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Tumor Biology |
Laboratory of Experimental Immunology, Center for Cancer Research [N. S., J. W., R. H. W.] and Intramural Research Support Program, Science Applications International Corporation-Frederick Inc. [A. D. B., T. J. S.], National Cancer Institute at Frederick, Frederick, Maryland 21702-1201; Cancer Immunology, Peter MacCallum Cancer Institute, East Melbourne, Victoria 3002, Australia [Y. H., J. E. T., M. J. S.]; and Department of Immunology, Juntendo University, School of Medicine, Bunkyo-ku, Tokyo 113-8421, Japan [H. Y.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Death receptor-mediated apoptosis plays an important role in tissue homeostasis. This is particularly evident in the immune system, where mutations in the death receptor Fas or its ligand (FasL)3
result in massive lymphoproliferation in both mice and humans (6, 7, 8)
. Furthermore, death ligands such as TNF-
, FasL, or TRAIL may be used by immune cytotoxic effector cells to eliminate virally infected cells or tumor cells (9)
. Several studies have suggested that a reduction in tumor cell sensitivity to apoptosis via death receptors can promote tumor growth and development of metastases in vivo (10, 11, 12, 13)
. More recently, it has been proposed that TRAIL may act as a tumor suppressor molecule in vivo (14
, 15)
, and TRAIL restricted the ability of several tumors to form experimental liver metastases in vivo. IFN-
was shown to be essential for the antimetastatic effects of TRAIL, and liver NK cells were shown to express TRAIL, particularly after activation with IFN-. Because tumors are heterogeneous, it would be predicted that the degree of sensitivity or resistance of individual tumor cells to TRAIL-mediated apoptosis could significantly influence their ability to form metastases, particularly in the liver.
Using the murine renal cancer cell line Renca, we isolated clones that had a growth rate similar to that of the parental tumor in vitro yet were much more sensitive to death receptor-mediated apoptosis. We have used one of these clones (R331) as a model to determine the relative importance of sensitivity to death receptor-mediated apoptosis in controlling tumor metastases in vivo. We therefore compared R331 with the parental Renca tumor for their ability to grow in different primary tissue locations as well as their propensity to form liver metastases after i.s. injection or lung metastases after i.v. injection. A thorough analysis of the importance of death receptor-mediated apoptosis in vivo was performed using death receptor-resistant c-FLIP transfectants of Renca and R331 tumor cells, neutralizing Abs to TRAIL and FasL, and appropriate gene-targeted or mutant mouse strains.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells and Reagents.
Renca is a BALB/c-derived renal adenocarcinoma cell line. R331 is the one of the clones that was established from Renca by a limiting dilution method in 96-well tissue culture plates (Costar, Acton, MA). 2PK-3 cells transfected with mouse TRAIL (mTRAIL/2PK-3) have been described previously (17)
. The d11S hybridoma cells kindly provided by Dr. P. Henkart (National Cancer Institute, NIH, Bethesda, MD) use FasL to mediate their cytotoxic activity. All of the cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1x nonessential amino acids, 1 mM sodium pyruvate, penicillin (100 units/ml) and streptomycin (100 µg/ml), 10 mM HEPES, and 5 x 10-5 M 2-mercaptoethanol (pH 7.4) [complete medium]. Recombinant murine IFN- and murine TNF- were purchased from BD PharMingen (San Diego, CA). Recombinant human FasL that is fused to a FLAG tag and cross-linked with anti-FLAG was purchased from Kamiya Biomedical Co. (Seattle, WA). Soluble mouse recombinant TRAIL was purchased from Biomol (Plymouth Meeting, PA) and cross-linked with anti-6x histidine Ab (R&D Systems, Minneapolis, MN). The neutralizing antimouse TRAIL mAb (N2B2) and antimouse FasL mAb (MFL-1) were prepared as described previously (17)
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Establishment of c-FLIP-transfected R331 and Renca Clones and Western Blot Analysis.
