The P53-Cathepsin Axis Cooperates with ROS to Activate Programmed Necrotic Death Upon DNA Damage

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The p53-cathepsin axis cooperates with ROS to activate programmed necrotic death upon DNA damage

Ho-Chou Tua,1, Decheng Rena,1, Gary X. Wanga, David Y. Chena, Todd D. Westergarda, Hyungjin Kima, Satoru Sasagawaa, James J.-D. Hsieha,b, and Emily H.-Y. Chenga,b,c,2

aDepartment of Medicine, Molecular Oncology, bSiteman Cancer Center, and cDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved November 25, 2008 (received for review August 19, 2008) Three forms of cell death have been described: apoptosis, autophagic cells that are deprived of the apoptotic gateway to mediate cyto- cell death, and necrosis. Although genetic and biochemical studies chrome c release for caspase activation (Fig. S1) (9–11, 19, 20). have formulated a detailed blueprint concerning the apoptotic net- Despite the lack of caspase activation (20), DKO cells eventually work, necrosis is generally perceived as a passive cellular demise succumb to various death signals manifesting a much slower death resulted from unmanageable physical damages. Here, we conclude an kinetics compared with wild-type cells (Fig. 1A, Fig. S2, and data active de novo genetic program underlying DNA damage-induced not shown). To investigate the mechanism(s) underlying BAX/ necrosis, thus assigning necrotic cell death as a form of ‘‘programmed BAK-independent cell death, we first examined the morphological cell death.’’ Cells deﬁcient of the essential mitochondrial apoptotic features of the dying DKO cells. Electron microscopy uncovered effectors, BAX and BAK, ultimately succumbed to DNA damage, signature characteristics of necrosis in DKO cells after DNA exhibiting signature necrotic characteristics. Importantly, this geno- damage, including the loss of plasma membrane integrity, the toxic stress-triggered necrosis was abrogated when either transcrip- spillage of intracellular contents, the swelling of organelles, the tion or translation was inhibited. We pinpointed the p53-cathepsin appearance of translucent cytosolic compartment, and the forma- axis as the quintessential framework underlying necrotic cell death. tion of intracellular vacuoles (Fig. 1B). We next determined one of p53 induces cathepsin Q that cooperates with reactive oxygen species the biochemical hallmarks of necrosis-the release of high mobility (ROS) to execute necrosis. Moreover, we presented the in vivo group box I (HMGB1) protein, a proinflammatory cytokine (21). evidence of p53-activated necrosis in tumor allografts. Current study lays the foundation for future experimental and therapeutic discov- HMGB1 functions in the nucleus as an architectural chromatin- eries aimed at ‘‘programmed necrotic death.’’ binding factor that bends DNA, stabilizes nucleosomes, and facil- itates transcription, whereas outside the cell it is a potent mediator necrosis ͉ BAX ͉ BAK ͉ apoptosis ͉ caspase-independent cell death of inflammation, cell migration and metastasis (22). Indeed, HMGB1 was released into the culture medium when DKO cells died upon DNA insults (Fig. 1C). Analyses revealed a 2-step release ells constantly interpret environmental cues before making a of HMGB1-it first translocated from the nucleus to the cytosol Ϸ8 correct life versus death decision to assure proper operation of C h after genotoxic stress (Fig. 1D), followed by the diffusion to the all biological processes (1). Impaired or overactive death results in extracellular milieu once the plasma membrane integrity was lost human illness including cancer, autoimmune disorders, neurode- C generative diseases, stroke, and myocardial infarction (2). Research (Fig. 1 ). Double staining of necrotic cells with Annexin-V and PI focused on the cell death/survival control provides a vantage point have been shown in dsRNA- and TNF-induced necrosis (ref. 23 and T. Vanden Berghe and P. Vandenabeele, personal communica- for the development of therapeutic agents that specifically trigger CELL BIOLOGY or prevent cell death (3, 4). Based on the morphological features, tions). By analogy, the dying Bax and Bak DKO cells were stained 3 forms of cell death have been described: apoptosis, autophagic cell positively with both Annexin-V and propidium iodide (Fig. S2). death, and necrosis (5–7). Among the 3 distinct forms of cell death, Although some cells displayed increased double-membrane auto- apoptosis is best studied. The evolutionarily conserved signaling phagosomes and conversion of LC3-I to LC3-II was observed (Fig. cascade, consisting of the BCL-2 family, the adaptor protein Apaf-1, S3), knockdown of Beclin1 and ATG5 provided minimal protection and the caspase family, outlines the essential apoptotic network (8). at 3 days