The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, H.
Right arrow Articles by Fan, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, H.
Right arrow Articles by Fan, Z.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH
The Journal of Immunology, 2006, 177: 1171-1178.
Copyright © 2006 by The American Association of Immunologists

Granzyme M Directly Cleaves Inhibitor of Caspase-Activated DNase (CAD) to Unleash CAD Leading to DNA Fragmentation1

Hongxia Lu, Qiang Hou, Tongbiao Zhao, Honglian Zhang, Qixiang Zhang, Lianfeng Wu and Zusen Fan2

National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Granzyme (Gzm)M is constitutively highly expressed in NK cells that may play a critical role in NK cell-mediated cytolysis. However, the function of GzmM has been less defined. Just one report showed GzmM induces a caspase-independent death pathway. In this study, we demonstrate a protein transfection reagent Pro-Ject can efficiently transport GzmM into target cells. GzmM initiates caspase-dependent apoptosis with typical apoptotic nuclear morphology. GzmM induces DNA fragmentation, not DNA nicking. GzmM can directly degrade inhibitor of caspase-activated DNase to release the nuclease activity of caspase-activated DNase for damaging DNA. Furthermore, GzmM cleaves the DNA damage sensor enzyme poly(ADP-ribose) polymerase to prevent cellular DNA repair and force apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Perforin/Granzyme (Gzm)3 pathway is a key mechanism for cytotoxic lymphocytes to kill intracellular pathogens and tumor cells (1, 2). Perforin (also known as pore-forming protein) released from cytotoxic granules assists the entry of Gzms into the target cell cytosol (3, 4). Gzms are a group of serine proteases that initiate target cell apoptosis. They include GzmA, B, C, D, E, F, G, H, K, and M. Nine mouse and five human Gzms have been identified (5). GzmA and B are the most abundant Gzms in CTLs, and lymphokine-activated killer cells and their functions have been well defined (3, 6). GzmH, K, and M in humans are called orphan Gzms because their roles are less defined.

The five human Gzms differ in their chromosome locations and substrate specificities. GzmA and K are tryptases that locate on chromosome 5q11–12. GzmB is an aspase locating on 14q11.2. GzmH is a chymase that is colocated with GzmB. GzmM is a metase locating on chromosome 19p13.3 (7, 8). GzmA induces ssDNA nicks as well as apoptotic morphology and loss of cell membrane integrity (9). GzmA initiates a caspase-independent death pathway through targeting an endoplasmic reticulum-associated SET complex that contains three GzmA substrates: the nucleosome assembly protein SET, the DNA-bending protein HMG2, and the apurinic endonuclease Ape1 (10, 11). SET cleavage releases the GzmA-activated DNase (NM23H1) to nick DNA (12). The execution of apoptosis by GzmB generally occurs by activation of the caspase family of cysteine proteases that triggers caspase cascade (13). GzmB can directly process several procaspases and some key caspase pathway substrates, such as Bid, inhibitor of caspase-activated DNase (ICAD), DNA-PKcs, NuMa, poly(ADP-ribose) polymerase (PARP), and laminB (3). GzmB causes a caspase-dependent death pathway with DNA fragmentation. GzmB leads to DNA fragmentation through activation of the caspase-activated DNase (CAD) via degradation of its inhibitor ICAD (14, 15). CAD exists as a heterodimer with ICAD in a resting cell where CAD is inactive. ICAD is not cleaved in the induction of apoptosis in GzmB-deficient CTLs (16). The mechanism used by GzmB to initiate DNA fragmentation by inactivating the DNase inhibitor ICAD is reminiscent of the activation of nick-induced nuclease NM23H1 by GzmA cleavage of its inhibitor SET (12).

GzmM is an orphan Gzm that cleaves preferentially after methionone, leucine, or norleucine (17, 18). GzmM is constitutively highly expressed in NK cells, whereas it is not expressed in CD4+ or CD8+ T cells either constitutively or after stimulation (19). One study compared the cytolysis of three NK cell lines and showed that stronger cytotoxicity of NK cell lines is consistent with higher expression of GzmM (20). It suggests GzmM may play an important role in NK cell-mediated cytolysis for killing virally infected or transformed cells. A recent study reported that GzmM induces a novel form of perforin-dependent death without caspase activation and DNA fragmentation (21). In this study, we found that GzmM triggers caspase-dependent apoptosis with typical DNA laddering. The DNA fragmentation was confirmed by TUNEL assay. GzmM can directly cleave ICAD to activate CAD, leading to DNA damage. Furthermore, GzmM degrades the DNA damage sensor enzyme PARP to prevent cellular DNA repair and force apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines, Abs, and reagents

Jurkat cells were grown in RPMI 1640 medium supplemented with 10% FCS, 50 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. HeLa cells were grown in DMEM medium containing 10% FCS, 20 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Commercial Abs were rabbit antisera against caspase 3 (BD Pharmingen) and PARP (Cell Signaling Technology), mouse mAb against His-tag (Sigma-Aldrich), HRP-conjugated sheep anti-mouse IgG and HRP-conjugated sheep anti-rabbit IgG (Santa Cruz Biotechnology), and Alexa 488-conjugated donkey anti-mouse IgG (Molecular Probes). Rabbit antiserum against ICAD was provided by Dr. R. P. Sekaly (Laboratoire d’Immunologie, Université de Montreal, Montreal, Canada). Annexin VFITC was from BD Pharmingen, and PI from Sigma-Aldrich. ProLong Antifade kit was from Molecular Probes. In Situ cell death detection kit was from Roche Applied Science.

Production of active and inactive GzmM, rICAD, and rPARP

The cDNA encoding mature human GzmM was PCR amplified from full-length cDNA of GzmM (RZPD Deutsches Ressourcenzentrum) and subcloned into the yeast expression vector pPICZ{alpha}A (Invitrogen Life Technologies). The sequence of the forward primer containing XhoI site was 5'-ACTCTCGAGAAAAGAATCATCGGGGGCCGGGAGGTG-3'. The reverse primer containing XbaI site and polyhistidine tag for purification was 5'-ATCTCTAGATCAATGATGGTGGTGATGATGGGCCGATCGGCCGGTGACCTTC-3'. The resulting construct permitted the sequence of mature human GzmM to immediately follow the Kex2 signal cleavage site of the Saccharomyces cerevisiae {alpha}-factor secretion signal. The vector was linearized with SacI and transformed into the X33 strain of Pichia pastoris, according to the manufacturer’s instruction. Clones with the integrated human GzmM cDNA were selected by resistance to Zeocin (Invitrogen Life Technologies). After 3 days of induction with methanol, the conditioned medium from the shaker flask cultures was isolated and loaded onto the QuiStand benchtop system (Amersham Biosciences) for concentration and buffer exchange. One-step purification was performed using nickel affinity chromatography. The concentration of rGzmM was determined by bicinchoninic acid assay. GzmM catalytic site Ser182 was mutated to alanine by PCR mutagenesis. The mutated S-AGzmM sequence was subcloned to pPICZ{alpha}A as GzmM and verified by DNA sequencing. Recombinant S-AGzmM was expressed and purified, as above. Full-length cDNA coding human ICAD was provided by Dr. X. Wang (Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, Dallas, TX) and was subcloned into pET26b+. Full-length cDNA of human PARP was subcloned into pET15b. Recombinant human ICAD and PARP were expressed in BL21(DE3) and purified using nickel affinity chromatography.

