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Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsReferences |
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Apoptosis, or programmed cell death, is a major pathway of cell death described by its morphological characteristics, including chromatin condensation and nuclear fragmentation (6, 7). Recently, two major interacting pathways of apoptosis have been identified. In the first pathway, apoptosis is induced by the engagement of proapoptotic cytokines to specialized cell surface death receptors (CD95/Fas/Apo and tumor necrosis factor receptor; Refs. 8, 9). These death receptors specifically bind to their ligands such as FasL and lead to the activation of FADD (Fas-associated death domain) and then initiator caspases such as caspase-8 and -10 through death effector domain. Caspase-8 then activates downstream effector caspases (caspase-3, -6, and -7) leading to the terminal event, cell death. Another pathway of apoptosis is triggered by changes in the homeostasis of mitochondria (8, 10). Cytochrome c is released from the mitochondria into the cytosol after the induction of apoptosis (10–12). The released cytochrome c activates apoptosis activating factor-1 and caspase-9 through caspase-recruitment domain. Activated caspase-9 leads to the activation of effector caspases such as caspase-3 (CPP32), which results in proteolytic cleavage of numerous substrates including PARP, a nuclear enzyme involved in DNA repair (13–15).
In addition to the two highly investigated pathways of apoptosis, NO, an important signal transduction molecule, has been implicated in a variety of functions such as vasodilatation, neurotransmission, host defense, and apoptosis (16–18). NO also plays a key role in the cytostatic/cytotoxic function of the immune system. It is endogenously produced by NOS, a group of enzymes including three different isoenzymes in mammalian cells (18). Inducible isoenzyme (iNOS) was originally isolated from mouse macrophages, but it is now known to be expressed in a variety of cell types. The iNOS is not constitutively expressed in cells. Although the function of NO has been extensively studied in vivo, its role in apoptosis is still poorly understood. It has been shown that lipopolysaccharide or cytokines such as IFN-
and IL-1ß can induce iNOS in target cells, which then release copious amounts of NO in short period of time. NO then interacts with superoxide resulting in cellular cytotoxic activity (19, 20). In contrast, NO has also been reported to be a potent inhibitor of apoptosis through S-nitrosylation of the active cysteine residues conserved in all caspases (21, 22).
Because targeted immunotoxins and cytotoxins can mediate apoptosis in breast carcinoma and in head and neck tumor cells (23–25), and we have observed that IL-13 cytotoxin can induce apoptosis in tumor cells in vitro, in this study we have examined whether IL-13 cytotoxin can induce apoptosis in vivo in malignant glioma tumors. We demonstrate that IL-13 cytotoxin can induce significant apoptosis of glioma cells in a time-dependent manner. Even though IL-13 cytotoxin was rapidly metabolized in tumors and its level was nondetectable, some apoptotic activities continued to be detected, resulting in potent in vivo antitumor activity of IL-13 cytotoxin.
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Materials and Methods |
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Detection of Apoptosis in Vitro by Bis-Benzimide Staining.
Apoptotic cells were assessed morphologically by fluorescent microscopy. U251, A172, or T98G cells (2 x 106) were plated in 100-mm Petri dishes and exposed to medium only (control), IL-13 cytotoxin (100 ng/ml), or IL-13 (100 ng/ml) for 48 h. Cells were then washed and fixed with 1% glutaraldehyde, and stained with 1 mM bis-benzimide (Hoechst no. 33342; Sigma Chemical Co., St. Louis, MO). The cells were examined by fluorescent microscopy under UV filter.
Flow Cytometry.
Cells (8 x 105) were treated with control media or IL-13 cytotoxin (100 ng/ml) in 60-mm Petri dishes and harvested by trypsinization at 0, 6, 12, 24, 48, 72, and 96 h and fixed with cold 70% ethanol. Then, cells were washed with PBS and incubated with 0.5% RNase for 30 min at room temperature and stained with 25 µg/ml propidium iodide. DNA population was analyzed by flow cytometry.