Plasmid encoding murine FLAG-tagged c-FLIP under the control of a cytomegalovirus promoter was generously provided by Dr. J. P. Medema (Leiden University, Leiden, the Netherlands). Plasmids were transfected into R331 or Renca cells using LipofectAMINE Plus reagent (Life Technologies, Inc., Gaithersburg, MD). Briefly, Renca cells (2 x 104 cells/well) were plated in 24-well plates 24 h before transfection. Transfection was performed with 0.8 µg plasmid DNA/well with LipofectAMINE Plus reagent by the standard procedure (2 µl of LipofectAMINE and 4 µl of Plus reagents), and then stable clones were obtained by limiting dilution under the selection of G418 (400 µg/ml; Invitrogen, Carlsbad, CA). Cell lysates of clones were obtained, protein content was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) as described previously (18)
, and c-FLIP expression in clones was identified by Western blot analysis. In brief, 20 µg of cell lysate were subjected to SDS-PAGE using the NuPAGE Bis-Tris Electrophoresis system (Invitrogen) according to the manufacturer’s instructions, and Western blot analyses were performed using a standard protocol with anti-FLIP mAb (rat IgG2a, Dave-2; Kamiya Biomedical Co.) and horseradish peroxidase-conjugated goat antirat IgG polyclonal Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). To confirm the expression of FLAG-tagged c-FLIP, lysates of 2 x 107 cells were immunoprecipitated with FLAG-tagged protein immunoprecipitation kit (Sigma-Aldrich, St. Louis, MO), and FLIP expression was tested with precipitated samples by Western blot analysis as described above. Individual clones with high levels of c-FLIP expression (R331-FLIP or Renca-FLIP) were used for additional in vitro and in vivo experiments. R331 clones with a low expression of c-FLIP were used as transfection controls (R331-TC). For Renca, clones transfected with the plasmid containing the FLAG sequence but no FLIP were used as controls (Renca-TC). DR5 (TRAIL-R2) was detected by Western blotting using a rabbit anti-TRAIL-R2 Ab (R&D Systems), and actin was detected using anti-ß-actin mAb (mouse IgG2a; AC-74; Sigma-Aldrich).
Flow Cytometric Analysis.
Fas or MHC class I (H-2Kd) expressions of tumor cell lines were analyzed after overnight treatment with IFN- (500 units/ml) and TNF- (400 units/ml) or media alone by flow cytometry using Phycoerythrin-labeled mAbs or isotype-matched mAbs. Flow cytometry analysis was performed on a FACScan (BD Biosciences, Mountain View, CA) using CellQuest software. All mAbs were purchased from BD PharMingen.
Cytotoxicity Assays.
Target cells that had been incubated overnight in the presence or absence of IFN- (500 units/ml) and TNF- (400 units/ml) were labeled with [111In]Ox (Medi-Physics, Silver Spring, MD) as described previously (19)
. Briefly, 1 x 106 target cells were incubated with 10 µCi of [111In]Ox for 30 min at room temperature. Cells were then washed twice in complete medium, and labeled cells (1 x 104) were then incubated with effector cells at various E:T ratios for 16–18 h at 37°C in a final volume of 200 µl. To evaluate the sensitivities of tumor cell lines to FasL or TRAIL, d11S or mTRAIL/2PK-3 cells were used as effector cells. Alternatively, recombinant human FasL was added in the culture to evaluate susceptibility to Fas-induced apoptosis. After incubation, supernatants were harvested and counted on a gamma counter. Specific killing (percentage of cytotoxicity) was calculated as follows: [(experimental release - spontaneous release)/(maximal release - spontaneous release)] x 100. All groups were run in triplicate, and all experiments were performed three or more times with similar findings.
Fluorometric Assay of Caspase-3.