GzmM loading with PJ

Pro-Ject protein transfection reagent (PJ) kit (Pierce) was prepared and aliquoted, according to the manufacturer’s instruction. GzmM or S-AGzmM was diluted in loading buffer (HBSS, 1 mM MgCl2, 1 mM CaCl2, and 1 mg/ml BSA) and incubated with appropriate volume of PJ at room temperature for 5 min. Target cells were harvested and washed twice with cold HBSS. The indicated number of cells was cultured with PJ/GzmM complex in 50 µl of loading buffer at 37°C for 4 h. Cells were pelleted for subsequent assays.

Cytotoxicity assay

Target cells were labeled with 100 µCi of sodium 51Cr-chromate (Na2CrO4) at 37°C for 1 h. After being thoroughly washed, 1 x 104-labeled cells were incubated with 1 µM S-AGzmM or GzmM alone or with PJ in the presence or absence of 100 µM ZVAD-fmk at 37°C for 4 h. The supernatant from each tube was carefully transferred into a disposable tube. Specific 51Cr release was counted on the 1450 MicroBeta counter (Pharmacia) and was calculated as follows: percentage of 51Cr release = ((test release – spontaneous release)/(maximum release – spontaneous release)) x 100.

Detection of apoptosis

Jurkat cells were treated with 1 µM GzmM or S-AGzmM alone or with PJ at 37°C for 4 h in the presence or absence of 100 µM ZVAD-fmk. Jurkat cells cultured with 50 ng/ml staurosporine (STP) for 20 h served as a positive control. Treated cells were double stained with annexin V-Fluos and propidium iodide (PI) before being evaluated by flow cytometry using a FACSCalibur (BD Biosciences). Analysis was performed using CellQuest software (BD Biosciences).

Flow-TUNEL assay with In Situ cell death detection kit (Roche Applied Science) was performed on treated cells, according to the manufacturer’s instruction. Briefly, treated cells were washed three times with PBS, fixed with 2% paraformaldehyde in PBS for 1 h at room temperature, and permeabilized with freshly prepared 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. A total of 50 µl of TUNEL reaction mixtures was added into each sample and incubated for 1 h at 37°C in a humidified atmosphere in the dark. After washing twice, cells were visualized using immunofluorescent microscopy.

Jurkat cells treated as above were fixed, permeabilized as described, and incubated with 5 µg/ml Hoechst 33342 (Sigma-Aldrich) in PBS for 10 min in dark and plated on the slide for observation. Images were captured using a Nikon microscope camera.

Klenow incorporation assay

Klenow fragment of DNA polymerase I (New England Biolabs) was used to label DNA nicks, as described (22). Briefly, 2 x 105 Jurkat cells were buffer treated or treated with 1 µM S-AGzmM or GzmM pretreated with NPase lysis buffer for 4 h. Treated cells were incubated with 5 U of Klenow and 10 µCi of [32P]dATP at 37°C for 1 h. Radiolabeled nuclei were washed thoroughly, dotted, and detected with phosphor screen. The radioactivity counts were recorded with Storm program (Pharmacia) and analyzed with ImageQuant software. The values shown were comparative [32P]dATP incorporations and expressed as mean ± SEM. Radiolabeled DNA was also separated on 1% alkaline agarose gel electrophoresis, as described (12).

DNA fragmentation assay

A total of 5 x 105 Jurkat cells lysed with 0.5% Nonidet P-40 buffer was buffer treated or treated with 1 µM S-AGzmM and indicated doses of GzmM in 50 µl of HBSS supplemented with 1 mM MgCl2, 1 mM CaCl2, and 1 mg/ml BSA at 37°C for 4 h in the presence or absence of ZVAD-fmk. Genomic DNA was extracted and analyzed by agarose gel electrophoresis. Briefly, after incubation, 330 µl of buffer containing 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2 M NaCl, 0.2% w/v SDS, and 0.2 mg/ml proteinase K was added to each reaction and incubated at 37°C overnight. NaCl was then added to a final concentration of 1.5 M, and the nuclear debris was spun down for 15 min in a microcentrifuge at room temperature. The DNA in the supernatant was precipitated with an equal volume of 100% ethanol. The DNA precipitate was washed once with 70% (v/v) ethanol and resuspended in 20 µl of TE buffer (10 mM Tris·HCl (pH 7.4), 1 mM EDTA) supplemented with 200 µg/ml DNase-free RNase A at 37°C for 2 h, the DNA was loaded onto a 2% agarose gel with 2 µg/ml ethidium bromide, and electrophoresis was conducted at 5 V/cm for 2 h. Genomic DNA of Jurkat cells treated with 1 µM GzmM or S-AGzmM alone or with PJ at 37°C for 4 h in the presence or absence of 100 µM ZVAD-fmk was also extracted and analyzed by agarose gel electrophoresis.

Laser-scanning confocal microscopy

Jurkat cells were treated with 1 µM S-AGzmM or GzmM in the presence or absence of PJ for the indicated times, washed, and plated on polylysine-coated slides. After fixing with 4% paraformaldehyde for 20 min at room temperature and permeabilizing with 0.1% Triton X-100 for another 20 min, the cells were incubated at room temperature for 1 h with 5 µg/ml anti-His-tag mAb, 50 µg/ml donkey serum, and 100 µg/ml RNase I. After washing twice with cold PBS, the cells were stained with Alexa 488-conjugated donkey anti-mouse IgG and soaked for 5 min in PBS containing 0.1 µg/ml PI. The slides were mounted with ProLong Antifade reagent and observed using laser-scanning confocal microscopy (Olympus FV500 microscope).

