e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704

Available online at www.sciencedirect.com

jo ur nal homep age: www.elsev ier .com/ locate /e tap

Methyl methanesulfonate induces apoptosis inp53-deficient H1299 and Hep3B cells through acaspase 2- and mitochondria-associated pathway

Ying Jianga,b,c, Xiao-Yun Zhanga,b, Li Sune, Guang-Lin Zhanga,b,Penelope Duerksen-Hughesd, Xin-Qiang Zhub,∗∗, Jun Yanga,e,∗

a The First Affiliated Hospital, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, Hangzhou,Zhejiang 310003, Chinab Department of Toxicology, Zhejiang University School of Public Health, Hangzhou, Zhejiang 310058, Chinac Center Testing International Corporation, Shenzhen, Guangdong 518101, Chinad Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA 92354, USAe Department of Toxicology, Hangzhou Normal University School of Public Health, Hangzhou, Zhejiang 310036, China

a r t i c l e i n f o

Article history:

Received 7 March 2012

Received in revised form

19 September 2012

Accepted 30 September 2012

Available online 8 October 2012

Keywords:

Methyl methanesulfonate

p53

Caspase 2

a b s t r a c t

Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types

through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of

p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the

presence of p53-independent apoptotic mechanisms. To understand the p53-independent

mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced

apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated

that MMS at concentrations of 50, 100, 200, 400 and 800 �M induced the formation of

gammaH2AX foci, and that at higher concentrations, 400 and 800 �M, MMS treatment led

to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss

of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2,

release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and

gammaH2AX

Apoptosis

PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73

and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-

induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as

well as the mitochondria-mediated pathway.

ation, and esterification reactions (Wyatt and Pittman, 2006).

1. Introduction

Methyl methanesulfonate (MMS) is a typical methylatingagent which can be used as an experimental research

Abbreviations: MMS, methyl methanesulfonate; DSB, double strandtivation competent p73; DNp73, dominant negative p73.

∗ Corresponding author at: Department of Toxicology, Hangzhou NormaChina. Tel.: +86 571 8820 8140; fax: +86 571 8820 8140.∗∗ Corresponding author. Tel.: +86 571 8820 8143; fax: +86 571 8820 8143.

E-mail addresses: zhuxq@zju.edu.cn (X.-Q. Zhu), gastate@zju.edu.c1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.etap.2012.09.019

© 2012 Elsevier B.V. All rights reserved.

chemical and as a solvent catalyst in polymerization, alkyl-

break; MMP, mitochondrial membrane potential; TAp73, transac-

l University School of Public Health, Hangzhou, Zhejiang 310036,

n, gastate@yahoo.com (J. Yang).

MMS has also been tested as a cancer chemotherapeutic agent,and the monoester of methanesulfonic acid can be used asa human male contraceptive and an insect attractant and

p h a r

rpo(ctr(eirst(f(

i2parcaa(Jorihitrtcpeasataa

DItmpac(wa2iid(

gest that there exists a caspase 2-associated, but p53-

e n v i r o n m e n t a l t o x i c o l o g y a n d

epellent (Kunz et al., 2010). MMS is a known genotoxic com-ound that can directly react with guanine and adenine basesf DNA to generate interstrand and intrastrand cross-links

Hosseinimehr et al., 2010). In dividing cells, a replication forkan be stalled at the sites of DNA cross-links, resulting inhe formation of DNA double strand breaks (DSBs), which areegarded as one of the most detrimental forms of DNA damageYu et al., 2006). By disrupting the dynamic and structural prop-rties of DNA, DSBs affect many aspects of DNA metabolism,ncluding DNA replication, transcription, recombination andepair (Shanbhag et al., 2010). DSBs can also activate severalignal transduction pathways that can eventually lead to cellumorigenesis or, depending on the situation, to apoptosisSuwaki et al., 2011). This DSB-induced apoptosis is a key eventor many conventional chemotherapeutic agent applicationsWaxman and Schwartz, 2003).

p53 plays a pivotal role in DSB-induced apoptosis, whichs central to its function as a tumor suppressor (Chen et al.,011). In response to DSB formation, p53 is rapidly phos-horylated by the ataxia telangiectasia mutated (ATM) andtaxia telangiectasia and Rad3 related (ATR) protein kinases,esulting in its activation (Shiloh, 2006). Upon activation, p53an activate a series of pro-apoptotic proteins such as BCL2-ssociated X protein (BAX), p53 upregulated modulator ofpoptosis (PUMA), p53-induced protein with a death domainPIDD), and FAS receptor (Berube et al., 2005; Meulmeester andochemsen, 2008; Yee et al., 2009). However, the high frequencyf p53 mutations in human cancer cells makes the explo-ation of p53-independent cell signaling pathways becomencreasingly urgent (Kashiwazaki et al., 1997). Previous studiesave also demonstrated important roles for p53 in MMS-

nduced apoptosis (Tian et al., 2009; Ziv et al., 2006). Despitehese findings, it is recognized that p53 may only be partiallyesponsible for MMS-induced cytotoxicity. For instance, afterreatment with MMS, the p53 mutant human lymphoblastoidells (WTK1) exhibited a delayed apoptosis, as compared to the53 proficient human lymphoblastoid cells (TK6) (Greenwoodt al., 1998). In another case, both p53 knockout fibroblastsnd the corresponding wild-type cells displayed a similarensitivity to MMS-induced chromosomal aberrations andpoptosis (Lackinger et al., 2001). Thus, the apoptotic responseo MMS-induced DNA damage involves both p53-dependentnd -independent mechanisms. However, very little is knownbout the p53-independent mechanisms.

p73, as a p53 family member, is also responsive to theNA damage that leads to apoptosis (Roos and Kaina, 2006).

