Division of Molecular Medicine, Faculty of Medicine and Health Science, University of Auckland, New Zealand.
Correspondence to: Geoffrey W. Krissansen, Ph.D., Division of Molecular Medicine, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand (e-mail: gw.krissansen{at}auckland.ac.nz).
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ABSTRACT |
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INTRODUCTION |
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Programmed cell death involves an evolutionarily conserved cascade of events that are controlled by inhibitor of apoptosis (IAP) proteins, such as survivin, apollon, MLIAP (i.e., melanoma inhibitor of apoptosis), XIAP (i.e., X-linked inhibitor of apoptosis protein), cIAP1 (i.e., cellular inhibitor of apoptosis), cIAP2, and NAIP (i.e., neuronal apoptosis inhibitory protein) (4,5), and by Bcl-2 family members that either promote (i.e., Bax, Bak, Bcl-xS, Bad, and Bid) or counteract (i.e., Bcl-2, Bcl-xL, and Mcl-1) apoptotic cell death signals (68). IAP proteins inhibit the executors of apoptosis, including caspases 3 and 7 (9), and modulate the inducible transcription factor, NF-kappa B. IAP proteins contain variable numbers of a unique 80-amino-acid residue repeat, the baculovirus inhibitor of apoptosis repeat (BIR) domain, and a single carboxy-terminal RING Zn-finger motif (2). Survivin, a recently identified IAP member (10), exists primarily as a dimer in cells. Unlike other IAPs, survivin contains a single zinc-binding BIR domain at its amino-terminus, followed by a long amphipathic helix instead of a RING finger, which generates a "bow-tie" shape in the dimer (1113). Survivin is highly expressed during human embryonic and fetal development, but it is absent from all adult tissues except for the endometrium (14) and basal keratinocytes (15). Survivin is highly overexpressed in most common cancers of the lung, colon, pancreas, prostate, and breast, and its expression in these cancers is associated with an unfavorable prognosis (10,1620).
Survivin is the only apoptosis inhibitor yet described that is expressed in a cell cycle-dependent manner in G2 and M phases. During mitosis, survivin localizes to mitotic spindle microtubules, centrosomes, pericentrosomal microtubules, and the cytokinetic remnant. Survivin is required to preserve the integrity of the mitotic apparatus in situ, and in transformed cell lines, it forms a novel apoptotic checkpoint (21,22). Overexpression of survivin initiates cell division by inducing resistance to G1 arrest and thereby accelerating the entry into S phase. Overexpression of survivin also induces activation of the cyclin-dependent kinase 2 (Cdk2)cyclin E complex, phosphorylation of the retinoblastoma protein, and release of p21 from Cdk4. p21 then interacts with mitochondrial procaspase 3 to suppress Fas-mediated cell death (2325). Therefore, when overexpressed in the tumors of adult mammals, survivin has oncogenic potential because it can overcome the G2/M checkpoint to ensure mitotic progression (22).
Caspase-3, a protease that plays a key role in a proteolytic cascade within the apoptosis signal pathway, is activated by numerous cell death-inducing signals. Like other IAP proteins, survivin directly inhibits caspase-3, which is essential for suppression of caspase-mediated cleavage of centrosome-associated p21 (26). Survivin also binds to the IAP inhibitor Smac/Diablo, and that binding may free other IAP members to suppress caspases (27). Survivin's interaction with the mitotic spindle apparatus places it in a unique position to regulate the supramolecular assembly of cell death and cell cycle regulatory molecules present within the centrosome.
Antiapoptosis proteins are now regarded as important targets in cancer therapy. Antisense complementary DNA (cDNA) and oligonucleotides that reduce the expression of Bcl-2 inhibit the growth of certain tumor cell lines in vitro (2830). Similarly, antisense oligonucleotides that reduce survivin expression in tumor cells induce apoptosis and polyploidy, decrease colony formation in soft agar, and sensitize tumor cells to chemotherapy in vitro (3134). Cells that depend on survivin for their survival cannot complete cytokinesis without survivin, resulting in multinucleated cells (32). Survivin may be required for tumor angiogenesis, since it is highly expressed in newly formed blood vessels in response to vascular endothelial growth factor and basic fibroblast growth factor (35,36) and mediates angiopoietin inhibition of endothelial cell apoptosis (37). Hence, therapies that inhibit survivin expression and/or function may prevent tumors from acquiring an adequate blood supply.
Immunotherapy, the treatment of tumors with immunostimulators, including the lymphocyte costimulatory cell adhesion molecules B7-1, VCAM-1 (i.e., vascular cell adhesion molecule-1), and ICAM-1 (i.e., intercellular cell adhesion molecule-1), is often effective against small tumors but not against large tumors, perhaps because tumors become increasingly resistant to immune attack as they grow (3840). It is possible that barriers to cancer immunotherapy can be overcome by targeting a tumor's weapons of survival (e.g., proangiogenic factors), defense (e.g., antiapoptotic and immunosuppressive factors), and attack (e.g., Fas ligand [FasL] and receptor-binding cancer antigen expressed on SiSo cells [RCAS]) (41).
Here, we investigate whether the inhibition of survivin expression or function can inhibit tumor growth in situ. Because survivin potentially protects tumors from immune attack by preventing programmed cell death, we also examined the effects of survivin antagonists on the efficacy of cancer immunotherapy using B7-1, which has been shown to stimulate potent antitumor immunity when introduced into tumors in situ.