after DNA damage (Fig. S4). Furthermore, over- The ‘‘activator’’ BH3-only molecules, BID, BIM, and PUMA, expression of BCL-2 or BCL-XL had no effects, supporting that convey apoptotic signals to trigger homo-oligomerization of mul- DNA damage-induced death in DKO cells is not caused by mito- tidomain proapoptotic BAX and BAK, which in turn permeabilize chondrion-dependent apoptosis (Fig. S5A). Remarkably, DNA mitochondria, leading to the efflux of cytochrome c, the assembly damage-induced necrosis in DKO cells was completely blocked by of apoptosome, and the ultimate activation of caspases (9–11). either actinomycin D (ActD) or cycloheximide (CHX) that inhibits Autophagy serves as either a survival or a death mechanism transcription and translation, respectively (Fig. 2 A and B). More- depending upon the context of the signaling events (12). Although over, transient cycloheximide treatment enhanced the clonogenic necrosis is generally perceived as an unavoidable consequence of extreme physicochemical stress, recent studies have indicated a molecular control of tumor necrosis factor (TNF)/Fas and DNA Author contributions: H.-C.T., D.R., and E.H.-Y.C. designed research; H.-C.T., D.R., G.X.W., alkylation induced necrosis (13–18). However, whether necrosis D.Y.C., H.K., and S.S. performed research; H.-C.T., D.R., G.X.W., D.Y.C., T.D.W., J.J.-D.H., and triggered by other intrinsic death signals is similarly regulated and E.H.-Y.C. analyzed data; and E.H.-Y.C. wrote the paper. more importantly, whether necrotic cell death recruits de novo The authors declare no conﬂict of interest. genetic programs in analogy to apoptosis remain undetermined. This article is a PNAS Direct Submission. 1 H.-C.T. and D.R. contributed equally to this work. Results 2 To whom correspondence should be addressed. E-mail: [email protected]. DNA Damage Activates a de Novo Genetic Program to Execute This article contains supporting information online at www.pnas.org/cgi/content/full/ Necrotic Death in Bax and Bak DKO Cells. To interrogate nonapo- 0808173106/DCSupplemental. ptotic cell death, we used Bax and Bak double-knockout (DKO) © 2009 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808173106 PNAS ͉ January 27, 2009 ͉ vol. 106 ͉ no. 4 ͉ 1093–1098 Downloaded by guest on September 26, 2021 p53 Transactivates Cathepsin Q to Trigger Necrotic Death in Bax and Bak DKO Cells. To pinpoint the responsible transcription factor(s) in orchestrating this necrotic program, we investigated several candi- dates known to control cell death and identified p53 as the key necrotic programmer. Overexpression of a dominant negative mutant of p53 or retrovirus-mediated stable knockdown of p53 protected DKO cells from DNA damage-induced necrosis (Fig. 2 D and E and Fig. S6). To identify the molecular signatures underlying the observed necrosis, we performed gene expressing profiling on DKO cells upon DNA damage and recognized up- regulation of several lysosomal cysteine proteases including cathepsin F, H, and Q, but not cathepsin B and L (data not shown). The observed induction of cathepsin F, H, and Q was confirmed by quantitative RT-PCR analyses (Fig. 3A). Importantly, the up- regulation of these cathepsins was abrogated when p53 was knocked down, positioning p53 in orchestrating the cathepsin program (Fig. 3A). The importance of individual cathepsins in necrosis was investigated with shRNA-mediated stable knockdown of respective cathepsins (Fig. S7). Knockdown of cathepsin Q, but not F or H, protected DKO cells from DNA damage-induced necrosis activated by etoposide (Fig. 3B). Furthermore, deficiency of cathepsin Q also protected DKO cells from UV- and camptothecin-induced necrosis (Fig. 3C and data not shown). Although cathepsin Q was barely detected by anti-cathepsin Q Western blot, DNA damage induced up-regulation of cathepsin Q protein that was readily detectable (Fig. S7D and S8A). Real time RT-PCR revealed a ubiquitous, mostly low-level expression of cathepsin Q in all mouse tissues except lung where no message was detected (Fig. S8B), which contrasts the prior reported placenta-restricted expression (24). Indeed, the expression of cathepsin Q protein could be detected in bone marrow (Fig. S8C). To directly link p53 to the transactivation of cathepsin Q, we searched for consensus p53 binding sites based on the p53MH algorithm (25) and identified one candidate p53-binding site located at the intron 1 of cathepsin Q. Indeed, intron 1 but not the promoter of cathepsin Q responded to p53-mediated transactivation (Fig. 3D). Moreover, mutation of the identified p53-binding site abolished its responsiveness to p53 (Fig. 3D). Taken together, our data uncovered an active genetic program, the p53-cathepsin Q axis, which initiates ‘‘programmed necrotic death’’ in response to DNA damage.