Caspase-3 activities of tumor cells, induced on TRAIL exposure, were measured by a fluorometric method using a caspase-3 assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, 2 x 106 tumor cells were plated in 6-well plates and left to settle overnight. The wells were washed with media twice, followed by a 4-h incubation in the complete media with or without TRAIL (1000 ng/ml). Extracts of 2 x 105 cell equivalents were incubated in the reaction buffer [20 mM HEPES (pH 7.4), 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, 5 mM DTT, and 2 mM EDTA] containing fluorogenic synthetic substrates acetyl-Asp-Glu-Val-Asp-7-AMC (16 µM) at room temperature for 2 h. Free AMC was quantified fluorometrically at excitation and emission wavelengths of 360 and 460 nm, respectively, on a Cytofluor multiplate reader series 4000 (Perspective Biosystems, Framington, MA). Standard curves were generated with AMC.
Tumor Proliferation Assays in Vitro.
On day 0, tumor cells were plated in a 96-well tissue culture plate at the indicated dose in 100 µl of complete medium. On days 1, 2, and 5, viable cells were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Roche, Mannheim, Germany) or 3-[4,5-dimethylthiazol-2-yl-5]-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. Spectrophotometric absorbance at 595 nm or 490 nm was measured with absorbance at 655 nm for reference in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or 3-[4,5-dimethylthiazol-2-yl-5]-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium assay, respectively. All groups were run in triplicate, and all experiments were performed three or more times with similar findings. Proliferation of Renca or R331 tumor cells was also assessed using a soft agar colony assay (13)
. Briefly, single-cell suspensions of tumor cells in complete medium were mixed with agarose in a final concentration of 0.3% agarose and 1x complete medium. Aliquots of 2 ml containing 103 cells were seeded in triplicate in 35-mm culture dishes over a base layer of 0.6% agarose and 1x complete media and allowed to gel. The dishes were incubated at 37°C in 5% CO2 for 14 days and stained with 0.005% crystal violet solution.
Tumor Growth and Experimental Metastasis Assay in Vivo.
To examine primary tumor growth, male WT BALB/c mice (6–12 weeks of age) were inoculated s.c. or in the kidney capsule with Renca or R331 tumor cells on day 0 at the doses indicated. s.c. tumors were measured after tumor inoculation over the course of 27 days with a caliper square as the product of two perpendicular diameters (mm2). The mice in which tumor cells had been injected into the kidney were sacrificed on day 24, and the tumor-bearing kidneys were weighed. BALB/c WT and gene-targeted mice were injected i.s., followed by splenectomy, or i.v. with tumor cells at the indicated doses. Mice were killed 14 days after tumor inoculation, and liver (after i.s.) or lung (after i.v.) metastases were quantified with the aid of a dissecting microscope. Some mice also received injections of antimouse TRAIL mAb, control rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), or antimouse FasL mAb (250 µg) i.p. on days 0, 1, and 7 after tumor inoculation. Data were presented as the mean ± SE of 5–15 mice in each group. All data are representative of two to three independent experiments with similar results.
Statistical Analysis.
The significance of difference in the number of metastases and tumor sizes between experimental groups was determined by the Mann-Whitney t test. Two-sided Ps of <0.05 are considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Renca was not killed by the FasL-expressing hybridoma cell line d11S, whereas R331 displayed some sensitivity. Renca expressed small amounts of Fas on the cell surface, whereas R331 expressed more Fas constitutively as detected by fluorescence-activated cell-sorting analysis (Fig. 1B)
. Cytokine stimulation with the combination of IFN- and TNF- increased Fas expression on both Renca and R331 cells, although R331 cells still expressed significantly more Fas than Renca cells. These cytokine pretreatments sensitized Renca cells and enhanced the susceptibility of R331 cells to FasL-mediated apoptosis by d11S cells (Fig. 1A)
. After cytokine treatment, R331 was still more sensitive to d11S effector cells than Renca, suggesting that FasL sensitivity was at least partially dependent on the expression level of Fas on the tumor cell surface. R331 was also more sensitive to TRAIL-mediated apoptosis than Renca, although both Renca and R331 were killed by the murine TRAIL-transfected cell line mTRAIL/2PK-3. In contrast, TRAIL-mediated killing was not enhanced by cytokine pretreatment. TRAIL-R2/DR5 was detected constitutively at similar levels on both Renca and R331 cells by Western blot analysis, and cytokine treatment of these cells did not enhance TRAIL-R2 expression (Fig. 1C)
. These data were also confirmed by flow cytometric analysis of surface TRAIL-R2 expression (data not shown). Nonetheless, these findings clearly showed that R331 was much more susceptible to death receptor-mediated apoptosis. The differences in TRAIL sensitivity between Renca and R331 could not be explained by TRAIL-R2 expression level. Therefore, other downstream signaling events in Renca and R331 may account for differences in TRAIL sensitivity. Further study of Renca and R331 tumor cells will be required to address this issue.