n contrast to p53, p73 is rarely mutated in human cancers,hus, p73 as a new candidate tumor suppressor is getting

ore and more interest (Ozaki and Nakagawara, 2005). The73 gene encodes two isoforms that differ in their N-termini,nd which can be functionally classified as trans-activationompetent (TAp73) and dominant negative (DNp73) proteinsMoll and Slade, 2004). The TAp73 shares similarities with p53

ith regards to DNA damage-induced apoptosis through itsctivation of p53 responsive genes (Nieto-Rementeria et al.,009). The DNp73 has a very important regulatory role, ast exerts a dominant-negative effect on p53 and TAp73 by

nhibiting their transactivation activity. Thus, DNp73 confersrug resistance to tumor cells harboring p53 and/or TAp73

Stiewe et al., 2002). Its inhibitory function is exerted either

m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704 695

at the oligomerization level, or by competing for binding tothe p53/TAp73 DNA target sequence (Vossio et al., 2002). Inaddition, the DNp73 promoter contains an efficient p53/TAp73responsive element that can be transactivated by p53 and/orTAp73, and therefore creates a dominant-negative feedbackloop that regulates the function of p53 and/or TAp73 (Concinet al., 2004). Several lines of evidences indicate that thepresence and participation of different spliced forms of p73might be one reason for the different cell fates observed inresponse to DNA damage (Sayan et al., 2010). Moreover, pre-vious studies have also demonstrated in response to differentDNA-damaging stimuli, p73 isoforms can contribute to p53-dependent or -independent cell apoptotic signaling pathways(Costanzo et al., 2002; Murray-Zmijewski et al., 2006). However,it is still unknown whether TAp73 and/or DNp73 participate inMMS-induced apoptosis.

Caspase 2 is the only pro-caspase constitutively present inthe nucleus, and it is unique among the caspases in that ithas features of both upstream and downstream caspases (vanLoo et al., 2002). In recent years, accumulating evidence indi-cates that in response to DNA damage, caspase 2 can act asan initiator regulatory enzyme upstream of the mitochondria-dependent apoptosis pathway (Vakifahmetoglu et al., 2008).For example, treatment of cells with 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a well-known oxidative agent thatinduces DNA damage, resulted in the release of cytochromec from mitochondria, and this activity can be inhibitedby the caspase 2 selective inhibitor (zVD-VAD-fmk) (Tammet al., 2008). In addition, during cisplatin-induced apopto-sis, using siRNA to inhibit caspase 2 expression causeddecreased cytochrome c release, and reduced caspase 3 andcaspase 9 activities (Vakifahmetoglu et al., 2008). Moreover,several observations suggested in some cell lines, caspase 2is required for p53-mediated apoptosis induced by genotoxicagents (Ren et al., 2005). But the link between p73 isoformsand caspase 2 activation remains to be elusive. Despite theabove evidence supporting roles for caspase 2 in stress-induced apoptosis, there is currently no model that links agiven type of DNA damage with nuclear caspase 2 activationand mitochondria-dependent apoptosis. Furthermore, mostreports have only investigated either the role of caspase 2 onthe activation of down-stream genes, or the effect of genotoxicagents on the expression of caspase 2 in whole-cell lysates,rather than in specific sub-cellular compartments (Guo et al.,2002; Sidi et al., 2008).

In this current study, we sought to analyze the p53-independent mechanisms induced during MMS-inducedapoptosis. Using the p53-deficient H1299 and Hep3B cells,we first established an MMS-induced apoptotic model. Then,we carried out a set of analyses assessing cell viabil-ity, percentages of apoptotic cells, extent of DNA damage,and mitochondrial membrane potentials (MMP). Finally, weanalyzed the protein levels of TAp73, DNp73, pro-caspase2, cleaved caspase 2, pro-caspase 9, cleaved caspase 9,pro-caspase 3, cleaved caspase 3, pro-PARP, cleaved PARP,and cytochrome c. Our results, as detailed below, sug-

and p73-independent, apoptotic response to MMS-inducedDNA damage, which is mediated through the mitochondrialpathway.

d p h

696 e n v i r o n m e n t a l t o x i c o l o g y a n

2. Materials and methods

2.1. Materials

RPMI 1640, Dulbecco’s Modified Eagle’s Medium (DMEM)and fetal bovine serum (FBS) were purchased from GibcoInvitrogen Corp. (Gibco Laboratories, Grand Island, NY, USA).3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT), methyl methanesulfonate (MMS), 4,6-diamidino-2-phenylindole (DAPI) and rhodamine123 werepurchased from Sigma–Aldrich (St. Louis, MO, USA). Thenucleus/nucleus-free cytosol protein isolation kit was sup-plied by KeyGEN (Nanjing, China). TAp73, DNp73, caspase2 and �-actin antibodies were obtained from Santa CruzBiotechnology (Santa Cruz, CA, USA). Caspase 9 and cas-pase 3 antibodies, as well as IRDye-conjugated anti-rabbitand anti-mouse secondary antibodies were purchased fromBioworld Technology (St. Louis Park, MN, USA). Cytochromec antibody and the mitochondria-free cytosol protein iso-lation kit were supplied by Boster (Wuhan, China), andthe Annexin V-fluorescein isothiocyanate (FITC)/propidiumiodide (PI) apoptosis detection kit and �H2AX antibody wereobtained from MultiSciences Biotechnology (Hangzhou,China). FITC-conjugated anti-mouse secondary was obtainedfrom Zhongshan Biotechnology (Beijing, China).

2.2. Cell culture and treatment

H1299 cells were kindly provided by Professor Caicun Zhou(Shanghai Pulmonary Hospital, China). Hep3B cells were sup-plied by the Cell Bank of Type Culture Collection of the ChineseAcademy of Science (Shanghai, China). H1299 cells and Hep3Bcells were cultured in RPMI 1640 medium and in DMEMmedium, respectively. Both media contained 10% (v/v) FBS.The cells were cultured at 37 ◦C in a humidified 5% CO2 atmo-sphere. MMS was dissolved in sterile distilled water to make astock solution (final concentration: 800 mM). H1299 and Hep3Bcells were treated with MMS at concentrations of 50, 100, 200,400, and 800 �M. For the selection of concentration for MMS,we referred to a previous study which involved treatment ofhomozygous p53-deficient fibroblast cell line with MMS (MMSat concentrations of 0–1000 �M reduced the cell viability by0–40%) (Lackinger et al., 2001).