Cleavage assay

Cell lysates were prepared with 0.5% Nonidet P-40 lysis buffer at 4°C for 15 min and centrifuged at 13,000 rpm for 15 min to remove nuclei and cell debris. Cell lysates (equivalent to 2 x 105 cells) or 0.5 µg of recombinant protein were incubated with indicated doses of GzmM or S-AGzmM for indicated time intervals. For in vivo cleavage assay, cells loaded with GzmM/PJ were lysed with 0.5% Nonidet P-40 lysis buffer. The reactions were terminated in 1x SDS loading buffer before being run on SDS-PAGE gels and immunoblotted with specific Abs, as indicated.

For N-terminal sequencing, the cleaved products of rICAD were run on SDS-PAGE gels and transferred to polyvinylidene difluoride membrane. After staining with Coomassie R-250, the polyvinylidene difluoride band was cut down and analyzed using ABI Procise 492cLC sequencer. The data were analyzed using Model 610 software program supplied by Applied Biosystems.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PJ can directly load GzmM into target cells causing apoptotic nuclear morphological changes

Recombinant human active GzmM and inactive S-AGzmM were expressed in P. pastoris and purified by nickel affinity chromatography. The enzymatic activity of rGzmM was detected by hydrolysis of its synthetic tetrapeptide substrate Suc-AAPL-pNA (7). rGzmM showed strong proteolytic activity even at low nanomolar concentration, whereas the mutant form S-AGzmM had no activity (data not shown).

A recent report showed GzmM induces a novel form of cell death that depends on perforin (21). Perforin can assist all the Gzms into target cells to trigger death by a variety of assays (6, 23, 24). Perforin transfers Gzms into the target cells through endocytosis and causes release of Gzms into the cytosol of target cells (4). However, in vitro GzmB-loading experiments showed GzmB enters a target cell and induces apoptosis independently of perforin (25, 26). Bleackley and colleagues (25, 27) demonstrated that a replication-deficient adenovirus type V (Ad) can efficiently transport GzmB into target cells and trigger cytolysis. We wanted to determine whether PJ can substitute for perforin or Ad to directly deliver Gzms into the target cell cytosol and induce cell death. PJ is used to deliver biologically active proteins directly into a living cell. Proteins loaded by PJ remain biologically active because the interaction between a protein and PJ is noncovalent. We loaded GzmM into Jurkat cells in the presence of an optimal dose of PJ and observed the entry of GzmM. GzmM transferred into the cytosol and the nuclei of treated cells within 4 h (Fig. 1A). The nuclei appeared condensed, shrunken, and with morphological changes. By 6 h, the nuclei treated by GzmM with PJ occurred segmented and collapsed. PJ and GzmM alone failed to make any change as mock-treated cells. Hoechst DNA staining obtained the similar apoptotic nuclear morphology in GzmM- and PJ-loaded cells (Fig. 1B). PJ or GzmM alone was without effect. Apoptosis-inducing agent STP-treated cells appeared characteristic apoptosis as a positive control. Similar results were obtained by using Ad for GzmM loading (data not shown).


Figure 1
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 1. GzmM is transported into the cytosol and the nuclei of target cells, leading to typical apoptotic nuclear morphology. A, Jurkat cells were treated with 1 µM GzmM in the presence of an optimal dose of PJ for the indicated times or left untreated. The cells were fixed, permeabilized, and incubated with anti-His6 mAb, followed by staining with the second Alex 488-conjugated goat anti-mouse Ab and PI. GzmM staining green fluorescence was shown at left, PI staining red in the middle, and the merged image at right. B, After incubating with PJ, 1 µM GzmM, or 1 µM GzmM/PJ at 37°C for 4 h, Jurkat cells were stained with Hoechst 33342. STP-treated cells were used as a positive control.

 
GzmM induces a caspase-dependent death pathway

Kelly et al. (21) showed GzmM loaded with perforin caused 51Cr release and higher annexin V+/PI+ double-positive cells and comparable single annexin V+ cells. To detect the cytolysis by PJ direct loading of GzmM, we analyzed cytotoxicity in GzmM/PJ-treated cells by a standard 4-h 51Cr release assay. In three independent experiments, Jurkat cells loaded with GzmM plus PJ induced 74 ± 8% specific cytolysis (Fig. 2A). GzmM and PJ alone or S-AGzmM and PJ just got background lysis (generally <10%). Similar results were found by using HeLa cells (data not shown). Jurkat cells loaded with different concentrations of GzmM plus PJ led to dynamic increase of cell death, as shown in Fig. 2B. At the concentration of 0.2 µM GzmM, cells began to undergo death (6.9% single annexin V+ and 7.4% annexin V+/PI+). At 1 µM GzmM, death rate increased to the equivalent rate of annexin V+/PI+ cells by Kelly’s (21) observations (33.4 vs 36%). Moreover, 1 µM GzmM induced high rate of single annexin V+ cells (24.9%). By contrast, Kelly et al. (21) showed GzmM did not initiate single annexin V+ cells. PJ and GzmM alone or inactive S-AGzmM/PJ just got background annexin V+/PI+ and single annexin V+ cells. These results are representative of at least three independent experiments.


Figure 2
View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 2. GzmM induces cell death that can be blocked by the pan caspase inhibitor ZVAD-fmk. A, 51Cr-labeled Jurkat cells were incubated with 1 µM GzmM or S-AGzmM for 4 h in the presence or absence of PJ. Specific 51Cr release was counted. For ZVAD treatment, cells were pretreated with 100 µM ZVAD-fmk at 37°C for 30 min before loading with 1 µM GzmM/PJ. The values are mean ± SEM. B, Jurkat cells were treated with PJ, 1 µM GzmM, 1 µM S-AGzmM/PJ, or different doses of GzmM plus PJ for 4 h, followed by staining with annexin V and PI. Buffer and STP-treated cells served as negative and positive controls, respectively. Results shown are representative of three separate experiments.

 
To determine whether GzmM/PJ-induced death is dependent on caspase activation, cells were preincubated with the pan caspase inhibitor ZVAD-fmk before GzmM loading. A total of 100 µM ZVAD-fmk blocked GzmM/PJ-induced specific lysis measured by a standard 4-h 51Cr release assay (14 ± 2 vs 74 ± 8%) (Fig. 2A) and annexin V/PI staining (data not shown). These data are representative of at least three separate experiments.

