression, as expected (6.09-fold compared to the control vec- shN4 cells is due to decreased transcription of p21.
tor) right). However, we note that NEK4-suppressed Cell cycle arrest, but not acute senescence, is altered by sup-
cells are not refractory to p53 expression, since overexpression of pression of NEK4. Because we observed decreased levels of p21 in
a Flag epitope-tagged p53 in BJ shGFP no. 1, BJ shN4 no. 1, or BJ BJ shN4 cells, we were interested in determining whether acute shN4 no. 2 cells resulted in comparable levels of cell cycle senescence and/or stimulus-induced cell cycle arrest are globally arrest, as assessed from decreased BrdU incorporation defective in these cells. An acute senescence response has been These observations indicate that the p21 promoter is still respon- demonstrated upon oncogene activation, such as the expression of sive to p53 in these cells and suggest that Nek4 functions upstream an oncogenic allele of H-RAS (H-RasV12). To investigate if sup- of p53-mediated p21 transcription.
pression of NEK4 renders cells resistant to the acute oncogene- Consistent with p21 being a primary mediator of p53-depen- induced senescence response, we expressed H-RasV12 in BJ dent cell cycle arrest, loss of p21 has previously been linked to an shGFP or shN4 cells Expression of H-RasV12 in BJ extension of the cell life span We characterized BJ shN4 shGFP cells induced senescence, as determined from a dramatic cells during the extended life span period by assessing the status of decrease in BrdU incorporation, and suppression of NEK4 failed previously described markers of senescence in BJ shGFP and BJ to alter the percentage of BrdU-incorporating cells in response to shN4 cells at a time when the control cells (BJ shGFP) had begun H-RasV12 expression Thus, suppression of NEK4 is not to enter senescence and the BJ shN4 cells continued to proliferate sufficient for cells to escape from acute senescence triggered by (PD 64). Similar to what has been described for late-passage expression of H-RasV12.
p21⫺/⫺ human diploid fibroblasts (HDFs) we found that We also determined whether reversible cell cycle arrest, such as senescent BJ shGFP and proliferating BJ shN4 cells exhibited sim- that induced by DNA damage, was altered in these cells. Colony ilar levels of SA ␤-Gal activity Furthermore, we observed formation assays were performed on BJ shGFP and BJ shN4 cells that the levels of p21 gradually increase over progressive popula- that were treated with vehicle control or increasing concentrations tion doublings, coincident with the eventual proliferative arrest of the DNA-damaging agents etoposide and mitomycin C Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR FIG 5 Response of NEK4-suppressed cells to p53 overexpression. (A) Over-
expression of p53 in BJ cells. BJ shGFP no. 1 or BJ shN4 no. 1 and no. 2 cells
were infected with a control vector (⫺) or Flag-tagged p53 (⫹). The cell lineswere assessed for NEK4 suppression and p53 overexpression by immunoblot-ting. Actin is shown as a loading control. (B) BrdU incorporation assays toassess cell cycle arrest. BJ cells, characterized in panel A, were incubated withBrdU for 18 h postselection to assess the percentage of cells undergoing pro-liferation. At least 100 cells were scored for each triplicate experiment, and theerror bars represent SD.
with MMC middle), suggesting that NEK4 suppressioninterferes with the arrest induced by double-stranded DNA dam-age. To explore this finding further, we performed colony forma-tion assays using other double-stranded DNA-damaging drugs,bleomycin and neocarzinostatin, and observed similar decreasesin sensitivity (see Fig. S2 in the supplemental material). Moreover,following treatment with increasing doses of gamma irradiation,BJ shN4 no. 2 cells again displayed decreased sensitivity comparedto BJ shGFP no. 1 cells (P ⬍ 0.015); BJ shN4 no. 1 cells displayedsensitivity intermediate between those of BJ shGFP no. 1 and BJ FIG 4 Suppression of NEK4 alters the proliferation rate and p21 expression.
shN4 no. 2 cells, consistent with the intermediate phenotypes ob- (A) Effect of NEK4 suppression on doubling time. Proliferation was measured served throughout other assays bottom).
in BJ cells over 15 days and used to extrapolate the doubling time. The error To confirm that the observed changes in sensitivity were due to bars represent SD from triplicate experiments. *, P ⬍ 0.008 compared toshGFP no. 1 control. (B) Immunoblotting of cell cycle regulators in control BJ impaired cell cycle arrest, we performed short-term BrdU incor- shGFP no. 1 and no. 2 or BJ shN4 no. 1 and shN4 no. 2 cells. Actin is shown as poration assays. Specifically, BJ shGFP and BJ shN4 cells were a loading control. (C) qRT-PCR analysis of NEK4, p53, and p21 mRNA levels treated with vehicle control and 5 M etoposide or 0.05 g/ml in BJ shN4 no. 1 and shN4 no. 2 cells compared to control BJ shGFP no. 1 cells.