2.3. Assessment of cell viability

Cell viability was determined by the mitochondrial-dependentreduction of MTT as previously described (Mosmann, 1983).The cells were seeded in 96-well plates at 5 × 103 cells perwell. Twenty four hours after plating, cells were treated withthe indicated concentrations of MMS. At 2, 4, 8, 12 and 24 hafter treatment, the supernatant was removed and 100 �l(500 �g/ml) MTT solution was added to each well. After 4 hof incubation at 37 ◦C, the MTT solution from each well wasremoved by aspiration. A volume of 150 �l dimethyl sulfox-

ide (DMSO) was added and the plates were shaken for 5 minto dissolve formazan crystals. The optical density at 570 nmfor each well was determined using a microtiter plate reader(BioTek, Winooski, VT). Relative survival was represented

a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704

as the absorbance of treated sample/absorbance of controlgroup × 100%.

2.4. Measurement of cell death

The percentage of cells undergoing apoptosis was analyzedby staining with Annexin V-FITC and propidium iodide. Thecells were seeded in 6-well plates at 4 × 105 cells per well.After treatment with the indicated concentrations of MMS,cells were trypsinized and resuspended in buffer containing1% (v:v) Annexin V-FITC and 2% (v:v) propidium iodide, andincubated in the dark at room temperature for 10 min. Anal-ysis was performed using a flow cytometer (BD Biosciences,Franklin Lakes, NJ, USA), and the characteristics of cells wereanalyzed by ModFit software (Verity software House, Topsham,ME, USA).

2.5. Assessment of DNA damage

DNA damage was assessed by quantification of �H2AX foci(Kuo and Yang, 2008). Briefly, 1 × 105 cells were seeded into6-well plates that contained a glass coverslip in each well.At the end of the MMS treatment, cells were fixed with 4%paraformaldehyde for 15 min, washed with PBS, and perme-abilized by 0.2% Triton X-100. After blocking with blockingserum for 1 h, cells were incubated with a mouse monoclonalanti-�H2AX antibody (1:3000) overnight at 4 ◦C, followed byFITC-conjugated goat anti-mouse secondary antibody (1:300)for 1 h. To stain the nuclei, DAPI was added to the cells, whichwere then incubated for another 15 min. The coverslips werethen removed from the 6-well plates, mounted on glass slides,and observed with an Olympus DP70 fluorescence microscope(Olympus, Tokyo, Japan).

2.6. Measurement of mitochondrial membranepotential (MMP)

MMP was measured using the rhodamine 123 fluorescent dyeaccording to the method described previously (Han et al.,2007). Loss of MMP results in the release of rhodamine123 from the mitochondria into the cytosol and a resultingincrease in intracellular fluorescence (Satoh et al., 1997). Atthe end of the MMS treatment, a total of 1 × 106 cells were har-vested and incubated with rhodamine 123 (10 �g/�L) at 37 ◦Cfor 30 min in the dark. The rhodamine 123 stained cells werethen resuspended in PBS and analyzed directly by flow cytom-etry.

2.7. Western blotting analysis

Expression levels of caspase 2, TAp73, DNp73, cytochrome c,caspase 9, caspase 3 and PARP were analyzed by Westernblotting. After treatment with the indicated concentrationsof MMS, 1 × 107 cells were harvested for protein extraction.Nuclear, nucleus-free cytosolic and mitochondria-free pro-teins were extracted according to the manufactures’ protocols.

The whole-cell lysate was extracted with a RIPA buffer (1 MTris–HCl, 5 M NaCl, 1% Nonidet P-40, 1% sodium deoxycholate,0.05% SDS, 1 mM phenylmethyl sulfonyl fluoride). The pro-teins were denatured at 96 ◦C for 5 min after mixing with

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704 697

Fig. 1 – H1299 and Hep3B cells were treated with indicatedconcentrations of MMS for 2, 4, 8, 12 and 24 h. Cell viabilitywas determined by the MTT assay. (A) H1299 cells; (B)Hep3B cells. Mean absorption was normalized to controllevels with controls being 100%. The values representaverages of three independent experiments with sixrc

5spTnmwbMidOts

3

3

T2

Fig. 2 – H1299 and Hep3B cells were treated with indicatedconcentrations of MMS for 12 h. Cell apoptosis wasanalyzed by dual-parameter flow cytometry utilizingAnnexin V-FITC and PI. (A) Representative dot plot of H1299cells from three independent experiments; (B)representative dot plot of Hep3B cells from threeindependent experiments; (C) the results of threeindependent experiments were pooled and averaged

eplicate measurements (mean ± SD). *P < 0.05, compared toontrol group; **P < 0.01, compared to control group.

�L SDS-loading buffer, separated by 12% sodium dodecylulfate-polyacrylamide gel (SDS-PAGE) and transferred toolyvinylidene fluoride (PVDF) membranes (Millipore, USA).he membranes were then blocked with TBS containing 5%onfat milk at 4 ◦C for 1 h, incubated with the specific pri-ary antibodies (1:400 dilution for all) at 4 ◦C overnight, thenith the corresponding IRDye-conjugated secondary anti-odies (1:5000 dilution for all) at room temperature for 1 h.embranes were visualized using the Odyssey Infrared Imag-

ng System and Odyssey v1.2 (LI-COR, NE, USA). The relativeensities of the protein bands were analyzed using Quantityne software (Bio-Rad, CA, USA). The relative expressions of

arget proteins were normalized to the corresponding inten-ities of �-actin.