ROS Cooperates with p53-Cathepsin Q to Execute DNA Damage- Induced Necrotic Death. Because cathepsin Q is homologous to cathepsin L and no specific cathepsin Q inhibitors are available, we used a cathepsin L inhibitor to chemically probe the cathepsin Q executed necrosis. Cathepsin L inhibitor, z-FY-CHO, significantly inhibited necrotic death in DKO cells despite the minor toxicity associated with the chemical (Fig. 3E). Of note, other cathepsin Fig. 1. DNA damage activates necrotic death in Bax and Bak DKO cells. (A) inhibitors including E64, CA-074 Me (cathepsin B inhibitor), or Bax and Bak DKO cells eventually succumb to DNA damage in a much slower pepstatin A (cathepsin D inhibitor) did not protect DKO cells (Fig. death kinetics compared with wild-type cells. Wild-type or Bax and Bak DKO S9 A–C). Furthermore, knockdown of cathepsin L or cathepsin B cells were treated with etoposide (10 ␮g/ml) for the indicated times. Cell death provided minimal protection (Fig. S9 D–F). With this inhibitor in was quantiﬁed by Annexin-V and propidium iodide staining. Data are mean Ϯ hand, we investigated at which time point when inhibition of SD from 3 independent experiments. (B) Electron microscopy demonstrates cathepsin Q no longer provides protection. We added this inhibitor necrotic features of Bax and Bak DKO cells after treatment with etoposide (10 at 6, 12, or 24 h after etoposide treatment. Z-FY-CHO no longer ␮ g/ml) for 2 days. (C) HMGB1 is released extracellularly during DNA damage- prevented necrotic death when added at 12 h after etoposide induced necrosis. Anti-HMGB1 immunoblot was performed on cell lysates (C) treatment (Fig. 3F), which correlated with the peak of cathepsin Q or culture medium (M) after treatment with etoposide for indicated time. (D) HMGB1 translocates from nucleus to cytosol upon DNA damage. Fluorescence induction by DNA damage (Fig. S8A). These data suggest that microscopy of Bax and Bak DKO cells treated with 10 ␮g/ml etoposide for 8 h. cathepsin Q needs to be induced to execute necrotic death. How- Green, HMGB1 immunostaining; blue, Hoechst staining of DNA. ever, the Ϸ24-h lag between cathepsin Q induction and the detectable onset of cell death (Fig. 1A), and the inability of cathepsin Q overexpression alone to trigger cell death indicate the existence of survival of DKO cells in response to DNA damage (Fig. 2C). Of additional death effector(s) that cooperates with cathepsin Q to note, cycloheximide did not prevent necrotic death triggered by induce death. Because ROS is one of the most important secondary hydrogen peroxide (Fig. S5 B and C). These findings indicate the messengers implicated in necrotic death (6, 13, 14, 26), we inves- essence of de novo gene expression in executing necrotic signaling tigated whether ROS is such a candidate. Indeed, both the antiox- cascade upon DNA damage, which contrasts the general perception idant N-acetyl cysteine (NAC) and flavin-dependent oxidoreduc- of necrosis as passive, unorganized cellular demise. tase inhibitor, diphenylene iodonium (DPI), protected DKO cells

1094 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808173106 Tu et al. Downloaded by guest on September 26, 2021 Fig. 2. DNA damage activates a de novo genetic program that is orchestrated by p53 to execute necrotic death in Bax and Bak DKO cells. (A) DNA damage-induced cell death in Bax and Bak DKO cells re- quires de novo gene expression. SV40-transformed Bax and Bak DKO cells were treated with etoposide or etoposide plus actinomycin D (ActD, 1 ␮M) or etoposide plus cycloheximide (CHX, 2 ␮g/ml). Cell death was quantified by Annexin-V and propidium iodide staining the at indicated times. (B) E1A/Ras transformed Bax and Bak DKO cells were treated with etoposide (5 ␮g/ml) or etoposide plus cycloheximide (2 ␮g/ml). Cell death was quantified by Annexin-V staining after 4 days. (C) Cycloheximide enhances clonogenic survival of DKO cells in response to DNA damage. Three independent clones of SV40-transformed Bax and Bak DKO cells were treated with etoposide or etoposide plus cycloheximide for 24 h. Colonies were stained with