MMC in the presence of BrdU for 20 h. In parallel, BJ shGFP and *, P ⬍ 0.0005 compared to shGFP no. 1 control. (D) Luciferase assay in tran- BJ shN4 cells were treated with 1 Gy of irradiation and cultured siently transfected 293T cells to assess transcription off the p21 promoter in thepresence of control shGFP no. 1 or shN4 no. 1 and 2 (left) and in the presence with BrdU for 20 h. As expected, the proliferation of BJ shGFP no.
of empty vector, ectopic Nek4, or ectopic p53 (right). *, P ⬍ 0.04, and **, P ⬍ 1 cells was arrested or decreased upon treatment with all of the 0.0006 compared to shGFP no. 1 control (left); *, P ⬍ 0.0006 compared to DNA-damaging agents top). However, even in the con- empty-vector control (right).
tinuous presence of etoposide, approximately 40 to 50% of BJshN4 cells continued to proliferate (P ⬍ 0.03 compared to BJshGFP no. 1), further suggesting that NEK4-suppressed cells do (MMC). Etoposide inhibits DNA topoisomerase II, leading pri- not arrest as expected in response to double-stranded DNA dam- marily to double-stranded DNA breaks, while MMC is a DNA age. Similar results were observed in IMR-90 cells expressing cross-linker. BJ shN4 cells displayed decreased sensitivity to eto- shGFP or shN4 and treated with etoposide, showing that the phe- poside treatment top) (P ⬍ 0.015), but not to treatment notype is not specific to BJ fibroblasts (see Fig. S3 in the supple- October 2012 Volume 32 Number 19 Nguyen et al.
FIG 6 Characterization of extended-life-span NEK4-suppressed cells. (A) (Left) SA ␤-Gal staining. BJ shGFP no. 1 and no. 2 and BJ shN4 no. 1 and no.
2 cells were fixed and stained with X-Gal to identify the percentages of cells with SA ␤-Gal activity at the indicated population doublings. At least 100 cells
were scored for each time point. (Right) Parallel BrdU incorporation assays. Cells were also incubated with BrdU for 48 h to assess the percentages of
proliferating cells at the indicated population doublings. At least 100 cells were scored for each time point. (B) Immunoblotting (IB) of markers of
senescence. Steady-state levels of p53, p21, Nek4, and actin were assessed via immunoblotting in BJ shGFP no. 1 or BJ shN4 no. 1 and no. 2 cells at the
indicated PD. Control cells senesced prior to PD 88, as can be seen in panel A. The last time points for shN4 no. 1 and no. 2, PD 92 and 101, respectively,
were assessed together in the bottom row.
mental material). No appreciable difference was seen in BrdU in- metric analysis. We expressed the full-length NEK4 cDNA as a corporation rates upon treatment of the cells with MMC, in Flag epitope-tagged protein (F-Nek4) in 293T cells, isolated im- agreement with the colony formation assays middle). In mune complexes with Flag M2-conjugated agarose beads, and response to gamma irradiation, BJ shN4 cells exhibited a higher eluted bound proteins with excess Flag peptide. Upon SDS-PAGE, BrdU incorporation rate than control BJ shGFP cells (approxi- we readily detected a band corresponding to the expected size of mately 10 to 30% higher; P ⬍ 0.016), again in accordance with a Nek4 by silver staining in lysates derived from 293T cells express- defective cell cycle arrest in response to double-stranded DNA ing F-Nek4, but not in lysates derived from control transfected damage bottom).
293T cells . Furthermore, differentially displayed bands To confirm that the decreased level of Nek4 is specifically re- corresponding to potential interacting proteins were seen in sponsible for the observed impairment in DNA damage-induced the F-Nek4 immune complexes We excised these cell cycle arrest, Nek4 was ectopically expressed in BJ shGFP and bands from a replicate colloidal blue-stained gel and per- BJ shN4 no. 1 cells Upon expression of Nek4 in these formed mass spectrometry analysis. We cross referenced the cells, we found no appreciable change in basal BrdU incorporation list of putative interacting proteins with a list of common con- compared to cells containing a control vector. When treated with taminants of Flag-M2 IPs from 293T cells and, based upon etoposide, however, reintroduction of Nek4 into BJ shN4 no. 1 this information, compiled a list of putative Nek4-interacting cells restores the cell cycle arrest induced by DNA damage to levels similar to those observed in empty-vector- or Nek4-overexpress- The putative interactions with DNA-PK(cs), Ku70, and Ku80 ing BJ shGFP cells Together, these observations show were of particular interest to us, since we had found a role for Nek4 that suppression of NEK4 does not interfere with acute senescence in cell cycle arrest in response to DNA damage. Together, these responses but does disrupt double-stranded DNA damage-medi- proteins make up the active DNA-PK kinase, a key mediator of ated cell cycle arrest.
