Serpinb2 Protection of Retinoblastoma Protein from Calpain Enhances Tumor Cell Survival

Research Article

SerpinB2 Protection of Retinoblastoma Protein from Calpain Enhances Tumor Cell Survival

Laura Tonnetti,1 Sarah Netzel-Arnett,1 Grant A. Darnell,2 Tamara Hayes,1 Marguerite S. Buzza,1 Ian E. Anglin,1 Andreas Suhrbier,2 and Toni M. Antalis1

1Center for Vascular and Inflammatory Diseases and the Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland and 2Immunovirology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland, Australia

Abstract antiapoptotic activities of Rb include its ability to repress E2F gene transcription (5) and its direct inhibition of proapoptotic The tumor suppressor retinoblastoma protein (Rb) plays a signal transduction (1, 6). Rb ablation leads to up-regulation of pivotal role in the regulation of cell proliferation and E2F, which can sensitize cells to apoptosis (7). Gene expression sensitivity to apoptosis through binding to E2F transcription profiling studies and direct transcription experiments show that factors. Loss of Rb in response to genotoxic stress or up-regulation of E2F1 induces transcription of cell cycle genes, inflammatory cytokines can enhance cell death,in part,by such as the G -S cyclins, and also genes encoding proapoptotic eliminating Rb-mediated repression of proapoptotic gene 1 cell-death machinery, including Apaf-1, p73, procaspase-3, and transcription. Here we show that calpain cleavage of Rb procaspase-7 (8, 9). Because Rb is capable of regulating the facilitates Rb loss by proteasome degradation and that this expression of cell cycle and apoptotic gene targets, additional may occur during tumor necrosis factor A–induced apoptosis. factors in association with Rb likely contribute to ultimate cell fate The cytoprotective,Rb-binding protein SerpinB2 (plasmino- decisions. gen activator inhibitor type 2) protects Rb from calpain The activities of Rb are determined by the cellular proteins that cleavage,increasing Rb levels and enhancing cell survival. interact with Rb and the functional consequences of these Chromatin immunoprecipitation assays show that the interactions. We recently identified the intracellular serine protease increased Rb levels selectively enhance Rb repression of inhibitor SerpinB2 (also called plasminogen activator inhibitor proapoptotic gene transcription. This cytoprotective role of type 2) as a Rb-binding protein that colocalizes with Rb in the SerpinB2 is illustrated by reduced susceptibility of SerpinB2- nucleus and protects Rb from proteolytic degradation, resulting in deficient mice to multistage skin carcinogenesis,where Rb- enhanced Rb protein levels (10). SerpinB2 is a multifunctional dependent cell proliferation competes with apoptosis during protein of the serine protease inhibitor (serpin) family, which is initiation of papilloma development. These data identify synthesized by a variety of cells (11), and promotes cell survival SerpinB2 as a cell survival factor that modulates Rb (12–14). Transgenic overexpression of SerpinB2 in proliferating repression of proapoptotic signal transduction and define a basal keratinocytes of mice inhibits apoptosis and promotes new posttranslational mechanism for selective regulation of keratinocyte survival during skin carcinogenesis (15). SerpinB2 the intracellular levels of Rb. [Cancer Res 2008;68(14):5648–57] protects cells from the cytolytic effects of cytopathic viruses (16, 17) and mycobacterial infection (18, 19) and confers resistance to death Introduction induced by the inflammatory cytokine tumor necrosis factor a The retinoblastoma tumor suppressor protein (Rb), the product (TNFa). SerpinB2 biosynthesis is an acute-phase response to TNFa of the Rb1 susceptibility gene, was the first tumor suppressor gene (20) and SerpinB2 inhibits the characteristic morphologic changes to be identified and has emerged to play a central role in limiting and DNA fragmentation patterns associated with TNFa-induced cell cycle progression through regulation of the E2F family of apoptosis (18). Although the cell survival activity of SerpinB2 is now transcription factors. In addition to its antiproliferative function, well established, the molecular mechanism of SerpinB2 cytopro- Rb possesses prosurvival activity, which is mediated by the ability tection is not known. SerpinB2 protection from TNFa apoptosis of Rb to suppress apoptosis directly, independent of growth cannot be explained by loss of TNFa receptors, impaired ability of suppression (1, 2). In mice, genetic deletion of Rb1 results in TNFa to bind to receptors, impaired TNFa receptor signal excessive apoptosis associated with abnormal degeneration of transduction, or direct inhibition of caspases (18). neurons and lens fiber cells (3). Rb1 inactivation in epidermis Ablation of Rb through either inactivation or protease-induced causes increased apoptosis at an early stage of epidermal degradation is associated with apoptosis initiated by genotoxic cell oncogenic progression, rendering mice less susceptible to skin stress or TNFa (6). To date, only a few proteolytic mechanisms carcinogenesis (4). Mechanisms implicated in mediating the targeting Rb degradation have been identified. Rb may be targeted by viral oncoproteins, including the human papillomavirus E7, which accelerate Rb degradation by proteasomes (21, 22). Alternatively, in response to death receptor signals, Rb may be Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). cleaved and inactivated through the action of caspases (23). L. Tonnetti and S. Netzel-Arnett contributed equally to this work. Cleavage of Rb at the major caspase consensus DEAD886-G site Current address for L. Tonnetti: Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. within the COOH terminus of Rb, and its subsequent degradation, Requests for reprints: Toni M. Antalis, University of Maryland School of Medicine, occurs during TNF receptor I–induced apoptosis (24). Mice Biopark Building One, Room 220, 800 West Baltimore Street, Baltimore, MD 21201. engineered to express the caspase-resistant Rb mutant (Rb-MI) Phone: 410-706-8222; Fax: 410-706-8121; E-mail: [email protected]. a I2008 American Association for Cancer Research. show resistance to TNF -induced apoptosis in several tissues (25). doi:10.1158/0008-5472.CAN-07-5850 Notably, caspase-resistant Rb could be eliminated under certain