crystal violet after 12 days. (D) A dominant negative mutant of p53 or knockdown of p53 protects DKO cells from DNA damage-induced necrotic death. SV40- transformed DKO cells transduced with control retrovirus (MIG or pSuper-Retro) or retrovirus expressing either a dominant negative mutant of p53 or shRNA against p53, were treated with etoposide for 3 days. Cell death was quantified by Annexin-V. (E) E1A/Ras transformed Bax and Bak DKO cells transduced with control retrovirus (MIG) or retrovirus expressing a dominant negative mutant of p53, were treated with etoposide (5 ␮g/ml) for the indicated times. Cell death was quantified by Annexin-V. Data in A, B, D, and E are mean Ϯ SD from 3 independent experiments. *, P Ͻ 0.01.

from etoposide-induced necrosis (Fig. 4A). More importantly, DPI any discernable features of apoptosis were evident (Fig. S11). On further protected DKO cells with stable knockdown of cathepsin Q the contrary, HMGB1 remained in the nucleus upon apoptosis from necrotic death (Fig. 4B), supporting the notion that cathepsin triggered by staurosporine (Fig. S11). These data suggest that Q and ROS are the necrotic death effectors. Although p53 was wild-type cells are equipped to mount a full-blown necrotic event reported to up-regulate a series of redox genes to produce ROS once extreme damages compromise the integrity of the plasma (27), expression of p53 DN or knockdown of p53 did not prevent membrane. Since there is no direct human orthologues of cathepsin the accumulation of ROS in DKO cells upon genotoxic stress (Fig. Q been recognized so far, we used protease inhibitors to probe S10 and data not shown). By contrast, both cycloheximide and DPI whether the same de novo necrotic pathway operates in human significantly reduced the ROS production (Fig. S10B). Accordingly, cells. Indeed, the combination of caspase inhibitor (z-VAD-FMK) CELL BIOLOGY DPI further protected cells expressing p53 DN from necrotic death and cathepsin L inhibitor (Z-FY-CHO) provided a synergistic (Fig. 4C). DPI also protected Bax, Bak and p53 triple knockout protection of DNA damage induced cell death in human cancer cell MEFs (data not shown). In summary, our data are consistent with lines, including A549 (lung carcinoma) and U031 (renal cell carci- a model in which p53 coordinates the induction of cathepsin Q that noma) (Fig. 4 E and F and Fig. S12A). The combination of caspase cooperates with ROS to execute ‘‘programmed necrotic death’’ inhibitor (z-VAD-FMK) and cathepsin L inhibitor (Z-FY-CHO) also provided a synergistic protection of DNA damage induced cell (Fig. 5D). death in E1A/Ras transformed wild-type MEFs (Fig. S12B). These data indicate that apoptotic and necrotic programs work together DNA Damage-Induced Necrotic Program Is Present in Wild-Type Cells. to execute cellular demise. As apoptotic machinery efficiently executes cellular demise, the necrotic component is easily masked in cells with functional BAX Chemotherapy Induces p53-Dependent Necrotic Death in Tumor Al- or BAK. To investigate DNA damage-induced necrosis in the lografts. To demonstrate the DNA damage-activated, p53- presence of apoptosis, cathepsin Q was knockdowned in wild-type dependent necrotic death in vivo, we examined chemotherapy- cells. As expected, knockdown of cathepsin Q provided minor yet induced cell death in tumor allografts using E1A/Ras transformed significant protection of wild-type cells against DNA damage- DKO MEFs. Consistent with our in vitro studies, etoposide induced induced cell death (Fig. 4D). These findings are consistent with the necrosis in tumors derived from DKO cells expressing GFP control fact that apoptosis dominates over necrosis with its high efficiency but not p53 DN (Fig. 5A). Accordingly, the tumor volume was and fast kinetics. It was reported that HMGB1 binds tightly and significantly lower in tumors derived from control DKO cells than irreversibly to the condensed chromatin of apoptotic cells such that those from p53 DN-expressing DKO cells in response to etoposide its extracellular release is only observed in necrotic but not apo- treatment (Fig. 5B). All of the tumors were within the same size ptotic cells (22). Our observation of early cytoplasmic translocation range before chemotherapy (Fig. 5C). of HMGB1 in DKO cells after DNA damage raises a possibility that HMGB1 might translocate to the cytosol before chromatin con- Discussion densation in wild-type cells. Similar to DKO cells, wild-type cells Emerging evidence indicates that in response to a given death also exhibited early cytoplasmic translocation of HMGB1 before stimulus, there is often a continuum of apoptosis and necrosis (6,