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MATERIALS AND METHODS |
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EL-4 thymic lymphoma cells, which were derived from C57BL/6 (H-2b) mice, were purchased from the American Type Culture Collection (ATCC), Manassas, VA, and cultured at 37 °C in Dulbecco's modified Eagle medium (DMEM) (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) supplemented with 10% fetal calf serum, 50 U/mL penicillinstreptomycin, 2 mM L-glutamine, and 1 mM pyruvate. Rat hybridomas secreting monoclonal antibodies (MAbs) against mouse CD8 (hybridoma 536.72), CD4 (hybridoma Gk1.5), and natural killer (NK) cells (hybridoma PK136) were purchased from the ATCC. Hybridomas 536.72 and PK136 were grown in RPMI-1640 medium (Life Technologies, Inc.), whereas Gk1.5 was grown in DMEM; other conditions were the same as those used to culture EL-4 cells. Hybridomas were injected intraperitoneally into pristane-primed nude mice. We purified MAbs from ascites by ammonium sulfate precipitation and dialysis against phosphate-buffered saline (PBS). The titer of the purified MAbs was determined at a maximum dilution of 1 : 2000 by flow cytometry with the use of a FACScan (Becton Dickinson, San Jose, CA).
Animals and Experimental Tumor Model
Female C57BL/6 mice, 69 weeks old, were obtained from the Animal Resource Unit, Faculty of Medicine and Health Science, University of Auckland, Auckland, New Zealand. The tumors were established by the subcutaneous injection of 2 x 105 EL-4 cells into the left flank of the mice. Tumor size was determined by measuring two perpendicular diameters. The animals were killed when their tumors reached 11.5 cm in diameter, in accord with Animal Ethics Approval (University of Auckland). All experiments included five or six mice per treatment group, and each experiment was repeated at least once.
Construction of Expression Plasmids
Antisense survivin plasmid. Total RNA was extracted from EL-4 cells with the use of 4 M guanidine (Fluka Biochemika Ltd., Schweiz, Switzerland) as described previously (42), and 1 µg of RNA was reverse transcribed with the use of SuperScript II Reverse Transcriptase (Life Technologies, Inc.) to generate complementary DNA (cDNA). The cDNA was used as a template to amplify a 509-base-pair DNA fragment encoding mouse survivin with the use of the polymerase chain reaction (PCR) and the oligonucleotide primers 5`-GAGTCGTCTTGGCGGAGG-3` (sense primer) and 5`-CTTAGATGTGGCATGTCACTC-3` (antisense primer). The resulting PCR product, which contained the entire coding region of mouse survivin (nucleotides 75583; GenBank accession No. NM_009689) (43), was subcloned into pGEM-T (Promega Corp., Madison, WI) by A-T ligation, liberated by digestion with ApaI and NotI, and directionally cloned into the ApaI and NotI sites of expression vector pCDNA3 (Invitrogen, Carlsbad, CA) in an antisense orientation with respect to the promoter on the plasmid.
Dominant-negative mutant survivin plasmid. We used PCR to construct a dominant-negative mutant of survivin by using the survivin cDNA as a template. Two overlapping complementary oligonucleotide primers, C84AF (5`-CACTCCCCGGGCGCAGCCTTCCTCAC-3`) and C84AR (5`-GGCTGCGCCCGGGGAGTGCTT-3`), which produced a T-to-G substitution at nucleotide 354 that introduced a diagnostic SmaI site, were used to change the cysteine residue at amino acid 84 to an alanine. The resulting PCR product was cloned into the XbaI and NotI sites of pCDNA3B, a plasmid identical to pCDNA3 except with a reversed polylinker. All constructs were verified by DNA sequencing.
B7-1 expression plasmid. A pCDM8-based expression plasmid encoding mouse B7-1 has been described previously (38).
Intratumoral Gene Transfer of Expression Plasmids
All plasmids were prepared by alkaline lysis, purified by cesium chloride gradient centrifugation at 270 000g for 22 hours at 20 °C, and diluted to 5 µg/µL in a solution of 5% glucose in 0.01% Triton X-100. The diluted plasmids were mixed at 1 : 1 (wt/wt) with a 0.1% solution of N-[1-(2,3-dioleoyloxy)propyl]N,N,N-trimethylammonium methyl sulfate cationic liposomes (Boehringer Mannheim, Mannheim, Germany) to form a DNAliposome complex. For single-vector transfer, small tumors (0.20.3 cm in diameter) were injected at multiple sites with 100 µg of DNA, whereas large tumors (0.40.6 cm in diameter) were injected at multiple sites with 180 µg of DNA, unless otherwise stated. For therapy using combinations of the B7-1- and survivin-based plasmids, 100 µg and 180 µg of the B7-1 plasmid DNAliposome complex were injected at multiple sites into small tumors (0.20.3 cm in diameter) and large tumors (0.40.6 cm in diameter), respectively. Twenty-four hours later, 100 µg and 180 µg of the DNAliposome complex containing either the antisense or dominant-negative mutant survivin vectors were injected into the same tumors, respectively.
Rechallenge of Mice With Parental Tumor Cells
Treated mice that rejected their tumors and subsequently remained tumor free were re-injected subcutaneously 7 weeks later with 2 x 106 EL-4 cells (in 0.1 mL) in the right flank.
Measurement of Tumor-Specific Cytotoxic T Lymphocytes
Splenocytes (effector cells) were harvested from mice 28 days after injection with expression plasmids, mixed with EL-4 target cells in graded effector cell-to-target cell ratios, and then incubated at 37 °C in 96-well, round-bottom plates. After a 4-hour incubation, 50 µL of supernatant was collected from each well, and cell lysis was measured with the use of the CytoTox 96 Assay Kit (Promega Corp.). Controls for nonspecific lysis of the target and effector cells were included in each assay. After subtraction of the values produced by those controls, the percentage of cell lysis was calculated with the use of the formula: 100 x (experimental spontaneous effector spontaneous target)/(maximum target spontaneous target).