To further verify GzmM-induced death is caspase dependent, we analyzed caspase 3 cleavage by immunoblotting. Caspase activation is a key regulator of the apoptotic pathway for multiple inducers of cell death (28). Procaspases are processed at aspartic acid residues between the subunits of a heterotetramer and can be activated by other caspases or by GzmB (29, 30). Caspase 3 is a central executioner in the induction of apoptosis. Caspase 3 can be activated by processing its proenzyme procaspase 3 undergoing apoptosis. We found procaspase 3 was processed in GzmM-treated cell lysates with a dose- and time-dependent fashion (Fig. 3A). Caspase 3 began to be processed with a low dose of 10 nM GzmM at very early time of 5 min. The active form p17 band was detectable at high dose of 1 µM GzmM, which was comparable to 0.3 µM GzmB treatment. The cleavage patterns by GzmM and B are different. Inactive S-AGzmM was without effect. To verify the caspase activation is physiologically relevant, Jurkat cells were loaded with GzmM in the presence of PJ. Procaspase 3 was cleaved to produce the active form, a p17 product in GzmM/PJ-loaded cells (Fig. 3B). PJ or GzmM alone or S-AGzmM/PJ failed to cut procaspase 3. beta-Actin was unchanged as a loading control. Taken together, GzmM-induced death is required for caspase activation.


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 3. Caspase 3 is activated after GzmM treatment. A, Jurkat cell lysates (equivalent to 2 x 105 cells) were treated with different concentrations of GzmM from 0.01 to 1.0 µM (lanes 3–8) or GzmB from 0.3 to 0.5 µM (lanes 9–12) for 2 h (top panel), or with 1.0 µM GzmM for the indicated times (bottom panel). The reaction products were probed with anti-caspase 3 Ab. B, A total of 2 x 105 Jurkat cells was treated with buffer, 1.0 µM GzmM, or S-AGzmM alone or with PJ at 37°C for 4 h. The whole cell lysates were electrophoresed on a 15% SDS-PAGE gel and probed for caspase 3. beta-Actin served as a negative control.

 
GzmM fails to nick DNA

DNA damage is another feature of cell death. GzmA induces caspase-independent death with ssDNA nicking (6, 9). DNA nicking is supposed to be an early stage of cell death induced by cytolytic lymphocytes. GzmM initiates a faster death pathway of target cells consistent with Kelly’s report (21). We next wanted to look at whether GzmM initiates ssDNA nicking. The Klenow polymerase labeling was used to assess DNA strand breaks, as described (12). DNA nicks were not induced from GzmM-treated Jurkat nuclei, even with 1 µM GzmM (Fig. 4A), whereas extensive DNA nicking occurred with 0.5 µM GzmA treatment under the same condition, which is in concert with our previous reports (12). PJ- or S-AGzmM-treated nuclei were not nicked. These results were confirmed via denaturing alkaline gel electrophoresis, as shown in Fig. 4B. These data are representative of three separate experiments.


Figure 4
View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 4. GzmM does not cause ssDNA nicking. A, Jurkat nuclei were treated with GzmM (1 µM) or GzmA (1 µM) at 37°C for 4 h and radiolabeled by Klenow DNA polymerase. The radioactivity counts were determined, as described in Materials and Methods. The values shown represent mean ± SEM. B, Genomic DNA treated above was separated with 1% denaturing alkaline gel electrophoresis and visualized by autoradiography. The data shown are representative of three separate experiments.

 
GzmM triggers DNA fragmentation

To determine the form of DNA damage in GzmM-induced death, genomic DNA was extracted from GzmM-treated nuclei. Characteristic apoptotic DNA ladders were visualized on agarose gels in GzmM-treated nuclei (Fig. 5A). DNA laddering occurred in a dose-dependent manner. Mock- or S-AGzmM-treated nuclei were without effect. To further assess the DNA fragmentation induced by GzmM is physiologically relevant, GzmM was loaded into Jurkat cells in the presence of PJ. Genomic DNA was extracted and assayed to visualize DNA fragmentation from GzmM-loaded cells. DNA ladders appeared in GzmM/PJ-treated cells (Fig. 4B). GzmM and PJ alone or S-AGzmM/PJ failed to damage DNA. A total of 100 µM ZVAD-fmk nearly blocked GzmM-induced DNA laddering that is consistent with the above observations. To further confirm DNA damage induced by GzmM, fluorescein-dUTP-labeled TUNEL was used to detect the free 3'-OH DNA ends at a single cell level by fluorescence microscopy. GzmM/PJ-treated cells appeared TUNEL positive with strong staining (Fig. 5C). PJ or GzmM alone was TUNEL negative.


Figure 5
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5. GzmM mediates DNA fragmentation. A, The isolated Jurkat nuclei were untreated or treated with 1 µM S-AGzmM, or 0.5 µM or 1 µM GzmM, respectively. Genomic DNA was extracted and visualized by 2% agarose gel electrophoresis with ethidium bromide staining. B, Jurkat cells were incubated with 1 µM GzmM or S-AGzmM alone or with PJ for 4 h. Cells were pretreated with 100 µM ZVAD-fmk at 37°C for 30 min before being loaded with GzmM/PJ, while DMSO pretreatment was used as a control. C, Jurkat cells were incubated with PJ, 1 µM GzmM, or 1 µM GzmM/PJ at 37°C for 4 h. Treated cells were fixed and permeabilized, and TUNEL staining was performed. The images shown are representative of three independent experiments.

 
GzmM directly cleaves the ICAD

CAD, also known as DFF40, has been verified to be directly responsible for DNA fragmentation. CAD exists as a heterodimer with its inhibitor ICAD (also called DFF45) that serves as both a specific molecular chaperone to mediate the correct folding of DFF40 and an inhibitor of the nuclease activity of DFF40 in resting cells (31, 32, 33). CAD can be activated by cleavage of its inhibitor ICAD to initiate DNA fragmentation after a cell receives a death signal. To further determine whether ICAD is degraded by GzmM treatment, ICAD was probed by immunoblotting with anti-ICAD Ab. ICAD was cleaved to generate a predominant 30-kDa fragment in GzmM-treated cell lysates in a dose-dependent manner (Fig. 6A). S-AGzmM had no proteolytic activity. ICAD began to degrade within 30 min and was almost completely processed by 2 h with 0.5 µM GzmM treatment. S-AGzmM did not cause ICAD degradation. To investigate whether ICAD degradation occurs in GzmM-loaded cells, ICAD was probed in Jurkat cells loaded with GzmM plus PJ. ICAD was cleaved in GzmM/PJ-loaded cells (Fig. 6B). GzmM or PJ alone or S-AGzmM plus PJ failed to degrade ICAD. The same blot was stripped and reprobed for beta-actin. beta-Actin was unchanged as a good loading control.


