Suppression of Apoptosis by PIF1 Helicase in...

12
Tumor and Stem Cell Biology Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells Mary E. Gagou, Anil Ganesh, Ruth Thompson, Geraldine Phear, Cyril Sanders, and Mark Meuth Abstract Defining the processes that sustain telomere maintenance is critical to our understanding of cancer and longevity. PIF1 is a nonprocessive 5 0 ->3 0 human DNA helicase that exhibits broad substrate specificity. In vitro studies have implicated PIF1 in maintaining telomeres and processing stalled DNA replication forks, but disruption of the murine Pif1 gene did not yield any apparent phenotype. In this study, we evaluated the function of the PIF1 gene in human cells by using siRNA knockdown strategies to gauge its role in the response to DNA replication stress. We found that PIF1 depletion reduced the survival of both p53-deficient and p53-proficient human tumor cells by triggering apoptosis. In contrast, nonmalignant cells were unaffected by PIF1 depletion. Apoptosis induction in tumor cells was augmented by cotreatment with replication inhibitors (thymidine, hydroxyurea, or gemcitabine). When sensitive PIF1-depleted cells were released from a thymidine-induced S-phase arrest, there remained a subpopulation of cells that failed to enter S-phase. This cell subpopulation displayed an increase in levels of cyclin E and p21, as well as a deficiency in S-phase checkpoint markers that were induced with thymidine in PIF1 expressing cells. Specifically, CHK1 activation was suppressed and we detected no consistent changes in ATM S1981 autophosphorylation, g H2AX induction, or RPA hyperphosphorylation. Death in PIF1-depleted cells was detected in late G 1 /early S-phase and was dependent on caspase-3 activity. Taken together, our findings suggest roles for PIF1 in S-phase entry and progression that are essential to protect human tumor cells from apoptosis. Cancer Res; 71(14); 49985008. 2011 AACR. Introduction DNA helicases are essential for the unwinding of DNA that occurs during replication, repair, and transcription. Human cells encode a number of helicases that reflect this broad range of functions. Several of these, involved in replication or transcription, are essential for the growth and survival of cells, whereas others are required to maintain genetic stability. Inherited mutations of helicases involved in cellular response to replication fork stress predispose to premature aging and early onset of various forms of cancer. Helicases affected in these disorders include the human RecQ-related helicases BLM (mutated in Blooms syndrome; ref. 1), WRN (Werners syndrome; ref. 2), RecQ4 (RothmundThomson syndrome; ref. 3), and the 5 0 ->3 0 helicase FancJ/BACH1 (Fanconi's Anemia; ref. 4). The highly conserved protein Pif1 is another such helicase implicated in the maintenance of genome stability. Sacchar- omyces cerevisiae Pif1 (ScPif1) is a nonprocessive 5 0 ->3 0 heli- case. It was first identified in a screen for genes that affect the frequency of recombination between rþ and r mitochon- drial DNA (5). Extensive work has shown that ScPif1 plays a role in a wide range of DNA transactions including the regulation of telomere length (6), Okazaki fragment matura- tion (7), replication of ribosomal DNA (8), and G-quadruplex DNA resolution (9). S. cerevisiae also encodes a second PIF1- like protein called RRM3. This helicase has a more prominent role at paused replication forks, where it disrupts proteinDNA complexes at specific (RRM3-sensitive) sites that could slow the translocation of the replicative helicase complex (10). Despite this wide range of functions, ScPif1 and RRM3 are not essential genes in S. cerevisiae although cell-cycle progression and growth rate are slowed in double mutants (8). All mammalian cells studied thus far have only a single PIF1-like protein that has nearly equal identity and similarity to ScRRM3 and ScPIF1 (11). Although PIF1 has been found in both nuclei and mitochondria (12), its role in mammalian cells is less certain. Although antibodies to PIF1 immunoprecipi- tate telomerase activity from cultured human cells (11), knockouts of the mouse Pif1 gene do not affect telomere length, chromosome stability, or mitochondrial function in Authors' Affiliation: Institute for Cancer Studies, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M.E. Gagou and A. Ganesh contributed equally this work. Corresponding Author: Mark Meuth, Institute for Cancer Studies, Uni- versity of Sheffield, Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom. Phone: 44-114-271-3288; Fax: 44-114-271-3515; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-10-4404 2011 American Association for Cancer Research. Cancer Research Cancer Res; 71(14) July 15, 2011 4998 on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Transcript of Suppression of Apoptosis by PIF1 Helicase in...

Page 1: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

Tumor and Stem Cell Biology

Suppression of Apoptosis by PIF1 Helicase inHuman Tumor Cells

Mary E. Gagou, Anil Ganesh, Ruth Thompson, Geraldine Phear, Cyril Sanders, and Mark Meuth

AbstractDefining the processes that sustain telomere maintenance is critical to our understanding of cancer and

longevity. PIF1 is a nonprocessive 50->30 human DNA helicase that exhibits broad substrate specificity. In vitrostudies have implicated PIF1 in maintaining telomeres and processing stalled DNA replication forks, butdisruption of the murine Pif1 gene did not yield any apparent phenotype. In this study, we evaluated thefunction of the PIF1 gene in human cells by using siRNA knockdown strategies to gauge its role in theresponse to DNA replication stress. We found that PIF1 depletion reduced the survival of both p53-deficientand p53-proficient human tumor cells by triggering apoptosis. In contrast, nonmalignant cells wereunaffected by PIF1 depletion. Apoptosis induction in tumor cells was augmented by cotreatment withreplication inhibitors (thymidine, hydroxyurea, or gemcitabine). When sensitive PIF1-depleted cells werereleased from a thymidine-induced S-phase arrest, there remained a subpopulation of cells that failed to enterS-phase. This cell subpopulation displayed an increase in levels of cyclin E and p21, as well as a deficiency inS-phase checkpoint markers that were induced with thymidine in PIF1 expressing cells. Specifically, CHK1activation was suppressed and we detected no consistent changes in ATM S1981 autophosphorylation,gH2AX induction, or RPA hyperphosphorylation. Death in PIF1-depleted cells was detected in late G1/earlyS-phase and was dependent on caspase-3 activity. Taken together, our findings suggest roles for PIF1 inS-phase entry and progression that are essential to protect human tumor cells from apoptosis. Cancer Res;71(14); 4998–5008. ’2011 AACR.

Introduction

DNA helicases are essential for the unwinding of DNA thatoccurs during replication, repair, and transcription. Humancells encode a number of helicases that reflect this broadrange of functions. Several of these, involved in replication ortranscription, are essential for the growth and survival of cells,whereas others are required to maintain genetic stability.Inherited mutations of helicases involved in cellular responseto replication fork stress predispose to premature aging andearly onset of various forms of cancer. Helicases affected inthese disorders include the human RecQ-related helicasesBLM (mutated in Blooms syndrome; ref. 1), WRN (Wernerssyndrome; ref. 2), RecQ4 (Rothmund–Thomson syndrome; ref.