Identification of Specific Leukocyte Subsets Responsible for Antitumor Immunity by Leukocyte Depletion
Mice bearing EL-4-derived tumors received intraperitoneal and intravenous injections every other day of 300 µg (in 0.1 mL) of the 536.72 (anti-CD8), Gk1.5 (anti-CD4), or PK136 (anti-NK cell) MAbs to deplete them of CD8-positive (CD8+) T cells, CD4+ T cells, and NK cells, respectively. Rat immunoglobulin G (IgG) (Sigma Chemical Co., St. Louis, MO) was injected as a control antibody. Seven days after the first MAb injections, the mice received an injection of the various expression plasmids. Depletion of individual leukocyte subsets was more than 90% effective, as determined by flow cytometry analysis of isolated splenocytes from leukocyte-depleted mice compared with mice whose leukocytes were not depleted.
Detection of Apoptosis
In situdetection of apoptotic cells by the TUNEL assay. Large tumors were injected with plasmids, and after 2 days, the tumors were excised, immediately frozen in dry ice, and stored at -70 °C. Serial sections (6 µm thick) of the frozen tumors were prepared to detect apoptosis by TUNEL (i.e., terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling) staining with the use of the In Situ Apoptosis Detection Kit from Boehringer Mannheim. Briefly, frozen sections were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, incubated with 20 µL TUNEL reagent for 60 minutes at 37 °C, and then examined by fluorescence microscopy. Adjacent sections were counterstained with hematoxylin and mounted onto poly-L-lysine-coated slides to allow the total number of nucleated cells to be counted. The percentage of apoptotic cells was assessed in 10 randomly selected fields viewed at x40 magnification. The apoptotic index (A/I) was calculated as follows: A/I = number of apoptotic (TUNEL-positive) cells x (100/total number of nucleated cells).
In situdetection of apoptotic cells by annexin-V-biotin staining. Mice received an injection via the tail vein of 0.5 mg of annexin-V-biotin (Apoptest Biotin, product B500; Nexins Research, Hoeven, The Netherlands) 2 days after the injection of plasmids into large tumors (44). The tumors were excised 30 minutes after administration of annexin-V-biotin, fixed in 4% formalin in HEPES buffer (i.e., 10 mM HEPES [pH 7.4], 137.5 mM NaCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 6 mM KCl, 0.4 mM NaH2PO4, 5.6 mM glucose, and 0.1% bovine serum albumin) at 4 °C, decalcified in distilled water containing 4% EDTA and 4% formalin for 4 weeks, and embedded in paraffin. Sections (6 µm) were dewaxed in xylene and rehydrated, incubated with 3% H2O2 in PBS for 5 minutes to block endogenous peroxidase activity, and blocked with 30% normal goat serum (Vector Laboratories, Burlingame, CA). The sections were then incubated at 37 °C for 30 minutes with avidinbiotinperoxidase complex (VECTASTAIN ABC Elite Kit; Vector Laboratories), developed in Sigma Fast DAB (3,3`-diaminobenzidine; Sigma Chemical Co.), and then mounted with coverslips in 50% glycerol in PBS and photographed. In some cases, the coverslips were removed in buffer, and the sections were restained with hematoxylin. The A/I was measured with the use of five sections and was calculated as follows: A/I = number of apoptotic cells x (100/total number of cells).
Detection of apoptotic cells by annexin-V-FLUOS staining. Large tumors were excised 2 days after intratumoral injection of plasmids, minced, and digested with collagenase for 60 minutes at 37 °C to isolate tumor cells, as described previously (45). Cell preparations were filtered through a stainless-steel strainer to remove debris and matrix, and erythrocytes were removed by ammonium chloride lysis. The viability of the isolated tumor cells, as determined by trypan blue dye exclusion, was greater than 95%. The cells were stained for 10 minutes with annexin-V-FLUOS (green dye) (Boehringer Mannheim) to identify apoptotic cells and counterstained with methylene blue to determine the total number of tumor cells per field. The A/I was calculated as above.
Analysis of B7-1 and Survivin Protein Expression by Flow Cytometry
Tumors were excised 2 days after plasmid injection and digested with collagenase to produce single-cell suspensions, and the cells were cultured for 2 days with the use of the same conditions used to culture EL-4 cells. For B7-1 analysis, the cells were stained sequentially with 1G10 MAb (anti-B7-1; Pharmingen, San Diego, CA) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG (Sigma Chemical Co.) or with 2G5 MAb (anti-human HML-1, integrin E
7), an FITC-labeled isotype-matched control IgG2a MAb (Pharmingen). Antibody staining was then evaluated by flow cytometry with the use of a Becton Dickinson FACScan. For survivin analysis, the cells were harvested from EL-4 tumors that were injected previously with antisense and dominant-negative vectors or empty vector. They were permeabilized with 0.3% Triton X-100 and stained for survivin expression with the use of SURV11-A, a polyclonal rabbit anti-human survivin antibody that recognizes mouse survivin (Alpha Diagnostic, San Antonio, TX), or with rabbit IgG (Sigma Chemical Co.) as a negative control, followed by FITC-conjugated goat anti-rabbit IgG1. Stained cells were analyzed by flow cytometry.