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, indicating that R331 may have more tumorigenic potential in vivo. After s.c. injection, R331 developed larger tumors at a more rapid rate than parental Renca (Fig. 3)
. In the renal capsule, the growth rates of Renca and R331 were not significantly different (data not shown). Next, these tumor cells were compared for their ability to form metastases in vivo. Four different doses of Renca cells and R331 cells were inoculated i.s. (Fig. 4A)
, and four doses were injected i.v. (Fig. 4B)
, and after 2 weeks, liver or lung metastases were quantified, respectively. Surprisingly, in contrast to the local growth in vivo (Fig. 3)
and in vitro data shown in Fig. 2
, Renca developed many more metastases than R331 in both the liver and lungs (P < 0.01).
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). In contrast, a mAb reactive with FasL did not significantly increase the number of liver metastases. These findings are consistent with TRAIL, and not FasL, being the key proapoptotic protein that controlled R331 metastases in the liver. For a more detailed and comparative analysis of antitumor effector functions, perforin-deficient (pfp-/-) and TRAIL-deficient (TRAIL-/-) mice were used. Perforin is a major effector mechanism used by both NK cells and T cells to kill tumor target cells, and it plays an important tumor surveillance role in a number of mouse tumor models in vivo (16
, 20
, 21)
. The number of Renca liver metastases was increased in pfp-/- (P < 0.05) or TRAIL-/- (P < 0.05) mice compared with WT mice as reported previously (22)
. The Renca tumor displayed a similar number of liver metastases in WT mice injected with anti-FasL mAb or control Ab (Fig. 5B)
, suggesting that FasL did not play a major role in limiting liver metastases of either Renca or R331. Much more strikingly, TRAIL-/- mice developed 15 times more R331 liver metastases than WT mice (P < 0.01). Pfp-/- mice and WT mice treated with anti-FasL mAb showed minor increases in R331 liver metastases as compared with the control WT mice, but these differences were not statistically significant (Fig. 5B)
. Furthermore, there was no significant difference in the number of liver metastases of R331 between WT mice and gld mice that possess a nonfunctional FasL (data not shown). These findings clearly demonstrate that TRAIL, rather than either perforin or FasL, is the major factor controlling R331 metastases in the liver.
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. Interestingly, the number of R331 lung metastases was not significantly increased in pfp-/- mice, TRAIL-/- mice, or WT mice treated with anti-FasL mAb. This suggests that the number of lung metastases of R331 is not influenced by local levels of perforin, FasL, or TRAIL. By contrast, the number of Renca lung metastases was significantly increased in pfp-/- mice, confirming that perforin was an important regulator of Renca metastases in the lung (Fig. 5C)
. Overall, these data indicated that TRAIL was not an important effector molecule against Renca or R331 tumor cell metastasis to the lung, despite its obvious activity in the liver. It remains to be determined whether any other immune effector mechanism can be detected against R331 tumor cells in the lung.
Transfection of R331 with c-FLIP Reduces Death Receptor-mediated Apoptosis and Increases the Number of Liver Metastases.