nonhomologous end joining (NHEJ) and, thus, the recognition Identification of Nek4-interacting proteins. To gain insight
and repair of double-stranded DNA breaks (reviewed in reference into Nek4 function, we isolated Nek4 immune complexes and To confirm that these proteins interact with Nek4, we isolated identified putative Nek4-interacting proteins by mass spectro- immune complexes using antibodies specific for either endoge- Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR Normally, DNA-PK(cs) is recruited to sites of DNA damage via a Ku70/Ku80 heterodimer that assembles at the site, and the acti-vated kinase subsequently signals to and recruits downstream fac-tors necessary for DNA repair (reviewed in reference To in-vestigate whether these functions are altered in NEK4-suppressedcells, BJ shGFP and BJ shN4 cells were treated with 50 M etopo-side and fractionated into cytoplasmic, soluble nuclear, and insol- uble nuclear (chromatin) lysates, as previously described The nuclear fractions were separated by SDS-PAGE and immuno-blotted with antibodies specific for DNA-PK(cs), Ku70, and his-tone variant H2A as a loading control. Upon treatment with eto-poside, we failed to identify changes in the proportion of solubleor insoluble chromatin-bound Ku70 in BJ shGFP or BJ shN4 cellsHowever, although the chromatin-bound fraction ofDNA-PK(cs) increased upon etoposide treatment in BJ shGFP cells, as expected, we found that this increase was diminished in BJshN4 cells To assess this in a quantitative manner, weadapted previously described assays in which soluble nuclearDNA-PK(cs) is extracted from cells prior to fixation in order toanalyze the percentage of insoluble DNA-PK(cs) that remainsbound to DNA BJ shGFP and BJ shN4 cells weretreated with vehicle control or 50 M etoposide and fixed eitherprior to or following extraction. These cells were incubated withan antibody specific for DNA-PK(cs) and a fluorescent secondary antibody to analyze the amount of bound, insoluble DNA-PK(cs)using flow cytometry. Using this method, we found that, following FIG 7 Effect of NEK4 suppression on oncogene-induced senescence. (A) Sup-
pression of NEK4 in BJ cells plus H-RasV12 overexpression. BJ cells were
double-stranded DNA damage, the observed percentage of DNA- infected with a control shRNA (GFP no. 1) or each of two shRNAs targeting PK(cs) that is bound to DNA was decreased in BJ shN4 cells com- NEK4 (shN4 no. 1 and shN4 no. 2), followed by infection with a control vector pared to control BJ shGFP cells (61.1% in BJ shN4 no. 1 cells and (⫺) or H-RasV12 (⫹). These cell lines were assessed for NEK4 suppression and 77.8% in BJ shN4 no. 2 cells; P ⬍ 0.002) These obser- H-RasV12 expression, p53 levels, and p21 levels by immunoblotting. ␤-Actin vations are in agreement with our immunoblot analyses and, to- is shown as a loading control. (B) BrdU incorporation as a measure of prolif-eration. Five days postinfection with H-RasV12, the cells shown in panel A gether, indicate that NEK4 suppression interferes with recruit- were incubated with BrdU for 24 h to assess the percentage of proliferating ment of DNA-PK(cs) to damaged DNA.
cells. At least 100 cells were scored for each triplicate experiment, and the error To examine the consequences of impaired recruitment of bars represent SD.
DNA-PK to sites of DNA damage, we examined the activation ofp53 in NEK4-suppressed cells. Following 50 M etoposide treat-ment, phosphorylation of p53 at serine 15 was reduced in BJ shN4 nous Nek4 or Ku80 in BJ fibroblasts Nek4 and Ku80 no. 1 and no. 2 cells compared to BJ shGFP cells In immune complexes isolated from BJ cells and resolved via SDS- addition, we found decreased levels of HDM2 mRNA, a transcrip- PAGE were immunoblotted with antibodies specific for endoge- tional target of p53, in BJ shN4 no. 1 and no. 2 cells compared to BJ nous DNA-PK(cs), Ku70, and Ku80. We detected each of these shGFP cells, following etoposide treatment (P ⬍ 0.045) proteins in complex with Nek4 To eliminate the possi- We also examined another primary target of DNA-PK, the histone bility that the interaction of the Nek4 and Ku proteins was medi- variant H2AX. In untreated asynchronous BJ shGFP or BJ shN4 ated by DNA, we isolated Nek4 immune complexes in the pres- cells, we failed to observe a notable difference in the numbers of ence of ethidium bromide and found that interaction between ␥-H2AX foci (see Fig. S4 in the supplemental material). However, Nek4 and Ku70 was intact Furthermore, we also con- when BJ shGFP and BJ shN4 cells were treated with 50 M etopo- firmed that this interaction remains intact following etoposide side, a decreased level of phosphorylated H2AX (␥-H2AX) was treatment Together, these observations indicate that present in treated BJ shN4 cells compared to control BJ shGFP Nek4 associates with the NHEJ proteins DNA-PK(cs), Ku70, and cells, as evidenced via immunofluorescence To exam- ine this more quantitatively, we analyzed the cells using flow cy- Impairment of DNA-PK upon suppression of NEK4. Since
tometry and found that shN4 no. 1 cells and shN4 no. 2 cells our observations implicated Nek4 in the response to double- contain only 72.8% and 77.7% of the amount of ␥-H2AX ob- stranded DNA damage, we investigated the effects of suppressing served in BJ shGFP cells, respectively (P ⬍ 0.03) More- NEK4 on the DNA-PK complex. We examined the steady-state over, these differences occur despite the fact that the control and levels of DNA-PK(cs), Ku70, and Ku80 in asynchronous BJ shGFP shN4 cells acquire similar levels of DNA damage as assessed via a and BJ shN4 cells but did not observe any notable differences traditional comet assay Additionally, ATM, another Furthermore, the overall nuclear localization of these pro- key regulator of the DNA damage response (DDR), is similarly teins was not altered upon suppression of NEK4 (data not shown), activated in BJ shGFP and BJ shN4 cells upon treatment with and the interaction between Ku70 and Ku80 is not impaired in BJ etoposide, as measured by autophosphorylation at serine 1981 Together, these data support the notion that recogni- October 2012 Volume 32 Number 19 Nguyen et al.