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Tumor Survival by Protection of Rb from Calpain experimental conditions, suggesting the existence of additional (29). Assay of calpain activity using the fluorogenic peptide substrate (Suc- intracellular proteolytic mechanisms for eliminating Rb. Leu-Tyr-AMC) is described in Supplementary data. Calpains are Ca2+ dependent, nucleocytoplasmic cysteine pro- Transfection. Human SerpinB2 in pRC/cytomegalovirus (CMV) has teases that have emerged to play important roles in cell death been described previously (12, 18). The mammalian expression vector encoding full-length CAPN4 was purchased from ATCC (IMAGE clone no. signaling via the cleavage and/or degradation of a number of 8578411 cloned into pCMV-SPORT6). The Rb-810 W mutant was generated regulatory proteins and transcription factors (26, 27). The calpain from pcDNA3.1-Rb by site-directed mutagenesis. Primer sequences are A À À family includes the ubiquitously expressed calpain-1 ( -calpain) given in Supplementary data. Transient transfections in CAPN4 / cells and calpain-2 (m-calpain), as well as a number of tissue-specific were done with the Amaxa Nucleofector Kit R (Program U-30). HeLa calpains (26). Both calpain-1 and calpain-2 are heterodimers cells were transfected using Lipofectamine 2000 (Invitrogen). Stable clonal containing a large f80-kDa subunit, encoded by the genes capn1 cell lines were selected with hygromycin B (Invitrogen). and capn2, respectively, and a common 28-kDa regulatory small TNFA-induced apoptosis. Cells were treated with 10 ng/mLTNF a subunit, encoded by capn4. Here we report that Rb is a calpain (R&D Systems) in the presence of 10 Ag/mLcycloheximide or with substrate. Calpain cleavage of Rb precedes proteasome degradation cycloheximide alone. Cell death was assessed after the indicated time by and participates in the regulation of Rb turnover. We identify the counting live cells using trypan blue exclusion, by flow cytometry after propidium iodide staining, or by immunoblotting for procaspase-3 calpain cleavage site within the COOH-terminal domain of Rb, activation and PARP cleavage. which overlaps the SerpinB2 binding site contained within this Chromatin immunoprecipitation assays. Chromatin immunoprecipi- domain (10). SerpinB2 blocks calpain cleavage of Rb, enhancing Rb tation was done using the Chromatin Immunoprecipitation Assay Kit levels and Rb antiapoptotic activities including repression of (Upstate). After stimulation, cells were fixed in formaldehyde at a final proapoptotic gene transcription and promotion of keratinocyte concentration of 1%. Chromatin was sheared by sonication (4 Â 10 s at 30% survival. We conclude that SerpinB2 functions as a cytoprotective maximum potency). Immunoprecipitations were done at 4jC overnight factor that influences cell death signaling pathways through Rb with anti-Rb mAb (G3-245). Immune complexes were collected with protein protection. A and protein-DNA cross-links were reverted by heating at 65jCfor6h. Immunoprecipitated DNA was isolated and used for PCR amplification. The sequences of the promoter-specific primers used are given in Supplemen- Materials and Methods tary data. Cell culture. HeLa cells and the HeLa cell line stably expressing SerpinB2 Quantitative PCR. RNA was extracted with the RNeasy mini kit À À (S1a) have previously been described (18). SV40-transformed CAPN4 / and (Qiagen). For quantitative real-time PCR, specific primers and fluorescence- CAPN4+/+ mouse embryonic fibroblasts (MEF) were provided by Dr. John labeled probes for cyclin A2 (Hs 00153138_m1), p73 (Hs 00232088_m1), Elce (Queen’s University, Kingston, Canada; ref. 28). Jurkat cells and thymidine kinase (Hs 00177406_m1), caspase-7 (Hs 00169152_m1), caspase- RAW264.7 cells were obtained from the American Type Culture Collection 8 (Hs 00154256_m1), caspase-10 (Hs 00154268_m1), or h-actin (Hs (ATCC). Cell viability and cell cycle analyses were done by flow cytometry 99999903_m1) were obtained from Applied Biosystems Assay-on-Demand following propidium iodide staining. Gene Expression products and assayed using the ABI PRISM 7900HT Rb cleavage assays. For cell-free assays, Jurkat lysates were incubated at sequence detector system. À À 30jC for 1 h with the indicated enzymes and inhibitors. For in vitro Rb Chemical carcinogenesis. The SerpinB2 / mice on the C57BL/6 cleavage, purified recombinant Rb (QED Biosciences) was incubated with background (backcrossed seven generations) were provided by D. Ginsburg calpain-1 (>90% purity) or recombinant calpain-2 (>98% purity; Calbiochem) (Department of Internal Medicine and Howard Hughes Medical Institute, in the presence of 25 mmol/LHEPES (pH 7.4), 5 mmol/LCaCl 2, 1 mmol/L University of Michigan, Ann Arbor, MI; ref. 30). Both gender-matched wild- DTT for 15 min at 30jC. Where indicated, Rb was incubated with caspase-3 type littermates from heterozygote crosses and C57BL/6 mice (The Jackson (R&D Systems), calpain inhibitor II or III (Calbiochem), or PD145305 or Laboratory) were used as controls. For the skin carcinogenesis experiments, PD150606 (Calbiochem), or preincubated with recombinant human the dorsal area of 8- to 14-wk-old mice were shaved and treated with a single SerpinB2 (Biotech Australia, Pty. Ltd.). application of 7,12-dimethylbenz(a)anthracene (DMBA; 25 Ag) followed by Immunoblot analyses. Lysates were prepared in ice-cold lysis buffer promotion with phorbol-12-myristate-13-acetate (PMA; 12.5 Ag), twice [50 mmol/LTris (pH 7.4), 2 mmol/LEDTA, 150 mmol/LNaCl, 1% NP40] weekly (experiment 1) or thrice weekly for 3 wk and then twice weekly with protease inhibitor cocktail (Roche). To dephosphorylate Rb proteins, (experiment 2). Mice were monitored weekly for formation of papillomas all lysates were incubated with 10 units of calf intestine alkaline (z1 mm). No papillomas formed as a result of treatment with vehicle alone. phosphatase (New England Biolabs) at 37jC for 30 min. These conditions Animals were maintained and all experiments done under approved Uni- were sufficient to completely dephosphorylate all Rb species, verified with versity of Maryland Institutional Animal Care and Use Committee protocols. anti–phospho-Rb antibodies (not shown). Immunoblotting was done as Immunohistochemistry on formalin-fixed tissues was done using described (18) with antibodies specific for calpain-1 (2H2D7C2), calpain-2 standard protocols with antibodies specific for keratin 5 (AF-138, Covance) (107-82), and the calpain small subunit (8E9) (Calbiochem); glyceraldehyde- and murine SerpinB2 (protein G affinity-purified rabbit polyclonal gene- 3-phosphate dehydrogenase (GAPDH) and Rb (C-15; detecting the COOH rated as described; ref. 30). Apoptosis was monitored using the DeadEnd terminus of Rb amino acids 913–928) (Santa Cruz); caspase-3, caspase-7, Colorimetric terminal deoxyribonucleotidyl transferase–mediated dUTP caspase-8, caspase-9, and poly(ADP-ribose) polymerase (PARP) (Cell nick end labeling (TUNEL) system (Promega). Signaling); SerpinB2 monoclonal antibody (mAb) (American Diagnostica); Statistical analyses. Student’s t test was used to compare averages of and Rb (G3-245; detecting Rb amino acids 332–344) and G99-2005 normally distributed data with equal variance. m2 analysis was used for