Figure 6
View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 6. ICAD is directly cleaved by GzmM. A, Jurkat cell lysates (equivalent to 2 x 105 cells) were incubated with different doses of GzmM at 37°C for 2 h (lanes 1–6) or with 1.0 µM GzmM for the indicated times (lanes 7–12). Untreated or 1.0 µM S-AGzmM-treated sample served as a negative control. The products were electrophoresed on SDS gels and probed by anti-ICAD Ab. B, 2 x 105 Jurkat cells were treated with 1.0 µM GzmM, S-AGzmM alone, or PJ at 37°C for 4 h. The whole cell lysates were immunoblotted with anti-ICAD serum. beta-Actin was unchanged as a negative control. C, rICAD (0.5 µg) was treated with different doses of GzmM ranging from 5 to 100 nM (lanes 1–7) and 300 nM recombinant active caspase 3 for 2 h (lane 8). A total of 0.5 µg of rICAD was incubated with 100 nM GzmM for the indicated times (lanes 9–12). The reaction products were probed, as described above.

 
To further evaluate whether ICAD can be directly processed by GzmM, human rICAD was treated with different concentrations of GzmM. GzmM can directly cut rICAD to produce 11- and 30-kDa fragments beginning at low nanomolar concentrations (Fig. 6C). rICAD was completely degraded with 50 nM GzmM. rICAD cleavage occurred very rapidly within 5 min at 100 nM concentration of GzmM. rICAD was completely degraded by 15 min. S-AGzmM did not lead to rICAD degradation. Caspase 3 was used as a positive control. We performed N-terminal sequencing of the cleaved products and showed the cleavage site was after Ser107 (TAWIS{downarrow}QESFD). This is different from GzmB.

PARP is degraded by GzmM

In cells, a NAD+-dependent signal transduction mechanism serves to protect cells against the genome-destabilizing effects after DNA breaks. The mechanism involves two nuclear enzymes, PARP-1 and PARP-2, that sense DNA strand breaks. When activated by DNA breaks, these PARPs catalyze the transfer of ADP-ribose moiety from NAD+ to nuclear target substrates. Through recruitment of specific proteins at the site of damage and regulation of their activities, these polymers may either directly participate in the DNA repair process or coordinate repair through chromatin unfolding, cell cycle progression, and cell death (34, 35). PARP is a sensor of DNA damage, which is a downstream substrate of caspase 3 and can be directly degraded by GzmB (36, 37). We wanted to determine whether PARP is degraded after GzmM treatment. Human rPARP was incubated with different doses of GzmM at 37°C for 2 h. PARP was cleaved beginning at a low concentration of 30 nM GzmM (Fig. 7A). A total of 50 nM GzmM can completely degrade PARP. PARP cleavage appeared rapidly within 15 min by 50 nM GzmM treatment. To further investigate whether PARP is a physiological substrate of GzmM, Jurkat cells were loaded with GzmM plus PJ and probed by immunoblotting with anti-PARP serum. PARP was completely cleaved to generate a 45-kDa product in GzmM-loaded cells within 4 h (Fig. 6B). GzmM and PJ alone or S-AGzmM/PJ was without effect. Therefore, PARP is a direct physiological substrate of GzmM.


Figure 7
View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 7. PARP is a direct substrate of GzmM. A, 0.5 µg of rPARP was untreated or treated with different doses of GzmM ranging from 0.01 to 0.5 µM for 2 h at 37°C (upper panel), or with 0.3 µM GzmM for 15–120 min (lower panel). The products were run on SDS-PAGE gels and probed for PARP. B, A total of 2 x 105 Jurkat cells was treated with 1.0 µM GzmM or S-AGzmM and/or PJ at 37°C for 4 h. The products were immunoblotted with anti-PARP serum. NS, Nonspecific band.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Perforin was originally thought to generate pores in the target cell plasma membrane through which Gzms would get into cytosol (38, 39). However, this model for Gzm entry has been questioned. At low concentrations, perforin delivers Gzms to trigger apoptosis, and at high concentrations perforin induces necrotic death. Actually, at a sublytic concentration the plasma membrane integrity is not disrupted; small dyes do not enter the cell (40, 41). Froelich et al. (25, 26, 27) showed that a replication-deficient Ad can efficiently transport GzmB into targets and lead to apoptosis. Recent reports verified Gzms are taken into cells by endocytosis and perforin or Ad causes release of Gzms into the cytosol (4, 42). Transient expression of GzmB or direct transfer of GzmB by microinjection can initiate cell death (27, 43). It indicates that GzmB exerts its proapoptotic function inside a target cell independently of perforin. In this study, we showed a cationic lipid-based PJ can efficiently transport GzmM into target cells and induce cell death. PJ binds to proteins without covalent formation that moves proteins into cells, where proteins remain their physiological activities. GzmM alone did not enter the target cells. GzmM entry needs assistance of PJ or Ad that is consistent with perforin-dependent entry of GzmM (21). Actually, an optimal dose of PJ was used to load GzmA or B into target cells, which led to similar apoptotic features as perforin- or Ad-delivered GzmA or B (data not shown).

A recent study showed that GzmM causes a third major death pathway besides GzmA and B (21). GzmM-induced death is independent of caspase activation and mitochondrial damage. GzmM does not trigger chromosomal DNA fragmentation by analyzing DNA content via PI staining. However, we found that GzmM initiates a caspase-dependent death pathway. The pan caspase inhibitor ZVAD (100 µM) can completely block GzmM-induced cytolysis, DNA fragmentation, and procaspase 3 processing. Our observations are different from those of Kelly et al. (21). They showed that even high concentration of 200 µM ZVAD failed to inhibit GzmM-induced death. GzmM-treated cells did not cause caspase activation and phosphatidylserine externalization. We found GzmM initiated high rate of single annexin V+ cells and caspase activation. Although we used the different concentrations of GzmM and different delivery agents, we obtained the similar death rates by GzmM (1 µM)/PJ and GzmM (83 nM)/perforin treatment. It indicates that the 1 µM concentration of GzmM we used is equal in activity to 83 nM concentration used by Smyth group. Different GzmM activity might be due to the different expression systems. We produced GzmM in P. pastoris yeast, whereas Smyth group generated GzmM from baculovirus sf21 cells. The same death rates induced by GzmM (1 µM)/PJ and GzmM (83 nM)/perforin might be also attributable to the delivery efficiency of the two different delivery agents. Caspases are central regulators of apoptotic pathway for multiple inducers of cell death (13). Caspases are synthesized as a single polypeptide, act as a heterotetramer consisting of two large and two small subunits, and can be initiated by other caspases or GzmB (29, 30). Although a number of caspases are processed by GzmB, caspases 3 and 7 are cleaved most rapidly in cells loaded GzmB with perforin (44, 45). Caspase 3 is a central execution member of the caspase family.