3), and the 50->30 helicase FancJ/BACH1 (Fanconi's Anemia;ref. 4).

The highly conserved protein Pif1 is another such helicaseimplicated in the maintenance of genome stability. Sacchar-omyces cerevisiae Pif1 (ScPif1) is a nonprocessive 50->30 heli-case. It was first identified in a screen for genes that affect thefrequency of recombination between rþ and r� mitochon-drial DNA (5). Extensive work has shown that ScPif1 plays arole in a wide range of DNA transactions including theregulation of telomere length (6), Okazaki fragment matura-tion (7), replication of ribosomal DNA (8), and G-quadruplexDNA resolution (9). S. cerevisiae also encodes a second PIF1-like protein called RRM3. This helicase has a more prominentrole at paused replication forks, where it disrupts protein–DNA complexes at specific (RRM3-sensitive) sites that couldslow the translocation of the replicative helicase complex (10).Despite this wide range of functions, ScPif1 and RRM3 are notessential genes in S. cerevisiae although cell-cycle progressionand growth rate are slowed in double mutants (8).

All mammalian cells studied thus far have only a singlePIF1-like protein that has nearly equal identity and similarityto ScRRM3 and ScPIF1 (11). Although PIF1 has been found inboth nuclei and mitochondria (12), its role in mammalian cellsis less certain. Although antibodies to PIF1 immunoprecipi-tate telomerase activity from cultured human cells (11),knockouts of the mouse Pif1 gene do not affect telomerelength, chromosome stability, or mitochondrial function in

Authors' Affiliation: Institute for Cancer Studies, School of Medicine andBiomedical Sciences, University of Sheffield, Sheffield, United Kingdom

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M.E. Gagou and A. Ganesh contributed equally this work.

Corresponding Author: Mark Meuth, Institute for Cancer Studies, Uni-versity of Sheffield, Medical School, Beech Hill Road, Sheffield S10 2RX,United Kingdom. Phone: 44-114-271-3288; Fax: 44-114-271-3515;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-4404

’2011 American Association for Cancer Research.

CancerResearch

Cancer Res; 71(14) July 15, 20114998

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 2: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

cells and mPif1�/� mice have no obvious phenotype (13).However in vitro studies of purified PIF1 protein indicate thatit can bind and unwind DNA structures resembling stalledreplication forks to produce free 30ssDNA ends, suggestingthat PIF1 could play a role in the response to DNA replicationstress (14). The aim of the work reported here was to inves-tigate the potential role of PIF1 in the cellular response to DNAreplication stress. Our results suggest that PIF1 is essential toprotect a number of human tumor cell lines from apoptosis,particularly after the inhibition of DNA replication.

Materials and Methods

Cell lines and culturesHuman tumor cell lines and the human embryonic kidney

(HEK) 293 line were from American Type Culture Collection.p21�/� and p53�/� derivatives of HCT116 were provided byDr. Bert Vogelstein (Johns Hopkins University, Baltimore,MD). The immortalized human fibroblast cell line MRC5VA was from the CRUK London Laboratories Cell Repository.Identity of the lines used in these experiments was confirmedby DNA fingerprinting (LGC Standards). Cells were carried forno longer than 2months before rethawing from verified frozenstocks. Cells were routinely tested for Mycoplasma and foundto be free of contamination. HCT116 strains overexpressingFLAG-tagged (HPF7 and HPF8) or untagged (PH15 and PH16)PIF1 were obtained by transfection of the tagged or untaggedPIF1 cDNA cloned into the expression vector pCAG-Flox.Stable isolates expressing PIF1 were obtained by selectionin puromycin.Cells were maintained in Dulbecco's modified Eagle's med-

ium supplemented with 10% FBS. For experiments usingthymidine, dialyzed FBS was used to remove interferingdeoxynucleosides. Thymidine and hydroxyurea (HU; Sigma-Aldrich) were used at a concentration of 2 mmol/L. Gemci-tabine (Tocris Bioscience) was used at a concentration of 25mmol/L, and Aphidicolin (Sigma-Aldrich) at 0.2 mmol/L. Atotal of 100 mmol/L of the Caspase-3 Inhibitor II Z-DEVD-FMK(Calbiochem) was added to cultures 1 hour prior to treatmentwith replication inhibitors.

siRNA transfectionPIF1 siRNAs, sense-strand sequences GGCCAGAGCAU-

CUUCUUCATT (siRNA1), GGGAAGUCAUAUCUGCUAATT(siRNA 2), and CCCUUCAGAGCCUAACCAATT (siRNA3),were obtained from Applied Biosystems (Ambion). TheCHK1 siRNA (sense strand: GAAGCAGUCGCAGUGAAGA)was designed by J. Blackburn and C. Smythe. Control siRNAs,containing nonspecific sequences that do not have homologyin the human genome, were provided by Eurogentec (OR-0030-NEG). siRNA duplexes were transfected into cells byusing Lipofectamine 2000 (Invitrogen).

Cell-cycle analysisCell-cycle analysis was done as described previously (15).

Stained nuclei were analyzed on a FACScan (BD Biosciences)by using CellQuest software. Bromodeoxyuridine (BrdU)incorporation was analyzed as previously described (16).

Detection of apoptosisCaspase-3 activation was analyzed by flow cytometry as

described previously (15). Annexin V assays were done asdirected by the manufacturer (BD Biosciences).

Cyclin E and p21 analysisAfter indicated treatments, cells were fixed with 70% ice-

cold ethanol, washed twice with PBS, and incubated for 10minutes in PBS-T (PBS/0.1% BSA/0.2% Tween 20) beforeincubation with a 1:50 dilution of the primary antibody inPBS-T (anti-cyclin E, Invitrogen 32-1600, or anti-p21 Phar-mingen) for 30 minutes. After 2 washes with PBS-T, cellswere incubated with fluorescein isothiocyanate–conjugatedanti-mouse immunoglobulin G (1:10; Dako) for 30 minutes,washed with PBS, and resuspended in PBS containing5 mg/mL PI (Sigma) and 100 mg/mL RNase A (Sigma). Aftera 30-minute incubation, cells were analyzed by flowcytometry.

Protein extraction and Western blottingWestern blots were done as described (17). Proteins were

detected with the ECL Detection System (GE Healthcare) byusing the following antibodies: gH2AX (2577; Cell Signaling),CHK1 (2360; Cell Signaling), b-actin (A-5060; Sigma-Aldrich),RPA34 (NA19L; Calbiochem), cleaved caspase-3 (ab32042;Abcam), phospho-ATM (Ser1981; 2152–1; Epitomics), phos-pho-CHK1 (Ser345; 2349; Cell Signalling), and FLAG M2(F1804; Sigma). Antibodies to PIF1 were raised in rabbits.Amino acids 1 to 205 of PIF1 were expressed as a GST fusionprotein, using pET11c in E. coli BL21(DE3). The fusion proteinwas bound to GST sepharose and the PIF1 moiety cleaved offthe beads by using thrombin after extensive washing of thebeads. Further purification was achieved by size exclusionchromatography (Superdex 75). Band intensities on the Wes-tern blots were quantified by densitometry by using theImageJ software (18).