(detecting the NH2 terminus of Rb amino acids 1–240) (BD Bioscience). analysis of frequency distributions. A threshold of P < 0.05 was considered NH2-terminal amino acid sequence analysis. Purified recombinant Rb significant. was incubated with calpain, and Rb protein fragments were separated by The oligonucleotide primer sequences for construction of the Rb mutant SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and the and for the chromatin immunoprecipitation assay are included in transferred proteins stained with Coomassie blue. The f10-kDa fragment Supplementary Methods. was excised from the membrane, and the NH2-terminal amino acid residues were identified by 8 to 12 Edman cycles on an Applied Biosystems Procise 494 Results sequencer. Calpain activity assays. Calpain zymography was done on cell lysates Proteolytic cleavage of Rb precedes Rb degradation by using calpain zymograms incorporating 0.2% casein in the gel as described proteasomes. We previously reported that SerpinB2 increases Rb www.aacrjournals.org 5649 Cancer Res 2008; 68: (14). July 15, 2008

protein levels, resulting from inhibition of Rb degradation (10). NH2-terminal Rb mAb (N-240; Fig. 1C, left and right), occurred very Whereas Rb is ultimately degraded via the proteasome pathway rapidly and was accompanied by the release of a f10-kDa Rb (22), a number of considerations indicate that it is unlikely that fragment (Fig. 1C, left, arrow) shown to be COOH-terminal by SerpinB2 is a direct inhibitor of the protease activity of the immunoreactivity with the COOH-terminal specific Rb (C-15) proteasome. Therefore, we postulated that proteasome-mediated antibody (Fig. 1C, middle, arrow). Experiments using calpain-2 Rb degradation may be preceded by a separate, SerpinB2- generated similar Rb cleavage products (data not shown). When inhibitable, proteolytic cleavage event. If this was the case, then SerpinB2 was added to Rb before addition of calpain, the inhibition of proteasome activity could enable a normally transient generation of the 95-kDa Rb was specifically abolished, showing Rb cleavage intermediate to be stabilized. Indeed, when Jurkat that this calpain-mediated proteolytic cleavage was directly cells were treated with the proteasome inhibitors, lactacystin or inhibitable by SerpinB2 (Fig. 1D, lane 5 versus lane 4). Whereas MG-132, the accumulation of a faster-migrating Rb intermediate of calpain was capable of cleaving Rb at multiple sites under these f95 kDa was detected, which was accompanied by the loss of full- cell-free conditions, the 95-kDa Rb fragment was the only cleavage length 110-kDa Rb (Fig. 1A). Rb proteolytic cleavage was assessed product observed in cultured cells in vivo (Fig. 1A and data not specifically by treatment of lysates with alkaline phosphatase to shown). dephosphorylate Rb, thus eliminating bands due to multiphos- Identification of the SerpinB2-protected calpain cleavage phorylated Rb species (10). Detection of the f95-kDa species site at Rb-Lys810. The identity of the calpain cleavage site within required the presence of the caspase inhibitor Boc-D-FMK to inhibit the COOH-terminal domain of Rb was determined by NH2-terminal caspases activated by proteasome inhibitors (31), which may also sequence analysis of the purified 10-kDa calpain cleavage product 811 cleave and degrade Rb (23). This ‘‘trapped’’ 95-kDa Rb intermediate (Fig. 1C, arrow). Ser was identified at the NH2 terminus of this 810 showed strong immunoreactivity with an NH2-terminal specific Rb peptide fragment, revealing the calpain cleavage site at P1 Lys . mAb (Rb amino acids 1–240); however, immunoreactivity was lost This cleavage is consistent with calpain substrate preference for a with a COOH-terminal specific Rb mAb (C-15), indicating loss of a basic amino acid at the P1 position (27, 32). Using fluorogenic f10- to 15-kDa COOH-terminal Rb peptide. These data suggested peptide assays of calpain protease activity, SerpinB2 did not inhibit the occurrence of a COOH-terminal, caspase-independent proteo- calpain protease activity directly (data not shown). Considering lytic cleavage of the Rb protein, producing a 95-kDa Rb intermediate that Lys810 is centered within the SerpinB2-Rb binding domain targeted for proteasomal degradation. previously mapped to Rb amino acids 768 to 785 and 825 to 840 Rb is COOH-terminally cleaved by calpain,distinct from (Fig. 1D; ref. 10), these data may suggest that SerpinB2-Rb binding caspase cleavage. To investigate whether intracellular calpains interaction may interfere with accessibility of calpain to its cleave Rb, Jurkat cell extracts containing Rb were treated with cleavage site at Rb-Lys810. calpain and immunoblotted for detection of Rb protein. Mild Calpain inhibitors stabilize Rb,decreasing Rb turnover. Our calpain treatment resulted in a decrease in full-length 110-kDa Rb data provided evidence for calpain cleavage of Rb in cell-free and the appearance of a 95-kDa Rb cleavage product [Fig. 1B, Rb systems, which could control Rb levels by regulating Rb turnover. (G3-245)], which was identical to the Rb intermediate trapped by To determine whether inhibition of calpain affects Rb turnover in proteasome inhibition. An additional 100-kDa Rb band was also cells, we exposed several different cell types to a series of cell- observed, but subsequent experiments showed that this cleavage permeable calpain inhibitors (Fig. 2A). Calpain inhibition specif- product was generated only under cell-free conditions (data not ically enhanced the accumulation of full-length Rb protein (Fig. 2A, shown), indicating that it was not relevant in vivo. The 95-kDa Rb left), which was not accompanied by changes in Rb mRNA levels was not immunoreactive with the Rb (C-15) antibody [Fig. 1B, Rb (measured by Q-PCR; data not shown), consistent with posttran- (C-15), lane 2], showing that proteolytic cleavage occurred near the scriptional stabilization of Rb protein and inhibition of Rb Rb COOH terminus. Cleavage was calpain and Ca2+ dependent turnover. The increased Rb was not a general stress response to because cleavage did not occur in the presence of Ca2+ alone (data calpain inhibitors because cell viability did not change throughout not shown) or when Ca2+ was chelated by EGTA (Fig. 1B, lane 4). the time period of the experiments (data not shown). Time course Cell extracts treated with caspase-3 did not produce the same Rb studies in Jurkat cells revealed that Rb stabilization was time cleavage products but instead generated 105-kDa Rb (Fig. 1B, dependent, being maximally effective during the first 4 to 8 hours lane 5), which was expected from the COOH-terminal release of a after exposure to calpain inhibitors (Fig. 2A, right). 5-kDa peptide (24). These data show that calpain can mediate the Calpain regulates Rb turnover directly through a mecha- cleavage of Rb near the COOH terminus to generate a 95-kDa Rb nism inhibitable by SerpinB2. To further investigate the cleavage product. participation of calpain in the regulation of Rb turnover, we used À À When recombinant SerpinB2 was added to the Jurkat cell extract calpain-deficient (CAPN4 / ) MEFs (28). Genetic deletion of the before calpain addition, SerpinB2 inhibited the appearance of Capn4 gene abolishes both calpain-1 and calpain-2 activities (28). À À 95-kDa Rb [Fig. 1B, Rb (G3-245), lane 3 versus lane 2] and stabilized CAPN4 / MEFs had substantially increased Rb protein levels full-length 110-kDa Rb [Fig. 1, Rb (C-15), lane 3 versus lane 2]. The compared with control CAPN4+/+ MEFs (Fig. 2B), which could not cleavage of Rb by caspase was unaffected by SerpinB2 (Fig. 1B, be accounted for by increased Rb gene transcription (data not lane 6). These data suggested that SerpinB2 could protect Rb from shown), implicating calpain deficiency with stabilization of Rb. We À À COOH-terminal cleavage by calpain. attempted to rescue the CAPN4 / MEF phenotype by transfecting COOH-terminal Rb cleavage by calpain is inhibited by an expression plasmid encoding CAPN4 (pCMV-CAPN4) into À À SerpinB2. To determine whether calpain cleaves Rb directly, we CAPN4 / MEFs (Fig. 2C). Restoration of calpain activity in À À used a purified system in which purified recombinant Rb protein CAPN4 / MEFs was shown by the presence of CAPN4 protein was treated with catalytic amounts of calpain-1. Multiple cleavage expression (Fig. 2C, lanes 2 and 4, CAPN4) and detection of restored fragments were detected (Fig. 1C), showing that Rb is a direct calpain-1 proteolytic activity by calpain zymography (Fig. 2C, calpain substrate. Generation of the 95-kDa Rb, recognized by the lanes 2 and 4, Zymogram). Restoration of calpain activity resulted