Tu et al. PNAS ͉ January 27, 2009 ͉ vol. 106 ͉ no. 4 ͉ 1095 Downloaded by guest on September 26, 2021 Fig. 3. p53 transactivates cathepsin Q to trigger necrotic death in Bax and Bak DKO cells. (A) p53-dependent transactivation of cathepsin expression upon DNA damage. DKO cells with or without p53 knockdown were treated with etoposide for 6 h. Levels of various cathepsins were analyzed by qRT-PCR and presentedas mean Ϯ SD. (B) DKO cells transduced with control retrovirus (pSuper-Retro) or retrovirus expressing scrambled shRNA or shRNA against indicated cathepsins were treated with etoposide for 3 days. (C) DKO cells transduced with retrovirus expressing scrambled shRNA or shRNA against cathepsin Q were irradiated with UV and cell death was assessed at day 4. (D) Intron 1 of cathepsin Q is responsive to p53. Luciferase reporter constructs containing the intron 1 of cathepsin Q (CtsQ I1-Luc), the intron 1 of cathepsin Q with a mutated p53 binding site (CtsQ I1m-Luc), or the promoter of cathepsin Q (CtsQ prom-Luc) were assayed for transactivation by wild-type p53 in Saos-2 cells 24 h after transfection with the indicated reporters and either p53 or vector control. Data are presented as mean fold activation Ϯ SD from 3 independent experiments. (E) Necrotic death in Bax and Bak DKO cells can be blocked by a cathepsin L inhibitor. Bax and Bak DKO cells were treated with etoposide or etoposide plus z-FY-CHO (100 ␮M) for 3 days. (F) Bax and Bak DKO cells were treated with etoposide, followed by addition of z-FY-CHO (100 ␮M) at 0, 6, 12, or 24 h. Cell death was assessed after 3 days. Cell death was quantiﬁed by Annexin-V. Data are mean Ϯ SD from 3 independent experiments. *, P Ͻ 0.01.

26). Many insults induce apoptosis at lower doses and necrosis at and necrotic features in neurons (6). Here, we demonstrated that higher doses. Certain death stimuli such as ischemia-reperfusion DNA damage activates p53 to trigger a necrotic program (Fig. 5D) injury and excitotoxicity even concurrently induce both apoptotic in addition to the well-characterized mitochondrion-dependent

Fig. 4. ROS cooperates with p53-Cathepsin Q to execute DNA damage-induced necrotic death and DNA damage-induced necrotic program is present in wild-type cells. (A) Bax and Bak DKO cells were treated with etoposide or etoposide plus N-acetylcysteine (NAC, 20 mM) or etoposide plus diphenylene iodonium (DPI, 100 ␮M) for indicated time. *, P Ͻ 0.01. (B) DKO cells transduced with retrovirus expressing scrambled shRNA or shRNA against cathepsin Q were treated with etoposide or etoposide plus diphenylene iodonium for 4 days. *, P Ͻ 0.01 between CtsQ KD and CtsQ KD plus DPI or between DPI and DPI plus CtsQ KD. (C) DKO cells transduced with control retrovirus (MIG or pSuper-Retro) or retrovirus expressing a dominant negative mutant of p53, were treated with etoposide or etoposide plus diphenylene iodonium for 4 days. *, P Ͻ 0.01. (D) Knockdown of cathepsin Q protects wild-type cells from DNA damage-induced cell death. Wild-type MEFs with or without cathepsin Q knockdown were treated with etoposide for 18 h. *, P Ͻ 0.01. (E) A549 cells were irradiated with UV plus the indicated caspase (z-VAD-FMK, 50 ␮M) or cathepsin inhibitors (z-FY-CHO, 100 ␮M) and cell death was assessed after 4 days. *, P Ͻ 0.01. (F) U031 cells were irradiated with UV plus the indicated caspase (z-VAD-FMK, 50 ␮M) or cathepsin inhibitors (z-FY-CHO, 50 ␮M) and cell death was assessed after 3 days. *, P Ͻ 0.01. Cell death was quantiﬁed by Annexin-V. Data are mean Ϯ SD from 3 independent experiments.