. Results

.1. Effect of MMS on cell viability

he cytotoxic effects of different concentrations (0, 50, 100,00, 400 and 800 �M) of MMS on H1299 and Hep3B cells were

values are shown graphically. Data are presented asmean ± SD. **P < 0.01, compared to control group.

assessed using the MTT reduction assay at 2, 4, 8, 12 and 24 hpost-treatment. As shown in Fig. 1, viabilities of H1299 (Fig. 1A)and Hep3B (Fig. 1B) cells were both significantly reduced by 400and 800 �M MMS at 12 h, and by 200, 400 and 800 �M MMS at24 h (P < 0.01). However, there was no significant change in theviability at 2, 4 and 8 h for all MMS-treated groups in the twocell lines (P > 0.05).

3.2. Effect of MMS on cell death

To determine the mode of cell death in MMS-treated H1299and Hep3B cells at 12 and 24 h, an Annexin V-FITC/PI

698 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704

Fig. 3 – H1299 and Hep3B cells were treated with the indicated concentrations of MMS for 12 h. DNA DSB induction wasexamined by assessing formation of �H2AX foci. (A) Representative images of H1299 cells from one of three independentexperiments (�H2AX: green; DAPI: blue; X200); (B) representative images of Hep3B cells from one of three independentexperiments (�H2AX: green; DAPI: blue; X200); (C) semi-quantification of �H2AX-positive cells from (A); (D)semi-quantification of �H2AX-positive cells from (B). The values represent averages of three independent experiments withtriplicate measurements, mean ± SD (n = 100 cells). **P < 0.01, compared to control group. (For interpretation of the references

b ve

to color in this figure legend, the reader is referred to the we

double-staining assay was performed using flow cytometry.By staining cells with Annexin V-FITC and PI, apoptotic cells

(Annexin V positive, PI negative) can be distinguished fromnecrotic cells (Annexin V positive, PI positive) and viable cells(Annexin V negative, PI negative). As shown in Fig. 2, highlevels of apoptotic H1299 (Fig. 2A) and Hep3B (Fig. 2B) cells

rsion of this article.)

were detected in the 400 and 800 �M MMS-treated groups at12 h, and these numbers were significantly higher than those

observed in the control groups (P < 0.01) (Fig. 2C). However, onlynecrotic H1299 and Hep3B cells were detected in 200, 400 and800 �M MMS-treated groups at 24 h (data not shown). Thesedata were consistent with the cell viability results. Based on

p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704 699

ta

3

TuetwtaqiuaHto1Mct5fo2f8oo

3

TmwFHtadgu

3

TioDPdHsi24nc

Fig. 4 – H1299 and Hep3B cells were treated with theindicated concentrations of MMS for 12 h. Themitochondrial membrane potential (MMP) was determinedusing rhodamine 123 and flow cytometry. (A)Representative dot plot of H1299 cells from threeindependent experiments; (B) representative dot plot ofHep3B cells from three independent experiments; (C) theresults of three independent experiments were pooled andaveraged values are shown graphically. Mean fluorescencewas normalized to control levels with controls being 100%.Data were presented as mean ± SD. **P < 0.01, compared to

e n v i r o n m e n t a l t o x i c o l o g y a n d

he above results, the 12 h time point was chosen for furthernalyses.

.3. Effect of MMS on DNA damage

he extent of MMS-induced DNA damage was determinedsing �H2AX foci formation as an indicator for DSB. To thisnd, the appearance of �H2AX foci in H1299 and Hep3B cellsreated with and without indicated concentrations of MMSas assessed by immunofluorescence. Fig. 3A and B shows the

ypical immunofluorescent images corresponding to H1299nd Hep3B cells, respectively. Fig. 3C and D shows the semi-uantitative representations of the number of �H2AX foci

n H1299 and Hep3B cells, respectively. We found that inntreated H1299 cells, less than 10% of cells had 10–20 foci/cellnd more than 90% of cells had 1–10 foci/cell; however, in1299 cells treated with 50, 100 and 200 �M MMS, about 50% of

he cells contained 10–20 foci/cell, 30–40% of cells containedver 20 foci/cell, and the remaining 10–20% of cells contained–10 foci/cell, and in H1299 cells treated with 400 and 800 �MMS, 30–40% of cells contained 10–20 foci/cell, 55–70% of cells

ontained over 20 foci/cell, and the remaining 5% of cells con-ained 1–10 foci/cell (Fig. 3C). For untreated Hep3B cells, about% of cells had 10–20 foci/cell and about 95% of cells had 1–10oci/cell; in Hep3B cells treated with 50 �M MMS, about 30%f cells contained 10–20 foci/cell, 10% of cells contained over0 foci/cell, and the remaining 60% of cells contained 1–10oci/cell, and in Hep3B cells treated with 100, 200, 400 and00 �M MMS, 50% of cells contained 10–20 foci/cell, 15–30%f cells contained over 20 foci/cell, and the remaining 5–20%f cells contained 1–10 foci/cell.

.4. Effect of MMS on MMP

o determine whether MMS-induced apoptotic cell death wasitochondria associated, the MMP of H1299 and Hep3B cellsas measured using rhodamine 123 staining. As shown in

ig. 4, increased rhodamine 123 positive H1299 (Fig. 4A) andep3B (Fig. 4B) cells were observed in 400 and 800 �M MMS-

reated groups. The quantitative results are shown in Fig. 4C,nd indicate that in both cell lines, the populations of rho-amine 123-stained cells in the 400 and 800 �M MMS-treatedroups were significantly higher than in the correspondingntreated groups (P < 0.01).