RNA purification and quantitative analysis of totalmRNA levels

RNA was extracted from adherent cells by using theRNeasy Plus Kit (QIAGEN). RNA was treated with DNase IRNase-free (Roche) and purified over an RNeasy column.Two micrograms of RNA were used for first-strand cDNAsynthesis by using oligo(dT)18 (Bioscript kit; Bioline). qPCRs(quantitative PCR; Quantace) were run on a 7900HT FastReal-Time PCR system (Applied Biosystems). Samples wereheated for 10 minutes at 95�C and amplified for 45 cycles (15sec 95�C/15 sec 59�C/25 sec 72�C). All samples were ana-lyzed in duplicate. Data were evaluated by using SDS 2.2.1data analysis software (Applied Biosystems) and the Pfafflmethod (19). PIF1 primers were designed by using Primer3-plus (20) and NCBI (21) programs: PIF1 50-ccctggattgtgtg-gagatt-30 (forward) and 50-actccagactgaggctcctg-30 (reverse).Primers for the U1 snRNA internal control were from Haut-bergue (22).

Immunofluorescence analysisRPA foci were analyzed as described previously (16).

PIF1 and Tumor Cell Death

www.aacrjournals.org Cancer Res; 71(14) July 15, 2011 4999

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 3: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

Results

PIF1 depletion sensitizes HCT116 cells to DNAreplication stress

Given our previous work showing that static fork-likestructures could serve as efficient substrates for purifiedPIF1 (14), we investigated the effects of siRNA-mediateddepletions of PIF1 on cells exposed to DNA replication stress.The effect of the siRNA treatment on PIF1 levels was firstdetermined by Western blotting. PIF1 is present at very lowlevels in mammalian cells (11) and large amounts of extractshad to be loaded onto gels to visualize the protein by Western

blotting. At 24 hours posttransfection, PIF1 levels werereduced 3-fold relative to control siRNA-transfected cellsand this level of depletion was maintained through 48 hours(Fig. 1A). At 72 hours, PIF1 levels remained low in PIF1 siRNA-transfected cells although the levels of this protein alsodecreased in cultures treated with the control siRNA as theseapproached confluence. In addition, DNA expression arrayanalysis showed a highly significant (P ¼ 2.69 � 10�4) 3-folddepletion of the PIF1 transcript 48 hours after transfection ofthe siRNA. To further confirm the effectiveness of the siRNA,HCT116 cells overexpressing FLAG-tagged PIF1 were trans-fected with PIF1 siRNA. Western blot analysis showed a

A

C

D

E

Bh

siRNA :PIF1

PIF1

PIF1

β-Actin

control

control

siRNA:

PIF1

P = 0.03

P = 0.0001

P = 0.002

P = 6.8 x 10–5

SubG1

SubG1

SubG1 4.051.5

18.225.1

15.355.3

14.714.2

siRNA

– +

Pif1Control

Pif1Control

24.510.1

46.528.8

TdR 48 h:

3.452.1

35.45.6

11.545.7

32.77.5

2.2

57.8

12.027.8

15.9

62.9

7.613.3

4.6

21.9

55.517.4

28.7

29.6

25.013.3

control PIF1 con+24TdR PIF1+24TdR control PIF1 con+48TdR PIF1+48TdR

G1

G1

S

S

G2/M

G2/M

SubG1

G1

SG2/M

ns ns

80

60

40

20

0

50

40

30

20

10

0

80

60

40

20

0

HCT116

Control

siRNA:

PIF1

Control

siRNA:

Surv

ivin

g fra

ctio

n

0

0 24 h

0 50 100 150

TdR [µmol/L] HU [µmol/L] Gemcitabine [µmol/L]200 0 50 100 150 200 1.00.50

No thymidine +thymidine

48 h 0 24 h 48 h

24

34.5 35.5 50 % remaining

48 72 1.0

0.1

0.01

1.0

0.1

0.01

1.0

0.1

0.01

controlsiRNA:

PIF1

% c

ells

Annex

in V

+

% c

ells

Figure 1. Apoptosis is increasedin PIF1-depleted HCT116 cells inthe presence or absence of DNAreplication inhibitors. A, HCT116cells were transfected with controlor PIF1 siRNAs and harvested at24, 48, and 72 hours for Westernblot analysis of PIF1. Cellstransfected with PIF1 siRNA showa 2.9-fold knockdown of the targetprotein level at 24 or 48 hours. At72 hours, this low level of PIF1 wasmaintained whereas it declined incontrol siRNA-treated cells asthey reached confluence. b-Actinlevels are presented as loadingcontrols. The % remaining valuespresented were corrected forloading. B, HCT116 cellstransfected with control or PIF1siRNAs were plated in mediumcontaining increasingconcentrations of thymidine (TdR),HU, or gemcitabine. Survivingcells were allowed to formcolonies that were scored.Surviving fractions presented arenormalized to the survivalobtained in the control siRNA-transfected cells in the absence ofthe replication inhibitors. Underthese conditions, survivingfractions of PIF1 siRNA-treatedHCT116 cultures were about 10%of those obtained with controlsiRNA. C and D, HCT116 cellstransfected with control or PIF1siRNAs and treated or not treatedwith 2 mmol/L thymidine wereharvested at 24 or 48 hours foranalysis of DNA content by flowcytometry. Representative cell-cycle profiles (C) and cell-cycledistributions (D) determined fromthese profiles are presented. E,Annexin V levels in PIF1-depletedHCT116 cells treated as indicated.Results in D and E representmeans of at least 3 independentexperiments with SDs indicatedby error bars. ns, not significant.

Gagou et al.