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Figure 1. Rb is a calpain substrate. A, trapping of a 95-kDa Rb proteolytic cleavage intermediate by proteasome inhibition. Jurkat cells were incubated for 16 h with proteasome inhibitors lactacystin (10 Amol/L) or MG-132 (3 Amol/L) in the presence of 25 Amol/L of the caspase inhibitor Boc-D-FMK. Harvested cell lysates were heated to 90jC for 10 min to inactivate enzymes, and then proteins were dephosphorylated by calf intestine alkaline phosphatase treatment. Cell lysates were immunoblotted for Rb (1–240 and C-15 antibodies) or cyclin A loading control. B, Rb cleavage by calpain generates 95-kDa Rb and is distinct from caspase cleavage. Lanes 1 to 4, Rb-containing Jurkat cell extracts were treated with 0.2 unit of calpain-1 in the presence of 2 mmol/L CaCl2 and 25 Amol/L Boc-D-FMK in combination with 300 mmol/L EGTA or 1.5 Ag recombinant SerpinB2. Lanes 5 and 6, treatment with 100-ng caspase-3 in the absence or presence of 1.5-Ag recombinant SerpinB2. Reactions were heated to 90jC for 10 min to inactivate enzymes, and then proteins were dephosphorylated by calf intestine alkaline phosphatase treatment and analyzed by immunoblotting with Rb antibodies (C-15 and G3-245) and GAPDH. In three separate experiments, the magnitude of the protection of Rb by SerpinB2 in the presence of calpain averaged 56 F 3%, as determined by densitometric analyses. C, calpain cleaves Rb to directly generate 95-kDa Rb. Recombinant Rb (1 Ag) was incubated without or with 0.02 or 0.04 unit of recombinant calpain-1 for 15 min at 30jC. Cleavage peptides were separated by 4% to 12% SDS-PAGE and visualized by silver staining (left), by immunoblotting for Rb with the COOH-terminal specific antibody (C-15; middle), or with an anti-Rb antibody that detects the Rb NH2 terminus (G99-2005, right). Arrow, the 10-kDa polypeptide fragment that was gel purified and subjected to NH2-terminal sequence analysis. The NH2-terminal sequence was determined to be S-P-Y-K-I-S-E-G-L, identifying cleavage at Lys810-Ser811. D, SerpinB2 inhibits Rb cleavage by calpain. Recombinant Rb (1 Ag) was incubated with a 10-fold molar excess of recombinant SerpinB2 for 15 min and then 0.02 unit of calpain-1 for 5 min. The reactions were visualized by silver staining (left) or by immunoblot with the COOH-terminal Rb antibody C-15 (right). SerpinB2 blocked the cleavage associated with the appearance of the 95-kDa Rb peptide. Full-length uncleaved Rb is indicated by immunoreactivity with the C-15 antibody. The bottom diagram illustrates the COOH-terminal calpain and caspase cleavage sites. The calpain cleavage site is situated at the SerpinB2-Rb interaction site (10). in a dramatic decrease in Rb protein levels [Fig. 2C, lane 2 versus was rescued (Fig. 2C, lane 4, CAPN4 and Zymogram). SerpinB2 lane 1, Rb (G3-245)], showing that calpain specifically mediates Rb expression alone in the absence of calpain did not affect Rb levels turnover in these cells. Strikingly, when an expression plasmid [Fig. 2C, lane 3 versus lane 1, Rb (G3-245)]. Together, these data encoding SerpinB2 (pCMV-SerpinB2) was cotransfected along with provide compelling evidence for regulation of Rb turnover through À À the pCMV-CAPN4 into CAPN4 / MEFs, Rb was stabilized and calpain cleavage by a mechanism inhibitable by SerpinB2. detected at a level indistinguishable from control transfected cells Rb cleavage by calpain occurs during TNFA-induced cell (Fig. 2C, Rb (G3-245), lane 4 versus lane 1), although calpain activity death. We previously identified SerpinB2 as a cytoprotective factor www.aacrjournals.org 5651 Cancer Res 2008; 68: (14). July 15, 2008

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research that confers resistance to TNFa-induced apoptosis in HeLa cells TNFa-induced cell death compared with CAPN4+/+ MEFs (Fig. 3B). À À (18). Calpains have been implicated in orchestrating the induction When calpain activity was restored in CAPN4 / MEFs by À À of apoptosis (33), and Rb loss is associated with enhanced transfection of CAPN4, the sensitivity of CAPN4 / MEFs to susceptibility to apoptosis when cells are exposed to TNFa (6). TNFa-induced death was rescued (Fig. 3B, CAPN4). Coexpression To determine whether SerpinB2 may confer resistance to apoptosis of SerpinB2 with CAPN4 could block calpain-dependent cell death, through the calpain-Rb pathway, we investigated the effect of resulting in resistance to TNFa (Fig. 3B, CAPN4 + SerpinB2). These SerpinB2 on Rb levels during apoptosis induced by TNFa in HeLa data show that calpain cleavage of Rb contributes to initiation of cells. Exposure of HeLa cells to TNFa and cycloheximide initiates TNFa-induced death and that this activity can be blocked by cell death (18) as evidenced by activated caspase-3 and the SerpinB2. À À appearance of the marker of caspase activation, PARP cleavage Resistance to TNFa-induced cell death in CAPN4 / MEFs was within 3 hours (Fig. 3A). The HeLa cell line expressing SerpinB2 associated with delayed caspase activation, evidenced by delayed À À (S1a) shows resistance to TNFa-induced death (18) and further PARP cleavage in CAPN4 / MEFs compared with CAPN4+/+ MEFs showed delayed caspase activation (Fig. 3A). These TNFa-resistant on exposure to TNFa (Fig. 3C, left, lane 6 versus lane 3). In a similar S1a cells showed increased levels of COOH-terminally intact Rb way, pretreatment of TNFa-sensitive HeLa cells (18) with calpain [Fig. 3A, Rb (C-15)], suggesting that resistance to TNFa-induced inhibitor III to inhibit calpain activity stabilized Rb and delayed death could be mediated by SerpinB2 inhibition of Rb loss via caspase activation in response to TNFa (Fig. 3C, right, lane 5 versus protection of Rb from calpain cleavage. In contrast, full-length Rb lane 4). begins to disappear in HeLa cells after exposure to TNF for 3 hours These data implicate regulation of Rb by calpain during (Supplementary Fig. S1). To specifically investigate the role of induction of the TNFa-induced cell death response. To explore calpain and SerpinB2 in protecting Rb during TNFa-induced this directly, a mutant Rb (Rb-810W) was generated, which, when À À apoptosis, we investigated calpain-deficient MEFs. CAPN4 / expressed in HeLa cells (Supplementary Fig. S2), showed resistance MEFs, which have elevated Rb (Fig. 2B), show resistance to to cleavage by exogenous calpain at Rb-Lys810 compared with