Immunohistochemistry
Tumors were excised from mice 2 days after plasmid injection, frozen on dry ice, and stored at -70 °C in isopentane. Sections (10 µm thick) of the frozen tumors were prepared and mounted on poly-L-lysine-coated slides. The slides were then incubated for 30 minutes in 0.3% H2O2 in methanol to block endogenous peroxidases. Tumor sections were incubated with 1G10 (anti-B7-1 MAb), 53-6.72 (anti-CD8 MAb), Gk1.5 (anti-CD4 MAb), PK136 (anti-NK cell MAb), or the antisurvivin antibody. The bound antibodies were detected with the use of the VECTASTAIN ABC Elite Kit and 3,3`-diaminobenzidine tetrahydrochloride (Sigma Fast DAB) with metal enhancer (CoCl2) tablets. The sections were counterstained with hematoxylin. As controls, rat IgG and rabbit IgG (Sigma Chemical Co.) were used as the primary antibodies.
Immunofluorescence Staining and Confocal Microscopy
Tumor sections (6 µm) prepared 2 days after plasmid injection from frozen tumors were stained with 4D11, a rat MAb (purchased from the ATCC) that recognizes large granular lymphocyte protein-1 (LGL-1) (46), followed by FITC-conjugated anti-rat IgG (Sigma Chemical Co.); with rabbit anti-human survivin antibody followed by tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit IgG (Sigma Chemical Co.); or with the anti-NK cell MAb (PK136) followed by TRITC-conjugated anti-rat IgG. As a control, rat IgG was used as the primary antibody. Sections were examined by confocal microscopy with the use of a Leica TCS 4D microscope (Leica Microsystems, GmbH, Heidelberg, Germany). Images were compiled from sets of three to five consecutive single optical sections with the use of Leica ScanwareTM 4.2A software (Leica Lasertechnik, GmBH, Heidelberg).
Western Blot Analysis
The tumors were excised and homogenized in lysis buffer (i.e., 50 mM Tris [pH 7.4], 100 µM EDTA, 0.25 M sucrose, 1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, and 100 µM phenylmethylsulfonylfluoride) at 4 °C with the use of a motor-driven homogenizer (VirTis, Gardiner, NY). Tumor lysates from each group of mice were pooled and centrifuged at 10 000g for 10 minutes at 4 °C to remove tissue debris. Protein concentrations of the resulting supernatants were determined as described previously (47), and 100 µg of each supernatant was resolved on 10% polyacrylamide gels containing SDS under reducing conditions and then electrophoretically transferred to Hybond C Extra nitrocellulose membranes (Amersham Life Science, Amersham, U.K.). The membranes were blocked with 3% bovine serum albumin in TBS-T (i.e., 20 mM Tris and 137 mM NaCl [pH 7.6] containing 0.1% Tween 20) for 2 hours at room temperature and then incubated in TBS-T containing the rabbit anti-human survivin antibody diluted 1 : 200 for 60 minutes at room temperature. The membranes were washed three times with TBS-T and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma Chemical Co.) diluted 1 : 5000 in TBS-T. Immunoreactivity was detected by Enhanced Chemiluminescence (Amersham International, Buckingham, U.K.) and autoradiography.
Statistical Analysis
All in vivo and in vitro experiments were performed at least twice. To compare the change in size of the small and the large tumors over time in response to treatment with plasmids encoding B7-1, antisense survivin, or a dominant-negative mutant of survivin, we used a mixed linear model fitted with number of days and treatment as a repeated measure and treatment groups and initial (day 0) tumor size group (small or large) as explanatory variables. Tumor size at day 0 (the day of injection of plasmid encoding B7-1, antisense survivin, or a dominant-negative mutant of survivin) was included as a covariate. Because the change in tumor size over time was not monotonic, day was treated as a categorical variable. The interaction of day with treatment group and initial tumor size group was examined to determine whether the effect of treatment was independent of tumor size group, with the difference in treatment effects due to antisense survivin or dominant-negative mutant survivin, with and without B7-1 being of interest.
To compare changes in tumor size during the 21 days after mice were depleted of leukocyte subsets (T cells, B cells, or NK cells), we fitted a mixed linear model, where day 0 and day 21 were repeated measures and the interaction of day and depletion group was the outcome of interest. The particular contrasts that we investigated were the change in tumor size over time for mice that had not undergone leukocyte depletion contrasted with that for mice that had been depleted, for each type of leukocyte. This analysis was performed separately for the four treatment groups (i.e., antisense survivin, dominant-negative survivin, B7-1 plus antisense survivin, and B7-1 plus dominant-negative survivin).
All other statistical comparisons were made with the use of Student's t tests. All statistical tests were two-sided.