1096 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0808173106 Tu et al. Downloaded by guest on September 26, 2021 DKO cells did not lose Lysotracker staining until they became AV-positive, indicating that LMP is a consequence of dying (data not shown). In contrast, cathepsin Q appears to initiate the necrotic program before the appearance of discernible dying features (Fig. 3F). Although lysosomal cysteine cathepsins were believed to be involved primarily in intralysosomal protein degradation, genetic studies using knockout mouse models start to uncover their important roles in various biological processes (34, 35). Cathepsin B is important in TNF-induced apoptosis of hepatocytes, cathepsin K in bone remodeling, cathepsin S in MHC Class II antigen presen- tation, cathepsin C in the activation of granzyme A and B, and cathepsin L in positive selection for CD4ϩ T cells and terminal differentiation of keratinocytes (34, 35). Cathepsin Q is clearly not a placenta-restricted protease and is induced by DNA damage through p53. How cathepsin Q executes necrotic death and what is the human counterpart of mouse cathepsin Q require further experimentation. Modern biomedical research profoundly impacts the development of targeted therapeutics. Indeed, the studies on apoptosis have led to the development of drugs that modulate apoptosis in treating human illness (3, 4). Recently, a small molecule inhibitor of RIP1 kinase, necrostatin-1, was reported to provide protection in a murine ischemic brain injury model (36, 37). Of note, necrostatin-1 failed to prevent DNA damage-induced necrotic death in DKO cells (Fig. S12D), suggesting that DNA damage triggered necrotic program is different from ‘‘necroptosis’’ that is activated upon death receptor signaling (36). Further elucidation of the necrotic signaling cascades will certainly enable targeted therapeutics in eliminating cancer cells that evade apoptosis (38). Noticeably, Fig. 5. In vivo evidence of p53-activated necrotic death upon DNA damage. the BAX/BAK-independent necrotic program appears to be more (A) Electron microscopy demonstrates necrotic features in the tumor allo- pronounced in transformed cells, suggesting that this pathway could grafts derived from Bax and Bak DKO cells expressing GFP but not p53 DN after be a promising cancer-specific target. Our study demonstrates a treatment with etoposide. (B) Tumor weights from groups of 7 mice injected p53-initiated genetic program in executing ‘‘programmed necrotic with Bax and Bak DKO cells expressing GFP or p53 DN after etoposide treat- death,’’ which assigns DNA damage-induced necrosis as a form of ment. *, P ϭ 0.0011 (n ϭ 7 vs. 7). (C) Tumor volume from groups of 7 mice ‘‘programmed cell death’’ and opens a new avenue for rationally injected with Bax and Bak DKO cells expressing GFP or a dominant negative designed therapeutics aimed at the necrotic pathways. mutant of p53 before etoposide treatment. (D) Model depicts that DNA damage activates the p53-cathepsin axis and ROS to execute ‘‘programmed Materials and Methods necrotic death.’’ Plasmid Construction and Retrovirus Production. Retrovirus-mediated knockdown constructs were generated using pSuper-Retro-Puro or pSuperior-Retro-Puro according to the manufacture’s instruction (Oligoengine). A dominant negative apoptotic program (28, 29). Necrosis is clearly a fail-safe cell death mutant of p53 (amino acids 1–14 and amino acids 303–390) was dually tagged with mechanism for apoptosis. Together, they ensure the clearance of FLAG and HA at the N-terminus and cloned into MSCV-IRES-GFP (pMIG) or MSCV-