Figure 2. Calpain induces Rb degradation in calpain-deficient MEFs. A, inhibition of calpain activity stabilizes Rb and inhibits Rb turnover. Left, Jurkat cells were incubated in the presence of the cell-permeable competitive inhibitors calpain inhibitor II [N-acetyl-leu- leu-methional (aLLM), 230 nmol/L], calpain inhibitor III (carbobenzoxy-valyl-phenylalanal; 8 nmol/L), the noncompetitive selective calpain inhibitor PD150606 [3-(4-iodophenyl)- 2-mercapto-(4Z)-sphingenine or (Z)-11- tetradecenoic (Z)-2-propenoic acid; 400 nmol/L], which targets the Ca2+ binding sites of calpains and its noninhibitory PD145305 analogue, or DMSO vehicle control. HeLa, RAW 264.7, and MEFs were cultured in the presence of 10 Amol/L calpain inhibitors. After 7 h, cell lysates were immunoblotted for Rb (G3-245) or GAPDH control. Right, time course of Rb stabilization in Jurkat cells. Jurkat cells were incubated in the presence of the different cell-permeable calpain inhibitors for up to 24 h. Rb protein was detected by immunoblot and signal intensity normalized to the untreated control at each time point. B, Rb protein levels are enhanced in calpain-deficient MEFs. Lysates from CAPN4+/+ and CAPN4À/À cells were immunoblotted for Rb (G3-245 or C-15) or GAPDH control. C, calpain mediates cleavage and degradation of Rb. CAPN4À/À cells were transfected with expression plasmids encoding the calpain regulatory small subunit CAPN4, SerpinB2, both of these plasmids, or vector alone. Transfected cells were cultured in complete medium containing 25 Amol/L Boc-D-FMK (ICN), which was added fresh every 12 h. Forty-eight hours after transfection, cells were treated with 1 Amol/L of the Ca2+ ionophore A23187 for 1 h to activate calpain before immunoblot analysis. Restored calpain-1 proteolytic activity was detected by calpain zymography after incubation of the cells with A23187 for 1 h.

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Figure 3. Rb cleavage by calpain occurs during TNFa-induced apoptosis and is protected by SerpinB2. A, SerpinB2 delays caspase activation in HeLa cells by stabilizing Rb. HeLa and HeLa-expressing SerpinB2 (S1a; ref. 18) cells were treated with cycloheximide (CHX) alone or in combination with 1 ng/mL (*) or 10 ng/mL (**) of human TNFa for 3 h. Cell lysates were immunoblotted with the indicated antibodies. B, CAPN4À/À MEFs are more resistant to TNFa-induced death and can become sensitive by restoration of calpain. CAPN4À/À and CAPN4+/+ cells were treated with mouse TNFa and cycloheximide for 4 h, and live cells quantitated by trypan blue exclusion. Each treatment was assessed in duplicate; bars, SD. In addition, CAPN4À/À cells were transfected with plasmids encoding CAPN4 or SerpinB2 or cotransfected with both plasmids. Twenty-four hours after transfection, cells were exposed to cycloheximide alone or TNFa and cycloheximide for 3 h, and live cells counted by trypan blue exclusion. Columns, mean percent live cells relative to treatment with cycloheximide alone in each sample from triplicate experiments; bars, SD. C, calpain deficiency in CAPN4À/À MEFs or inhibition of calpain in TNFa-sensitive HeLa cells decreases caspase activation. Left, CAPN4+/+ or CAPN4À/À cells were exposed to mouse TNFa and cycloheximide for 3 h and cell death was monitored by immunoblotting for Rb (G3-245) and PARP cleavage. Right, HeLa cells were incubated for 1 h with 10 Amol/L calpain inhibitor III and then subjected to TNFa and cycloheximide treatment for 2 h. Cell lysates were immunoblotted for Rb (G3-245) or GAPDH for loading control. Induction of cell death was monitored by immunoblotting for PARP cleavage. D, the dominant-negative Rb-810W mutant shows resistance to calpain cleavage and delays caspase activation in response to TNFa-induced cell death. Left, lysates from HeLa cells stably producing Rb or Rb (K810W) mutant were subjected to 0.2-unit calpain-1 digestion for the indicated times. Proteins were separated and immunoblotted for Rb (G3-245). Right, HeLa cells and HeLa cell lines stably expressing wild-type Rb or two independent clonal cell lines expressing the Rb (K810W) mutant were exposed to human TNFa and cycloheximide for 2 h. Immunoblots for PARP and active caspase-3 were quantified by densitometry using Scion Image analysis software, and the latter normalized for protein levels using GAPDH. *, relative to TNFa-treated samples. wild-type Rb (Fig. 3D, left). HeLa cells transfected with Rb-810W mediate antiapoptotic activity through repression of the transcrip- were more resistant to caspase activation after exposure to TNFa, tion of proapoptotic E2F target genes. A Rb-containing signal compared with HeLa cells transfected with wild-type Rb or transduction module present on selective E2F-regulated proapop- parental HeLa cells, evidenced by significantly less PARP cleavage totic gene promoters (e.g., Apaf-1, p21, p73, and several caspases) and severalfold less active caspase-3 (Fig. 3D, right). These studies has been associated with repression of apoptotic cell death (9, 34). suggest that regulation of Rb by calpain contributes to induction of To determine whether SerpinB2 stabilization of Rb contributes TNFa-induced apoptotic responses and that inhibition or blocking to Rb-mediated repression of proapoptotic genes, HeLa and S1a of calpain cleavage of Rb can delay caspase activation and confer cells were compared for differential Rb binding to promoters of resistance to TNFa-induced death. E2F-regulated genes during induction of TNFa-induced death SerpinB2 sustains Rb-mediated repression of proapoptotic using chromatin immunoprecipitation assays. Rb binding to the genes. How does SerpinB2 stabilization of Rb cause antiapoptotic promoters of the proapoptotic genes p73, caspase-7, and p21 was activity? Increased stabilization of Rb might be expected to substantially decreased after exposure of HeLa cells to TNFa, www.aacrjournals.org 5653 Cancer Res 2008; 68: (14). July 15, 2008