Cancer Res; 71(14) July 15, 2011 Cancer Research5000

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 4: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

pronounced reduction of the tagged protein at 24 or 48 hoursposttransfection by using either PIF1 or anti-FLAG antibodies(Supplementary Fig. S1).To determine the effect of PIF1 depletion on cell survival

during replication stress, HCT116 cells were treated withcontrol or PIF1 siRNAs for 24 hours before plating in thepresence of the replication inhibitors thymidine or hydro-xyurea (HU) or the therapeutic deoxycytidine analog gemci-tabine for colony formation. Interestingly, cells depleted ofPIF1 showed reduced frequencies of colony formation relativeto cells treated with control siRNA and these were furtherreduced by exposure to the replication inhibitors (Fig. 1B).Most notably, PIF1-depleted cells showed a 270-fold decreasein survival in 1.0 nmol/L gemcitabine relative to cells treatedwith the control siRNA.We next determined the effect of the replication inhibitors

on cell-cycle distribution following PIF1 depletion. HCT116cells transfected with control or PIF1 siRNAs were treatedwith thymidine and analyzed for DNA content by flow cyto-metry (Fig. 1C and D). PIF1-depleted cultures that were nottreated with thymidine showed a significant increase in thelevel of cells with a subG1 DNA content by 24 hours relative tocontrol. Cells transfected with the control siRNA beforethymidine treatment accumulated in early S-phase at 24 hoursand progressed slowly through S-phase, characteristic of theslowing of DNA replication during thymidine-induced dCTPstarvation. PIF1-depleted cells showed a similar accumulationin early S-phase after the 24-hour thymidine treatment butafter 48 hours, there was a marked decrease in the proportionof cells in S-phase, accompanied by a significant increase ofcells with a subG1 DNA content. The fraction of "subG1 cells" inPIF1-depleted cultures after exposure to thymidine was sig-nificantly greater than that seen in cells depleted of PIF1 alone.Similar increases in the proportion of cells showing the earlyapoptotic marker Annexin V were found in PIF1-depleted cellsin the presence or absence of thymidine (Fig. 1E).To investigate potential off-target effects, individual siRNAs

in the pool used here were tested and all had similar effects ondepletion of PIF transcript, as measured by qPCR and cell

death (Supplementary Fig. S2A and B). In addition, the PIF1siRNA had a substantially reduced apoptotic effect on 2HCT116 strains overexpressing PIF1 (SupplementaryFig. S3). Other replication inhibitors had similar effects onPIF1-depleted cells. Treatment of PIF1-depleted HCT116 cul-tures with HU or gemcitabine produced an increased level ofsubG1 cells relative to controls (Supplementary Fig. S4A andB). Taken together, these results indicate that PIF1 protectsHCT116 cells from apoptosis, particularly during DNA repli-cation stress induced by several replication inhibitors.

Effects of PIF1 depletion on other cell linesGiven that knockouts of mPif1 in mouse cells did not

produce a similar effect on cell death (13), we next determinedthe effect of PIF1-depletion on other human cell lines. Both thep53-deficient colon cancer cell line SW480 and the p53-profi-cient breast cancer line MCF7 responded similarly to HCT116with respect to the production of subG1 cells following PIF1depletion in the presence or absence of thymidine (Table 1).MCF7 cells were particularly sensitive to PIF1 depletion withmore than 80% dying even in the absence of the replicationinhibitor. In contrast, 2 immortalized nontumor cell lines(MRC5VA and HEK293) showed little effect.

PIF1-depleted cells accumulate at the G1/S border andin late S-phase after release from thymidine block

To further examine the effect of PIF1 depletion on S-phasetransition, HCT116 cells transfected with control or PIF1siRNAs were treated with thymidine for 24 hours beforerelease into thymidine-free medium. Cells were labeled withBrdU for 1 hour before transfer to deoxynucleoside-freemedium. At various times after release, cells were harvestedand analyzed for DNA and BrdU content by flow cytometry(Fig. 2A). A lower fraction of released PIF1-depleted cellsincorporated BrdU relative to cells treated with the controlsiRNA (Fig. 2B and C). Noticeably, about 20% of PIF1-depletedcells failed to enter S-phase and incorporate BrdU (Fig. 2C).Unlike cells treated with the control siRNA, PIF1-depleted cellsincorporating BrdU also accumulated in late S-phase at 4 to 6

Table 1. Effects of PIF1 depletion on the induction of apoptosis in tumor and nontumor cell lines in thepresence or absence of thymidine

Sub-G1 population (%)

siRNA Control PIF1 Control PIF12 mmol/L TdR (h) 0 0 48 48

HEK293 4.32 � 4.00 4.55 � 3.05 4.04 � 1.79 7.26 � 5.62 Nontumor, kidney, immortalizedMRC5VA 3.39 � 1.72 4.85 � 3.26 3.87 � 2.35 5.24 � 2.42 Nontumor, lung, immortalizedSW480 6.47 � 2.14 15.39 � 6.12 8.10 � 2.99 18.93 � 6.10 Colon p53�

HCT116 2.15 � 0.50 15.95 � 4.00 4.57 � 1.44 28.74 � 4.13 Colon p53þ, hMLH1�

MCF7 20.88 � 9.32 83.41 � 9.35 NT NT Breast p53þ

NOTE: The indicated cells were transfected with control or PIF1 siRNAs and 24 hours later were exposed or not exposed to 2 mmol/Lthymidine. After a 48-hour treatment, cells were harvested for analysis of DNA content by flow cytometry. Proportions of cells with asubG1 DNA content are presented. Results presented are an average of at least 3 independent experiments � SDs.Abbreviation: NT, not tested.

PIF1 and Tumor Cell Death

www.aacrjournals.org Cancer Res; 71(14) July 15, 2011 5001

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 5: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

A

B

C

siRNA

24 hTdR

G1/S S G2/M

1 hBrdU

Brd

Uce

llsce

llsB

rdU

cells

cells

DNA

DNA

Total cells

40.43 ±3.74%

BrdU + cells

Total cells

BrdU + cells

83.42 ±3.81%

84.58 ±2.85%

80.98 ±4.13%

80.73 ±4.09%

79.02 ±1.50%

43.28 ±0.33%

69.3 ±1.97%

71.75 ±0.07%

68.61 ±0.86%

66.68 ±1.77%

67.77 ±1.9%

2 h

Release in TdR-free medium

Control siRNA

PIF1 siRNA

Release in BrdU-free medium

4 h 6 h 20 h

W W

Figure 2. PIF1 is required for efficient entry into S-phase and S-phase progression. A, HCT116 cells, transfected with control or PIF1 siRNAs, were treatedwith 2 mmol/L thymidine for 24 hours. Thymidine-containing medium was removed and cultures were washed (W) with thymidine-free medium beforetransfer to BrdU (10 mmol/L)-containingmedium. After 1 hour, cultures were washed with BrdU-free medium and restored to deoxynucleoside-freemedium. Atindicated times postrelease, cells were harvested and analyzed for DNA content and BrdU incorporation by flow cytometry. B and C, top panels in eachpresent scatter plots of analysis of BrdU incorporation and DNA content. Middle panels present DNA content of all the cells analyzed, whereas thebottom panels present the DNA content of cells labeled with BrdU (gated into cells with G1, S, or G2/M DNA contents in top panels). Percentages presented inbottom panels represent the proportion of cells incorporating BrdU. Left-most panels present randomly growing cultures labeled for 1 hour with BrdUbefore harvest. The next panel from left presents the cells labeled for 1 hour with BrdU after thymidine release. The remaining panels present cells released fromboth deoxynucleosides for the indicated times. Closed arrowheads in C indicate PIF1-depleted cells that do not enter S-phase after release, whereasopen arrowheads indicate those delayed from entering G2-M. The data presented are representative of 3 independent experiments. Values for percentages ofcells incorporating BrdU represent means of values obtained from 3 experiments � SDs.