Animals and Treatments.
Four-week-old (body weight, 
20–22 g) male athymic nu/nu mice were obtained from Frederick Cancer Center Animal Facilities (National Cancer Institute, Frederick, MD). The mice were housed in sterilized filter-topped cages and maintained in a pathogen-free animal facility. Animal care was in accordance with the guidelines of the NIH Animal Advisory Committee. U251 cells were implanted by s.c. injection of 5 x 106 cells in 150 µl of PBS with 0.2% HSA into the dorsal surface of mice. The tumors were developed in 4–5 days to 100 mm3 volume and then i.t. administration with excipient only (0.2% HSA/PBS) or with IL-13 cytotoxin at a concentration of 100 µg/kg was performed. After administration of IL-13 cytotoxin, mice were killed at 0.5, 1, 2, 4, 6, 24, 48, and 72 h, and tumors were excised for lysates and tissue sections.
Apoptosis Detection by H&E or TUNEL Staining in Vivo.
Tumors that developed in nude mice were excised and sectioned to 5-µm thickness. IL-13 cytotoxin (100 µg/kg) or 0.2% HSA/PBS for control had been previously injected i.t. Apoptotic cells in tumor sections were identified and counted after staining with H&E or with the TUNEL-based ApopTag in situ hybridization detection kit (Intergen, Purchase, NY). Previously established cytomorphological criteria were used to assess apoptosis. These include chromatin condensation, cytoplasmic budding to form membrane-bound fragments (apoptotic bodies), and nuclear fragmentation (6, 7). Apoptotic cells in tumors were counted with a light microscope and percentages were calculated. Cancer cells (n = 1000) were counted under x400 magnification in 10 chosen fields from tissue sections derived from entire tumor tissue. The number of apoptotic cell counts was expressed as a percentage of 100 viable tumor cells.
Caspase Activity Assay. The excised tumors were homogenized in lysis buffer (BioVision Inc., Palo Alto, CA) by grinding with a pestle and then passing several times through 22-gauge needles. The whole tumor lysates were kept on ice for 10 min and were clarified by centrifugation at 10,000 rpm for 1 min at 4°C. Caspase activity in the supernatant was determined using a caspase colorimetric assay kit (BioVision) as described previously (27, 28). Briefly, 100 µg of total protein as determined by the Bio-Rad protein assay (Bio-Rad, Richmond, VA) in the tumor lysate, was incubated with 5 µl of each caspase substrate (4 mM; BioVision), DEVD (N-acetyl-Asp-Glu-Val-Asp)-pNA (caspase-3), IETD (N-acetyl-Ile-Glu-Thr-Asp)-pNA (caspase-8), and LEHD (N-acetyl-leu-Glu-His-Asp)-pNA (caspase-9) in a total 100 µl of caspase assay reaction buffer (final concentration of substrate, 200 µM). After incubation at 37°C for 2 h, caspase activity was determined by measuring the cleavage of these substrates. The light absorption was monitored using a MRX II microplate reader (DYNEX, Chantilly, VA) at 405 nm, which measures pNA cleaved from the labeled substrates by activated caspases.
Preparation of Subcellular Fractions. The procedure described in previous reports was adopted with modifications (29, 30). Treated cells or tumors were homogenized in isotonic sucrose buffer containing 250 mM sucrose, 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA (pH 7.1) plus 0.025% digitonin and complete protease inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN) with 1% BSA. After removing nuclear proteins by centrifugation, lysates were centrifuged then supernatant was designated "cytosolic fraction." The pellet was resuspended in isotonic sucrose buffer containing 0.5% Triton X-100 and complete protease inhibitor mixture and was kept for 10 min on ice, after which the Triton X-100-insoluble fraction (including mitochondria) was separated by centrifugation. Fractions were resolved in 15% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and probed with rabbit antihuman cytochrome c Ab (Santa Cruz Biotechnology).