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RESULTS |
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Tumors become increasingly immune resistant as they grow. Here, we sought to determine whether survivin expression increases as tumors grow and to determine whether increased immune resistance was associated with increased survivin expression. Increased survivin expression might also reflect a tumor's dependence on survivin to overcome increasingly poor nutrient conditions at an ectopic site. Syngeneic EL-4 tumors were established by injection of EL-4 cells into the left flank of C57BL/6 mice, and the expression of survivin within the tumors at this ectopic site was examined. Survivin was poorly expressed in small tumors (i.e., tumors 0.2 cm in diameter) (Fig. 1
, A). However, survivin expression increased dramatically with increasing tumor size. Tumors 1.0 cm in diameter had approximately 10-fold more survivin than 0.2-cm tumors, as measured by immunohistochemical staining of tumor sections (Fig. 1
, AC), western blot analysis of tumor homogenates (Fig. 1
, J), and flow cytometry analysis of cells isolated from 0.5-cm and 1.0-cm tumors (Fig. 1
, K). Survivin was expressed uniformly throughout all tumors (Fig. 1
, AC). LGL-1, the antigen recognized by 4D11, is normally expressed on EL-4 cells, on a subset (50%) of C57BL/6 NK cells, and on fewer than 2% of T cells (46). We used 4D11 to detect EL-4 cells within tissue sections. Large tumors (1 cm in diameter) were composed almost entirely of 4D11-staining EL-4 cells, which coexpressed survivin (Fig. 1
, DF). Only small numbers of NK cells (Fig. 1
, H) infiltrated tumors, and some of those appeared to express LGL-1 (Fig. 1
, GI). Thus, survivin expression increases uniformly throughout tumors as they enlarge, suggesting that it may be a critical factor for the survival of large tumors. We have found that survivin is not expressed in EL-4 cells in culture, presumably because the culture conditions are optimal for cell growth and survival.
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We next tested the effects of two molecular reagents designed to inhibit survivin expression or function on the growth of established EL-4 tumors. One reagent, an antisense survivin expression plasmid, was expected to block the translation of endogenous survivin messenger RNA transcripts and, consequently, survivin expression. The other reagent, an expression plasmid encoding a dominant-negative mutant form of survivin, was constructed on the basis of the finding that mutation of Cys84 to Ala in the extreme C-terminal region of the BIR domain completely abrogates survivin's ability to inhibit apoptosis (22). The dominant-negative mutant form of survivin was expected to block endogenous survivin function by competitively binding to survivin effectors. We examined the effects of these two reagents on 0.2-cm EL-4 tumors, which express little endogenous survivin, and on 0.4- to 0.6-cm tumors, which express high levels of survivin. Small (0.2-cm) EL-4-derived tumors injected with 100 µg of plasmid encoding either antisense survivin or dominant-negative mutant survivin showed a 40%50% reduction in tumor diameter 28 days after plasmid injection compared with small tumors injected with empty plasmid. For example, tumors injected with empty plasmid had a mean size of 1.21 cm (95% CI = 0.92 cm to 1.50 cm) 28 days after injection, whereas tumors injected with antisense survivin or dominant-negative mutant survivin had mean sizes of 0.62 cm (95% CI = 0.34 cm to 0.90 cm; P = .006) and 0.50 cm (95% CI = 0.34 cm to 0.66 cm; P = .0018), respectively, at 28 days after injection (Fig. 2, A). In contrast, large (0.4- to 0.6-cm) EL-4-derived tumors injected with 180 µg of plasmid encoding either antisense survivin or dominant-negative mutant survivin showed an 80% reduction in size 14 days after plasmid injection compared with large tumors injected with empty plasmid (Fig. 2
, B). For example, large tumors injected with empty plasmid had a mean size of 0.86 cm (95% CI = 0.66 cm to 1.06 cm) 14 days after injection, whereas tumors injected with antisense survivin or dominant-negative mutant survivin had mean sizes of 0.18 cm (95% CI = 0.12 cm to 0.24 cm; P<.001) and 0.18 cm (95% CI = 0.11 cm to 0.25 cm; P<.001), respectively, at 14 days after injection (Fig. 2
, B). These effects were specific for plasmids that expressed either antisense survivin or dominant-negative mutant survivin; empty plasmids (i.e., those containing no survivin-related sequences) had, as shown previously (38), no effect on tumor growth.
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B7-1 is a molecule expressed on dedicated antigenpresenting cells that interacts with CD28 on responding T cells and costimulates T-cell activation and proliferation (48). Gene transfer of B7-1 into established tumors induces the generation of tumor-specific cytotoxic T lymphocytes (CTLs) and systemic immunity that results in the rejection of small tumors (38,39). While the mechanism involved in this antitumor immunity is not fully understood, B7-1 may induce tumor rejection by stimulating the proliferation of antitumor T cells or by serving as an NK cell receptor. To determine whether inhibiting survivin expression would render tumors more susceptible to lysis by CTLs following B7-1-mediated immunotherapy, we injected established small and large tumors with two plasmids, one that encodes either antisense survivin or the dominant-negative mutant survivin and one that encodes B7-1, and measured tumor growth. Small (0.2- to 0.3-cm) tumors were injected with 100 µg of each plasmid (Fig. 2, A), and large (0.4- to 0.6-cm) tumors were injected with 180 µg of each plasmid (Fig. 2
, B). As reported previously (38), small tumors regressed rapidly and completely after injection with a plasmid encoding B7-1. We observed the same result for small tumors that were injected with plasmids encoding B7-1 and either antisense survivin or the dominant-negative mutant survivin (Fig. 2
, A). Because the effects of the different plasmids were dependent on initial tumor size, we analyzed the effects of plasmid injections on the sizes of the small and large tumors separately. At 14 days after plasmid injection, the mean change in size for small tumors was -0.08 cm (95% CI = -0.06 cm to -0.10 cm) for those treated with B7-1, -0.14 cm (95% CI = -0.11 cm to -0.17 cm) for those treated with B7-1 plus antisense survivin, -0.13 cm (95% CI = -0.10 cm to -0.16 cm) for those treated with B7-1 plus dominant-negative mutant survivin, 0.21 cm (95% CI = 0.14 cm to 0.28 cm) for those treated with antisense survivin, 0.09 cm (95% CI = 0.02 cm to 0.16 cm) for those treated with dominant-negative mutant survivin, and 0.38 cm (95% CI = 0.24 cm to 0.52 cm) for those treated with empty plasmid. The mean change in size for tumors treated with empty plasmid was statistically significantly different from that for tumors treated with B7-1 in the absence or presence of either survivin plasmid (P<.001 for each comparison), as well as for tumors treated with the dominant-negative mutant survivin plasmid (P = .03) but not for those treated with the antisense survivin plasmid (P = .188). In contrast, there was no statistically significant difference in the mean change in size for tumors treated with B7-1 compared with that for tumors treated with B7-1 plus either survivin plasmid (P = .15) or between tumors treated with B7-1 plus antisense survivin and those treated with B7-1 plus dominant-negative survivin (P = .63). In summary, while each of the survivin-based plasmids inhibited the growth of small tumors, neither plasmid enhanced or impaired B7-1-mediated rejection of small tumors.