DNA damage is a key feature of cell death. We previously showed GzmA triggers cell death with ssDNA nicks by targeting an endoplasmic reticulum-associated SET complex (6, 9). The SET complex contains the GzmA-activated DNase NM23H1 that binds to its inhibitor SET in a resting cell. Cleavage of SET by GzmA unleashes the nuclease activity of NM23H1 to nick DNA (12). DNA nicking is thought to be an initial stage of death induced through cytotoxic lymphocytes, followed by DNA fragmentation by other Gzms. In addition to GzmA, GzmC and K have been reported to cause DNA nicks (23, 46). Only GzmB has been defined to make DNA fragmentation (3). We found GzmM did not cause DNA nicking, but led to typical DNA laddering. The DNA fragmentation induced by GzmM was verified by TUNEL assay. To further gain insight into the mechanism, we demonstrated that ICAD/DFF45 can be directly cleaved by GzmM. CAD/DFF40 has been defined to be directly responsible for DNA fragmentation. CAD remains in an inactive form with its inhibitor ICAD of normal cells (31, 32, 33). Once a cell receives a death signal, caspase 3 is activated and it in turn cleaves ICAD, which activates the nuclease activity of CAD. ICAD acts as both an inhibitor of CAD and a specific nuclear chaperone to assist the correct folding of CAD. ICAD-deficient mice have a reduced ability to make DNA fragmentation, which is partially resistant to in vitro and in vivo induced apoptosis (16). These indicate that CAD/ICAD complex plays an essential role in DNA fragmentation undergoing apoptosis. ICAD degradation by GzmB releases the nuclease activity of CAD to result in DNA fragmentation (14, 15). GzmM also directly degrades ICAD to activate CAD activity. ICAD was cleaved to generate 11- and 30-kDa fragments, which were from one cut after Ser107 (TAWIS{downarrow}QESFD). The cleavage pattern by GzmM is different from that of GzmB, producing 28- and 31-kDa fragments at residues of D117 and D224. It has been reported GzmM prefers to cleave after methionine, leucine, or norleucine by measuring of synthetic peptides (18). The cleavage sites of GzmM for physiological substrates remain to be further investigated.

PARP is a sensor enzyme of DNA damage. It has three functional domains, an N-terminal DNA binding domain, a C-terminal catalytic domain, and an automodification domain in the middle part. Once DNA breaks, PARP is activated to bind to the DNA strand interruptions through its DNA binding domain. Activated PARP catalyzes formation of long and branched polymers of ADP-ribose using NAD as a substrate (47). These polymers recruit specific proteins at the DNA damage site and enhance their activities to participate in DNA repair or coordinate repair via regulation of other cellular DNA-related functions (34, 35). PARP is specifically activated at a very early stage of apoptosis, while the cell continues to retain membrane integrity and the majority of the proteins are not cleaved (48). PARP is directly processed to produce a 89-kDa product by caspase 3-like activity undergoing apoptosis (37), whereas PARP is cleaved to generate 89- and 64-kDa fragments in GzmB-mediated cytolysis. The dual pattern for cleavage of PARP by GzmB indicates GzmB not only activates caspases, but also enters the nucleus to directly degrade PARP. GzmM can directly cleave PARP to produce a 45-kDa fragment, which is different from that generated by caspase 3-like activity or GzmB.

Unlike GzmB, GzmM is not expressed in CD4+ or CD8+ T cells either constitutively or after activation (19). However, GzmM is constitutively highly expressed in NK cells as is perforin. A recent report showed a human NK cell line KHYG-1 appears to have greater killing activity that is consistent with higher GzmM expression (20), although GzmA and B are undetectable. It suggests that GzmM may play a crucial role in NK cell-mediated cytolysis. In fact, reports by us and Kelly et al. (21) demonstrated that GzmM-induced rapid cell death is in concert with the kinetics of cytolysis by NK cells. But we found that GzmM-induced cell death is dependent on caspase activation and DNA fragmentation reminiscent of GzmB. NK cells are the effectors of the innate immunity, acting as the first line of defense against viral infection and tumors (49). A recent report from NK cell depletion experiments showed NK cells are critical for tumor immunity (50). They exert their effector function without prior sensitization. Manipulating NK cells to eradicate tumors may be a powerful approach for cancer immunotherapy. Therefore, to fully understand the biology and function of NK cells will provide the basis for their potential clinical applications.


   Acknowledgments
 
We thank Drs. X. Wang and R. P. Sekaly for their reagents, and Chunchun Liu and Yan Teng for their technical help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by the National Natural Science Foundation of China and the Outstanding Youth Grant (30525005 and 30470365) and the Hundred Talents Program of Chinese Academy of Sciences (to Z.F.). Back

2 Address correspondence and reprint requests to Dr. Zusen Fan, National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China. E-mail address: fanz{at}moon.ibp.ac.cn Back

3 Abbreviations used in this paper: Gzm, granzyme; Ad, adenovirus; CAD, caspase-activated DNase; ICAD, inhibitor of CAD; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PJ, Pro-Ject protein transfection reagent; STP, staurosporine. Back