FIG 8 Effect of NEK4 suppression on DNA damage-mediated cell cycle arrest. (A) Colony formation assays to examine the sensitivity of BJ shGFP no. 1 or BJ
shN4 no. 1 and no. 2 fibroblasts to treatment with etoposide (top), mitomycin C (middle), or gamma irradiation (bottom). Survival is expressed as the percentage
of colonies formed following treatment compared to mock-treated cells. The error bars indicate the standard deviations of triplicate experiments. *, P ⬍ 0.015
compared to shGFP no. 1 control. (B) BrdU incorporation assays to assess cell cycle arrest of cells mentioned in part A in response to treatment with etoposide
(top), mitomycin C (middle), or gamma irradiation (bottom). The bars indicate the percentages of BrdU-positive cells following treatment with the indicated
drug or irradiation, normalized to the percentage of BrdU-positive mock-treated cells. The error bars represent the standard deviations of triplicate experiments.
*, P ⬍ 0.03; **, P ⬍ 0.004; †, P ⬍ 0.016; ‡, P ⬍ 0.0003 compared to the shGFP no. 1 control. (C) Reexpression of Nek4 restores etoposide-induced cell cycle arrest.
A BrdU incorporation assay was performed as shown in panel B using BJ shGFP no. 1 or BJ shN4 no. 1 cells stably transfected with empty vector or Flag-tagged
Nek4. Below the bar graph is an immunoblot confirming overexpression of Flag-tagged Nek4 in the appropriate cell lines; actin is shown as a loading control. *,
P ⬍ 0.024 compared to shN4 no. 1 cells expressing the empty vector.
tion of double-stranded DNA damage by DNA-PK and the con- stood. Here, we used a loss-of-function approach to identify genes sequent downstream signaling are impaired in NEK4-suppressed involved in regulating entry into replicative senescence and iden- tified the NIMA-related kinase gene, NEK4, as a gene whose ex-pression is required for timely entry into replicative senescence.
Further characterization of NEK4-suppressed cells revealed a re- NEK4 and replicative senescence. Entry into replicative senes-
duction in the levels of p21, a protein well described as an impor- cence occurs with extended passage of explanted human cells de- tant mediator of cell cycle arrest and replicative senescence (re- rived from normal tissues While the p53 and pRB path- viewed in reference The extension of replicative capacity ways play key roles in enforcing the senescent state, the beyond the normal replicative barrier has been described for sup- mechanism for triggering replicative senescence is poorly under- pression of the primary p21 trans activator, p53 Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR FIG 9 Identification of Nek4-interacting proteins. (A) IP of Nek4 complexes. Representative silver-stained SDS-PAGE gel containing Flag peptide eluates
of mock (⫺)- or Flag-Nek4 (F-Nek4)-transfected 293T cell lysates immunoprecipitated for the Flag epitope tag. Size markers (in kilodaltons) are
indicated on the left. F-Nek4 is indicated on the right. (B) Validation of Nek4 interactions by endogenous co-IPs. Immune complexes were immuno-
precipitated using antibodies specific for endogenous Nek4 or Ku80 and were subjected to SDS-PAGE, together with whole-cell lysate (WCL). The
antibody used for subsequent immunoblotting is indicated. (C) IP of Nek4 complexes in the presence of 0.1 mg/ml ethidium bromide (EtBr), as indicated.
Immunoblots were performed as described in the legend to panel B. (D) IP of Nek4 complexes in mock- or 50 M etoposide-treated cells, as indicated.
Immunoblots were performed as described in the legend to panel B.
and for direct suppression of p21 itself Notably, extended- are fundamentally different than acute senescence, in a way that life-span BJ shN4 cells phenocopy late-passage p21⫺/⫺ and uniquely requires the presence of Nek4. Indeed our results suggest p21⫹/⫺ cells strongly supporting the idea that reduced that Nek4 may play a role specifically in the double-stranded DNA p21 levels are responsible for the extended life span of BJ shN4 damage response, which is likely not primarily responsible for oncogenic Ras-induced senescence.