Western Analysis. Whole-cell extracts from tumor tissues were mixed with SDS sample buffer and heated for 5 min at 70°C. Proteins (20–50 µg/lane) were separated by 4–12% SDS/PAGE and transferred onto a polyvinylidene difluoride membrane (Invitrogen, Carlsbad, CA). Membranes were blocked for 1 h in TBST [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Tween 20] and 5% skim milk. After blocking, membranes were incubated for 1 h with mouse antihuman procaspase-3 monoclonal Ab (Transduction Laboratories, Lexington, KY), rabbit antihuman PARP monoclonal Ab (Boehringer Mannheim, Indianapolis, IN), rabbit anti-PE polyclonal Ab (Sigma), or rabbit antimouse iNOS polyclonal Ab (Santa Cruz Biotechnology) in TBST. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary Ab in TBST for 30 min and were developed by using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia).
Antitumor Activity in Vivo. Mice with established tumors, by s.c. injection of 5 x 106 U251 cells in 150 µl of PBS plus 0.2% HSA, received either the excipient (0.2% HSA in PBS) or the IL-13 cytotoxin i.t. (30 µl) using a 27-gauge needle. Tumor sizes were carefully measured by a Vernier caliper. The tumor volume on a given day was calculated by the formula: (length of the tumor) x (width of the tumor)2/2. Each treatment group consisted of seven mice.
Immunohistochemistry. Frozen sections (5-µm) were prepared and fixed in acetone at -20°C for 5 min. Nonspecific binding was blocked by treatment with 10% goat serum for 1 h followed by incubation with Ab or isotype control. Slides were stained with 1:500 rabbit antimouse NOS2 polyclonal Ab (M-19; Santa Cruz Biotechnology) and developed using a DAB (3,3'-diaminobenzidine) substrate biotinylated peroxidase kit (Vector Laboratories, Inc., Burlingame, CA). Tissues were counterstained with hematoxylin (Sigma, St. Louis, MO).
Statistical Analysis. The statistical significance of tumor regression and various parameters was calculated by Student’s t test.
Results
IL-13 Cytotoxin Causes Apoptosis in GBM Cells in Vitro.
To investigate whether IL-13 cytotoxin causes apoptosis in human GBM tumor cells in vitro, three GBM cell lines (U251, A172, and T98G) were incubated with IL-13 cytotoxin, and morphological changes, including chromatin condensation, were examined by bis-benzimide staining. As shown in Fig. 1A, after treatment with IL-13 cytotoxin, apoptotic cells were detected in U251 and A172 cell lines that overexpressed IL-13R, whereas T98G cells not expressing high levels of IL-13R and all control cell lines incubated with PBS did not show apoptosis. The percentage of apoptotic cells significantly increased as a result of the IL-13 cytotoxin treatment in U251 and A172 cell lines compared with respective untreated control cells (P < 0.05; Fig. 1B). In contrast, no increase in apoptotic cells was observed in the T98G cell line. Furthermore, IL-13 (100 ng/ml) did not cause apoptosis in any of the three cell lines (data not shown).
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3-fold higher number of apoptotic cells were detected in IL-13 cytotoxin-treated tumors compared with tumors at time 0.
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Induction of Release of Cytochrome c from Mitochondria to the Cytosol in U251 Cells by IL-13 Cytotoxin in Vitro and in Vivo. To investigate whether IL-13 cytotoxin-induced apoptosis involves mitochondrial homeostasis, we studied the release of cytochrome c from mitochondria to cytosol in U251 tumors after the incubation of tumor cell lines with IL-13 cytotoxin in vitro or i.t. injection of IL-13 cytotoxin in vivo. As shown in Fig. 5A, incubation of U251 tumor cells with IL-13 cytotoxin (100 ng/ml) caused a release of cytochrome c into the cytosol that was clearly detected after 24 h of incubation. Cytochrome c release continued to increase up to 72 h in a time-dependent manner. The mitochondrial fraction of treated U251 cells did not show any change in cytochrome c. Similarly, on a single i.t. administration of IL-13 cytotoxin (100 µg/kg) in vivo, cytochrome c release into the cytosol was detected by 2 h and increased to a maximum level at 6 h and maintained this level up to 72 h after IL-13 cytotoxin administration (Fig. 5B). These results suggest that IL-13 cytotoxin can use the mitochondrial pathway of programmed cell death in glioma tumor cells in vitro and in vivo.