.5. Effect of MMS on apoptosis-associated proteins

o investigate the mechanism of MMS-induced apoptosisn the two cell lines, the distributions or/and inductionsf several apoptosis-associated proteins including TAp73,Np73 caspase 2, cytochrome c, caspase 9, caspase 3 andARP were examined by Western blotting, and the relativeensity of the bands was calculated. After treatment of1299 (Fig. 5) and Hep3B (Fig. 6) cells with MMS for 12 h,imilar results were obtained. As shown in Figs. 5A and 6A,n the nuclear extracts, an obvious decrease of pro-caspase

and a corresponding increase of cleaved caspase 2 in the00 and 800 �M MMS-treated groups was observed; in theucleus-free cytosolic extracts, the protein levels of cleavedaspase 2 were increased, while no significant change in

control group.

pro-caspase 2 levels was found in the 400 and 800 �M MMS-treated groups (Figs. 5B and 6B). On the other hand, neitherTAp73 nor DNp73 was induced by MMS treatment in either ofthe two cell lines (Figs. 5C and 6C). In the whole-cell lysate,MMS markedly increased cleaved caspase 9, caspase 3 andPARP and slightly decreased pro-caspase 9, pro-caspase 3 andpro-PARP in the 400 and 800 �M MMS-treated groups of thetwo cell lines (Figs. 5C–D and 6C–D). Similarly, exposure to 400

and 800 �M MMS of the two cell lines induced accumulationof Cytochrome c in the mitochondria-free cytosolic extracts(Figs. 5E and 6E).

700 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704

Fig. 5 – H1299 cells were treated with indicated concentrations of MMS for 12 h. Western blotting analysis was performedusing the corresponding antibodies to check expression and distribution of the proteins. (A) Pro-caspase 2 and cleavedcaspase 2 in the nucleus; (B) pro-caspase 2 and cleaved caspase 2 in nucleus-free cytosolic extracts; (C) TAp73, DNp73,pro-caspase 9, and cleaved caspase 9 in whole-cell lysate; (D) pro-caspase 3, cleaved caspase 3, pro-PARP and cleaved PARPin whole-cell lysate; (E) cytochrome c in mitochondria-free cytosolic extracts. Densitometry data of three independentexperiments, standardized by �-actin were presented below the band. Data were presented as mean ± SD. **P < 0.01,compared to control group.

Fucpiec

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r

ig. 6 – Hep3B cells were treated with indicated concentrations osing the corresponding antibodies to check expression and distaspase 2 in the nucleus; (B) pro-caspase 2 and cleaved caspase

ro-caspase 9, and cleaved Caspase 9 in whole-cell lysate; (D) prn whole-cell lysate; (E) cytochrome c in mitochondria-free cytosoxperiments, standardized by �-actin were presented below the

ompared to control group.

m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704 701

f MMS for 12 h. Western blotting analysis was performedribution of the proteins. (A) Pro-caspase 2 and cleaved2 in nucleus-free cytosolic extracts; (C) TAp73, DNp73,o-caspase 3, cleaved caspase 3, pro-PARP and cleaved PARPlic extracts. Densitometry data of three independentband. Data were presented as mean ± SD. **P < 0.01,

d p h

702 e n v i r o n m e n t a l t o x i c o l o g y a n

4. Discussion

MMS is an alkylating agent used in cancer therapy, and hasbeen reported to induce apoptosis through the activationof p53-dependent and/or -independent pathways (Lackingeret al., 2001; Ryu et al., 2001). In agreement with these studies,we found that MMS at concentrations of 400 and 800 �M sig-nificantly reduced the viability of the p53-deficient H1299 andHep3B cells 12 h post-treatment. This result was further con-firmed by flow cytometry, which demonstrated that MMS atconcentrations of 400 and 800 �M induced apoptosis in H1299and Hep3B cells after 12 h of treatment.

DSBs are one of the most serious DNA lesions, and can initi-ate genomic instability, ultimately leading to cancer (Jeggo andLobrich, 2007). Paradoxically, however, induction of DSBs isalso used to kill cancer cells, and most anticancer agents act byintroducing sufficient DSBs into the cancer cells to activate cellapoptosis (Helleday et al., 2008). Some anticancer agents cangenerate DSBs directly. However, many others (such as MMS)generate various types of non-DSB DNA damage that can ulti-mately lead to DSBs during the repair process (Takahashiand Ohnishi, 2005). �H2AX foci formation has been widelyaccepted as a sensitive marker for DSB formation, and is appli-cable even under conditions where only a few DSBs are elicited(Mah et al., 2010). Thus, �H2AX detection to determine theextent of DSB induction is a sensitive method for recogniz-ing precancerous cells (live cells with unrepaired DSB), andto evaluate the effectiveness of anticancer agents (percentageof apoptotic cells) (Sedelnikova and Bonner, 2006). Our datashowed that for both cell lines, after 12 h treatment with MMS(50–800 �M), �H2AX foci formation was induced in a dose-dependent manner. However, cell apoptosis was only inducedat higher concentrations of MMS (400–800 �M). These results,combined with the disruption of MMP, release of cytochromec from mitochondria, and cleavage of pro-caspase 9, pro-caspase 3 and pro-PARP, indicate that the accumulation ofDSBs contributes to the induction of mitochondria-dependentcell apoptosis under these experimental conditions.

p53 plays pivotal roles in connecting the DNA damageresponse to mitochondria-dependent apoptosis (Kumari et al.,2004). p73, a p53 family member, has also been predictedto act as a “functional link” due to its significant sequencesimilarity to p53. It has been suggested that the balancebetween the levels of its intracellular pro-apoptotic isoform(TAp73) and its anti-apoptotic isoform (DNp73) may play animportant role in regulating cell sensitivity to anticanceragents (Emmrich et al., 2009). However, because most of thepublished reports have only investigated the effect of p73isoforms in conditional over-expression cell models, the func-tions of p73 isoforms under basal conditions have not beenadequately investigated (Levy et al., 2008; Rosenbluth et al.,2008). In the present study, we found that MMS-inducedDNA DSBs failed to induce either TAp73 or DNp73 protein inthe two p53-deficient cell lines, indicating that MMS-inducedapoptosis is p73-independent. However, one recent studyfound that, although the protein levels of the TAp73 isoforms

(p73� and p73�) in unstressed, p53-mutated, Jurkat cells werealmost undetectable, thymoquinone (a DNA-damaging agent)induced significant increases in TAp73 protein expression,

a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704

and triggered mitochondria-dependent apoptosis (Alhosinet al., 2010). In addition, our previous studies found thatbenzo(a)pyrene-induced DNA damage activated p53 at theprotein level in p53-proficient MRC-5 cells. Interestingly, how-ever, it activated p73 only at the mRNA level in both thep53-proficient MRC-5 cells and the p53-deficient H1299 cells(Jiang et al., 2010). Together with our current results, these dataindicate that p73 and p53 may represent distinct mechanismsfor the cellular response to DNA damage, and p73 activationmight be cell-type-specific or stress-type-dependent.