Suppression of NEK4 expression did not affect the acute senes- The role of Nek4 in the response to DNA damage. Mass spec-
cence response induced by oncogenic H-RasV12 expression, al- trometry analysis of Nek4-interacting proteins identified the mu- though we observed an effect on cell cycle arrest induced by vari- tually interacting proteins DNA-PK(cs), Ku70, and Ku80. The ous DNA-damaging drugs. We believe this discrepancy may be Ku70/Ku80 heterodimer localizes to sites of double-stranded due to the possibility that senescence-inducing phenotypes, such DNA breaks and recruits DNA-PK(cs), becoming the active as oncogenic Ras expression, might be more potent and therefore DNA-PK kinase complex that is responsible for the cascade of more difficult to mitigate than temporary cell cycle arrest or even events necessary to repair the breaks (reviewed in reference replicative senescence, in which reduction of the proliferative rate The observed association between Nek4 and all components of the is more gradual. In other words, NEK4-suppressed cells are not DNA-PK complex suggests that this interaction may occur at sites refractory to p53- or p21-mediated proliferative arrest, but in of DNA damage; however, it also remains possible that Nek4 may cases where the induction of p21 is low level or gradual, the de- interact with each protein individually. Nevertheless, we note that creased basal p21 levels observed in NEK4-suppressed cells cause in NEK4-suppressed cells, we observed a defect in the recruitment the cell cycle arrest to be delayed or dampened, requiring higher of DNA-PK(cs) to chromatin upon the induction of double- activation of p21 than usual. Another explanation for this obser- stranded DNA damage. We thus hypothesize that Nek4 plays a vation is that temporary cell cycle arrest and replicative senescence role in this recruitment or in the stability of the interaction be-tween DNA-PK(cs) and Ku70/Ku80. Although we observed only apartial disruption in DNA-PK(cs) recruitment, this may be due to TABLE 2 Mass spectrometry resultsa
incomplete suppression of NEK4 in these fibroblasts. Therefore, Interacting protein Peptide coverage (%) the remaining amounts of Nek4 may be able to mediate DNA-PKcomplex formation, albeit at reduced efficiency. Alternatively, it is possible that Nek4 regulates a subpopulation of DNA-PK or plays a role in complex formation in specific contexts.
In line with the decreased recruitment of DNA-PK(cs) to DNA, fibroblasts containing shRNAs specific for NEK4 also display decreased activation of p53 and decreased levels of ␥-H2AX upon treatment with etoposide. Although several ki- nases are known to phosphorylate H2AX, it has been reported that DNA-PK is the primary kinase responsible for H2AX phosphorylation in the context of double-stranded DNA dam- Bands excised from a replicate of the gel depicted in Fig. 9A were submitted to mass age These results correlate with the observation that NEK4 spectrometry. After comparison with known contaminants, the list of interactingproteins and the peptide coverage of each was compiled.
suppression decreases the sensitivity of fibroblasts to double- October 2012 Volume 32 Number 19 Nguyen et al.
FIG 10 Effect of NEK4 suppression upon DNA-PK activity. (A) Immunoblotting of Nek4, DNA-PK(cs), Ku80, Ku70, and actin (loading control) in BJshGFP
no. 1 or BJshN4 no. 1 and 2 cells. (B) IP of Ku70 complexes in BJ shGFP no. 1, BJ shN4 no. 1, and BJ shN4 no. 2 cells to examine the interaction between Ku70
and Ku80. The antibody used for subsequent immunoblotting is indicated. (C) Immunoblotting of cellular fractionation assays. The soluble nuclear fractions
(10%) and chromatin fractions (10%) of dimethyl sulfoxide (DMSO)- or etoposide-treated BJ shGFP no. 1 and BJ shN4 no. 1 and no. 2 cells were separated by
SDS-PAGE and immunoblotted to examine levels of DNA-PK(cs), Ku70, XRCC4, and H2A. (D) Flow cytometric analysis of chromatin-bound DNA-PK(cs)
levels. The bars indicate the percentages of DNA-PK(cs) bound to chromatin following etoposide treatment normalized to DMSO-treated levels. The error bars
represent SD from triplicate experiments. *, P ⬍ 0.0005, and **, P ⬍ 0.002 compared to shGFP no. 1 control.
stranded DNA-damaging drugs, but not to a DNA cross-linker, in reference Nek1 appears to be activated in response to mitomycin C. We also showed that the decreased sensitivity is a ionizing radiation and plays a role in the consequent cell cycle result of defective cell cycle arrest in NEK4-suppressed cells, arrest Nek10 is a mediator of the G2/M cell cycle arrest likely linked to defective signaling by DNA-PK. We thus believe that occurs in response to UV irradiation Nek11 is impli- that Nek4 is a novel mediator of the response to double- cated in the infrared (IR)-induced degradation of CDC25A and stranded DNA damage.
Several lines of evidence implicate DNA damage as a significant Relevance of Nek4 to cancer. Both replicative and oncogene-
contributor to the induction of replicative senescence. In agree- induced senescence have been implicated as tumor-suppres- ment with this, we identified Chek2, a key substrate of the DNA sive mechanisms (reviewed in reference and bypass of se- damage kinase ataxia-telangiectasia mutated (ATM), as a regula- nescence may be an important step in tumorigenesis.
tor of replicative senescence, as has been previously described Furthermore, it is clear that defective DNA damage repair fa- Moreover, it was shown that inactivation of Chek2 also re- cilitates the accumulation of tumor-promoting mutations.
sulted in decreased p21 expression in human fibroblasts due to Therefore, one would expect that a gene required for timely defective p53 activation It was previously shown that DNA- entry into replicative senescence and for the proper recogni- PK-deficient scid mouse embryonic fibroblasts display delayed tion and repair of double-stranded DNA breaks might be sup- and attenuated induction of p21 expression in response to ioniz-ing radiation Thus, our findings that Nek4 associates with pressed or lost in human cancers. Indeed, a survey of lung DNA-PK and is involved in the response to double-stranded DNA cancer samples showed that the genomic region containing damage is consistent with the extension of life span acquired upon NEK4, chromosome 3p, is frequently deleted Although NEK4 suppression. We believe that inefficient DNA-PK signaling, clearly recurrently deleted in many cancers, a key tumor sup- which may otherwise be occurring sporadically or at low levels in pressor gene located in this region has not yet been identified.
normal cells, is likely responsible for the observed decrease in p21 Based on the data that we provide here, NEK4 may be one target levels of NEK4-suppressed cells, leading to delayed replicative se- of this region of recurrent deletion.