Gagou et al.

Cancer Res; 71(14) July 15, 2011 Cancer Research5002

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 6: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

A

B

E F

D

C

TdR (h):

Control

24

G1/S S G2/M

G1/S S G2/M

24

2

24

24

6Release (h):

Total cells

Total cells

Control PIF1 HCT116

Untreated

Control

2.1

4.6

15.9

28.7

3.7

11.5

39.1

59.4

PIF1 Control PIF1

48 h TdR

HCT116 p21–/–

Untreated

48 h TdR

Total

G1

S

G2

Total

G1

SG2

* P = 0.048

**

**

*

*

*

***

**** P = 0.01

* P = 0.018** P = 0.016

70

60

50

40

30

20

10

0

80

60

40

20

0

Total cells

cells

cells

cells

cells

DNA

DNA

% p

21

+ c

ell

s

% S

ub

G1

DNA

PIF1

PIF1

DNA

Total cells

cy

cli

n E

cy

cli

n E

p2

1p

21

TdR (h):

Control

–Release (h):

TdR (h): 00

12.1

6.3

0.5

5.3

35

2.9

21.2

10.8

33.9

2.7

1.2

29.9

37.8

30

1.1

6.7

55.7

22.3

21.2

12.3

42.5

16.9

2.1

23.5

240

246

00

240

246Release (h):

Figure 3. PIF1-depleted cells arrested at G1/S show increased cyclin E and p21. A and B, HCT116 cells transfected with control (A) or PIF1 (B) siRNAswere exposed to 2 mmol/L thymidine for 24 hours before release into thymidine-free medium. Control (thymidine-untreated), thymidine-treated, andthymidine-treated cultures released into thymidine-free medium for 2 hours were harvested and analyzed for cyclin E and DNA content by flow cytometry.Top panels present cyclin E levels and DNA content whereas bottom panels present DNA content of all cells. Closed arrowhead in B indicates cyclinE level in PIF1-depleted cells arrested at G1/S. Cells gated in top panels indicate those with elevated cyclin E levels relative to control cultures. C and D,HCT116 cultures, transfected with control (C) or PIF1 (D) siRNAs, treated with thymidine and released for 6 hours as above, were analyzed for p21 andDNA content. Top panels in each present p21 and DNA content whereas bottom panels present DNA content of all cells. Closed arrowhead in D indicates thep21 level of cells that fail to enter S-phase. E, percentages of cells in the indicated phases of the cell cycle containing elevated levels of p21 (gated in C and D).Results represent means of 3 independent experiments � SDs. F, p21�/� HCT116 cells depleted of PIF1 show enhanced apoptosis. Cells transfectedwith control or PIF1 siRNAs and treated or not treated with 2 mmol/L thymidine for 48 hours were analyzed for DNA content by flow cytometry for the fractionof subG1 cells. Average percentages of cells with subG1 DNA contents from 3 independent experiments are presented � SDs.

PIF1 and Tumor Cell Death

www.aacrjournals.org Cancer Res; 71(14) July 15, 2011 5003

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 7: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

hours after release from thymidine, suggesting a delay in S-phase progression. Similar cell-cycle delays at the G1/S borderand in late S-phase were observed with PIF1-depleted SW480cells (Supplementary Fig. S5).

To clearly distinguish cells entering S-phase from those thatdid not, thymidine-released HCT116 cells were labeled withBrdU for 6 hours. Under these conditions, unlabeled G1 cellspersisted at least 24 hours. There was a weak increase in thelevel of unlabelled cells with a subG1 DNA content (Supple-mentary Fig. S6). However, the level of BrdU-labeled cells witha subG1 DNA content increased more rapidly, suggesting thatcycling cells may be more prone to apoptosis.

Flow cytometric measurements of DNA content and cyclinE in PIF1-depleted HCT116 cells released into thymidine-freemedium indicate that cells failing to enter S-phase have anelevated level of cyclin E (Fig. 3A and B). However, levels of thecyclin-dependent kinase inhibitor p21 were also high in thePIF1-depleted cells relative to controls, particularly in cellsretained at the G1/S border (Fig. 3C–E). To further address thepotential role of p21 in the induction of death in PIF1-depletedcells, p21�/� HCT116 cells were depleted of PIF1 in thepresence or absence of thymidine. The level of subG1 cellsin PIF1-depleted p21�/� cultures increased relative to controlsiRNA-treated cells (Fig. 3F). Furthermore, the level of death inPIF1-depleted p21�/� HCT116 cells was higher than thatfound in p21þ/þ HCT116 cells. Taken together, these resultssuggest that p21 protects cells from entry into S-phase andapoptosis triggered by PIF1 depletion.

S-phase checkpoints in PIF1-depleted cells afterthymidine treatment

Cells exposed to DNA-damaging agents or replication stressrapidly activate ATR- and ATM-mediated signaling cascades.CHK1 is a key component of the ATR-mediated cascade and isquickly activated in response to ssDNA that is formed by thedisruption of DNA replication (23) to suppress inappropriateorigin firing, facilitate replication restart, and prevent apop-tosis. Given our in vitro data suggesting a potential role forPIF1 in the generation of ssDNA at stalled forks (14), we nextdetermined the effect of PIF1 depletion on signaling triggeredby thymidine treatment. Cell-free extracts prepared fromPIF1-depleted HCT116 cells were assayed for checkpointactivation by Western blotting by using antibodies for phos-phorylated forms of CHK1, ATM, or H2AX. pSer345 CHK1 wasdetected in cells treated with the control siRNA by 1 hour afterthymidine treatment, whereas pSer1981 ATM and gH2AXwere only weakly detected (Fig. 4A) as previously reported(16, 24). Despite the increased level of apoptosis in PIF1-depleted cells, phosphorylated forms of these checkpointproteins were not detected in the absence of the replicationinhibitor (Fig. 4A). After thymidine treatment, the level ofpSer345 CHK1 was noticeably lower at all times in the PIF1-depleted cells relative to cells transfected with the controlsiRNA. Interestingly, the fraction of cells showing RPA foci wasalso significantly decreased in PIF1-depleted cells treated withthe replication inhibitor aphidicolin (Fig. 4B) relative to cellstreated with the control siRNA, suggesting that ssDNA for-mation may be suppressed in PIF1-depleted cells. RPA hyper-

phosphorylation (that occurs after some forms of DNAdamage or replication stress; ref. 25) was not evident. pSer1981ATM and gH2AX formation were not significantly increased at24 hours in PIF1-depleted cells relative to controls.