The preceding data demonstrated that TRAIL could limit liver metastases of R331 but did not confirm whether the major effect of TRAIL expression in vivo was due to the apoptotic destruction of tumor cells. In recent years, several mechanisms have been proposed to interfere with death receptor-induced apoptosis. The antiapoptotic protein c-FLIP protects cells from Fas- and TRAIL-induced apoptosis (10)
. To determine whether death receptor-induced apoptosis was directly relevant for the development of experimental metastases, c-FLIP-transfected Renca and R331 clones were generated. Both Renca and R331 cells expressed endogenous c-FLIP at low but detectable levels on Western blot analysis (Fig. 6A)
. Transfectants of Renca and R331 were generated that expressed high levels of c-FLIP (Renca-FLIP.1 and R331-FLIP.1), some of which could be immunoprecipitated using anti-FLAG Abs. In contrast, R331 and Renca cells had undetectable levels of FLAG-tagged c-FLIP (Fig. 6A)
. The sensitivity of the R331 transfectants to FasL and TRAIL was tested after IFN- and TNF- treatment in cytotoxicity assays with recombinant FasL and mTRAIL/2PK-3 cells (Fig. 6B)
. Apoptosis of R331-TC (transfection controls) was similar to that of R331, whereas R331-FLIP.1 was almost completely resistant to TRAIL-mediated apoptosis. Although cytokine-pretreated R331-FLIP.1 showed some weak sensitivity to recombinant FasL at the highest dose tested, apoptosis of untreated R331-FLIP.1 was completely abrogated (data not shown). After TRAIL treatment, there was a large difference in caspase-3 activation between R331-FLIP.1 and R331 or R331-TC (Fig. 6C)
. R331 and R331-TC showed much higher caspase-3 activity after exposure to TRAIL than R331-FLIP.1. R331, R331-TC, and R331-FLIP.1 all expressed identical amounts of Fas and MHC class I by fluorescence-activated cell-sorting analysis, and growth rates of these cells were also identical in vitro (data not shown).
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. R331-FLIP.1 cells developed much larger numbers of liver metastases than R331 (P < 0.01 with 1 x 104 cells as initial dose and P < 0.0001 with 5 x 104 cells as initial dose; Fig. 7, A and C
), whereas the number of metastases after injection of R331-TC was similar to that seen after injection of R331. Furthermore, Abs to TRAIL enhanced the number of R331 liver metastases but had little effect on the number of R331-FLIP.1 metastases (Fig. 7C)
. Several other independent R331 clones that expressed FLIP at high levels (R331-FLIP.2 and R331-FLIP.3) also produced larger numbers of metastases after i.s. injection (Fig. 7A)
. With regard to the parental Renca cell line, a similar (albeit more modest) increase in the number of Renca-FLIP.1 and Renca-FLIP.2 liver metastases over Renca and control transfectant Renca-TC cells was observed (Fig. 7D)
. Renca-FLIP transfectants also expressed elevated levels of FLIP and were much less sensitive to TRAIL-mediated apoptosis than controls (data not shown). Interestingly, the number of metastases of R331-FLIP clones was even significantly greater than that of parental Renca (P < 0.05 with 1 x 104 cells as initial dose and P < 0.0001 with 5 x 104 cells as initial dose). In addition, the FasL- and TRAIL-resistant clone R331-FLIP.1 produced a larger number of liver metastases in WT, pfp-/-, TRAIL-/-, and WT mice treated with anti-FasL mAb, whereas R331-TC showed a pattern of metastasis development similar to that of R331 in these mice (Fig. 8)
. These findings clearly demonstrate that sensitivity of tumor cells to TRAIL-induced apoptosis can dramatically influence the development of liver metastasis in vivo, and TRAIL sensitivity is the dominant mechanism that restricts R331 liver metastases in vivo.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An important implication from these studies was that sensitivity to TRAIL-mediated apoptosis played a major role in the development of R331 metastases in the liver, but not at other sites, including metastases in the lung. Although R331 tumor cells form fewer metastases than parental Renca cells in the lungs, death receptor (TRAIL or FasL)-mediated cytolysis did not appear to be the molecular basis for restriction of R331 lung metastases. Presumably, another death receptor pathway, other factors intrinsic to the R331 cells themselves, or the lung tissue environment restricts the development of R331 metastases at this organ site. The former hypothesis will now be tested by examining metastases of R331-FLIP tumor cells in the lung. A number of previous studies have suggested that sensitivity to death receptor-mediated apoptosis can impede tumor growth and metastasis in vivo. Therefore, in vivo passage of tumor cells often selects for variants that express low levels of the death receptor Fas (13) . Conversely, overexpression of the Fas protein in tumor cells by gene transfection reduces the rate of tumor development in vivo (12) . In addition, overexpression of c-FLIP, which blocks death receptor-mediated apoptosis, resulted in tumors that grew more aggressively than vector controls in WT mice (10 , 11) . In a similar manner, we observed that FLIP-expressing Renca or R331 tumors metastasized more effectively in the liver. Clearly, just as normal hepatocytes can protect themselves by c-FLIP (24) , the ability of liver metastases to express FLIP-like molecules could have a significant bearing on their survival in vivo.