damaged cells to avoid accumulation of detrimental mutations. Puro (Clontech). The production of retroviruses was described in ref. 10. The pro- CELL BIOLOGY This further highlights the paramount importance of p53 as the moter (Ϫ2150 to ϩ 51) and Intron 1 of cathepsin Q were cloned into pGL2-basic and guardian of genome stability and a tumor suppressor (30, 31). DNA pGL2-promoter (Promega), respectively. The mutation of the p53 binding site iden- damage triggered by topoisomerase inhibitors (etoposide and tified at the intron I of cathepsin Q was generate by PCR based site-directed mu- camptothecin) or UV apparently activates a necrotic signaling tagenesis. The sequence was changed from ‘‘agaCatGcccaccaCttGtta’’ to ‘‘agaTatTc- ccaccaTttTtta.’’ All of the constructs were confirmed by DNA sequencing. The target pathway that is different from necrosis induced by DNA alkylating sequences of shRNA are listed in SI Materials and Methods. agents. DNA alkylation-induced necrosis does not involve p53 nor recruits a de novo genetic program (18). Consistent with a previous Antibodies, Reagents, and Immunoblot Analysis. Antibodies and reagents are report (18), PARP inhibitors protected DKO cell from necrosis listed in SI Materials and Methods. Cell lysates were resolved by 10% or 4–12% induced by alkylating DNA damage but not by DNA double-strand NuPAGE (Invitrogen) gels, transferred onto PVDF membrane (Immobilon-P; Mil- break (Fig. S12C). Moreover, neither knockdown of cathepsin Q lipore). Antibody detection was accomplished using enhanced chemilumines- nor z-FY-CHO provided protection against alkylating DNA dam- cence method (Western Lightning, PerkinElmer) and LAS-3000 Imaging system age (data not shown). Interestingly, a recent study indicated a role (FUJIFILM). of DRAM (damage-regulated autophagy modulator), a p53 down- Cell Culture and Viability Assay. Mouse embryonic fibroblasts (MEFs) were stream autophagy inducer, in assisting p53-initiated apoptosis (32). generated from E13.5 embryos. The 3 clones used in Fig. 2C were derived from 3 By contrast, deficiency of ATG5 or Beclin1 provided minimal different Bax and Bak DKO embryos. SV40 transformation was performed as protection against necrosis in DKO cells of which the mitochon- described in ref. 10. For E1A/Ras transformation, primary DKO MEFs were in- drion-dependent apoptotic machinery is disabled (Fig. S4). fected with retrovirus expressing E1A-IRES-Ras as described in ref. 39, followed by Lysosomes were described as ‘‘suicide bags’’ of the cells by the selection under 2 ␮g/ml puromycin. Pools of puromycin-resistant cells were used discoverer, De Duve, due the inherent danger of releasing hydro- by the indicated experiments. Saos-2 osteosarcoma cells were maintained in lytic enzymes through lysosomal membrane permeabilization McCoy’s 5A medium supplemented with 15% FBS. A549 cells and U031 cells were maintained in RPMI-1640 medium supplemented with 10% FBS. For UV-induced (LMP) (33). Our observation that knockdown of cathepsin Q but cell death, DKO cells were irradiated with UV-C (1500 J/m2) at day 0 and day 1 and not other lysosomal proteases such as cathepsin B and L blocked cell death was assessed at day 4; A549 cells were irradiated with UV-C (250 J/m2) DNA damage-induced necrotic death in DKO cells suggests that at day 0 and day 1 and cell death was assessed at day 4; and U031 cells were LMP is unlikely an initiating factor of death. Indeed, we found that irradiated with UV-C (500 J/m2), and cell death was assessed after 3 days. Cell

Tu et al. PNAS ͉ January 27, 2009 ͉ vol. 106 ͉ no. 4 ͉ 1097 Downloaded by guest on September 26, 2021 death was quantified by Annexin-V (BioVision) or propidium iodide (Sigma) Luciferase Assay. Saos-2 cells, plated in a 6-well plate (3 ϫ 105 cells per well), staining according to manufacturer’s protocols, followed by flow cytometric were transfected with 1 ␮g of the indicated reporter constructs, 1 ␮gof analyses using a FACSCalibur (BD Biosciences) and CellQuest Pro Software. P lacZ-expressing CH110 (Amersham Pharmacia), or 50 ng of mouse p53 cloned values for statistical analyses were obtained using Student’s t test. into pCDNA3 (Invitrogen) using FUGENE6 (Roche) according to the manufacturer’s protocol. Cells were harvested and lysed in cell lysis buffer (BD Bio- sciences) at 24 h after transfection. Cell lysates were assayed for luciferase Clonogenic Assays. SV40-transformed DKO cells were treated with etoposide (10 activity using an Enhanced Luciferase Assay Kit (BD Biosciences). The data ␮ ␮ 4 ϫ 4 g/ml) or etoposide plus cycloheximide (2 g/ml) for 24 h. A total of 10 or 2 10 cells obtained by a luminometer were normalized for the transfection efficiency were washed with PBS and plated in 24-well tissue culture plates. Colonies were fixed based on the beta-galactosidase activity. with methanol and stained with 0.5% crystal violet in 25% methanol after 12 days. Electron Microscopy. Electron Microscopy was performed by the Electron Measurement of ROS. Production of ROS was monitored by flow cytometric Microscopy Facility at the Department of Cell Biology and Physiology, Wash- analyses using the redox-sensitive dye, 2Ј,7Ј-dichlorofluorescein diacetate ington University, St. Louis. Cells were thin sectioned on a Reichert-Jung (H2DCF-DA, Invitrogen). Cells were incubated with 1 ␮MH2DCF-DA at 37 °C for Ultracut, poststained in uranyl acetate and lead citrate, viewed on a Zeiss 902 30 min, followed by flow cytometric analyses using a FACSCalibur (BD Bio- Electron Microscope, and recorded with Kodak E.M. film. sciences). Mean fluorescence detected by FL1 channel was assessed by FlowJo (Tree Star). Fold increase of ROS was determined by dividing the mean Indirect Immunofluorescence Microscopy. Cells, fixed in 4% paraformaldehyde fluorescence after etoposide treatment for 2 days with the untreated controls. and permeabilized with 0.1% Triton X-100, were sequentially incubated with anti-HMGB1 antibody (BD Biosciences), Alexa Fluro488 conjugated goat anti- rabbit secondary antibody (Invitrogen), and Hoechst 33342 (Invitrogen). Im- Reverse-Transcription and Quantitative PCR. Total RNA was extracted from cells ages were acquired with a SPOT camera (Diagnostics Instruments) mounted using TRIZOL (Invitrogen) according to the manufacturer’s instruction. Reverse on an Olympus EX51 microscope. transcription was performed with oligo(dT) plus random decamer primers (Am- bion) using SuperScript II (Invitrogen). Quantitative PCR was performed with Tumor Allografts. Tumors were induced in 5-week-old NOD/SCID/IL2Ry-null SYBR green master mix (Applied Biosystems) in duplicates using indicated gene mice by s.c. injection of 107 E1A/Ras transformed Bax and Bak DKO MEFs specific primers (listed in SI Materials and Methods). Quantitative PCR was infected with control retrovirus (GFP) or retrovirus expressing a dominant performed on an ABI Prism 7300 sequence detection system (Applied Biosystems). negative mutant of p53. Before injection, cells were resuspended in DMEM Data were analyzed as described previously by normalization against GAPDH with 33.3% Matrigel. After 13–17 days, tumors reached an average size of (39). GAPDH was detected using a rodent-specific GAPDH Taqman probe (Ap- 80–150 mm3. Tumor size was measured in 2 dimensions by caliper and volume plied Biosystems). was calculated as (4␲/3) ϫ (width/2)2 ϫ (length/2). Etoposide was administered by i.p. injection at 15 mg/kg/day for 6 or 7 consecutive days (3 groups for 6 days HMGB1 Release Assay. Cells with or without etoposide (10 ␮g/ml) treatment and 4 groups for 7 days). Finally, the tumors were removed and subjected to histological (hematoxylin and eosin staining) or electron microscopic analyses. grown in DMEM plus 1% FBS were pelleted and lysed in PBS containing 1% Electron Microscopy was performed by the Electron Microscopy Facility at the Triton X-100 plus complete protease inhibitors (Roche Applied Science). Me- Department of Pathology and Immunology, Washington University in St. dium was harvested and spun at 800 ϫ g for 5 min of which the supernatant Louis. was then filtered through a 0.45-␮m centrifugal filter device (Millipore) to remove cell debris. Proteins in the filtrates were precipitated with trichloro- ACKNOWLEDGMENTS. This work was supported by National Cancer Institute/ acetic acid (Sigma). Proteins from either cell lysates or cell-free-medium were National Institutes of Health Grants K01CA98320 and R01CA125562 (to E.H.- analyzed by anti-HMGB1 Western blots. Y.C.) and the Searle Scholars Program.

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