A

B

HCT116

siRNA

TdR

h

Control PIF1

siRNA:

Untreated

RPA34 RPA34

RPA34 RPA34

0.2 µmol/Laphidicolin

*<10 foci

>10 foci

<10 foci

>10 foci

12

10

8

6

4

2

0

* P = 0.0015

ControlsiRNA:APH (24 h): –

3.62

2.31

2.38

2

6.82

9.93

4.94

3.25

PIF1–

Control0.2 µmol/L

PIF10.2 µmol/L

% c

ells

with

RPA

34

foci

*

Control PIF1

pATM

pCHK1

CHK1

RPA34

γH2AX

β-Actin

0

– – – – + + + – – – – + + +

1 2 24 1 2 24 0 1 2 24 1 2 24

Figure 4. Suppression of DNA damage responses in PIF1-depleted cellstreated with thymidine. A, HCT116 cells transfected with control or PIF1siRNAs were treated or not treated with 2 mmol/L thymidine for indicatedtimes. Cultures were harvested for Western blot analysis of indicatedproteins. b-Actin levels are presented as loading control. B, siRNA-treatedHCT116 cells were exposed or not exposed to 0.2 mmol/L aphidicolin for24 hours before being fixed and stained for RPA34 foci. Top panels showrepresentative immunofluorescence images of RPA foci-containing cells.Bottom panels present the percentages of HCT116 cells having low (<10foci/cell) or high (>10 foci/cell) levels of RPA foci. Results represent themeans of 3 independent experiments � SDs.

Gagou et al.

Cancer Res; 71(14) July 15, 2011 Cancer Research5004

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 8: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

Apoptosis in PIF1-depleted cells is caspase-3 dependentIn CHK1-depleted cells, apoptosis has been shown to

proceed by either caspase-3 or caspase-2–dependent path-ways following replication inhibition (15) or DNA damage (26).PIF1-depleted HCT116 cells showed detectable levels of acti-vated caspase-3 at 24 hours in the presence or absence ofthymidine (Fig. 5A). Levels of activated caspase-3 were furtherincreased in PIF1-depleted cells after a 48-hour treatment withthymidine. Activated caspase-2 was not detected under any ofthese conditions (data not presented).Given the effects of the PIF1 depletion on thymidine-treated

cells in G1 or S-phase, we next determined where caspase-3activation occurred in the cell cycle by flow cytometry. Thelevel of PIF1-depleted cells showing caspase-3 activation wassignificantly elevated after a 48 hour-thymidine treatment(Fig. 5B and C). The percentage of PIF1-depleted cells showingactivated caspase-3 was also increased at 48 or 72 hoursposttransfection in the absence of thymidine treatment, butthis did not reach significance after 72 hours (P¼ 0.06) relativeto cells treated with the control siRNA. PIF1-depleted cellshaving increased levels of activated caspase-3 predominantlyshowed a G1/early S-phase DNA content. In addition, the

caspase-3 inhibitor II (Z-DEVD-FMK) suppressed the accu-mulation of cells with a subG1 DNA content in PIF1-depletedcultures in the presence or absence of thymidine (Fig. 5D).

Codepletion of CHK1 and PIF1 does not enhance orsuppress apoptosis in response to replication inhibitors

Given the importance of CHK1 in the apoptotic response toreplication inhibitors (27), we next determined the effect ofcodepletion of these 2 proteins. HCT116 cultures, transfectedwith CHK1 or PIF1 siRNAs singly or in combination and thentreated or not treated with thymidine for 48 hours, wereanalyzed for DNA content by flow cytometry. The resultingcell-cycle distributions were compared with those of cellstreated with the control siRNA (Fig. 6A and B). Culturesdepleted of CHK1 or PIF1 showed increased levels of cellswith a subG1 DNA content and a corresponding decrease inthe level of S-phase cells after thymidine treatment relative tocells treated with the control siRNA. Codepleted culturesshowed a virtually identical response to those depleted ofeither protein alone. Unlike CHK1, PIF1-depleted cells showan increased level of subG1 cells in the absence of thymidineand this is also evident in the codepleted cultures.

Figure 5. Apoptosis in PIF1-depleted cells is caspase-3dependent. A, Western blotanalysis of cleaved caspase-3(cleaved c3) in extracts obtainedfrom HCT116 cells transfectedwith control or PIF1 siRNAs andtreated or not treated with 2mmol/L thymidine for indicated times.b-Actin levels are presented asloading controls. B, HCT116 cellstransfected with control or PIF1siRNAs were harvested andanalyzed for cleaved caspase-3 orDNA content by flow cytometry.Top panels present scatter plotsof cleaved caspase-3 and DNAcontent. Bottom panels presentDNA content of all cells analyzedin the experiment. C, averagepercentages of cells havingactivated caspase-3 in indicatedconditions from 3 independentexperiments as presented in B. D,HCT116 cells transfected withcontrol or PIF1 siRNAs treated ornot treated with thymidine wereexposed to caspase-3 inhibitorand analyzed for DNA content byflow cytometry. Averagepercentages of cells showingsubG1 DNA contents in thepresence or absence of caspase-3 inhibitor from these assays arepresented. Values represent themeans obtained from 3experiments � SDs.

A

B

C D

siRNA:

Cleaved casp3

β-Actin

0 24–TdR

48 0 24–TdR

480 24+TdR

48 0 24+TdR

48 h

Control PIF1

siRNA Control

DNA DNA

Cel

ls

Cle

aved

casp

ase-

3

Cel

ls

Cle

aved

casp

ase-

3

PIF1

siRNA

Control

P = 0.04

P = 0.06

% c

aspa

se-3

act

ivat

ion

% S

ub G

1 po

pula

tion25

20

15

10

5

0

35

30

25

20

15

10

5

0

PIF1

siRNA

Control

PIF1

24

0

48

0

72

0

48

24

72

48

Posttran (h):

TdR (h):

72

0

72

0

72

48

72

48

Posttran (h):

TdR (h):– + – +Casp-3 inh.

Control PIF1

No thymidine 48 h thymidine

PIF1 and Tumor Cell Death

www.aacrjournals.org Cancer Res; 71(14) July 15, 2011 5005

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 9: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

Discussion

DNA helicases have been shown to play key roles in theprotection of cells from DNA replication stress and DNAdamage (4). Consistent with a role for PIF1 in the maintenanceof replication fork integrity, we show that PIF1 depletion inhuman tumor cells can prevent entry into S-phase and slow S-phase progression upon release from thymidine-induced repli-cation arrest. CHK1 activation following replication stress issuppressed in PIF1-depleted tumor cells. Cell viability isreduced and there is an increased level of apoptosis in lateG1/early S-phase that is enhanced by treatment with replica-tion inhibitors (Fig. 6C). PIF1-depleted cells arrested at G1/Shave elevated levels of cyclin E as well as p21, suggesting thatthey are prevented from entering S-phase and, to some extent,may be protected from death. Consistent with this conclusion,p21�/� cells that lack this G1/S checkpoint show increasedlevels of cell death.