In addition to the contributions made by p53 family mem-bers, caspase 2 has been reported to act as a candidatetransducer that may be involved in the transduction of theDNA damage signal to the mitochondria (Olsson et al., 2009).Activation of caspase 2 has been suggested to occur in ahigh molecular weight complex called the PIDDosome, con-taining RAIDD (RIP associated ICH/CED3 homologous proteinwith death domain) and PIDD, with the later functioning asa transcriptional target of p53 (Cuenin et al., 2008). Moreover,it has been shown that increased PIDD levels promote activa-tion of caspase-2, and that the inhibition of PIDD expressionattenuates p53-induced mitochondria-dependent apoptosis(Vakifahmetoglu et al., 2008). Thus, it has been proposedthat the caspase 2-associated apoptosis in response to DNAdamage is p53-dependent. However, in our model, in bothof the two p53 deficient cell lines, MMS-induced DSBs acti-vated nuclear caspase 2 rather than cytosolic caspase 2, asindicated by the detectable decreases of pro-caspase 2 in thenuclear fractions but undetectable changes in the cytosolicfractions. Moreover, we showed for the first time a nuclear-cytosolic translocation of active caspase 2 in response toMMS-induced DNA DSBs. This result clearly shows a p53-independent role for the nuclear caspase 2 in the regulation ofmitochondria-dependent apoptosis in response to MMS. Ourfindings are indirectly supported by the observations demon-strating that loss of either PIDD or RAIDD did not affectnuclear caspase 2 activation (Manzl et al., 2009). These pre-vious studies, together with the present data, suggest thatthere exists an alternative p53 (or PIDDosome)-independentmechanism for caspase 2 activation in the nucleus in responseto DNA damage. However, it is still unclear whether thep53-dependent and -independent mechanisms contribute tonuclear caspase 2 activation simultaneously, or if they havecell and/or DNA damage type specificity. As a matter offact, most anti-cancer drugs are genotoxic agents that elicittheir cell-killing effect through the p53-dependent apoptosispathway. Unfortunately, many tumor cells lack the func-tional p53, and thus become drug-resistant. In the currentstudy, what we found indicated that it is possible to activatethe p53-independent apoptotic pathway to reverse the drug-resistance. Detailed elucidation of such pathway will providepotentially useful targets for new drug development in cancertherapy.

In conclusion, our study demonstrates that MMS treatmentinduces DNA DSB accumulation in the p53-deficient H1299and Hep3B cells, which in turn trigger apoptosis through themitochondrial pathway. This MMS-induced mitochondria-

dependent apoptosis is associated with nuclear-cytosolictranslocation of active caspase 2, and is independent of p53and p73. Further studies need to be conducted to characterize

p h a r

tcm

C

T

A

TeNaero

r

A

B

C

C

C

C

E

G

G

e n v i r o n m e n t a l t o x i c o l o g y a n d

he p53-independent mechanisms responsible for nuclearaspase 2 activation, as well as subsequent events leading toitochondria-dependent apoptosis in response to MMS.

onflict of interest

he authors declare that they have no conflict of interest.

cknowledgements

his work was supported by grants from National Natural Sci-nce Foundation of China (No. 81172692); Zhejiang Provincialatural Science Foundation (R2100555); Ministry of Sciencend Technology, China (2009DFB30390); and Post Doctor Sci-nce Foundation of China (No. 2011M501356). J. Yang is aecipient of the Zhejiang Provincial Program for the Cultivationf High-level Innovative Health Talents.

e f e r e n c e s

lhosin, M., Abusnina, A., Achour, M., Sharif, T., Muller, C., Peluso,J., Chataigneau, T., Lugnier, C., Schini-Kerth, V.B., Bronner, C.,Fuhrmann, G., 2010. Induction of apoptosis by thymoquinonein lymphoblastic leukemia Jurkat cells is mediated by ap73-dependent pathway which targets the epigeneticintegrator UHRF1. Biochem. Pharmacol. 79, 1251–1260.

erube, C., Boucher, L.M., Ma, W., Wakeham, A., Salmena, L.,Hakem, R., Yeh, W.C., Mak, T.W., Benchimol, S., 2005.Apoptosis caused by p53-induced protein with death domain(PIDD) depends on the death adapter protein RAIDD. Proc.Natl. Acad. Sci. U.S.A. 102, 14314–14320.

hen, X., Zhu, H., Yuan, M., Fu, J., Zhou, Y., Ma, L., 2011.G-protein-coupled receptor kinase 5 phosphorylates p53 andinhibits DNA damage-induced apoptosis. J. Biol. Chem. 285,12823–12830.

oncin, N., Becker, K., Slade, N., Erster, S., Mueller-Holzner, E.,Ulmer, H., Daxenbichler, G., Zeimet, A., Zeillinger, R., Marth,C., Moll, U.M., 2004. Transdominant deltaTAp73 isoforms arefrequently up-regulated in ovarian cancer, Evidence for theirrole as epigenetic p53 inhibitors in vivo. Cancer Res. 64,2449–2460.

ostanzo, A., Merlo, P., Pediconi, N., Fulco, M., Sartorelli, V., Cole,P.A., Fontemaggi, G., Fanciulli, M., Schiltz, L., Blandino, G.,Balsano, C., Levrero, M., 2002. DNA damage-dependentacetylation of p73 dictates the selective activation ofapoptotic target genes. Mol. Cell 9, 175–186.