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Cancer Research whereas Rb binding to these same proapoptotic promoters in Because SerpinB2 is abundantly expressed by proliferating basal SerpinB2-expressing S1a cells was unaffected (Fig. 4A). We keratinocytes (Fig. 5A), we postulated a role for SerpinB2 as a observed no significant changes in Rb promoter occupancy protector of Rb during skin tumor surveillance. SerpinB2+/+ mice À À on treatment with TNFa for the cell cycle–regulated CDC24B, (n = 11) and SerpinB2 / mice (n = 12) were treated with DMBA CDC2, or thymidine kinase gene promoters. Interestingly, SerpinB2 followed by twice weekly applications of PMA. After 9 weeks, also sustained repression of the cell cycle gene cyclin A2, which hyperplasia and then the formation of papillomas were observed À À has previously been reported to be subject to regulatory in SerpinB2+/+ control mice, whereas SerpinB2 / mice showed a mechanisms similar to the proapoptotic genes (9). PCR amplifica- delay in onset of these features. After 21 weeks, when all of the tion of the h-actin promoter (a control to monitor specificity of SerpinB2+/+ control group had developed papillomas (example À À the immunoprecipitation) did not amplify any DNA (data not illustrated in Fig. 5A, right), only 42% (5 of 12) of the SerpinB2 / shown). Measurement of transcription of several of these E2F- mice developed papillomas (P < 0.01, m2 test). Tumor multiplicity regulated genes and additional caspases by quantitation of mRNA (mean number of tumors per mice) was markedly reduced in À À (Fig. 4B) confirmed their repression by Rb in SerpinB2-expressing SerpinB2 / mice [mean, 0.8 F 0.3 tumors; range, 0–3] compared S1a cells. with control SerpinB2+/+ mice [mean, 5.8 F 0.8 tumors; range, SerpinB2 deficiency reduces susceptibility to Rb-dependent 3–10]. skin carcinogenesis. Constitutive Rb-mediated repression of In a second experiment, where a more aggressive regimen of proapoptotic gene promoters is postulated to be a host PMA application was followed, there was again a significant delay À À surveillance mechanism whereby cells that have lost Rb function in the onset of tumor incidence in the SerpinB2 / animals activate a cell death response, thereby eliminating would-be (Fig. 5B, left). By week 15, 56% of the SerpinB2+/+ mice had formed À À tumor cells early in the transformation process (2). Accordingly, papillomas, compared with 11% of the SerpinB2 / mice (P < 0.03, Rb inactivation in epidermal keratinocytes renders mice less m2 test). Consistent with the delay in tumor onset, the numbers of À À susceptible to DMBA/PMA–induced skin carcinogenesis (4). papillomas present on SerpinB2 / mice were initially reduced; however, with repeated weekly PMA applications, the numbers of tumors eventually reached the levels present in the wild-type counterparts (Fig. 5B, middle). Tumor volumes were consistently À À reduced in SerpinB2 / mice (Fig. 5B, right), most likely due to a delay in the initiation of tumor formation and not due to an altered tumor growth rate. Once initiated, tumors progressed at compa- rable rates in both genotypes (data not shown). The response of À À the SerpinB2 / mice is very similar to the keratinocyte-targeted À À Rb / phenotype (35), with genetic loss of Rb or SerpinB2 leading to fewer and smaller papillomas. SerpinB2 deficiency is associated with increased apoptosis. In the initiation stages of tumor development during DMBA/PMA skin carcinogenesis, cell proliferation competes with apoptosis (36), and Rb plays a critical role (35). Quantitation of apoptotic cells À À in hyperplastic lesions of SerpinB2 / mice revealed a marked increase in apoptotic index (Fig. 5C), analogous to enhanced apoptosis detected in hyperplastic lesions of keratinocyte-targeted À À Rb / mice (4). These data suggest that SerpinB2 deficiency decreases keratinocyte survival early during the transformation process, resulting in delayed onset of papilloma development. We can speculate that TNFa induced by PMA during skin carcinogenesis (37) could initiate a calpain-dependent pathway to eliminate Rb, which would be blocked in cells expressing SerpinB2. These findings suggest that sustained SerpinB2 expression is important for the continued survival of basal keratinocytes and acts by reducing their sensitivity to apoptosis during stress, thereby enabling better survival and, paradoxically, promoting tumor development.

Figure 4. SerpinB2 stabilization of Rb sustains transcriptional repression of proapoptotic genes. A, Rb repression of proapoptotic genes is enhanced in the presence of SerpinB2. In vivo detection of promoter occupancy by Rb with Discussion chromatin immunoprecipitation assay. HeLa and HeLa-expressing SerpinB2 The elimination of Rb is a cellular response to stress induced by cells were treated with 2.5 Ag/mL cycloheximide with or without 10 ng/mL human TNFa for 3 h. Lysates were immunoprecipitated with Rb (G3-245) antibody, genotoxic agents and TNFa (6). Depending on the biological and precipitated chromatin samples were amplified with primers specific for context, Rb loss can precipitate induction of a cell death response E2F-regulated gene promoters. Input, PCR reactions done on total DNA before immunoprecipitation to ensure that equal amounts of DNA were present in and apoptotic cell death. Here we show that the cytoprotective samples. B, SerpinB2 enhances Rb repression of transcription of E2F-regulated protein SerpinB2 protects cells from Rb loss and delays cell death. genes and caspase genes after apoptotic stimuli. Quantitative PCR analysis The mechanism of SerpinB2 protection involves inhibition of of mRNA in HeLa and HeLa-expressing SerpinB2 cells following treatment with TNFa and cycloheximide for 30 min. mRNA levels are normalized to h-actin. calpain cleavage of Rb and subsequent proteasome degradation, Columns, mean from triplicate experiments; bars, SD. thus promoting elevated Rb levels and enhancing Rb-mediated