As reported previously (38), large tumors continued to grow after injection with a plasmid encoding B7-1. At 14 days after plasmid injection, the mean change in size for large tumors was 0.18 cm (95% CI = 0.04 cm to 0.32 cm) for those treated with B7-1, -0.43 cm (95% CI = -0.36 cm to -0.50 cm) for those treated with B7-1 plus antisense survivin, -0.23 cm (95% CI = -0.20 cm to -0.26 cm) for those treated with B7-1 plus dominant-negative mutant survivin, -0.29 cm (95% CI = -0.18 cm to -0.40 cm) for those treated with antisense survivin, -0.25 cm (95% CI = -0.21 cm to -0.29 cm) for those treated with dominant-negative mutant survivin, and 0.43 cm (95% CI = 0.34 cm to 0.52 cm) for those treated with empty plasmid. As we observed for small tumors, the mean change in size over time for large tumors treated with empty plasmid was statistically significantly different from that for tumors treated with B7-1 in the presence of either survivin plasmid and from that for tumors treated with either of the survivin-based plasmids alone (P<.001 for each comparison). The mean change in size over time for large tumors treated with empty plasmid was also statistically significantly different from that for tumors treated with plasmid encoding B7-1 alone (P = .02). However, in contrast to our observations for small tumors, the mean change in size over time for large tumors treated with B7-1 was statistically significantly different from that for tumors treated with B7-1 plus either survivin plasmid (P = .004 for each comparison), as well as for tumors treated with B7-1 plus antisense survivin compared with those treated with B7-1 plus dominant-negative survivin (P = .02). These results suggest that the combined actions of the B7-1 and antisense survivin plasmids contributed more to the reduction in the size of large tumors than did the action of either plasmid alone. For example, at day 14 after plasmid injection, large tumors injected with a combination of the B7-1 and antisense survivin plasmids had a statistically significantly (P<.001) smaller mean size (0.09 cm; 95% CI = 0.04 cm to 0.14 cm) than large tumors injected with empty plasmid (0.44 cm; 95% CI = 0.37 cm to 0.51 cm), with B7-1 plasmid alone (0.62 cm; 95% CI = 0.48 cm to 0.76 cm), or with antisense survivin plasmid alone (0.18 cm; 95% CI = 0.12 cm to 0.24 cm). On day 14, the mean size of the tumors injected with B7-1 plasmid alone was not statistically significantly (P = .06) smaller than the mean size of large tumors injected with empty plasmid, whereas the mean size of the tumors injected with antisense survivin plasmid was (P<.001). In contrast, the combined actions of the B7-1 and dominant-negative mutant survivin plasmids did not contribute more to the reduction in the size of large tumors than did the action of the dominant-negative mutant survivin plasmid alone. For example, when compared with the mean size of large tumors at day 14 after injection with empty plasmid, large tumors injected with B7-1 and dominant-negative mutant survivin had a mean size of 0.23 cm (95% CI = 0.16 cm to 0.30 cm; P<.001) at day 14 after injection, whereas large tumors injected with the dominant-negative mutant survivin plasmid alone had a mean size of 0.18 cm (95% CI = 0.11 cm to 0.25 cm; P<.001). These results suggest that, although each of the survivin-based plasmids appears to disrupt survivin function in vivo, these plasmids vary in their ability to reduce tumor growth when used in combination therapy with B7-1.
To determine whether B7-1 immunotherapy in combination with antisurvivin therapy would generate heightened systemic antitumor immunity observed previously with B7-1 alone, we challenged mice that had rejected their tumors with parental EL-4 cells. Animals previously cured of small tumors after injection with the B7-1 plasmid alone or in combination with antisense survivin or dominant-negative mutant survivin constructs remained tumor free for 26 days after they were re-injected with 2 x 106 parental EL-4 cells (Fig. 2, A). Intratumoral injection of the B7-1 plasmid led to de novo expression of B7-1 in approximately 90% of the tumor cells, as assessed by immunohistochemical staining of tumor sections prepared 2 days after gene transfer (Fig. 3
; compare panels G and H). This result was confirmed by flow cytometry analysis of tumor cells extracted from B7-1-treated tumors by collagenase digestion (Fig. 3
, I). These results suggest that combining plasmids that reduce endogenous survivin expression or activity with a plasmid that expresses B7-1 does not impair the generation of systemic antitumor immunity that results from B7-1-mediated immunotherapy.