Since NEK4-suppressed cells eventually enter replicative senes- Interestingly, other NIMA-related kinases have been impli- cence and do not proliferate indefinitely, it is likely that loss of NEK4 cated in cell cycle arrest in response to DNA damage (reviewed requires the concomitant alteration of other oncogenes and tumor Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR FIG 11 Effect of NEK4 suppression on events downstream of DNA-PK. (A) Immunoblotting of phosphoserine 15 (pSer15) p53, total p53, and actin
(loading control) in BJ shGFP no. 1, BJ shN4 no. 1, and BJ shN4 no. 2 cells treated with vehicle control or 50 M etoposide. (B) qRT-PCR analysis of
HDM2 mRNA levels in BJ shGFP no. 1 and BJ shN4 no. 1 and no. 2 cells treated with 50 M etoposide. *, P ⬍ 0.045 compared to shGFP control. The fold
change is shown. The error bars indicate SD. (C) Immunofluorescence staining of ␥-H2AX foci in BJ shGFP no. 1 or BJ shN4 no. 1 and no. 2 fibroblasts
following 50 M etoposide treatment. The arrows indicate cells that display reduced ␥-H2AX levels compared to control shGFP no. 1 cells. (D) Flow
cytometric analysis of ␥-H2AX levels. The bars indicate the percentages of ␥-H2AX fluorescence levels in etoposide-treated BJ shGFP no. 1 or BJ shN4 no.
1 and no. 2 cells normalized to DMSO-treated cells. *, P ⬍ 0.03, and **, P ⬍ 0.009 compared to shGFP control. (E) Comet assay to assess the amount of
DNA damage acquired. (Left) Fluorescence images of representative control and etoposide-treated cells after electrophoresis. (Right) The bars indicate
the percentages of DNA found in the comet tail of DMSO or etoposide-treated BJ shGFP no. 1 and BJ shN4 no. 1 and no. 2 cells. At least 100 cells were
analyzed per condition for each triplicate experiment. (F) Immunoblot analysis of ATM activation. Whole-cell lysates from DMSO- or etoposide-treated
BJ shGFP no. 1 and BJ shN4 no. 1 and no. 2 cells were separated by SDS-PAGE and immunoblotted to examine levels of phosphoserine 1981 (pSer1981)
ATM, total ATM, and actin as a loading control.
suppressors to initiate transformation. While Nek4 may be involved age occurs through other means, and extension of the time to repli- in the efficient recognition and repair of DNA damage, loss of Nek4 cative senescence might allow the prolonged expansion of premalig- should not necessarily result in a hypermutagenic state. Nevertheless, nant cells, further allowing the possibility of subsequent mutations loss of Nek4 could decrease the efficiency of repair when DNA dam- and selection for abnormal variants.
October 2012 Volume 32 Number 19 Nguyen et al.
tein significantly extends the life span of normal mammalian cells in cul-
ture. J. Virol. 71:6629 – 6640.
We thank the members of the Hahn and Cichowski laboratories for assis- 24. Gire V, Roux P, Wynford-Thomas D, Brondello JM, Dulic V. 2004.
tance and helpful comments and Matthew Meyerson, Kumiko Tanaka, DNA damage checkpoint kinase Chk2 triggers replicative senescence.
Jordi Barretina, Heidi Greulich, Hongbin Ji, and Kwok-Kin Wong for EMBO J. 23:2554 –2563.
assistance with lung cancer cell line data and reagents. We thank Daosong 25. Gire V, Wynford-Thomas D. 1998. Reinitiation of DNA synthesis and
Xu and Martin Hemler for the HT-1080 cell line.
cell division in senescent human fibroblasts by microinjection of anti-p53 C.L.N. was supported by an American Cancer Society postdoctoral antibodies. Mol. Cell. Biol. 18:1611–1621.
fellowship (PF-09-117-01-CCG), and R.P was supported by a Howard 26. Hahn WC. 2002. Immortalization and transformation of human cells.
Hughes predoctoral fellowship. This work was supported in part by NIH/ Mol. Cells 13:351–361.
27. Hahn WC, et al. 1999. Creation of human tumour cells with defined
NIA grant R01 AG023145 and by the H. L. Snyder Medical Foundation.
genetic elements. Nature 400:464 – 468.
28. Harley VR, et al. 1990. Vaccinia virus expression and sequence of an avian
influenza nucleoprotein gene: potential use in diagnosis. Arch. Virol. 113:
1. An J, et al. 2010. DNA-PKcs plays a dominant role in the regulation of
29. Hayflick L, Moorhead PS. 1961. The serial cultivation of human diploid
H2AX phosphorylation in response to DNA damage and cell cycle pro- cell strains. Exp. Cell Res. 25:585– 621.
gression. BMC Mol. Biol. 11:18.