uenin, S., Tinel, A., Janssens, S., Tschopp, J., 2008. p53-Inducedprotein with a death domain (PIDD) isoforms differentiallyactivate nuclear factor-kappaB and caspase-2 in response togenotoxic stress. Oncogene 27, 387–396.

mmrich, S., Wang, W., John, K., Li, W., Putzer, B.M., 2009.Antisense gapmers selectively suppress individual oncogenicp73 splice isoforms and inhibit tumor growth in vivo. Mol.Cancer 8, 61.

reenwood, S.K., Armstrong, M.J., Hill, R.B., Bradt, C.I., Johnson,T.E., Hilliard, C.A., Galloway, S.M., 1998. Fewer chromosomeaberrations and earlier apoptosis induced by DNA synthesisinhibitors, a topoisomerase II inhibitor or alkylating agents in

human cells with normal compared with mutant p53. Mutat.Res. 401, 39–53.

uo, Y., Srinivasula, S.M., Druilhe, A., Fernandes-Alnemri, T.,Alnemri, E.S., 2002. Caspase-2 induces apoptosis by releasing

m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704 703

proapoptotic proteins from mitochondria. J. Biol. Chem. 277,13430–13437.

Han, Y.H., Kim, S.Z., Kim, S.H., Park, W.H., 2007. Arsenic trioxideinhibits growth of As4.1 juxtaglomerular cells via cell cyclearrest and caspase-independent apoptosis. Am. J. Physiol.Renal Physiol. 293, F511–F520.

Helleday, T., Petermann, E., Lundin, C., Hodgson, B., Sharma, R.A.,2008. DNA repair pathways as targets for cancer therapy. Nat.Rev. Cancer 8, 193–204.

Hosseinimehr, S.J., Azadbakht, M., Tanha, M., Mahmodzadeh, A.,Mohammadifar, S., 2010. Protective effect of hawthorn extractagainst genotoxicity induced by methyl methanesulfonate inhuman lymphocytes. Toxicol. Ind. Health 27, 363–369.

Jeggo, P.A., Lobrich, M., 2007. DNA double-strand breaks: theircellular and clinical impact? Oncogene 26, 7717–7719.

Jiang, Y., Rao, K., Yang, G., Chen, X., Wang, Q., Liu, A., Zheng, H.,Yuan, J., 2012. Benzo(a)pyrene induces p73 mRNA expressionand necrosis in human lung adenocarcinoma H1299 cells.Environ. Toxicol. 27, 202–210.

Kashiwazaki, H., Tonoki, H., Tada, M., Chiba, I., Shindoh, M.,Totsuka, Y., Iggo, R., Moriuchi, T., 1997. High frequency of p53mutations in human oral epithelial dysplasia and primarysquamous cell carcinoma detected by yeast functional assay.Oncogene 15, 2667–2674.

Kumari, A., Schultz, N., Helleday, T., 2004. p53 protects fromreplication-associated DNA double-strand breaks inmammalian cells. Oncogene 23, 2324–2329.

Kunz, K., Guth, O., Benoit, H.a.T., Seitz, 2010. Pesticidepyrimidinyloxy substituted phenylamidine derivatives UnitedStates Patent (Patent No: US7727986B2), 1-17.

Kuo, L.J., Yang, L.X., 2008. Gamma-H2AX – a novel biomarker forDNA double-strand breaks. In Vivo 22, 305–309.

Lackinger, D., Eichhorn, U., Kaina, B., 2001. Effect of ultravioletlight, methyl methanesulfonate and ionizing radiation on thegenotoxic response and apoptosis of mouse fibroblastslacking c-Fos, p53 or both. Mutagenesis 16, 233–241.

Levy, D., Reuven, N., Shaul, Y., 2008. A regulatory circuitcontrolling Itch-mediated p73 degradation by Runx. J. Biol.Chem. 283, 27462–27468.

Mah, L.J., El-Osta, A., Karagiannis, T.C., 2010. gammaH2AX: asensitive molecular marker of DNA damage and repair.Leukemia 24, 679–686.

Manzl, C., Krumschnabel, G., Bock, F., Sohm, B., Labi, V.,Baumgartner, F., Logette, E., Tschopp, J., Villunger, A., 2009.Caspase-2 activation in the absence of PIDDosome formation.J. Cell Biol. 185, 291–303.

Meulmeester, E., Jochemsen, A.G., 2008. p53: a guide to apoptosis.Curr. Cancer Drug Targets 8, 87–97.

Moll, U.M., Slade, N., 2004. p63 and p73: roles in development andtumor formation. Mol. Cancer Res. 2, 371–386.

Mosmann, T., 1983. Rapid colorimetric assay for cellular growthand survival: application to proliferation and cytotoxicityassays. J. Immunol. Methods 65, 55–63.

Murray-Zmijewski, F., Lane, D.P., Bourdon, J.C., 2006. p53/p63/p73isoforms: an orchestra of isoforms to harmonise celldifferentiation and response to stress. Cell Death Differ. 13,962–972.

Nieto-Rementeria, N., Perez-Yarza, G., Boyano, M.D., Apraiz, A.,Izu, R., Diaz-Perez, J.L., Asumendi, A., 2009. Bexaroteneactivates the p53/p73 pathway in human cutaneous T-celllymphoma. Br. J. Dermatol. 160, 519–526.

Olsson, M., Vakifahmetoglu, H., Abruzzo, P.M., Hogstrand, K.,Grandien, A., Zhivotovsky, B., 2009. DISC-mediated activationof caspase-2 in DNA damage-induced apoptosis. Oncogene 28,1949–1959.

Ozaki, T., Nakagawara, A., 2005. p73, a sophisticated p53 family

member in the cancer world. Cancer Sci. 96, 729–737.

Ren, J., Shi, M., Liu, R., Yang, Q.H., Johnson, T., Skarnes, W.C., Du,C., 2005. The Birc6 (Bruce) gene regulates p53 and the

d p h

Lukas, J., Bekker-Jensen, S., Bartek, J., Shiloh, Y., 2006.