Effect of Gene Dose on Therapeutic Efficacy
We reported previously (38) that injection of tumors with a dose of 60 µg of plasmid encoding either B7-1 or other costimulatory cell adhesion molecules was the most effective dose for inducing antitumor immunity. Gene dose dependency may reflect transfection efficiency or the degree of T-cell costimulation that results from plasmid expression, since excessive antigen/costimulation induces T-cell anergy instead of proliferation. To investigate the optimal dose requirements for tumors treated with the combination of B7-1 and antisense survivin plasmids, we first injected established tumors with different amounts of B7-1 plasmid (60140 µg for small tumors [Fig. 4, A]; 100220 µg for large tumors [Fig. 4
, B]). Twenty-four hours later, the tumors were injected with an amount of antisense survivin plasmid equivalent to the amount of B7-1 plasmid that was injected. We found that injection of either 100 or 140 µg each of the antisense survivin and B7-1 expression plasmids caused the rapid and complete regression of small tumors, whereas injection of 60 µg of each reagent did not (Fig. 4
, A). In contrast, injection of either 180 or 220 µg of both plasmids inhibited the growth of large tumors but did not cause complete tumor regression (Fig. 4
, B).
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To determine whether injection with the antisense survivin plasmids might stimulate antitumor immunity, we measured the antitumor CTL activity (as determined by the ability to lyse EL-4 cells) of splenocytes collected from mice 28 days after their large EL-4 cell-derived tumors were injected with the B7-1 or antisense survivin expression plasmids, either alone or in combination. We found that the anti-EL-4 CTL activity, as measured by the ratio of release of lactate dehydrogenase (LDH) from splenocyte-lysed EL-4 cells compared with the total LDH content of EL-4 target cells (expressed as percent cytotoxicity), was statistically significantly greater in animals whose tumors were injected with either the B7-1 plasmid alone (P<.001 at an effector-to-target cell ratio of 50 : 1) or B7-1 in combination with the antisense survivin plasmid (P<.001 at an effector-to-target cell ratio of 50 : 1) compared with the CTL activity from animals whose tumors were injected with empty plasmid (Fig. 5). For example, splenocytes from animals whose tumors were injected with either B7-1, antisense survivin, or the combination of B7-1 and antisense survivin plasmids showed 18.8% (95% CI = 12.6% to 25%; P<.001), 33.6% (95% CI = 28.3% to 38.9%; P<.001), and 36.8% (95% CI = 28.2% to 45.4%; P<.001) cytotoxicity against EL-4 cells at an effector-to-target cell ratio of 50 : 1, respectively compared with splenocytes from animals whose tumors were injected with empty plasmid and showed only 7.2% (95% CI = 4.5% to 9.9%) cytotoxicity.
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We sought to determine the particular leukocyte subsets that infiltrated tumors as well as those responsible for tumor regression in response to the various treatment regimens, since this information might provide insights into the mechanisms responsible for antitumor immunity. Only small numbers of leukocytes infiltrated uninjected tumors (data not shown) or tumors injected with empty expression plasmid (Fig. 6). Large tumors injected with antisense survivin had statistically significantly (P = .003) more infiltrating CD8+ leukocytes than large tumors injected with empty plasmid, whereas large tumors injected with dominant-negative mutant survivin were poorly infiltrated with all three types of leukocytes but had statistically significantly (P = .03) more CD4+ T cells than tumors injected with empty plasmid (Fig. 6
). Tumors injected with the B7-1 plasmid had statistically significantly more CD8+ T cells (15.2 cells per field; 95% CI = 13.3 to 17.1 cells per field; P<.0001), CD4+ T cells (6.4 cells per field; 95% CI = 4.8 to 8.0 cells per field; P = .003), and NK cells (11.7 cells per field; 95% CI = 9.6 to 13.8 cells per field; P = .002) than tumors injected with empty plasmid. Tumors injected with the B7-1 and antisense survivin plasmids had statistically significantly more CD8+ T cells (23.8 cells per field; 95% CI = 17.1 to 30.5 cells per field; P = .0006), CD4+ T cells (12.8 cells per field; 95% CI = 9.0 to 16.6 cells per field; P = .0009), and NK cells (18.8 cells per field; 95% CI = 15.8 to 22.0 cells per field; P<.0001) than tumors injected with empty plasmid. Tumors injected with the B7-1 and the dominant-negative mutant survivin plasmids had statistically significantly more CD8+ T cells (19.8 cells per field; 95% CI = 14.6 to 25.0 cells per field; P = .0006), CD4+ T cells (9.6 cells per field; 95% CI = 6.0 to 13.2 cells per field; P = .005), and NK cells (13.8 cells per field; 95% CI = 10.6 to 16.9 cells per field; P = .002) than tumors injected with empty plasmid. In general, more CD8+ T cells and NK cells than CD4+ T cells were present in all plasmid-injected tumors.
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Effects of Antisense and Dominant-Negative Mutant Forms of Survivin on Tumor Cell Apoptosis
We reported previously (38) that B7-1-mediated antitumor immunity is accompanied by augmented CTL activity involving both the perforin and FasL pathways, suggesting that programmed cell death is responsible for tumor rejection. We therefore performed in situ TUNEL staining of tumor sections to detect the DNA strand breaks that characterize apoptotic cells. We measured the A/I 2 days after tumors were injected with B7-1 alone, with B7-1 and either antisense survivin or dominant-negative mutant survivin, with antisense survivin and dominant-negative survivin mutant alone, or with empty plasmid (Fig. 7, K). TUNEL staining revealed that tumors expressing either antisense survivin or dominant-negative mutant survivin plasmids had greater percentages of apoptotic cells (15% [95% CI = 10% to 20%; P = .001] and 25% [95% CI = 17% to 33%; P<.001], respectively) than tumors injected with empty plasmid (Fig. 7
, K; compare Fig. 7
, panels A and B with panel D). Tumors injected with B7-1 and either antisense survivin or dominant-negative mutant survivin plasmids had even greater percentages of apoptotic cells (28% [95% CI = 19.2% to 36.8%; P<.001] and 35% [95% CI = 26.4% to 43.6%; P<.001], respectively) than tumors injected with empty plasmid (Fig. 7
, C and K). Similar results were obtained with the use of annexin-V-FLUOS to stain apoptotic cells extracted from tumors (Fig. 7
, GK) and annexin-V-biotin to stain apoptotic cells in tumor sections (Fig. 7
, K). The higher A/I values obtained by annexin-V staining compared with TUNEL staining presumably reflect the increased sensitivity of annexin-V, which detects a very early hallmark of apoptosis.