The development of experimental liver metastases is thought to first involve tumor cells mechanically trapped in the portal sinuses, where they adhere to sinusoidal endothelial cells before extravasation (3 , 4) . Whether tumor cells come in contact with TRAIL+ cells before or after extravasation is not known. Many lymphoid cells in the liver are also reported to be localized in the portal sinuses (25, 26, 27, 28) , making it likely that close contract between lymphoid cells and tumor cells can occur before extravasation of tumor cells into the organ parenchyma. Nonetheless, additional studies are required to determine the exact stage of the metastatic process in the liver at which TRAIL has its major effects. Although the lungs also have lymphoid cell populations, it is known that they do not constitutively express surface TRAIL (22) , and therefore R331 and Renca lung metastases may not be subjected to TRAIL-expressing effector cells.
With regard to the rational application of TRAIL for antitumor therapy of R331 metastases in this model, a number of important issues remain. Firstly, R331 cells can grow efficiently at several tissue sites tested, including s.c. and under the kidney capsule. This indicates that the number of TRAIL+ cells in these locations is too low to influence tumor growth. It would therefore be interesting to determine whether systemic administration of TRAIL would indeed result in reduction of R331 tumor growth at these sites, or whether TRAIL-expressing effector cells can be mobilized at these sites by immune activation using cytokines and other biological response modifiers. It is likely that the relative susceptibility of some individual tumor cells (such as R331 or Renca cells) to death ligands may be determined by an "apoptotic threshold." It remains unclear why R331 tumor cells are more sensitive to TRAIL than Renca cells, because both express equivalent levels of TRAIL-R2/DR5. Also, although apoptotic sensitivity and caspase activation of R331 cells can be blocked by overexpression of the antiapoptotic c-FLIP, endogenous levels of this protein in Renca and R331 cells were not dramatically different. Renca cells can be sensitized to FasL-mediated lysis by prior treatments with the cytokines IFN- and TNF-, which enhance expression of the Fas receptor (29)
. With regard to the sensitivity of cells to FasL-mediated apoptosis, it has been proposed that two types of cells exist (30
, 31)
. In type I cells, caspase activation occurs in the absence of mitochondrial involvement due to a robust activation of the signaling caspase-8. By contrast, in type II cells, caspase-8 activation is relatively weak, and therefore amplification of the apoptotic signal via mitochondrial disruption is required. We do not know at present which apoptotic pathway(s) is (are) predominant in Renca and R331 tumor cells.
In conclusion, a high sensitivity to TRAIL-mediated apoptosis can severely restrict a tumor’s ability to form liver metastases. TRAIL appeared to be a more important natural regulator of tumor growth in the liver than FasL; however, neither effector pathway regulated the same tumors metastasizing to the lung. A more detailed knowledge of the crucial molecular events involved in apoptotic signaling in individual tumor cells may provide for the identification of appropriate molecular targets, thus allowing for amplification of tumor cell apoptosis in response to TRAIL. The organ specificity of effector molecules is clearly a key concern when developing effective immunotherapies for reduction or prevention of metastatic tumor burden.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was funded in whole or in part with federal funds from the National Cancer Institute, NIH, under Contract N01-C0-12400. H. Y., Y. H., J. E. T., and M. J. S. were supported by a Human Frontiers in Science Program Grant. M. J. S. was supported by a National Health and Medical Research Council of Australia Research Fellowship. 