30. Herbig U, Sedivy JM. 2006. Regulation of growth arrest in senescence:
2. Balajee AS, Geard CR. 2001. Chromatin-bound PCNA complex forma-
telomere damage is not the end of the story. Mech. Ageing Dev. 127:
tion triggered by DNA damage occurs independent of the ATM gene product in human cells. Nucleic Acids Res. 29:1341–1351.
31. Kachnic LA, et al. 1999. The ability of p53 to activate downstream genes
3. Beausejour CM, et al. 2003. Reversal of human cellular senescence: roles
p21(WAF1/cip1) and HDM2, and cell cycle arrest following DNA damage of the p53 and p16 pathways. EMBO J. 22:4212– 4222.
is delayed and attenuated in scid cells deficient in the DNA-dependent 4. Bodnar AG, et al. 1998. Extension of life-span by introduction of telo-
protein kinase. J. Biol. Chem. 274:13111–13117.
merase into normal human cells. Science 279:349 –352.
32. Kim NW, et al. 1994. Specific association of human telomerase activity
5. Bond JA, et al. 1995. Mutant p53 rescues human diploid cells from
with immortal cells and cancer. Science 266:2011–2015.
senescence without inhibiting the induction of SDI1/WAF1. Cancer Res.
33. Kiyono T, et al. 1998. Both Rb/p16INK4a inactivation and telomerase
activity are required to immortalize human epithelial cells. Nature 396:
6. Bond JA, et al. 1999. Control of replicative life span in human cells:
barriers to clonal expansion intermediate between M1 senescence and M2 34. Levedakou EN, et al. 1994. Two novel human serine/threonine kinases
crisis. Mol. Cell. Biol. 19:3103–3114.
with homologies to the cell cycle regulating Xenopus MO15, and NIMA 7. Bond JA, Wyllie FS, Wynford-Thomas D. 1994. Escape from senescence
kinases: cloning and characterization of their expression pattern. Onco- in human diploid fibroblasts induced directly by mutant p53. Oncogene 35. Li J, Stern DF. 2005. DNA damage regulates Chk2 association with chro-
8. Braig M, et al. 2005. Oncogene-induced senescence as an initial barrier in
matin. J. Biol. Chem. 280:37948 –37956.
lymphoma development. Nature 436:660 – 665.
36. Li QL, et al. 2002. Causal relationship between the loss of RUNX3 expres-
9. Brown JP, Wei W, Sedivy JM. 1997. Bypass of senescence after disruption
sion and gastric cancer. Cell 109:113–124.
of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 37. Lundberg AS, Hahn WC, Gupta P, Weinberg RA. 2000. Genes involved
in senescence and immortalization. Curr. Opin. Cell Biol. 12:705–709.
10. Campisi J, d'Adda di Fagagna F. 2007. Cellular senescence: when bad
38. Mao JH, et al. 2004. Fbxw7/Cdc4 is a p53-dependent, haploinsufficient
things happen to good cells. Nat. Rev. Mol. Cell Biol. 8:729 –740.
tumour suppressor gene. Nature 432:775–779.
11. Chen GI, Gingras AC. 2007. Affinity-purification mass spectrometry
39. McConnell BB, Starborg M, Brookes S, Peters G. 1998. Inhibitors of
(AP-MS) of serine/threonine phosphatases. Methods 42:298 –305.
cyclin-dependent kinases induce features of replicative senescence in early 12. Chen Y, Chen CF, Riley DJ, Chen PL. 2011. Nek1 kinase functions in
passage human diploid fibroblasts. Curr. Biol. 8:351–354.
DNA damage response and checkpoint control through a pathway inde- 40. Melixetian M, Klein DK, Sorensen CS, Helin K. 2009. NEK11 regulates
pendent of ATM and ATR. Cell Cycle 10:655– 663.
CDC25A degradation and the IR-induced G2/M checkpoint. Nat. Cell 13. Chen Y, Chen PL, Chen CF, Jiang X, Riley DJ. 2008. Never-in-mitosis
related kinase 1 functions in DNA damage response and checkpoint con- 41. Michaloglou C, et al. 2005. BRAFE600-associated senescence-like cell
trol. Cell Cycle 7:3194 –3201.
cycle arrest of human naevi. Nature 436:720 –724.
14. Chen Z, et al. 2005. Crucial role of p53-dependent cellular senescence in
42. Misteli T, Soutoglou E. 2009. The emerging role of nuclear architecture in
suppression of Pten-deficient tumorigenesis. Nature 436:725–730.
DNA repair and genome maintenance. Nat. Rev. Mol. Cell Biol. 10:243–
15. Collado M, et al. 2005. Tumour biology: senescence in premalignant
tumours. Nature 436:642.
43. Moffat J, et al. 2006. A lentiviral RNAi library for human and mouse genes
16. Courtois-Cox S, et al. 2006. A negative feedback signaling network un-
applied to an arrayed viral high-content screen. Cell 124:1283–1298.
derlies oncogene-induced senescence. Cancer Cell 10:459 – 472.
44. Moniz L, Dutt P, Haider N, Stambolic V. 2011. Nek family of kinases in
17. Dickson MA, et al. 2000. Human keratinocytes that express hTERT and
cell cycle, checkpoint control and cancer. Cell Div. 6:18.
also bypass a p16(INK4a)-enforced mechanism that limits life span be- 45. Moniz LS, Stambolic V. 2011. Nek10 mediates G2/M cell cycle arrest
come immortal yet retain normal growth and differentiation characteris- and MEK autoactivation in response to UV irradiation. Mol. Cell. Biol.
tics. Mol. Cell. Biol. 20:1436 –1447.