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IL-13 cytotoxin induced several morphological changes in A172 and U251 cell lines that expressed high levels of IL-13R. These changes included chromatin condensation and nuclear fragmentation. However, the T98G cell line, which expresses low numbers of IL-13R, showed few morphological features, even after treatment with 100 ng/ml IL-13 cytotoxin. Because the T98G cell line does not express the IL-13R
2 chain and only demonstrates low or no sensitivity to IL-13 cytotoxin (IC50 = 200 to >1000 ng/ml; Refs. 5, 32), it is possible that IL-13 cytotoxin will induce apoptosis when the IL-13R2 chain is inserted in these cells. Thus, as seen in previous studies using PE-based immunotoxin/cytotoxins, IL-13 cytotoxin also seems to cause cell death by apoptosis (23–25). Although the percentage of the hypodiploid cell population increased in a time-dependent manner as determined by fluorescence-activated cell sorting analysis, nevertheless, the percentage did not reach 50%, even though cells were treated with IL-13 cytotoxin for 96 h. These findings suggest that IL-13 cytotoxin causes cell death not only by apoptosis but also by necrosis.
IL-13 cytotoxin also induced apoptosis in U251 tumor xenografts in vivo. A single i.t. administration of IL-13 cytotoxin induced both caspase-8 and cytochrome c pathways of apoptosis. Activities of caspase-3, -8, and -9 reached maximum levels within 4 h, whereas optimum mitochondrial cytochrome c release to the cytosol seemed to occur between 6 and 48 h. Interestingly, activity of caspase-8 and -9 returned to baseline; however, effector caspase-3 activity remained somewhat higher than baseline, even 72 h after the i.t. injection of IL-13 cytotoxin. These results suggest that the apoptotic process continues for a prolonged period of time (at least 72 h), even though peak apoptotic cells were detected at 24 h. Sensitivity of assays between TUNEL and Western blot analyses may account for these differences. Alternatively, apoptotic cells were rapidly phagocytized after peaking at 24 h. Finally, the induction of caspase activity correlated with the cleavage of the caspase substrates, procaspase-3 and PARP. Both were cleaved at similar time periods (within 4–6 h) when caspase-3, -8, and -9 were activated.
It is of interest to note that after a single administration of IL-13 cytotoxin in tumors in vivo, the IL-13 cytotoxin is rapidly degraded, absorbed, or undetectable by this assay. Four h after injection, levels of IL-13 cytotoxin were barely detectable. This observation is similar to that in our previous studies, in which IL-13 cytotoxin was administered in AIDS-associated Kaposi’s sarcoma tumors (33). Nevertheless, in the present study, apoptosis pathways were still active beyond the disappearance of IL-13 cytotoxin from the tumor. This is important information that can help develop the rationale for schedules of IL-13 cytotoxin administration for optimum glioma therapy.
Although, after a single i.t. administration, IL-13 cytotoxin induced the cascade of events leading to apoptosis, a single dose was not sufficient for prolonged and pronounced antitumor activity. This could be attributable to suboptimal drug or distribution and/or suboptimal apoptosis of a limited number of tumor cells. Two additional injections of IL-13 cytotoxin on alternate days produced remarkable antitumor effects. Approximately 40% (3 of 7) of the animals showed complete disappearance of their tumors. Two additional injections once a day every 3 days also showed similar tumor responses. These results suggest that, in clinical situations, one can dose the patient with a more tolerated schedule. A once-a-day-every-3-days schedule may be better tolerated and practical than an every-alternate-day schedule of administration. Because the i.t. administration of IL-13 cytotoxin by two schedules did not produce complete regression in all of the animals, it is possible that complete antitumor activity can be achieved in all of the animals by continuous administration of IL-13 cytotoxin, using Alzet micropumps. These studies are currently ongoing in our laboratory.