Yeast Pif1 helicases are involved in a wide range of DNAtransactions, including the regulation of telomere length andthe resolution of G-quadruplex DNA that are important for themaintenance of genomic stability. Studies of the single PIF1helicase in mammalian cells have not produced consistentevidence for similar roles. Knockouts of the mouse gene do notlead to genetic instability and mPif1�/� mice have no obviousphenotype (13). Thus, it was proposed that PIF1 function isredundant in mammalian cells. Nevertheless, in vitro analysisof purified full-length PIF1 suggested that it bound andunwound DNA structures that might form during DNA repli-cation stress, including structures resembling stalled replica-tion forks (14) or G-quadruplexes (28). The effects of PIF1depletion on S-phase transition, checkpoint activation, and

apoptosis in tumor cells are consistent with these in vitroresults; however, nontumor cell lines did not show similarresponses. Therefore, we propose that the redundancy thatprotects mouse cells from loss of mPif1 may be lost in humantumor cells. Furthermore, tumor cells may encounter higherlevels or different forms of stress. It has been reported thatoncogene activation and the loss of control of entry into S-phase may lead to chronic DNA replication stress in manytypes of tumor cells (29, 30). In such conditions, tumor cellsmay show increased dependence upon helicases such as PIF1to maintain fork integrity and viability. Other 50->30 helicasesfrom human cells have been characterized and these mayshare overlapping functions with PIF1. In particular, it hasrecently been reported that BACH1/FANCJ is required forATR-dependent phosphorylation events triggered by DNAreplication stress (31) and shares an affinity for G-quadruplexstructures (32). Thus, tumor cells may show varying depen-dence upon helicases determined by the level of expression orgenetic status of the various helicases and the level or natureof the replication stress they encounter.

In vitro assays of PIF1 binding and unwinding on artificialsubstrates resembling replication forks (14) suggested thatPIF1 could play a role in the generation of ssDNA that triggersactivation of CHK1. Consistent with this argument, CHK1activation, which is dependent upon ssDNA formation (23),was lower in PIF1-depleted cells exposed to thymidine. Otherdownstream events triggered by ssDNA, including RPA fociformation and RPA hyperphosphorylation (16, 33), were alsosuppressed in PIF1-depleted cells. Recent work has shown theimportance of CHK1 in the control of apoptosis during DNAreplication stress and other forms of DNA damage (15, 26, 27).The experiments reported here indicate several similarities in

siRNA: Control CHK1 PIF1 CHK1/PIF1

Untreated

48 h TdR

Untreated

25

00

Eve

nts

25

00

Eve

nts

25

00

Eve

nts

30

00

0 0 0 0 1,023

Eve

nts

FL3-H FL3-H FL3-H FL3-H

40

30

20

10

0

48 h TdR

siRNA: Control CHK1

2.65.9

3.233.4

PIF1

15.231.2

CHK1/PIF1

PIF1

–PIF1

+PIF1CHK1

activation

forkstress

Replication inhibitors

Oncogene activation

SuppressedCHK1

activation

Apoptosis

Apoptosis

FANCJ ?other

helicases

Tumorcells

11.834.0

Untreated

48 TdR

% s

ub G

1 p

opula

tion

A

B C

Figure 6. Apoptosis induced inPIF1-depleted cells is not affectedby CHK1 depletion. A, HCT116cells transfected with control,PIF1, or CHK1 siRNAs singly or incombination and exposed to 2mmol/L thymidine for 48 hourswere harvested and analyzed forDNA content by flow cytometry. B,average percentages of cells withsubG1 content measured in 3independent experiments aspresented in A � SDs. C, modelfor the role of PIF1 in the inductionof apoptosis in human tumor cellsduring replication stress.Replication stress in tumor cellslines may be induced byreplication inhibitors or may be aconsequence of oncogeneactivation or the loss of control ofentry into S-phase.

Gagou et al.

Cancer Res; 71(14) July 15, 2011 Cancer Research5006

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 10: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

the induction of apoptosis in CHK1 and PIF1-depleted cells.Both types of cells commit to apoptosis in late G1/early S-phase by a caspase-3–dependent mechanism in response toreplication inhibitors. Furthermore, codepletion of PIF1 andCHK1 show levels of apoptosis similar to those found in cellsdepleted of the 2 proteins alone. On the other hand, theinduction of death in PIF1-depleted cells did not strictlyrequire treatment with replication inhibitors. Further workis needed to investigate the relationship between PIF1, CHK1activation, and the induction of cell death.These findings also have potential therapeutic applications.

Therapies that specifically induce apoptosis in tumor cells, butnot nontumor cells, obviously have distinct advantages for thetreatment of cancer patients. Although mouse knockoutsindicate that mPif1 is not essential for development or tumorsuppression, our initial data suggest that tumor cell lines areparticularly responsive to the apoptotic effects of PIF1 deple-tion. Recently, an inhibitor of the WRN helicase was identifiedand shown to have growth inhibitory and proapoptotic activ-

ities in a number of tumor cell lines (34). Our findings indicatethat searches for inhibitors of other helicases with roles inreplication and/or repair may be warranted.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

We are thankful to Tresa George for PIF1 purification.

Grant Support

This work was supported by a program grant from Yorkshire CancerResearch.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 8, 2010; revised April 28, 2011; accepted May 18, 2011;published OnlineFirst May 26, 2011.

References1. Ellis NA, Groden J, Ye TZ, Straughen J, Lennon DJ, Ciocci S, et al. The

Bloom's syndrome gene product is homologous to RecQ helicases.Cell 1995;83:655–66.

2. Kudlow BA, Kennedy BK, Monnat RJ. Werner and Hutchinson-Gilfordprogeria syndromes: mechanistic basis of human progeroid diseases.Nat Rev Mol Cell Biol 2007;8:394–404.

3. Kitao S, Shimamoto A, Goto M, Miller RW, Smithson WA, Lindor NM,et al. Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. Nat Genet 1999;22:82–4.

4. Wu L, Hickson ID. DNA helicases required for homologous recombi-nation and repair of damaged replication forks. Annu Rev Genet2006;40:279–306.

5. Foury F, Kolodynski J. Pif mutation blocks recombination betweenmitochondrial rþ and r� genomes having tandemly arrayed repeatsunits in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A1983;80:5345–9.

6. Schulz VP, Zakian VA. The Saccharomyces PIF1 DNA helicase inhibitstelomere elongation and de novo telomere formation. Cell1994;76:145–55.

7. Budd M, Reis CC, Smith S, Myung K, Campbell JL. Evidence sug-gesting that Pif1 helicase functions in DNA replication with the Dna2helicase/nuclease and DNA polymerase d. Mol Cell Biol 2006;26:2490–500.

8. Ivessa AS, Zhou J-Q, Zakian VA. The Saccharomyces Pif1p DNAhelicase and the highly related Rrm3p have opposite effects onreplication fork progression in ribosomal DNA. Cell 2000;100:479–89.