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Figure 5. SerpinB2 deficiency reduces susceptibility to Rb-dependent skin carcinogenesis. A, histopathology of representative papillomas. In normal adult mouse epidermis, SerpinB2 is present in suprabasal and basal keratinocytes (left), where it colocalizes with basal K5 (middle), a predominant keratin of proliferating basal keratinocytes. Right, H&E-stained section of a representative SerpinB2+/+ papilloma. Inset, gross morphology. B, SerpinB2 deficiency reduces susceptibility to tumor formation during skin carcinogenesis. SerpinB2+/+ (n = 9) and SerpinB2À/À mice (n = 9) were treated with DMBA/PMA as per experiment 2 and the onset of papilloma formation was followed with time. Left, a significant delay in the onset of papilloma incidence was observed in the SerpinB2À/À animals. Middle, tumor multiplicity represented by the total number of papillomas per genotype (n = 9). Right, mean papilloma volume per papilloma-bearing mouse. Once formed, papillomas did not produce SerpinB2, and SerpinB2 deficiency had no effect on the rate of papilloma progression (data not shown). C, increased apoptosis in SerpinB2-deficient hyperplastic skin lesions. TUNEL staining of apoptotic keratinocytes in the basal layer of SerpinB2+/+ (left) and SerpinB2À/À (middle) epidermis. Arrows, TUNEL-positive basal keratinocytes localized along the basement membrane (dotted line). Right, percentage of apoptotic cells in the basal layer. The number of TUNEL-positive cells relative to the total number of cells along the basement membrane was counted. Data represent specimens from four mice of each genotype (two sections per mouse); bars, SD. P V 0.001, Student’s t test. repression of E2F-regulated proapoptotic genes. Loss of this consequence of stress-induced signaling, contributes to elimination cytoprotective pathway, such as occurs with SerpinB2 deficiency, of Rb and subsequent proapoptotic signaling events through loss of increases susceptibility to cell death. These data define a novel Rb-mediated transcriptional repression. pathway for rapid and selective modulation of Rb that affects cell Calpain cleaves Rb within the COOH-terminal domain (Rb- survival pathways. Lys810) and the calpain-cleaved Rb1–810 intermediate is targeted for Rb degradation is required for TNFa receptor 1 to signal proteasomal degradation. The Rb COOH-terminal domain is apoptosis (25). Our findings that Rb is a calpain substrate and that recognized to contribute to Rb-mediated growth suppression calpain regulates Rb levels provide an additional mechanism, in through binding to transcriptional regulators such as c-Abl (39) addition to caspase cleavage of Rb (25), for eliminating Rb. The role and E2F1 (40). It may be that differential protein-protein of calpains in cell death, however, is clearly complex. Calpains are interactions associated with this domain potentially dictate the reported to play both proapoptotic and antiapoptotic roles in diversity of Rb functions in transcription, chromatin remodeling, several different cell systems and in response to a wide range of differentiation, and cell survival. Further studies are required to stimuli (38). Some of the complexity in calpain functions may be determine whether calpain cleavage of Rb affects binding of due to cell-specific regulatory factors such as the presence of transcriptional regulators that associate with Rb and subsequent SerpinB2. Our data show that calpain, likely activated as a downstream signaling events. www.aacrjournals.org 5655 Cancer Res 2008; 68: (14). July 15, 2008

SerpinB2 protects Rb from calpain cleavage. SerpinB2 is a transformed keratinocytes during DMBA/PMA skin carcinogenesis member of the intracellular clade B or ov-serpin subfamily (41). (15), and we show here that SerpinB2 deficiency decreases their Many cells involved in the innate immune response produce clade survival by reducing their susceptibility to transformation. PMA B serpins. SerpinB2 is expressed in a limited number of cells, tumor promotion during skin carcinogenesis induces TNFa (37), placental trophoblasts, monocytes/macrophages, and keratino- which may initiate a calpain-dependent pathway to eliminate Rb cytes; however, it may be strongly up-regulated in multiple cell and thus initiate the activation of a cell death response unless types following exposure to inflammatory and cellular stress protective factors such as SerpinB2 are present. These data mediators including cytokines, growth factors, viruses, and provide new insight into cell-specific and serpin-dependent bacterial endotoxin (11). Several other clade B members protect mechanisms by which cell death versus prosurvival cell fates cells from exogenous and endogenous proteinase-mediated injury may be determined. triggered by various death-inducing stimuli using a wide range of mechanisms (42–47). In addition, clade B serpins are implicated in direct inhibition of the activity of specific proteolytic enzymes, Disclosure of Potential Conflicts of Interest mostly serine and/or papain-like cysteine proteases. We did not No potential conflicts of interest were disclosed. find that SerpinB2 inhibited calpain protease activity directly, suggesting a unique mechanism of inhibition that likely involves Rb Acknowledgments binding via the PENF motif within the C-D interhelical loop region Received 10/11/2007; revised 4/1/2008; accepted 4/18/2008. of SerpinB2 (10). Grant support: National Cancer Institute grant R01 CA098369 (T.M. Antalis) The induction of cytoprotective SerpinB2 could affect cell and the National Health and Medical Research Council, Australia (T.M. Antalis and A. Suhrbier). M.S. Buzza is supported by a CJ Martin Training Fellowship from the survival for multiple physiologic stress–induced pathways but, in National Health and Medical Research Council, Australia. addition, would likely be restricted to cells or situations where Rb The costs of publication of this article were defrayed in part by the payment of page is a determinant of the death response. DMBA/PMA–induced skin charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. carcinogenesis is an Rb-dependent process (4, 37, 48). Transgenic À/À We thank Dr. John Elce for CAPN4 MEFs and Brian Hampton for NH2-terminal overexpression of SerpinB2 in mouse skin increases survival of sequence analyses.