Received for publication January 17, 2006. Accepted for publication April 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Russell, J. H., T. J. Ley. 2002. Lymphocyte-mediated cytotoxicity. Annu. Rev. Immunol. 20: 323-370. [Medline]
  2. Lieberman, J.. 2003. The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat. Rev. Immunol. 3: 361-370. [Medline]
  3. Trapani, J. A., V. R. Sutton. 2003. Granzyme B: pro-apoptotic, antiviral and antitumor functions. Curr. Opin. Immunol. 15: 533-543. [Medline]
  4. Keefe, D., L. Shi, S. Feske, R. Massol, F. Navarro, T. Kirchhausen, J. Lieberman. 2005. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 23: 249-262. [Medline]
  5. Grossman, W. J., P. A. Revell, Z. H. Lu, H. Johnson, A. J. Bredemeyer, T. J. Ley. 2003. The orphan granzymes of humans and mice. Curr. Opin. Immunol. 15: 544-552. [Medline]
  6. Lieberman, J., Z. Fan. 2003. Nuclear war: the granzyme A-bomb. Curr. Opin. Immunol. 15: 553-559. [Medline]
  7. Mahrus, S., C. S. Craik. 2005. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem. Biol. 12: 567-577. [Medline]
  8. Pilat, D., T. Fink, B. Obermaier-Skrobanek, M. Zimmer, H. Wekerle, P. Lichter, D. E. Jenne. 1994. The human Met-ase gene (GZMM): structure, sequence, and close physical linkage to the serine protease gene cluster on 19p13.3. Genomics 24: 445-450. [Medline]
  9. Fan, Z. S., Q. X. Zhang. 2005. Molecular mechanisms of lymphocyte-mediated cytotoxicity. Cell Mol. Immunol. 2: 259-264. [Medline]
  10. Fan, Z., P. J. Beresford, D. Zhang, J. Lieberman. 2002. HMG2 interacts with the nucleosome assembly protein SET and is a target of the cytotoxic T-lymphocyte protease granzyme A. Mol. Cell. Biol. 22: 2810-2820. [Abstract/Free Full Text]
  11. Fan, Z., P. J. Beresford, D. Zhang, Z. Xu, C. D. Novina, A. Yoshida, Y. Pommier, J. Lieberman. 2003. Cleaving the oxidative repair protein Ape1 enhances cell death mediated by granzyme A. Nat. Immunol. 4: 145-153. [Medline]
  12. Fan, Z., P. J. Beresford, D. Y. Oh, D. Zhang, J. Lieberman. 2003. Tumor suppressor NM23–H1 is a granzyme A-activated DNase during CTL-mediated apoptosis, and the nucleosome assembly protein SET is its inhibitor. Cell 112: 659-672. [Medline]
  13. Los, M., S. Wesselborg, K. Schulze-Osthoff. 1999. The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity 10: 629-639. [Medline]
  14. Sharif-Askari, E., A. Alam, E. Rheaume, P. J. Beresford, C. Scotto, K. Sharma, D. Lee, W. E. DeWolf, M. E. Nuttall, J. Lieberman, R. P. Sekaly. 2001. Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation. EMBO J. 20: 3101-3113. [Medline]
  15. Thomas, D. A., C. Du, M. Xu, X. Wang, T. J. Ley. 2000. DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 12: 621-632. [Medline]
  16. Zhang, J., X. Liu, D. C. Scherer, L. van Kaer, X. Wang, M. Xu. 1998. Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45. Proc. Natl. Acad. Sci. USA 95: 12480-12485. [Abstract/Free Full Text]
  17. Kelly, J. M., M. D. O’Connor, M. D. Hulett, K. Y. Thia, M. J. Smyth. 1996. Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. Immunogenetics 44: 340-350. [Medline]
  18. Smyth, M. J., T. Wiltrout, J. A. Trapani, K. S. Ottaway, R. Sowder, L. E. Henderson, C. M. Kam, J. C. Powers, H. A. Young, T. J. Sayers. 1992. Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia. J. Biol. Chem. 267: 24418-24425. [Abstract/Free Full Text]
  19. Sayers, T. J., A. D. Brooks, J. M. Ward, T. Hoshino, W. E. Bere, G. W. Wiegand, J. M. Kelly, M. J. Smyth. 2001. The restricted expression of granzyme M in human lymphocytes. J. Immunol. 166: 765-771. [Abstract/Free Full Text]
  20. Suck, G., D. R. Branch, M. J. Smyth, R. G. Miller, J. Vergidis, S. Fahim, A. Keating. 2005. KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity. Exp. Hematol. 33: 1160-1171. [Medline]
  21. Kelly, J. M., N. J. Waterhouse, E. Cretney, K. A. Browne, S. Ellis, J. A. Trapani, M. J. Smyth. 2004. Granzyme M mediates a novel form of perforin-dependent cell death. J. Biol. Chem. 279: 22236-22242. [Abstract/Free Full Text]
  22. Beresford, P. J., D. Zhang, D. Y. Oh, Z. Fan, E. L. Greer, M. L. Russo, M. Jaju, J. Lieberman. 2001. Granzyme A activates an endoplasmic reticulum-associated caspase-independent nuclease to induce single-stranded DNA nicks. J. Biol. Chem. 276: 43285-43293. [Abstract/Free Full Text]
  23. Johnson, H., L. Scorrano, S. J. Korsmeyer, T. J. Ley. 2003. Cell death induced by granzyme C. Blood 101: 3093-3101. [Abstract/Free Full Text]
  24. Shi, L., C. M. Kam, J. C. Powers, R. Aebersold, A. H. Greenberg. 1992. Purification of three cytotoxic lymphocyte granule serine proteases that induce apoptosis through distinct substrate and target cell interactions. J. Exp. Med. 176: 1521-1529. [Abstract/Free Full Text]
  25. Froelich, C. J., K. Orth, J. Turbov, P. Seth, R. Gottlieb, B. Babior, G. M. Shah, R. C. Bleackley, V. M. Dixit, W. Hanna. 1996. New paradigm for lymphocyte granule-mediated cytotoxicity: target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis. J. Biol. Chem. 271: 29073-29079. [Abstract/Free Full Text]
  26. Wang, G. Q., E. Wieckowski, L. A. Goldstein, B. R. Gastman, A. Rabinovitz, A. Gambotto, S. Li, B. Fang, X. M. Yin, H. Rabinowich. 2001. Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells. J. Exp. Med. 194: 1325-1337. [Abstract/Free Full Text]
  27. Pinkoski, M. J., M. Hobman, J. A. Heibein, K. Tomaselli, F. Li, P. Seth, C. J. Froelich, R. C. Bleackley. 1998. Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis. Blood 92: 1044-1054. [Abstract/Free Full Text]
  28. Danial, N. N., S. J. Korsmeyer. 2004. Cell death: critical control points. Cell 116: 205-219. [Medline]
  29. Adrain, C., B. M. Murphy, S. J. Martin. 2005. Molecular ordering of the caspase activation cascade initiated by the cytotoxic T lymphocyte/natural killer (CTL/NK) protease granzyme B. J. Biol. Chem. 280: 4663-4673. [Abstract/Free Full Text]
  30. Darmon, A. J., D. W. Nicholson, R. C. Bleackley. 1995. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature 377: 446-448. [Medline]
  31. Liu, X., P. Li, P. Widlak, H. Zou, X. Luo, W. T. Garrard, X. Wang. 1998. The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc. Natl. Acad. Sci. USA 95: 8461-8466. [Abstract/Free Full Text]