704 e n v i r o n m e n t a l t o x i c o l o g y a n

mitochondrial pathway of apoptosis and is essential formouse embryonic development. Proc. Natl. Acad. Sci. U.S.A.102, 565–570.

Roos, W.P., Kaina, B., 2006. DNA damage-induced cell death byapoptosis. Trends Mol. Med. 12, 440–450.

Rosenbluth, J.M., Mays, D.J., Pino, M.F., Tang, L.J., Pietenpol, J.A.,2008. A gene signature-based approach identifies mTOR as aregulator of p73. Mol. Cell. Biol. 28, 5951–5964.

Ryu, J.C., Seo, Y.R., Smith, M.L., Han, S.S., 2001. The effect ofmethyl methanesulfonate (MMS)-induced excision repair onp53-dependent apoptosis in human lymphoid cells. Res.Commun. Mol. Pathol. Pharmacol. 109, 35–51.

Satoh, T., Enokido, Y., Aoshima, H., Uchiyama, Y., Hatanaka, H.,1997. Changes in mitochondrial membrane potential duringoxidative stress-induced apoptosis in PC12 cells. J. Neurosci.Res. 50, 413–420.

Sayan, B.S., Yang, A.L., Conforti, F., Tucci, P., Piro, M.C., Browne,G.J., Agostini, M., Bernardini, S., Knight, R.A., Mak, T.W.,Melino, G., 2010. Differential control of TAp73 and DeltaNp73protein stability by the ring finger ubiquitin ligase PIR2. Proc.Natl. Acad. Sci. U.S.A. 107, 12877–12882.

Sedelnikova, O.A., Bonner, W.M., 2006. GammaH2AX in cancercells: a potential biomarker for cancer diagnostics, predictionand recurrence. Cell Cycle 5, 2909–2913.

Shanbhag, N.M., Rafalska-Metcalf, I.U., Balane-Bolivar, C., Janicki,S.M., Greenberg, R.A., 2010. ATM-dependent chromatinchanges silence transcription in cis to DNA double-strandbreaks. Cell 141, 970–981.

Shiloh, Y., 2006. The ATM-mediated DNA-damage response:taking shape. Trends Biochem. Sci. 31, 402–410.

Sidi, S., Sanda, T., Kennedy, R.D., Hagen, A.T., Jette, C.A.,Hoffmans, R., Pascual, J., Imamura, S., Kishi, S., Amatruda, J.F.,Kanki, J.P., Green, D.R., D’Andrea, A.A., Look, A.T., 2008. Chk1suppresses a caspase-2 apoptotic response to DNA damagethat bypasses p53, Bcl-2, and caspase-3. Cell 133, 864–877.

Stiewe, T., Theseling, C.C., Putzer, B.M., 2002.Transactivation-deficient Delta TA-p73 inhibits p53 by directcompetition for DNA binding: implications for tumorigenesis.

J. Biol. Chem. 277, 14177–14185.

Suwaki, N., Klare, K., Tarsounas, M., 2011. RAD51 paralogs: rolesin DNA damage signalling, recombinational repair andtumorigenesis. Semin. Cell Dev. Biol. 22, 898–905.

a r m a c o l o g y 3 4 ( 2 0 1 2 ) 694–704

Takahashi, A., Ohnishi, T., 2005. Does gammaH2AX fociformation depend on the presence of DNA double strandbreaks? Cancer Lett. 229, 171–179.

Tamm, C., Zhivotovsky, B., Ceccatelli, S., 2008. Caspase-2activation in neural stem cells undergoing oxidativestress-induced apoptosis. Apoptosis 13, 354–363.

Tian, C., Xing, G., Xie, P., Lu, K., Nie, J., Wang, J., Li, L., Gao, M.,Zhang, L., He, F., 2009. KRAB-type zinc-finger protein Apakspecifically regulates p53-dependent apoptosis. Nat. Cell Biol.11, 580–591.

Vakifahmetoglu, H., Olsson, M., Tamm, C., Heidari, N., Orrenius,S., Zhivotovsky, B., 2008. DNA damage induces two distinctmodes of cell death in ovarian carcinomas. Cell Death Differ.15, 555–566.

van Loo, G., Saelens, X., Matthijssens, F., Schotte, P., Beyaert, R.,Declercq, W., Vandenabeele, P., 2002. Caspases are notlocalized in mitochondria during life or death. Cell DeathDiffer. 9, 1207–1211.

Vossio, S., Palescandolo, E., Pediconi, N., Moretti, F., Balsano, C.,Levrero, M., Costanzo, A., 2002. DN-p73 is activated after DNAdamage in a p53-dependent manner to regulate p53-inducedcell cycle arrest. Oncogene 21, 3796–3803.

Waxman, D.J., Schwartz, P.S., 2003. Harnessing apoptosis forimproved anticancer gene therapy. Cancer Res. 63,8563–8572.

Wyatt, M.D., Pittman, D.L., 2006. Methylating agents and DNArepair responses: methylated bases and sources of strandbreaks. Chem. Res. Toxicol. 19, 1580–1594.

Yee, K.S., Wilkinson, S., James, J., Ryan, K.M., Vousden, K.H., 2009.PUMA- and Bax-induced autophagy contributes to apoptosis.Cell Death Differ. 16, 1135–1145.

Yu, Y., Zhu, W., Diao, H., Zhou, C., Chen, F.F., Yang, J., 2006. Acomparative study of using comet assay and gammaH2AXfoci formation in the detection ofN-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage.Toxicol In Vitro 20, 959–965.

Ziv, Y., Bielopolski, D., Galanty, Y., Lukas, C., Taya, Y., Schultz, D.C.,

Chromatin relaxation in response to DNA double-strandbreaks is modulated by a novel ATM- and KAP-1 dependentpathway. Nat. Cell Biol. 8, 870–876.