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DISCUSSION |
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We have shown that inhibition of survivin expression or function cripples large tumors such that they either regress or have severely impaired growth. The tumors that regressed failed to grow back to their original size at the initiation of gene therapy for at least 6 weeks and only began to grow again after 28 days, possibly because, by that time point, the therapeutic vectors had been degraded. It is possible that repeated administration of antisurvivin plasmids may cause sustained inhibition of tumor growth, as has been achieved with antiangiogenic therapy (49). The inhibition of survivin expression with either antisense or dominant-negative mutant forms of survivin clearly illustrates the dependence of large tumors on high levels of survivin expression. In contrast, inhibition of survivin expression in small tumors, where it is weakly expressed, had little effect on tumor growth, although the small tumors continued to grow at slightly reduced rates compared with tumors injected with empty plasmid. It is possible that the antisense and dominant-negative mutant survivin expression plasmids were degraded by the time the small tumors had become dependent on the protective properties of survivin.
The increased expression of survivin in growing tumors parallels the acquisition of immune resistance of tumors as they enlarge. Thus, as reported here and previously (38), small tumors can be eradicated completely by B7-1-mediated immunotherapy, but large tumors are refractory to such treatment. A pivotal finding in the present work was that inhibiting survivin expression in large tumors rendered them susceptible to B7-1-mediated immunotherapy. This result would be expected if survivin was protecting tumor cells from immune attack. We found that the efficacy of combination treatment with B7-1 and antisense survivin plasmids was dependent on plasmid dose, with administration of the highest dose of therapeutic plasmids always resulting in the greatest reduction in tumor size. However, whereas high plasmid doses caused the complete eradication of small tumors, large tumors were not similarly affected by doses of each plasmid of 100 µg or greater.
Tumors become resistant to immunotherapy by a variety of mechanisms (50). They may secrete immunosuppressive factors (51), develop mutant or variant cells that can avoid the immune response (52), or develop the ability to counterattack the immune system with their own immune-based weapons. Alternatively, tumors may become resistant to CTL-mediated cell lysis (3) or infiltration by CTLs because of endothelial anergy (53). CTLs kill target cells through the interaction of FasL on T cells with Fas, a mediator of apoptosis, on target cells. There are now several examples of how tumor cells can act like CTLs and kill antitumor T cells. For example, tumor cells expressing FasL can potentially counterattack and kill Fas-expressing CTLs (54). In addition, some tumors and tumor cell lines express the membrane-associated molecule RCAS1, which is thought to bind a putative receptor on leukocytes, causing the inhibition of leukocyte cell growth and the induction of apoptosis (41).
We suggest that survivin renders tumor cells more resistant to antitumor CTLs, thus acting as an important immunosuppressive factor. Accordingly, we found that inhibiting survivin expression in combination with B7-1 immunotherapy leads to increased apoptosis of tumor cells, which could be due, at least in part, to an increased susceptibility to attack by pre-existing antitumor T cells.
The inhibition of survivin expression led to a slight increase in the antitumor CTL response, as measured by an increase in the number of splenocytes in treated animals able to lyse EL-4 cell targets. However, we think that it is unlikely that survivin has direct immunostimulatory properties per se. Rather, the increase in CTLs in response to survivin inhibition may arise if cell fragments from apoptotic or necrotic tumor cells were taken up by dendritic cells, and the tumor antigens on such fragments were presented to the immune system indirectly. Cell fragments may be generated from dying tumor cells deprived of survivin, a protein that would otherwise protect them against an increasingly unfavorable, nutrient-poor, ectopic environment. Survivin plays a similar role in protecting interleukin 3 (IL-3)-dependent pre-B cells against IL-3 withdrawal (10). This protective role could explain, in part, the increased tumor cell apoptosis after inhibition of survivin.
Our results suggest that survivin is an important therapeutic target. However, other antiapoptotic factors besides survivin, such as Bcl-2, and Bcl-xL, are also widely expressed in many tumors. It was reported previously that Bcl-2 is expressed in the EL-4 lymphoma (55). We presume that an arsenal of therapeutic agents that simultaneously target survivin, Bcl-2, and other immunosuppressive factors would hold even greater promise than the antisurvivin therapy described in our study. Because survivin, unlike other potential therapeutic targets, is expressed in few adult tissues (14,15), we predict that survivin-specific therapies are likely to produce few, if any, side effects. Our study indicates that inhibiting survivin expression to restore susceptibility to programmed cell death could break down this barrier to cancer immunotherapy and should be investigated as an adjunctive therapy for the treatment of large immune-resistant tumors.
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NOTES |
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We thank Joanna Stewart, Department of Community Health, University of Auckland, Auckland, New Zealand, for assistance with the statistical analysis.
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Manuscript received December 27, 2000; revised August 6, 2001; accepted August 20, 2001.
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