We also estimated the numbers of apoptotic cells in tumor sections as a measure of biological activity of IL-13 cytotoxin. By 24 h after IL-13 cytotoxin administration in vivo, the maximum number of apoptotic cells was detected. Their numbers had decreased after 72 h of treatment, but still remained 3-fold higher compared with the control groups. These observations suggest that IL-13 cytotoxin-induced early events (caspase activation) of apoptosis lead to morphological changes at a later time point. This observation can also help guide the design of clinical trials of glioma therapy in regard to the frequency of repeat administration of IL-13 cytotoxin.
To elucidate other cell death mechanisms of IL-13 cytotoxin-induced tumor regression, we investigated expression of iNOS producing NO in U251 tumors. NO has been shown to be associated with several pathways, including signal transduction, immune regulation, and regulation of cell death having two opposite effects; cell killing and the prevention of apoptosis (19, 20). We found iNOS was induced rapidly by IL-13 cytotoxin in U251 tumors. Interestingly, expression of iNOS appeared to be localized in phagocytes that surrounded dying cells after the administration of IL-13 cytotoxin. The expression of iNOS was maintained for 72 h after a single i.t. administration of IL-13 cytotoxin. Because NO is most commonly produced by activated macrophages (16), it is possible that infiltrating macrophages may play a role in tumor regression. Alternatively, it is possible that NO-producing macrophages were recruited by dying tumor cells (34). Additional studies are needed to elucidate the role of NO that is produced by infiltrating phagocytes.
In conclusion, we demonstrate that IL-13 cytotoxin can cause apoptosis in brain tumor cells in vitro and in vivo. IL-13 cytotoxin modulated several proapoptotic molecules in vivo involving two apoptotic pathways. IL-13 cytotoxin continued to mediate apoptosis and tumor regression even after it had disappeared from tumors. Furthermore, phagocytes expressing iNOS were induced and may play a role in the tumor regression caused by IL-13 cytotoxin.
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Acknowledgments |
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Footnotes |
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2 To whom requests for reprints should be addressed, at Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 2NN10, 29 Lincoln Drive MSC4555, Bethesda, MD 20892. Phone: (301) 827-0471; Fax (301) 827-0449; E-mail: puri{at}cber.fda.gov
3 The abbreviations used are: IL, interleukin; IL-13R, IL-13 receptor; PE, Pseudomonas exotoxin; GBM, glioblastoma multiforme; i.t., intratumoral/intratumorally; NO, nitric oxide; NOS, NO synthase; iNOS, inducible NOS; PARP, poly(ADP-ribose) polymerase; pNA, p-nitroanilide; HSA, human serum albumin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; TBS, Tris-buffered saline; TBST, TBS with Tween 20; Ab, antibody.
Received 2/14/02; revised 7/19/02; accepted 8/20/02.
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, control (PBS); 
, treatment (IL-13-PE38QQR); *, significantly different from control at P < 0.05; **, significantly different from control at P < 0.01.
, treatment (IL-13-PE38QQR); bars, mean ± SD of apoptotic cells in 10 tissue fields.
, control (IT day 5); •, treatment (IT day 5); 
, control (IT days 5, 8, and 11); 
, treatment (IT days 5, 8, and 11); arrows, the days of injections. Tumor volumes were carefully measured by a vernier caliper. Tumor volumes, means ± SD of 7 mice/group. Some symbols and error bars are overlapped. The experiment shown is representative of two experiments.