31:30 – 42.
18. Dimri GP, et al. 1995. A biomarker that identifies senescent human cells
46. Morgenstern JP, Land H. 1990. Advanced mammalian gene transfer:
in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. U. S. A. 92:9363–
high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:
19. DiRenzo J, et al. 2002. Growth factor requirements and basal phenotype
of an immortalized mammary epithelial cell line. Cancer Res. 62:89 –98.
47. Nguyen CL, Eichwald C, Nibert ML, Munger K. 2007. Human papillo-
20. Drouet J, et al. 2005. DNA-dependent protein kinase and XRCC4-DNA
mavirus type 16 E7 oncoprotein associates with the centrosomal compo- ligase IV mobilization in the cell in response to DNA double strand breaks.
nent gamma-tubulin. J. Virol. 81:13533–13543.
J. Biol. Chem. 280:7060 –7069.
48. Ramirez RD, et al. 2001. Putative telomere-independent mechanisms of
21. Dulic V, Beney GE, Frebourg G, Drullinger LF, Stein GH. 2000. Un-
replicative aging reflect inadequate growth conditions. Genes Dev. 15:
coupling between phenotypic senescence and cell cycle arrest in aging 398 – 403.
p21-deficient fibroblasts. Mol. Cell. Biol. 20:6741– 6754.
49. Rogan EM, et al. 1995. Alterations in p53 and p16INK4 expression and
22. el-Deiry WS, et al. 1993. WAF1, a potential mediator of p53 tumor
telomere length during spontaneous immortalization of Li-Fraumeni syn- suppression. Cell 75:817– 825.
drome fibroblasts. Mol. Cell. Biol. 15:4745– 4753.
23. Gallimore PH, et al. 1997. Adenovirus type 12 early region 1B 54K pro-
50. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. 1997. Onco-
Molecular and Cellular Biology Nek4 Regulates Replicative Senescence and DDR genic ras provokes premature cell senescence associated with accumula- 55. Vaziri H, Benchimol S. 1998. Reconstitution of telomerase activity in
tion of p53 and p16INK4a. Cell 88:593– 602.
normal human cells leads to elongation of telomeres and extended repli- 51. Shay JW, Wright WE, Werbin H. 1991. Defining the molecular
cative life span. Curr. Biol. 8:279 –282.
mechanisms of human cell immortalization. Biochim. Biophys. Acta 56. Voorhoeve PM, Agami R. 2003. The tumor-suppressive functions of the
human INK4A locus. Cancer Cell 4:311–319.
52. Spardy N, et al. 2007. The human papillomavirus type 16 E7 oncoprotein
57. Wei W, Sedivy JM. 1999. Differentiation between senescence (M1) and
activates the Fanconi anemia (FA) pathway and causes accelerated chro- crisis (M2) in human fibroblast cultures. Exp. Cell Res. 253:519 –522.
mosomal instability in FA cells. J. Virol. 81:13265–13270.
58. Wright WE, Shay JW. 1992. The two-stage mechanism controlling cel-
53. Tonon G, et al. 2005. High-resolution genomic profiles of human lung
lular senescence and immortalization. Exp. Gerontol. 27:383–389.
cancer. Proc. Natl. Acad. Sci. U. S. A. 102:9625–9630.
59. Yan Y, Ouellette MM, Shay JW, Wright WE. 1996. Age-dependent
54. van Steensel B, de Lange T. 1997. Control of telomere length by the
alterations of c-fos and growth regulation in human fibroblasts expressing human telomeric protein TRF1. Nature 385:740 –743.
the HPV16 E6 protein. Mol. Biol. Cell 7:975–983.
October 2012 Volume 32 Number 19
Multimodal Systemic and Adam Young, MDAsokumar Buvanendran, MDRush University Medical CenterChicago, Illinois Ambulatory surgery encompasses the majority of surgical proce- dures performed in the United States. The number of proceduresperformed on an ambulatory basis has increased owing to improve-ments in surgical technology, anesthetic techniques, and pharmacol-ogy—specifically analgesic agents. The latter is important as there is anincreasing trend of performing more painful procedures on anoutpatient Inadequate management of pain or side effects frommedications (such as opioids) can lead to decreased patient satisfactionand delayed discharge. Multimodal analgesia captures the effectivenessof individual agents in optimal dosages that maximize efficacy andminimize side effects. This important concept includes the theory thatagents with different mechanisms of may have synergisticeffects in preventing or treating pain. Joshi offered guidelines onconstructing a multimodal analgesia strategy that, in addition to regionalor local anesthesia, included scheduled administration of nonopioidanalgesics [eg, acetaminophen, nonsteroidal anti-inflammatory drugs(NSAIDs), or cyclooxygenase (COX)-2 inhibitors] using oral opioids onlyfor breakthrough pain.Successful regimens for outpatient (andinpatient) procedures have since been proposed that follow theseprinciples.These regimens must be tailored to individual patients,keeping in mind the procedure being performed, side effects ofindividual medications, and patients' preexisting medical conditions.