9. Ribeyre C, Lopes J, Boule P, Piazza P, Guedin A, Zakian VA, et al. Theyeast Pif1 helicase prevents genomic instability caused by G-quad-ruplex-forming CEB1 sequences in vivo. PLoS Genet 2009;5:e1000475.

10. Ivessa AS, Zhou JQ, Schulz VP, Monson EK, Zakian VA. The Sac-charomyces Rrm3p, a 50 to 30 DNA helicase that promotes replicationfork progression through telomeric and subtelomeric DNA. Genes Dev2002;16:1383–96.

11. Mateyak MK, Zakian VA. Human Pif helicase is cell cycle regulatedand associates with telomerase. Cell Cycle 2006;5:2796–804.

12. Futami K, Shimamoto A, Furuichi Y. Mitochondrial and nuclear loca-lization of human Pif1 helicase. Biol Pharm Bull 2007;30:1685–92.

13. Snow ES, Mateyak M, Paderova J, Wakeham A, Iorio C, Zakian VA,,et al. Murine Pif1 interacts with telomerase and is dispensable fortelomere function in vivo. Mol Cell Biol 2007;27:1017–26.

14. George T, Wen Q, Griffiths R, Ganesh A, Meuth M, Sanders CM.Human Pif1 helicase unwinds synthetic DNA structures resemblingstalled DNA replication forks. Nucleic Acids Res 2009;37:6491–502.

15. Rodriguez R, Meuth M. Chk1 and p21 cooperate to prevent apop-tosis during DNA replication fork stress. Mol Biol Cell 2006;17:402–12.

16. Gagou ME, Zuazua-Villar P, Meuth M. Enhanced H2AX phosphoryla-tion, DNA replication fork arrest, and cell death in the absence ofChk1. Mol Biol Cell 2010;21:739–52.

17. Bolderson E, Scorah J, Helleday T, Smythe C, Meuth M. ATM isrequired for the cellular response to thymidine induced replication forkstress. Hum Mol Genet 2004;13:2937–45.

18. O'Neill RR, Mitchell LG, Merril CR, Rasband WS. Use of imageanalysis to quantitate changes in form of mitochondrial DNA afterx-irradiation. Appl Theor Electrophor 1989;1:163–7.

19. Pfaffl MW. A newmathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res 2001;29:e45.

20. Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, LeunissenJAM. Primer3Plus, an enhanced web interface to Primer3. NucleicAcids Res 2007;35:W71–4.

21. Rosen S, Skaletsky HJ. Primer3 on theWWW for general users and forbiologist programmers. In: Krawetz S, Miserner S, eds. Bioinformaticsmethods and protocols: methods in molecular biology. Totowa, NJ:Humana Press; 2000. p. 365–86.

22. Hautbergue GM, Hung ML, Walsh MJ, Snijders AP, Chang CT, JonesR, et al. UIF, a new mRNA export adaptor that works together withREF/ALY, requires FACT for recruitment to mRNA. Curr Biol 2009;19:1918–24.

23. Zou L, Elledge SJ. Sensing DNA damage through ATRIP recognitionof RPA-ssDNA complexes. Science 2003;300:1542–8.

24. WenQ, Scorah J, Phear G, Rodgers G, Rodgers S, Meuth M. Amutantallele of MRE11 found in mismatch repair deficient tumor cells sup-presses the cellular response to DNA replication fork stress in adominant negative manner. Mol Biol Cell 2008;19:1693–705.

25. Zou Y, Liu Y, Wu X, Shell SM. Functions of human replication protein A(RPA): from DNA replication to DNA damage and stress responses. JCell Physiol 2006;208:267–73.

26. Sidi S, Sanda T, Kennedy RD, Hagen AT, Jette CA, Hoffmans R, et al.Chk1 suppresses a caspase-2 apoptotic response to DNA damagethat bypasses p53, Bcl-2, and caspase-3. Cell 2008;133:2110–8.

27. Meuth M. Chk1 suppressed cell death. Cell Div 2010;5:21.

PIF1 and Tumor Cell Death

www.aacrjournals.org Cancer Res; 71(14) July 15, 2011 5007

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 11: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

28. Sanders CM. Human Pif1 helicase is a G-quadruplex DNA bindingprotein with G-quadruplex DNA unwinding activity. Biochem J2010;430:119–28.

29. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N,et al. Oncogene-induced senescence is part of the tumorigenesisbarrier imposed byDNAdamage checkpoints. Nature 2006;444:633–7.

30. Di Micco R, Fumagalli M, Cicalese A, Bensimon A, Maestro R, PelicciPG, et al. Oncogene-induced senescence is a DNA damage responsetriggered by DNA hyper-replication. Nature 2006;444:638–42.

31. Gong Z, Kim J-E, Leung CCY, Glover JNM, Chen J. Bach1/FancJ actswith TopBP1 and participates early in DNA replication checkpointcontrol. Mol Cell 2010;37:438–46.

32. London TB, Barber LJ, Mosedale G, Kelly GP, Balasubramanian S,Hickson ID, et al. FANCJ is a structure-specific DNA helicase asso-ciated with the maintenance of genomic G/C tracts. J Bio Chem2008;283:36132–9.

33. Rodriguez R, Gagou ME, Meuth M. Apoptosis induced by replicationinhibitors in Chk1-depleted cells is dependent upon the helicasecofactor Cdc45. Cell Death Differ 2008;15:889–98.

34. Aggarwal M, Sommers JA, Shoemaker RH, Brosh RM. Inhibi-tion of helicase activity by a small molecule impairs Wernersyndrome helicase (WRN) in the cellular response to DNAdamage or replication stress. Proc Natl Acad Sci U S A 2011;108:1525–30.

Gagou et al.

Cancer Res; 71(14) July 15, 2011 Cancer Research5008

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404

Page 12: Suppression of Apoptosis by PIF1 Helicase in …cancerres.aacrjournals.org/content/71/14/4998.full.pdf · Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells ... Bioline).

2011;71:4998-5008. Published OnlineFirst May 26, 2011.Cancer Res   Mary E. Gagou, Anil Ganesh, Ruth Thompson, et al.   Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

  Updated version

  10.1158/0008-5472.CAN-10-4404doi:

Access the most recent version of this article at:

  Material

Supplementary

  http://cancerres.aacrjournals.org/content/suppl/2011/05/26/0008-5472.CAN-10-4404.DC1

Access the most recent supplemental material at:

   

   

  Cited articles

  http://cancerres.aacrjournals.org/content/71/14/4998.full#ref-list-1

This article cites 33 articles, 11 of which you can access for free at:

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  [email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/71/14/4998To request permission to re-use all or part of this article, use this link

on September 12, 2018. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 26, 2011; DOI: 10.1158/0008-5472.CAN-10-4404