2 To whom requests for reprints should be addressed, at Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Building 560, Room 31-93, Frederick, MD 21702-1201. Phone: (301) 846-1329; Fax: (301) 846-1673; E-mail: Sayers{at}mail.ncifcrf.gov
3 The abbreviations used are: FasL, Fas ligand; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; WT, wild-type; c-FLIP, cellular Fas-associated death domain-like interleukin-1ß-converting enzyme inhibitory protein; NK, natural killer; TNF, tumor necrosis factor; mAb, monoclonal antibody; Ab, antibody; AMC, amido-4-methylcoumarin; i.s., intrasplenic or intrasplenically; Ox, oxine.
Received 8/ 1/02. Accepted 10/29/02.
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R. Shao, D.-F. Lee, Y. Wen, Y. Ding, W. Xia, B. Ping, H. Yagita, B. Spohn, and M.-C. Hung E1A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis through Enhancement of Caspase Activation Mol. Cancer Res., April 1, 2005; 3(4): 219 - 226. [Abstract] [Full Text] [PDF]
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 C. Tecchio, V. Huber, P. Scapini, F. Calzetti, D. Margotto, G. Todeschini, L. Pilla, G. Martinelli, G. Pizzolo, L. Rivoltini, et al. IFN{alpha}-stimulated neutrophils and monocytes release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying apoptotic activity on leukemic cells Blood, May 15, 2004; 103(10): 3837 - 3844. [Abstract] [Full Text] [PDF]
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 Y.-M. Ham, K.-J. Choi, S.-Y. Song, Y.-H. Jin, M.-W. Chun, and S.-K. Lee Xylocydine, a Novel Inhibitor of Cyclin-Dependent Kinases, Prevents the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptotic Cell Death of SK-HEP-1 Cells J. Pharmacol. Exp. Ther., March 1, 2004; 308(3): 814 - 819. [Abstract] [Full Text] [PDF]
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 K. Takeda, N. Yamaguchi, H. Akiba, Y. Kojima, Y. Hayakawa, J. E. Tanner, T. J. Sayers, N. Seki, K. Okumura, H. Yagita, et al. Induction of Tumor-specific T Cell Immunity by Anti-DR5 Antibody Therapy J. Exp. Med., February 17, 2004; 199(4): 437 - 448. [Abstract] [Full Text] [PDF]
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J. Brady, Y. Hayakawa, M. J. Smyth, and S. L. Nutt IL-21 Induces the Functional Maturation of Murine NK Cells J. Immunol., February 15, 2004; 172(4): 2048 - 2058. [Abstract] [Full Text] [PDF]
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 J. C.L. Schuh Trials, Tribulations, and Trends in Tumor Modeling in Mice Toxicol Pathol, January 1, 2004; 32(1_suppl): 53 - 66. [Abstract] [PDF]
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T. J. Kemp, B. D. Elzey, and T. S. Griffith Plasmacytoid Dendritic Cell-Derived IFN-{alpha} Induces TNF-Related Apoptosis-Inducing Ligand/Apo-2L-Mediated Antitumor Activity by Human Monocytes Following CpG Oligodeoxynucleotide Stimulation J. Immunol., July 1, 2003; 171(1): 212 - 218. [Abstract] [Full Text] [PDF]
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M. Komatsu, M. Mammolenti, M. Jones, R. Jurecic, T. J. Sayers, and R. B. Levy Antigen-primed CD8+ T cells can mediate resistance, preventing allogeneic marrow engraftment in the simultaneous absence of perforin-, CD95L-, TNFR1-, and TRAIL-dependent killing Blood, May 15, 2003; 101(10): 3991 - 3999. [Abstract] [Full Text] [PDF]
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, P < 0.01 on days 10, 13, and 17 (1 x 105).
