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Original Studies |
Laboratory of Pathology, National Institutes of Health (N.M., V.P., V.K., M.T.), Bethesda, Maryland 20892; and the Endocrine Unit, Evgenidion Hospital (N.M., G.M., D.A.K.), and the Pathology Department, University of Athens (S.T.-B.), Athens, Greece
Address all correspondence and requests for reprints to: Nicholas Mitsiades, M.D., Massachusetts General Hospital, Molecular Pathology Unit, 7th Floor, 149 13th Street, Charlestown, Massachusetts 02129.
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Abstract |
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Introduction |
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FasL is a transmembrane protein of the tumor necrosis factor (TNF) family (13) that induces apoptosis by binding to and activating the Fas (APO-1/CD95) receptor, a member of the TNF/nerve growth factor receptor superfamily (14). The Fas/FasL system plays an important role in immune homeostasis (15, 16) and participates in T cell-mediated cytotoxicity (17). FasL is expressed in the testis (18), eye (19), brain (20), and placenta (21), where it presumably contributes to the immune-privileged status of these organs by eliminating infiltrating lymphocytes. Furthermore, it is expressed by melanomas (22), astrocytomas (23), lymphomas (24, 25, 26), Ewing’s sarcomas (27), and carcinomas of the colon (12, 28), liver (29), and lung (30). Thus, it has been suggested that tumor cells expressing FasL similarly use this cytolytic effector molecule to kill Fas-expressing infiltrating lymphocytes (counterattack model) (12).
Fas is present in normal thyroid tissue (31, 32, 33, 34), whereas FasL expression is weak in this tissue (31) and stronger in thyrocytes from patients with nontoxic goiter (35). Furthermore, Fas and FasL can be up-regulated in thyrocytes during the course of Hashimoto’s thyroiditis, possibly under the influence of lymphocyte-derived cytokines. This up-regulation leads to an apoptotic suicide/fratricide and contributes to the destruction of the gland in this disease (31, 35). Although Fas expression has been found in thyroid carcinomas (32), the presence of FasL in malignant thyroid lesions is currently unknown.
We investigated the presence of FasL and apoptosis in thyroid carcinomas, compared to that in benign thyroid tissues, and in thyroid carcinoma cell lines. We found that FasL is expressed in thyroid carcinoma specimens, and that apoptotic nuclei are present among the immune cells infiltrating FasL-expressing carcinomas. Furthermore, high levels of FasL in papillary carcinomas correlated with aggressive histology and unfavorable clinical presentation. FasL was present in all five thyroid carcinoma cell lines tested and could induce apoptosis in Fas-expressing cells of lymphocytic origin. This suggests that FasL may have a role in the progression and immune evasion of thyroid carcinomas. Apoptosis is scarce among thyroid carcinoma cells themselves in vivo, indicating that FasL expression does not lead them to apoptotic suicide. This is probably due to the presence of a short-lived protein inhibitor of the Fas pathway, as thyroid carcinoma cells in vitro are sensitive to Fas-mediated apoptosis only in the presence of the protein synthesis inhibitor cycloheximide.
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Materials and Methods |
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Archival formalin-fixed and paraffin-embedded thyroid specimens from 48 patients (12 men and 36 women) with thyroid carcinomas, aged 16–76 yr (mean ± SD, 50 ± 14) were retrieved retrospectively from the files of the Pathology Department, University of Athens (Athens, Greece). They represented 28 papillary, 5 follicular, 6 oxyphilic (Huerthle), and 9 medullary carcinomas.
Papillary carcinomas were classified histologically as those exhibiting the classic well differentiated histological picture (36) (n = 17; group A) and those showing extensive areas of moderate/low differentiation or squamous differentiation (n = 11; group B). The latter comprised 2 carcinomas of the highly aggressive tall cell variant (37). Papillary carcinomas were also characterized according to size and clinical spread (38). Twelve carcinomas were small (larger diameter <4 cm) and confined to the thyroid gland; 16 carcinomas were large or even disseminated (>4 cm and/or infiltration of the thyroid capsule or represented recurrence after initial surgery). The clinical prognoses for these two classes of carcinomas were characterized as favorable and unfavorable, respectively (38).
As controls, specimens of normal thyroid tissue were obtained from the contralateral lobe of 10 thyroid glands removed surgically for a nodule from spontaneously euthyroid patients. Furthermore, we studied five hyperplastic nodules with extensive oxyphilic metaplasia (metaplastic oxyphilic nodules) as benign controls for the study of the oxyphilic Huerthle cell carcinomas. All thyroid specimens were removed from patients followed at the Endocrine Unit of the Evgenidion Hospital (Athens, Greece).
Materials
The Vectastain Elite ABC kit for immunohistochemistry was obtained from Vector Laboratories, Inc. (Burlingame, CA); rabbit antihuman FasL antibody Q20 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); mouse antihuman CD45 antibody was obtained from DAKO Corp. (Carpinteria, CA); mouse antihuman FasL antibodies G247–4 and NOK-2 were obtained from PharMingen (San Diego, CA); and rabbit antihuman FasL antibody Ab-3 was purchased from Oncogene Research (Cambridge, MA). Blocking peptide used in immunohistochemistry and corresponding to amino acid residues 2–19 of FasL (7 µg/mL) was obtained from Santa Cruz Biotechnology, Inc.. ExTaq and the anti-Fas CH11 antibody were purchased from Panvera (Madison, WI). 3,3'-Diaminobenzidine, triethanolamine, acetic anhydride, formamide, Denhardt’s solution, cycloheximide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dithiothreitol, insulin, hydrocortisone, transferrin, somatostatin, glycyl-L-histidyl-L-lysine acetate, and TSH were obtained from Sigma Chemcial Co. (St. Louis, MI). Concanavalin (Con) A was purchased from Pharmacia Biotech (Piscataway, NJ). Normal human thyroid protein extract (human thyroid protein medley) was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). The SW579 and Jurkat cell lines were obtained from American Type Culture Collection (Manassas, VA). The in situ cell death detection kit with peroxidase and fluorescence, the DNA fragmentation enzyme-linked immunosorbent assay (ELISA) kit, Triton X-100, alkaline-phosphatase-conjugated anti-digoxigenin (anti-DIG) antibody, Complete-TM mixture of proteinase inhibitors, positively charged nylon membranes, nitroblue tetrazolium, yeast transfer ribonucleic acid (RNA), 5-bromo-4-chloro-3-indolyl-phosphate, the DIG RNA labeling kit (SP6/T7), the PCR DIG probe synthesis kit, and the DIG nucleic acid detection kit were purchased from Roche Molecular Biochemicals (Indianapolis, IN). IgG-free normal horse serum, Trizol reagent, salmon sperm DNA, HBSS, SDS, saline-sodium citrate (SSC) buffer, proteinase K, and the Superscript II kit were obtained from Life Technologies, Inc. (Gaithersburg, MD). Dextran sulfate was purchased from Oncor (Gaithersburg, MD), and the TA cloning kit was obtained from Invitrogen (San Diego, CA). The enhanced chemiluminescence kit, which includes the peroxidase-labeled antimouse and antirabbit secondary antibodies, was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL).
Methods
Generation of FasL probes. A fragment of the human FasL genomic sequence corresponding to nucleotides 668-1558 (GenBank D38122) was isolated by PCR and inserted at random orientation into the pCR2.1 vector with the use of the TA cloning kit. Verification of the sequence and determination of the orientation of the insert were performed by sequencing in an automated sequencer (375 ABI). A DIG-labeled, double stranded DNA probe was prepared by PCR with the PCR DIG probe synthesis kit, using as template the previously described plasmid, and was used in the RT-PCR described below. DIG-labeled RNA probes (antisense and sense) were prepared by in vitro transcription with T7 polymerase and the DIG RNA labeling kit on two reactions run in parallel, using as templates plasmids carrying the insert in the respective orientation, and were used in the in situ hybridization described below.
Studies on thyroid tissue specimens. The presence of FasL in thyroid tissue specimens was examined by immunohistochemistry and in situ hybridization. We also evaluated the presence of apoptosis among immune cells infiltrating FasL-positive tumors. Immunohistochemistry on consecutive carcinoma sections with mouse antihuman CD45 was employed to identify the cells of immune origin.
Immunohistochemistry: Immunohistochemistry was performed and evaluated as previously described (31). Briefly, 5-µm paraffin sections were deparaffinized, rehydrated, microwaved for 15 min in 10 mmol/L citrate buffer, treated for 30 min in methanol containing 0.5% H2O2, and then incubated for 1 h in 16% normal goat serum and overnight with the primary antibodies with or without the corresponding blocking peptide. The respective secondary antibody was then applied for 1 h at room temperature, followed by the Vectastain Elite ABC reagent for 30 min. The peroxidase reaction was developed with 3,3'-diaminobenzidine, and the slides were counterstained with hematoxylin. The intensity and distribution of positive staining were evaluated on a scale of 0–3 (0 and 3 corresponded to the absence and the highest degree of staining, respectively) and 0–4 (0 = 0%; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100% of cells), respectively, by two independent observers whose agreement was almost complete. The numbers used for the evaluation of this method represent the mean of the two scores given by each independent observer.
The primary antibodies used for immunohistochemistry were the anti-FasL antibodies Q20 (0.7 µg/mL), G247–4 (1:200 dilution) and Ab-3 (1:50 dilution), and the mouse anti-human CD45 (1:100 dilution).
In situ hybridization: Slides were dried at 37 C overnight, deparaffinized, and rehydrated. They were then washed in PBS containing 100 mmol/L glycine and subsequently in PBS containing 0.1% Triton X-100. The sections were treated with 7 µg/mL proteinase K (ribonuclease-free) for 10 min at 37 C, postfixed for 5 min with PBS-containing 4% paraformaldehyde at 4 C, washed twice in PBS, and incubated in 0.1 mol/L triethanolamine buffer, pH 8.0, containing 0.25% acetic anhydride for 10 min. The sections were incubated with prehybridization buffer (4 x SSC containing 50% deionized formamide) at 37 C for 10 min and subsequently in hybridization buffer (40% formamide, 10% dextran sulfate, 1 x Denhardt’s, 4 x SSC, 10 mmol/L dithiothreitol, 1 mg/mL yeast transfer RNA, and 1 mg/mL denatured and sheared salmon sperm DNA) containing 400 ng/mL DIG-labeled RNA probe (FasL antisense or sense probe) at 42 C overnight. Then the slides were washed in wash buffer A (1 mmol/L ethylenediamine tetraacetate, 40 mmol/L NaH2PO4, 5% SDS, and 0.5% BSA) four times for 5 min each time at 65 C and subsequently in wash buffer B (1 mmol/L ethylenediamine tetraacetate, 40 mmol/L NaH2PO4, and 5% SDS) twice for 10 min each time at 65 C. The slides were rinsed with prewarmed 1 mol/L NaH2PO4 for 5 min at room temperature and then incubated in 1 mol/L NaH2PO4 with 20 µg/mL ribonuclease A for 30 min at 37 C to digest the single stranded (unbound) RNA probe. The slides were incubated for 10 min in buffer 1 [0.1 mol/L Tris-HCl (pH 7.5), 0.1 mol/L NaCl, 2 mmol/L MgCl2, and 3% BSA]. The DIG-labeled probe was detected with an alkaline-phosphatase-conjugated anti-DIG antibody (dilution, 1:500; for 2 h at room temperature) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate, according to the instructions of the manufacturer. All buffers were prepared with diethylpyrocarbonate-treated, doubly distilled water.
Detection of apoptosis by terminal deoxynucleotidyl transferase deoxy-UTP nick end labelling (TUNEL): The in situ cell death detection kit with peroxidase was used according to the manufacturer’s instructions and the suggestions of Negoescu et al. (39). Apoptotic nuclei were visualized with diaminobenzidine. Apoptosis was evaluated subjectively by two independent observers using a x10 lens in 10 randomly selected fields of each specimen. Sections from thyroid glands with Hashimoto’s thyroiditis were stained as positive controls, because apoptosis among thyrocytes is intense in this disease, as previously described (31).
Studies on cell lines. Experimental protocol: The presence of FasL was examined by Western blotting and RT-PCR in cultures of five human thyroid carcinoma cell lines. The NPA, FRO, WRO, and ARO cell lines (gifts from Dr. James A. Fagin, University of Cincinnati School of Medicine, Cincinnati, OH) have been previously described (40). The SW579 cell line (American Type Culture Collection) is derived from a poorly differentiated human thyroid adenocarcinoma (poorly differentiated carcinoma with nuclear features of papillary carcinoma and squamous differentiation). As a control, FasL presence was examined with the same techniques in cultures of the T cell human leukemia cell line Jurkat after stimulation with Con A (10 µg/mL for 8 h). The ability of FasL-expressing thyroid carcinoma cells to induce apoptosis of infiltrating lymphocytes was evaluated by quantifying the amount of fragmented DNA in Jurkat cells (target) cocultured with SW579 cells (effector). The functional status of the Fas pathway in thyroid carcinoma cells was evaluated by treating SW579 cells with the Fas cross-linking antibody CH11 in the presence or absence of the protein synthesis inhibitor cycloheximide.
Cultures of cell lines: The SW579 cell line was grown in Coon’s modification of Ham’s F-12 medium supplemented with 5% bovine calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 µg/mL insulin, 10 nmol/L hydrocortisone, 5 µg/mL transferrin, 10 ng/mL somatostatin, 10 ng/mL glycyl-L-histidyl-L-lysine acetate (5H medium), and 0.01 IU/mL TSH. All other cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics.
Western blotting: Detection of FasL by immunoblotting was performed as previously described (27). Briefly, cells (1 x 106) were scraped, centrifuged briefly, and lysed for 30 min on ice in 50 mmol/L Tris-HCl, pH 8, containing 120 mmol/L NaCl and 1% Igepal, supplemented with the Complete-TM mixture of proteinase inhibitors. The samples were cleared by centrifugation (14,000 rpm, 30 min, 4 C) and assessed for protein concentration. SDS-PAGE (12%) was performed (30 µg protein/lane), and the proteins were electroblotted onto nitrocellulose membranes. After 1-h incubation in blocking solution (20% IgG-free normal horse serum in PBS), the membrane was exposed to the primary antibody overnight at 4 C. After washing in PBS, the secondary peroxidase-labeled antibody was added at a 1:10,000 dilution for 40 min at room temperature. The proteins were visualized with the enhanced chemiluminescence technique.
The primary anti-FasL antibodies used were the G247–4 monoclonal antibody (1:500 dilution) and the polyclonal antibodies Q20 (1:100 dilution) and Ab-3 (1:100 dilution). The secondary antibodies were peroxidase-labeled antimouse or antirabbit antibodies, respectively.
RT-PCR: RNA was prepared from the cell lines with the Trizol reagent. Two micrograms of RNA were used for first strand cDNA synthesis with oligo(deoxythymidine) primer and the Superscript II kit, and subsequently, 0.5 µg cDNA was used as a template for PCR amplification. The primers used were 5'-gtttttcatggttctggttgcc-3' (forward) and 5'- gcctctagtcttccttttccatcc-3' (reverse). PCR reactions of 50 µL were prepared with the use of ExTaq (Panvera) and processed in a Perkin Elmer Corp. 480 Thermocycler under the following conditions: 94 C for 4 min; 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min for 30 cycles; and 72 C at 5 min. The products were electrophoresed on 1% agarose, blotted onto a nylon membrane, and hybridized with the DIG-labelled DNA probe for FasL. Detection was performed with the DIG nucleic acid detection kit according to the manufacturer’s instructions.
Induction of Jurkat cell apoptosis by thyroid carcinoma cells: The ability of thyroid carcinoma effector cells to kill target lymphocytes in a Fas-dependent manner was evaluated with the DNA fragmentation ELISA kit. This method is a nonradioactive analogue of the [3H]thymidine DNA fragmentation assay. Jurkat cells (target) were labeled overnight with 5'-bromo-2'-deoxyuridine according to the manufacturer’s instructions and subsequently cocultured with viable SW-579 cell monolayers for 24 h in the presence or absence of the FasL-neutralizing NOK-2 antibody (10 µg/mL). The Jurkat cell suspension was collected with vigorous pipetting. The amount of fragmented DNA in the target cells was quantified by ELISA according to the manufacturer’s instructions. The results were expressed as percentages of the value of control cells (Jurkat cells incubated in the absence of SW579 cells).
Assessment of anti-Fas antibody (CH-11)-induced apoptosis of thyroid carcinoma cells by TUNEL: SW579 cells were grown to 70–80% confluence in six-well plates, washed in HBSS, and incubated for 18 h with or without the CH-11 anti-Fas antibody (500 ng/mL, in DMEM medium with 5% calf serum) with or without 10 µg/mL cycloheximide at 37 C. The experiment was performed in both the presence and the absence of 0.01 IU/mL TSH. The Fas-sensitive Jurkat cell line was used as a positive control. Subsequently, the cells were scraped, centrifuged onto positively charged slides, air-dried, labeled with the in situ cell death kit fluorescence according to the instructions of the manufacturer, and viewed with a Carl Zeiss (New York, NY) standard fluorescence microscope equipped with an epifluorescence illuminator and FITC narrow-band filter.
Assessment of anti-Fas antibody (CH-11)-induced apoptosis of thyroid carcinoma cells by MTT: To quantify the differences found by TUNEL, the same experiment was performed in 24-well plates. After treatment, as described above, the cells were incubated with 1 mg/mL MTT (Sigma Chemical Co.) in fresh medium for 4 h at 37 C. The formazan crystals were dissolved in a mixture of isopropanol and 1 N HCl (23:2, vol/vol), and dye absorbance was measured at 570 nm, with 630 nm as a reference wavelength. Cell survival was expressed as a percentage of the untreated control value.
Statistics. Quantitative comparisons were examined using ANOVA, followed by Duncan’s test. When differences in immunohistochemical evaluations were compared among different histological types of thyroid carcinomas, the histological type was taken as the independent factor. When differences in immunohistochemical evaluations of papillary carcinomas were examined between groups of different aggressiveness (histological or clinical), histological and clinical aggressiveness were taken as the independent factors. Statistical significance was set at 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroid carcinoma specimens. The presence of FasL in specimens
of thyroid carcinomas and control thyroid tissue was detected by
immunohistochemistry with the anti-FasL antibody Q20. The mean
intensity of FasL immunoreactivity is summarized in Fig. 1a
, and the mean distribution of FasL
immunoreactivity is summarized in Fig. 1b. Consistent with our previous
studies (31), the FasL presence in normal thyroid tissue was very weak
or absent (Fig. 2a). All papillary
carcinomas exhibited FasL immunopositivity (Fig. 2b), especially in
areas of poor (Fig. 2c) and/or squamous differentiation. Staining was
more intense in group B and in large/locally invasive/recurrent
carcinomas than in group A (P < 0.01; Fig. 3a) and small localized carcinoma
[P < 0.004 among all papillary carcinomas (Fig. 3b)
and P < 0.0018 when confined to group A (Fig. 3c)],
respectively. All oxyphilic Huerthle cell carcinomas (n = 6)
exhibited strong FasL immunopositivity (Fig. 2d) that was more intense
than in papillary (P < 0.006) and follicular
(P < 0.005) carcinomas. It was also significantly
stronger than that in their nonneoplastic counterpart, the oxyphilic
metaplastic nodules (P < 0.00005; Fig. 2e). All
follicular carcinomas (n = 5) exhibited detectable FasL
immunoreactivity (Fig. 2f). Follicular carcinomas stained less
intensely than group B, but not group A, papillary carcinomas
(P < 0.003 and P = 0.1, respectively).
Only three of nine medullary carcinomas exhibited FasL
immunoreactivity, which was of poor intensity, as well (Fig. 2g).
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To confirm the results obtained with the Q20 antibody, sections from the same specimens were stained with two more anti-FasL antibodies: the polyclonal Ab-3 and the monoclonal G247–4. Both antibodies showed immunoreactivity only for specimens positive for the Q20 antibody, confirming the specificity of the staining.
Furthermore, the immunohistochemical data were supported by in
situ hybridization. Specimens positive by immunohistochemistry
gave a cytoplasmic signal when the FasL antisense probe was used,
suggesting the presence of FasL messenger RNA (mRNA; Fig. 4a). This signal was absent when the
sense FasL probe was used (Fig. 4b), confirming the specificity of the
method.
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). The
presence of FasL in the thyroid cell lines was also verified with two
more antibodies, the polyclonal antisera Q20 (Fig. 5b) and Ab-3 (not
shown), which detect only the full-length FasL.
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)
and the Con A-activated Jurkat cell lines (not shown). Apoptosis detection
Thyroid carcinoma specimens. Apoptotic nuclei were present
among cells surrounding and/or infiltrating FasL-positive thyroid
carcinomas (Fig. 2, h and i) that were subsequently proven to be of
immune origin by immunostaining consecutive sections with anti-CD45
antibody. Apoptotic nuclei were very scarce or absent among thyroid
carcinoma cells, in contrast to sections from thyroid glands with
Hashimoto’s thyroiditis used as controls, in which apoptotic
thyrocytes are abundant, as previously described (31).
Thyroid carcinoma cells can kill target cells in a FasL-dependent manner. To evaluate the biological activity of the FasL produced by thyroid carcinomas, we used SW-579 cells as cytotoxic effectors in coculture experiments with target Fas-expressing Jurkat cells. Quantification of specific DNA fragmentation of target cells was based on previous labeling of Jurkat cells with 5'-bromo-2'-deoxyuridine and was performed by ELISA. Jurkat cells grown in the presence of SW-579 cells exhibited 440 ± 34% (mean ± SEM) the amount of fragmented DNA observed in Jurkat cells grown in the absence of SW-579 cells (P < 0.001, by ANOVA). The presence of the FasL-neutralizing NOK-2 antibody reduced the amount of fragmented DNA to 241 ± 11% of the control value (P < 0.01 vs without NOK-2). These results suggest that thyroid carcinomas can induce apoptosis that is mediated at least in part by FasL.
The Fas pathway is blocked by a short-lived protein in thyroid
carcinomas. Having shown that FasL is functional in thyroid
carcinomas, we hypothesized that thyroid carcinoma cells avoid an
apoptotic suicide by being Fas resistant. To investigate the functional
status of the Fas pathway in the thyroid carcinoma cell line SW579, we
treated SW579 cells with 500 ng/mL CH11, an apoptosis-inducing,
Fas-cross-linking antibody, and evaluated apoptosis by TUNEL on cell
cytospins. Strongly fluorescent nuclei were considered apoptotic.
Hardly any such nuclei were present in control (Fig. 6a) or CH11-treated (Fig. 6b) nuclei.
This result was the same in both the presence and absence of TSH.
Treatment with cycloheximide, a protein synthesis inhibitor,
induced the appearance of a few TUNEL-positive nuclei (Fig. 6c).
However, cells treated with both cycloheximide and CH11 exhibited
massive apoptosis (>50% of nuclei were TUNEL positive; Fig. 6d) in
both the presence and absence of TSH. To quantify this difference, we
used the MTT assay. We found that in the presence of TSH, the survival
of CH11-treated SW579 cells was 99.8 ± 0.5% that of control
cells (mean ± SEM; P = 0.89).
Cycloheximide-treated cells had a 73.1 ± 1.8% survival rate,
whereas cells treated with both CH11 and cycloheximide had a 43.9
± 0.9% survival rate (P < 0.0003 vs.
cycloheximide alone; Fig. 6e). Similar results were obtained in the
absence of TSH (Fig. 6f). The Jurkat cell line was very sensitive to
CH11-induced apoptosis and served as a positive control (not shown).
These results suggest that in thyroid carcinoma cells, the Fas pathway
is blocked by a short-lived protein inhibitor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the expression of FasL has been described in a number of different tumor types (12, 22, 23, 24, 25, 26, 27, 28, 29, 30). However, direct evidence for a prognostic significance of FasL expression in tumors and/or correlation with disease-free survival is missing. We found that metastatic tumors of the Ewing’s sarcoma family express this molecule more often and more strongly than primary ones (27). Similarly, FasL expression was weak in nonaggressive and strong in aggressive non-Hodgkin’s lymphomas (26). In the present study, aggressive histology and extensive/locally invasive/recurrent disease, both of which represent unfavorable prognostic factors in papillary carcinomas, correlated independently with strong FasL expression in this subtype of thyroid carcinomas. In some thyroid specimens, the peripheral areas of the carcinoma (i.e. the infiltrating, most invasive edge) showed higher FasL immunopositivity, similar to previous findings in Ewing’s sarcomas and lung carcinomas (30). It could be suggested that the role of FasL in that area is to promote tumor aggressiveness, although it cannot be excluded that cytokines produced by lymphocytic and/or fibrotic tissues contribute to FasL up-regulation. Taken together, our results suggest that FasL expression in thyroid carcinomas might correlate with an aggressive phenotype and that it is probably acquired during the transformation process. By altering the balance between the tumor and the host’s immune response, this counterattack could transform a dormant, occult neoplastic focus into a clinically significant aggressive cancer.
The interaction between thyroid carcinomas and the immune system is particularly intriguing. Lymphocytic infiltrates, sometimes intense enough to be diagnosed as Hashimoto’s thyroiditis, are a common finding around and/or within papillary carcinomas, and numerous studies have suggested that they are associated with a better prognosis (8, 9, 10, 11, 43, 44, 45). These findings raise the question of how neoplastic cells evade this attack and evolve into a progressive and often metastatic disease. Various mechanisms have been suggested (reviewed in Ref. 7), such as loss of the expression of major histocompatibility antigens by tumor cells or production of TGFß and other immune-suppressive factors. Our study identifies another immune-suppressive factor, namely FasL, as a potential protective mechanism for thyroid carcinoma cells.
Furthermore, we found that cells of the oxyphilic Huerthle cell carcinomas, known for their relatively more aggressive behavior, stained intensely for FasL. This should probably not be attributed to their oxyphilic nature, as their nonneoplastic counterparts, the oxyphilic metaplastic nodules, stained less intensely. This further supports the theory that FasL expression is associated with the malignant transformation process. However, it appears that FasL expression is limited to carcinomas originating from the follicular epithelium of the thyroid gland, as its immunoreactivity in medullary carcinomas was poor or absent.
The detection of FasL by thyroid carcinomas raises the interesting question of why FasL-expressing tumor cells do not undergo apoptotic suicide, especially as they express the Fas receptor (32). We found that apoptosis was scarce among thyroid carcinoma cells in tumor specimens, as previously reported (46). Therefore, contrary to Hashimoto’s thyroiditis in which FasL up-regulation initiates an apoptotic suicide/fratricide of follicular cells, it appears that the expression of FasL on the neoplastic thyrocyte is, instead, directed against the immune system and not the thyrocyte itself, possibly due to a defect in the tumor cell apoptotic pathway. Indeed, we found that thyroid carcinoma cells are resistant to the apoptosis-inducing activity of the Fas-cross-linking antibody CH11 in the presence or absence of TSH. However, the protein synthesis inhibitor cycloheximide sensitized them to cell death induced by CH11. Our results, which are in agreement with those of Arscott et al. (34, 47), suggest that a short-lived protein inhibits Fas-mediated apoptosis and possibly protects the cell from suicide.
The presence of FasL in the thyroid has been an issue of debate recently. Giordano et al. (35) reported FasL expression in their control thyroid tissue, which was derived from nontoxic goiters. However, we believe that this goiter tissue should not be considered truly normal. Furthermore, two groups have questioned the specificity of the antibody (clone 33, Transduction Laboratories, Lexington, KY) used by Giordano et al. to detect FasL (48). Although preliminary work in our laboratory showed that the monoclonal antibody clone 33 cross-reacts with a band of 37–40 kDa in thyroid carcinoma cell line lysates by Western blotting (data not shown), we cannot found our conclusions on this antibody. To address possible concerns, we have shown in this study the presence of FasL in thyroid carcinoma specimens and cell lines by using three other antibodies for immunohistochemistry and Western blotting and by detecting FasL mRNA by in situ hybridization and RT-PCR.
In conclusion, we have shown that FasL is present in thyroid carcinoma specimens and cell lines and is functional in inducing apoptosis of infiltrating lymphocytes. FasL expression was prognostically significant in papillary carcinomas, suggesting that this counterattack could confer a survival advantage to the malignant cell during the course of cancer progression. Thyroid carcinoma cells avoid Fas-mediated apoptotic suicide by expressing an inhibitor of the Fas pathway.
Received October 2, 1998.
Revised January 28, 1999.
Accepted May 13, 1999.
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 V. Poulaki, C. S. Mitsiades, A. M. Joussen, A. Lappas, B. Kirchhof, and N. Mitsiades Constitutive Nuclear Factor-{kappa}B Activity Is Crucial for Human Retinoblastoma Cell Viability Am. J. Pathol., December 1, 2002; 161(6): 2229 - 2240. [Abstract] [Full Text] [PDF]
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V. Poulaki, C. S. Mitsiades, V. Kotoula, S. Tseleni-Balafouta, A. Ashkenazi, D. A. Koutras, and N. Mitsiades Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells Am. J. Pathol., August 1, 2002; 161(2): 643 - 654. [Abstract] [Full Text] [PDF]
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 N. Mitsiades, C. S. Mitsiades, V. Poulaki, K. C. Anderson, and S. P. Treon Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells Blood, March 15, 2002; 99(6): 2162 - 2171. [Abstract] [Full Text] [PDF]
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 N. Mitsiades, V. Poulaki, C. Mitsiades, and M. Tsokos Ewing's Sarcoma Family Tumors Are Sensitive to Tumor Necrosis Factor-related Apoptosis-inducing Ligand and Express Death Receptor 4 and Death Receptor 5 Cancer Res., March 1, 2001; 61(6): 2704 - 2712. [Abstract] [Full Text]
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M. Marinò, L. Chiovato, N. Mitsiades, F. Latrofa, D. Andrews, S. Tseleni-Balafouta, A. B. Collins, A. Pinchera, and R. T. McCluskey Circulating Thyroglobulin Transcytosed by Thyroid Cells Is Complexed with Secretory Components of Its Endocytic Receptor Megalin J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3458 - 3467. [Abstract] [Full Text]
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N. Mitsiades, V. Poulaki, S. Tseleni-Balafouta, D. A. Koutras, and I. Stamenkovic Thyroid Carcinoma Cells Are Resistant to FAS-mediated Apoptosis But Sensitive to Tumor Necrosis Factor-related Apoptosis-inducing Ligand Cancer Res., August 1, 2000; 60(15): 4122 - 4129. [Abstract] [Full Text]
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J. R. Baker Jr. Editorial: Dying (Apoptosing?) for a Consensus on the Fas Death Pathway in the Thyroid J. Clin. Endocrinol. Metab., August 1, 1999; 84(8): 2593 - 2595. [Full Text]
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