References 14. Varro A, Noble PJ, Pritchard DM, et al. Helicobacter mice expressing caspase-resistant Rb. Nat Cell Biol 2002; pylori induces plasminogen activator inhibitor 2 in 4:757–65. 1. Du W, Pogoriler J. Retinoblastoma family genes. gastric epithelial cells through nuclear factor-nB and 26. Goll DE, Thompson VF, Li H, Wei W, Cong J. The Oncogene 2006;25:5190–200. RhoA. Implications for invasion and apoptosis. Cancer calpain system. Physiol Rev 2003;83:731–801. 2. Chau BN, Wang JY. Coordinated regulation of life and Res 2004;64:1695–702. 27. Sato K, Kawashima S. Calpain function in the death by RB. Nat Rev Cancer 2003;3:130–8. 15. Zhou HM, Bolon I, Nichols A, Wohlwend A, modulation of signal transduction molecules. Biol Chem 3. Jacks T, Fazeli A, Schmitt EM, Bronson RT, Goodell Vassalli JD. Overexpression of plasminogen activator 2001;382:743–51. MA, Weinberg RA. Effects of an Rb mutation in the inhibitor type 2 in basal keratinocytes enhances 28. Arthur JS, Elce JS, Hegadorn C, Williams K, Greer PA. mouse. Nature 1992;359:295–300. papilloma formation in transgenic mice. Cancer Res Disruption of the murine calpain small subunit gene, 4. Ruiz S, Santos M, Lara MF, Segrelles C, Ballestin C, 2001;61:970–6. Capn4. calpain is essential for embryonic development Paramio JM. Unexpected roles for pRb in mouse skin 16. Antalis TM, La Linn M, Donnan K, et al. The serine but not for cell growth and division. Mol Cell Biol 2000; carcinogenesis. Cancer Res 2005;65:9678–86. proteinase inhibitor (serpin) plasminogen activation 20:4474–81. 5. Young AP, Longmore GD. Differential regulation of inhibitor type 2 protects against viral cytopathic effects 29. Raser KJ, Posner A, Wang KK. Casein zymography. apoptotic genes by Rb in human versus mouse cells. by constitutive interferon a/h priming. J Exp Med 1998; a method to study A-calpain, m-calpain, and their Oncogene 2004;23:2587–99. 187:1799–811. inhibitory agents. Arch Biochem Biophys 1995;319: 6. Wang JY. Nucleo-cytoplasmic communication in 17. Losick VP, Isberg RR. NF-nB translocation prevents 211–16. apoptotic response to genotoxic and inflammatory host cell death after low-dose challenge by Legionella 30. Dougherty KM, Pearson JM, Yang AY, Westrick RJ, stress. Cell Res 2005;15:43–8. pneumophila. J Exp Med 2006;203:2177–89. Baker MS, Ginsburg D. The plasminogen activator 7. DeGregori J, Leone G, Miron A, Jakoi L, Nevins JR. 18. Dickinson JL, Bates EJ, Ferrante A, Antalis TM. inhibitor-2 gene is not required for normal murine Distinct roles for E2F proteins in cell growth control Plasminogen activator inhibitor type 2 inhibits tumor development or survival. Proc Natl Acad Sci U S A 1999; and apoptosis. Proc Natl Acad Sci U S A 1997;94: necrosis factor a-induced apoptosis. Evidence for an 96:686–91. 7245–50. alternate biological function. J Biol Chem 1995;270: 31. Chandra J, Niemer I, Gilbreath J, et al. Proteasome 8. Muller H, Bracken AP, Vernell R, et al. E2Fs regulate 27894–904. inhibitors induce apoptosis in glucocorticoid-resistant the expression of genes involved in differentiation, 19. Gan H, Newman GW, Remold HG. Plasminogen chronic lymphocytic leukemic lymphocytes. Blood 1998; development, proliferation, and apoptosis. Genes Dev activator inhibitor type 2 prevents programmed cell 92:4220–9. 2001;15:267–85. death of human macrophages infected with Myco- 32. Tompa P, Buzder-Lantos P, Tantos A, et al. On the 9. Nahle Z, Polakoff J, Davuluri RV, et al. Direct coupling bacterium avium, serovar 4. J Immunol 1995;155: sequential determinants of calpain cleavage. J Biol of the cell cycle and cell death machinery by E2F. Nat 1304–15. Chem 2004;279:20775–85. Cell Biol 2002;4:859–64. 20. Pytel BA, Peppel K, Baglioni C. Plasminogen activator 33. Johnson DE. Noncaspase proteases in apoptosis. 10. Darnell GA, Antalis TM, Johnstone RW, et al. inhibitor type-2 is a major protein induced in human Leukemia 2000;14:1695–703. Inhibition of retinoblastoma protein degradation by fibroblasts and SK-MEL-109 melanoma cells by tumor 34. Young AP, Longmore GD. Differences in stability interaction with the serpin plasminogen activator necrosis factor. J Cell Physiol 1990;144:416–22. of repressor complexes at promoters underlie inhibitor 2 via a novel consensus motif. Mol Cell Biol 21. Helt AM, Galloway DA. Mechanisms by which DNA distinct roles for Rb family members. Oncogene 2004; 2003;23:6520–32. tumor virus oncoproteins target the Rb family of pocket 23:814–23. 11. Kruithof EK, Baker MS, Bunn CL. Biological and proteins. Carcinogenesis 2003;24:159–69. 35. Ruiz S, Santos M, Paramio JM. Is the loss of pRb clinical aspects of plasminogen activator inhibitor type 22. Ying H, Xiao ZX. Targeting retinoblastoma protein essential for the mouse skin carcinogenesis? Cell Cycle 2. Blood 1995;86:4007–24. for degradation by proteasomes. Cell Cycle 2006;5: 2006;5:625–9. 12. Kumar S, Baglioni C. Protection from tumor necrosis 506–8. 36. Hennings H, Glick AB, Lowry DT, Krsmanovic LS, Sly factor-mediated cytolysis by overexpression of plasmin- 23. Tan X, Wang JY. The caspase-RB connection in cell LM, Yuspa SH. FVB/N mice. an inbred strain sensitive to ogen activator inhibitor type-2. J Biol Chem 1991;266: death. Trends Cell Biol 1998;8:116–20. the chemical induction of squamous cell carcinomas in 20960–4. 24. Tan X, Martin SJ, Green DR, Wang JY. Degradation the skin. Carcinogenesis 1993;14:2353–8. 13. Kasyapa CS, Kunapuli P, Hawthorn L, Cowell JK. of retinoblastoma protein in tumor necrosis factor- 37. Scott KA, Moore RJ, Arnott CH, et al. An anti-tumor Induction of the plasminogen activator inhibitor-2 in and CD95-induced cell death. J Biol Chem 1997;272: necrosis factor-a antibody inhibits the development of cells expressing the ZNF198/FGFR1 fusion kinase that is 9613–16. experimental skin tumors. Mol Cancer Ther 2003;2: involved in atypical myeloproliferative disease. Blood 25. Chau BN, Borges HL, Chen TT, Masselli A, Hunton IC, 445–51. 2006;107:3693–9. Wang JY. Signal-dependent protection from apoptosis in 38. Tan Y, Wu C, De Veyra T, Greer PA. Ubiquitous

Cancer Res 2008; 68: (14). July 15, 2008 5656 www.aacrjournals.org

calpains promote both apoptosis and survival signals in 42. Bird PI. Serpins and regulation of cell death. Results for the formation of intracellular high Mr protease response to different cell death stimuli. J Biol Chem Probl Cell Differ 1998;24:63–89. inhibitor 10-containing complexes. J Biol Chem 2000; 2006;281:17689–98. 43. Suminami Y, Nagashima S, Vujanovic NL, Hirabayashi 275:26385–9. 39. Whitaker LL, Su H, Baskaran R, Knudsen ES, K, Kato H, Whiteside TL. Inhibition of apoptosis in 46. Welss T, Sun J, Irving JA, et al. Hurpin is a selective Wang JY. Growth suppression by an E2F-binding- human tumour cells by the tumour-associated serpin, inhibitor of lysosomal cathepsin Land protects defective retinoblastoma protein (RB). contribution SCC antigen-1. Br J Cancer 2000;82:981–9. keratinocytes from ultraviolet-induced apoptosis. Bio- from the RB C pocket. Mol Cell Biol 1998;18: 44. Takeda A, Kajiya A, Iwasawa A, Nakamura Y, chemistry 2003;42:7381–9. 4032–42. Hibino T. Aberrant expression of serpin squamous 47. Bailey CM, Khalkhali-Ellis Z, Seftor EA, Hendrix MJ. 40. Dick FA, Dyson N. pRB contains an E2F1-specific cell carcinoma antigen 2 in human tumor tissues Biological functions of maspin. J Cell Physiol 2006;209: binding domain that allows E2F1-induced apoptosis to and cell lines. evidence of protection from tumor 617–24. be regulated separately from other E2F activities. Mol necrosis factor-mediated apoptosis. Biol Chem 2002; 48. Ruiz S, Santos M, Segrelles C, et al. Unique and Cell 2003;12:639–49. 383:1231–6. overlapping functions of pRb and p107 in the control of 41. Remold-O’Donnell E. The ovalbumin family of serpin 45. Schleef RR, Chuang TL. Protease inhibitor 10 inhibits proliferation and differentiation in epidermis. Develop- proteins. FEBS Lett 1993;315:105–8. tumor necrosis factor a-induced cell death. Evidence ment 2004;131:2737–48.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. SerpinB2 Protection of Retinoblastoma Protein from Calpain Enhances Tumor Cell Survival

Laura Tonnetti, Sarah Netzel-Arnett, Grant A. Darnell, et al.

Cancer Res 2008;68:5648-5657.

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