  32. Liu, X., H. Zou, C. Slaughter, X. Wang. 1997. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89: 175-184. [Medline]
  33. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu, S. Nagata. 1998. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391: 43-50. [Medline]
  34. Ame, J. C., E. L. Jacobson, M. K. Jacobson. 1999. Molecular heterogeneity and regulation of poly(ADP-ribose) glycohydrolase. Mol. Cell. Biochem. 193: 75-81. [Medline]
  35. D’Amours, D., S. Desnoyers, I. D’Silva, G. G. Poirier. 1999. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem. J. 342: 249-268. [Medline]
  36. Andrade, F., S. Roy, D. Nicholson, N. Thornberry, A. Rosen, L. Casciola-Rosen. 1998. Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity 8: 451-460. [Medline]
  37. Froelich, C. J., W. L. Hanna, G. G. Poirier, P. J. Duriez, D. D’Amours, G. S. Salvesen, E. S. Alnemri, W. C. Earnshaw, G. M. Shah. 1996. Granzyme B/perforin-mediated apoptosis of Jurkat cells results in cleavage of poly(ADP-ribose) polymerase to the 89-kDa apoptotic fragment and less abundant 64-kDa fragment. Biochem. Biophys. Res. Commun. 227: 658-665. [Medline]
  38. Podack, E. R., J. D. Young, Z. A. Cohn. 1985. Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules. Proc. Natl. Acad. Sci. USA 82: 8629-8633. [Abstract/Free Full Text]
  39. Tschopp, J., D. Masson, K. K. Stanley. 1986. Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis. Nature 322: 831-834. [Medline]
  40. Kawasaki, Y., T. Saito, Y. Shirota-Someya, Y. Ikegami, H. Komano, M. H. Lee, C. J. Froelich, N. Shinohara, H. Takayama. 2000. Cell death-associated translocation of plasma membrane components induced by CTL. J. Immunol. 164: 4641-4648. [Abstract/Free Full Text]
  41. Metkar, S. S., B. Wang, M. Aguilar-Santelises, S. M. Raja, L. Uhlin-Hansen, E. Podack, J. A. Trapani, C. J. Froelich. 2002. Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 16: 417-428. [Medline]
  42. Shi, L., D. Keefe, E. Durand, H. Feng, D. Zhang, J. Lieberman. 2005. Granzyme B binds to target cells mostly by charge and must be added at the same time as perforin to trigger apoptosis. J. Immunol. 174: 5456-5461. [Abstract/Free Full Text]
  43. Zhao, J., L. H. Zhang, L. T. Jia, L. Zhang, Y. M. Xu, Z. Wang, C. J. Yu, W. D. Peng, W. H. Wen, C. J. Wang, et al 2004. Secreted antibody/granzyme B fusion protein stimulates selective killing of HER2-overexpressing tumor cells. J. Biol. Chem. 279: 21343-21348. [Abstract/Free Full Text]
  44. Talanian, R. V., X. Yang, J. Turbov, P. Seth, T. Ghayur, C. A. Casiano, K. Orth, C. J. Froelich. 1997. Granule-mediated killing: pathways for granzyme B-initiated apoptosis. J. Exp. Med. 186: 1323-1331. [Abstract/Free Full Text]
  45. Yang, X., H. R. Stennicke, B. Wang, D. R. Green, R. U. Janicke, A. Srinivasan, P. Seth, G. S. Salvesen, C. J. Froelich. 1998. Granzyme B mimics apical caspases: description of a unified pathway for trans-activation of executioner caspase-3 and -7. J. Biol. Chem. 273: 34278-34283. [Abstract/Free Full Text]
  46. Shi, L., R. P. Kraut, R. Aebersold, A. H. Greenberg. 1992. A natural killer cell granule protein that induces DNA fragmentation and apoptosis. J. Exp. Med. 175: 553-566. [Abstract/Free Full Text]
  47. Duriez, P. J., G. M. Shah. 1997. Cleavage of poly(ADP-ribose) polymerase: a sensitive parameter to study cell death. Biochem. Cell Biol. 75: 337-349. [Medline]
  48. Kaufmann, S. H., S. Desnoyers, Y. Ottaviano, N. E. Davidson, G. G. Poirier. 1993. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 53: 3976-3985. [Abstract/Free Full Text]
  49. Fan, Z., P. Yu, Y. Wang, M. L. Fu, W. Liu, Y. Sun, Y. X. Fu. 2005. NK cell activation by LIGHT triggers tumor specific CD8+ T cell immunity to reject established tumors. Blood 107: 1342-1351. [Medline]
  50. Kelly, J. M., P. K. Darcy, J. L. Markby, D. I. Godfrey, K. Takeda, H. Yagita, M. J. Smyth. 2002. Induction of tumor-specific T cell memory by NK cell-mediated tumor rejection. Nat. Immunol. 3: 83-90. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L. Wu, L. Wang, G. Hua, K. Liu, X. Yang, Y. Zhai, M. Bartlam, F. Sun, and Z. Fan
Structural Basis for Proteolytic Specificity of the Human Apoptosis-Inducing Granzyme M
J. Immunol., July 1, 2009; 183(1): 421 - 429.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. P. Cullen, I. S. Afonina, R. Donadini, A. U. Luthi, J. P. Medema, P. I. Bird, and S. J. Martin
Nucleophosmin Is Cleaved and Inactivated by the Cytotoxic Granule Protease Granzyme M during Natural Killer Cell-mediated Killing
J. Biol. Chem., February 20, 2009; 284(8): 5137 - 5147.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Bovenschen, P. J. A. de Koning, R. Quadir, R. Broekhuizen, J. M. A. Damen, C. J. Froelich, M. Slijper, and J. A. Kummer
NK Cell Protease Granzyme M Targets {alpha}-Tubulin and Disorganizes the Microtubule Network
J. Immunol., June 15, 2008; 180(12): 8184 - 8191.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Hua, Q. Zhang, and Z. Fan
Heat Shock Protein 75 (TRAP1) Antagonizes Reactive Oxygen Species Generation and Protects Cells from Granzyme M-mediated Apoptosis
J. Biol. Chem., July 13, 2007; 282(28): 20553 - 20560.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Zhao, H. Zhang, Y. Guo, and Z. Fan
Granzyme K Directly Processes Bid to Release Cytochrome c and Endonuclease G Leading to Mitochondria-dependent Cell Death
J. Biol. Chem., April 20, 2007; 282(16): 12104 - 12111.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
V. R. Sutton, N. J. Waterhouse, K. A. Browne, K. Sedelies, A. Ciccone, D. Anthony, A. Koskinen, A. Mullbacher, and J. A. Trapani
Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis
J. Cell Biol., February 12, 2007; 176(4): 425 - 433.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
M. Bots and J. P. Medema
Granzymes at a glance
J. Cell Sci., December 15, 2006; 119(24): 5011 - 5014.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, H.
Right arrow Articles by Fan, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, H.
Right arrow Articles by Fan, Z.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*Protein
*UniGene
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS