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Original Studies |
Departments of Internal Medicine (P.L.A., T.S., A.M., J.R.B.), Pathology (T.J.G.), and Surgery (N.W.T), University of Michigan, Ann Arbor, Michigan 48109
Address correspondence and requests for reprints to: James R. Baker, Jr., M.D., University of Michigan Medical Center, 1150 West Medical Center Drive, Room 9220 MSRB III, Ann Arbor, Michigan 48109-0648. E-mail: jbakerjr{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and cycloheximide. Whereas both normal
and cancerous thyrocytes were induced to die after this treatment, the
cancerous thyrocytes were more sensitive to anti-Fas antibody. This
work demonstrates that the Fas antigen is expressed and functional on
papillary thyroid cancer cells and this may have potential therapeutic
significance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lymphocytic infiltrates in thyroid cancer contain cytotoxic T lymphocytes (CTL) (7, 8), and a major mechanism of target cell lysis by CTL is through Fas-mediated apoptosis (9). In this process, Fas ligand (FasL) expressed on CTL binds to Fas on tumor cells, initiating an apoptotic signal (10). Fas has been detected on some tumors and on several tumor cell lines, including pancreas (11), lung (12), and gastric mucosa (13). In contrast, it has been suggested that tumors that do not express Fas antigen escape immune surveillance (14).
Whether Fas-signaled apoptosis is involved in immune control of PTC is unknown. Previously published work has shown that Fas is expressed on normal thyroid follicular cells and may play a role in the cellular destruction seen in thyroiditis (15). In cases of differentiated thyroid cancer, however, the presence and function of Fas is unknown.
In this study, we have examined the expression of Fas on thyroid cancer cells. We found that Fas was up-regulated in many papillary cancers compared with normal thyroid follicles. We then evaluated the potential for activation of the Fas death pathway in vitro and found that PTC thyrocytes also display increased sensitivity to the induction of apoptosis through this mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded sections from different types of thyroid cancer were obtained after pathological examination and immunostained for the presence of Fas antigen. Each slide was deparaffinized with three rinses of xylene, followed by rehydration with ethanol, and finally rinsed with phosphate-buffered saline (PBS). To unmask antigenic determinants, slides were microwave-pretreated for 15 min in 0.01 M citrate buffer, then washed with PBS and blocked with 5% normal goat serum in PBS for 20 min. The slides were then incubated for 4 h with either rabbit anti-Fas polyclonal antibody (N-18; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rabbit IgG control antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 1 µg/mL in 1.5% normal goat serum. After washing with PBS, the slides were incubated with biotinylated goat antirabbit IgG and detected using an avidin-biotin complex detection kit with glucose oxidase substrate (Vectastain ABC-GO kit; Vector Laboratories, Inc. Burlingame, CA). Stained slides were briefly counterstained with eosin and mounted with permount (Sigma Chemical Co., St. Louis, MO).
Thyroid cell culture
To examine differences in Fas expression and function, thyroid tissue was obtained at the time of thyroidectomy from seven patients with PTC. Normal and cancerous tissue were separated at surgery and were treated in parallel. The tissue was digested overnight with 40 mg collagenase, 4 mg hyaluronidase, and 4 mg DNase I (all from Sigma Chemical Co.) in 40 mL RPMI 1640 (Life Technologies, Inc., Grand Island, NY). Red blood cells were lysed with ammonium chloride lysis buffer [0.15 M NH4Cl, 10 mM KPO4, and 1 mM EDTA (pH 7.3)] and cells were cultured in Cellgro Complete Media (Mediatech, Herndon, VA) with 20% NuSerum IV (Collaborative Biomedical Products, Becton Dickinson and Co. Labware, Bedford, MA) at 37 C in 5% CO2. After 24 h, nonadherent cells were removed by washing with media. Adherent thyrocytes were supplemented with 10 mIU/mL bovine TSH (Sigma Chemical Co.) every 2–3 days. Cells were stained for thyroglobulin to ensure that they were thyroid in origin (16).
Ribonuclease protection assay
RNA was isolated from thyrocytes using TriZol (Molecular Research Center, Inc., Cincinnati, OH). RiboQuantTM Multi-Probe RNase Protection Assay System (PharMingen, San Diego, CA) was used for the detection and quantitation of specific messenger RNA (mRNA) species. [32P]-labeled antisense RNA probes were prepared using the Human Apoptosis hAPO-3 Template Set (PharMingen), which included human Fas, FasL, Fas-associated phosphatase-1 (FAP-1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The probes were hybridized with 10 µg total thyrocyte RNA, 2 µg HeLa RNA (assay-positive control), and 2 µg yeast transfer RNA (background control). After hybridization, the samples were subjected to RNase treatment, followed by purification of RNase-protected probes. The protected probes were resolved on a 5% denaturing polyacrylamide gel. The quantity of specific transcript present was analyzed by autoradiography and densitometric analysis of scanned films using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA). Relative amounts of specific message were corrected for RNA loading by comparing with the GAPDH band intensity for each sample.
Flow cytometry analysis
Normal and cancer-derived thyrocytes were lifted from 10-cm culture dishes using trypsin/ethylenediaminetetraacetate (Life Technologies, Inc.). After washing with PBS, 1–2 x 106 cells/mL were fixed with 1% formaldehyde in PBS for 30 min on ice. Cells were pelleted and permeabilized with 0.1% Triton X-100 in PBS with 0.1% BSA (PBA). After washing with PBS, cells were dual-stained with a mouse anti-Fas monoclonal antibody (mAb) (clone UB2; MBL International, Watertown, MA) and a rabbit antithyroglobulin polyclonal antibody (DAKO Corp., Carpinteria, CA). Controls included cells stained using isotype control antibodies, as well as single-stained cells. After a 1-h incubation, cells were washed with PBA, then incubated with fluorescein isothiocyanate-conjugated antimouse IgG and phycoerythrin-conjugated antirabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) for 1 h. Finally, cells were washed with PBA and fluorescence was detected with a FACScan flow cytometer (Becton Dickinson and Co., San Jose, CA) and analyzed with CellQuest software (Becton Dickinson and Co., San Jose, CA).
Induction of apoptosis
Cultured thyrocytes were pretreated with and without 500 IU/mL
human interferon (IFN)- for 48 h. To initiate apoptosis,
thyrocytes were then treated for 24 h with 0.2–0.8 µg/mL mouse
IgM anti-Fas mAb, clone CH11 (Upstate Biotechnology, Inc.,
Lake Placid, NY) or purified mouse IgM (Sigma Chemical Co.) as a control. Some cells were also treated with
concentrations of cycloheximide (CHX) that do not yield complete
apoptosis, from 2.5–5 µg/mL, concomitantly with the mAb. Apoptosis
was evaluated by observation of morphological changes, and cell death
was measured using a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.
MTT quantitation of cell viability
Cultured thyrocytes were grown in 96-well plates at cell densities of 10,000 - 20,000 cells/well in 0.1 mL and were treated to induce apoptosis, as described. Metabolically active cells were then detected by adding MTT to a final concentration of 0.5 mg/mL for 2–4 h at 37 C. Isopropanol with 0.04N HCl was added, wells were mixed, and the plates were read at 595 nm with a 630-nm reference (17). Mean and SE for triplicate wells are reported.
Statistics
Differences between anti-Fas mAb or IgM control-treated thyrocytes were compared using a paired two-tailed t test. Differences were considered significant when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo Fas expression in thyroid cancer was
evaluated by immunohistochemical staining of thyroid sections using a
rabbit polyclonal antibody to the amino terminus of Fas (N-18). Thyroid
cancer specimens from 18 patients were examined and included seven PTC
specimens, four follicular variants of PTC, and seven follicular cancer
(FC) specimens. The interpretation of staining on normal and cancerous
thyroid cells was evaluated independently by two individuals. Some PTC
regions displayed intense Fas staining over broad areas of tumor,
whereas others showed either patchy staining, or less intense staining.
Three specimens did not contain a region of normal follicles for
comparison. The thyroid specimens shown in Fig. 1
demonstrate differential staining of
normal thyroid follicles adjacent to a papillary tumor. Although normal
follicular cells revealed low-level Fas expression, the tumor cells
demonstrated a higher density of Fas antigen (Fig. 1A, arrows). A substantial increase in Fas expression in tumor
cells compared with adjacent normal thyroid follicles was demonstrated
in three PTC specimens and three follicular variants of PTC. Five of
seven FC specimens demonstrated low levels of Fas expression, and in
only one specimen was a slight increase observed in the tumor cells
compared with normal regions.
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We examined Fas expression on paired normal and cancer-derived
thyrocytes from the same patient to see if the differences observed
in vivo were maintained in culture. Fas protein expression
on thyrocytes isolated from two patients with PTC was compared by flow
cytometric analysis using a mAb against Fas (clone UB2). Fas was
expressed on more than 90% of normal thyrocytes as compared with
isotype control stained cells. However, a shift in the amount of
Fas-specific fluorescence was evident in the cancer cell population
(Fig. 2A). For both patients, the mean
channel fluorescence of anti-Fas-stained cells was increased in
cancer-derived thyrocytes compared with normal thyrocytes (Fig. 2B).
Fas expression was confirmed by Western blot for thyrocytes from two
other patients, however, no difference in expression between normal and
cancer thyrocytes could be measured using this technique (data not
shown).
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). Fas
mRNA was present in both normal and cancerous thyrocytes from all four
patients. In two of the four paired samples (Fig. 3, sets 2 and 4), the
cancer-derived thyrocytes demonstrated levels of Fas mRNA that were
substantially higher than in normal thyrocytes from the same patient.
In the two other cases, differences were negligible.
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To determine whether increased Fas expression on thyroid cancer
cells could affect the propensity of thyrocytes to undergo Fas-mediated
apoptosis, paired thyrocytes were treated with a control mouse IgM
antibody or an anti-Fas mAb (clone CH11), which cross-links the Fas
receptor, initiating a death signal. When treated with anti-Fas mAb
alone, no cell death was observed in either normal or cancerous
thyrocytes as measured by an MTT cell viability assay (Fig. 4A, left). Because earlier
studies demonstrated that a labile inhibitor of the Fas pathway
rendered thyrocytes resistant to apoptosis by the anti-Fas mAb alone
(16), CHX was then included with mAb treatment. Cancer thyrocytes
treated with anti-Fas mAb in the presence of CHX demonstrated a 47%
reduction in cell viability compared with IgM control
antibody-treated cells (Fig. 4A, right) and were more
sensitive to death induced in this manner than similarly treated normal
thyrocytes (Fig. 4A, left).
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for 48 h can enhance
normal thyrocyte susceptibility to Fas-mediated apoptosis (16). This
was also observed in the cancer thyrocytes where the death induced by
anti-Fas mAb and CHX was significantly increased after exposure to
IFN- (Fig. 4B
). An even greater increase in cell death was observed
in the corresponding normal thyrocytes, to the point where the response
in both cell populations was similar. Normal and cancer thyrocytes from five PTC patients were compared for sensitivity to Fas-mediated cell death, as measured by MTT assay. Fas-mediated death in normal thyrocytes ranged from 6–49%, whereas cancer thyrocytes displayed 20–78% death. In four of the five cases, the cancer thyrocytes were more sensitive to death induced by anti-Fas mAb in the presence of CHX, demonstrating 1.5–7 times more death than observed in normal thyrocytes.
FAP-1 mRNA in normal and cancer thyrocytes in vitro
The requirement of CHX for both normal and cancerous thyrocytes to
respond to Fas-mediated apoptosis suggests that inhibitors of this
pathway may also play a role in determining sensitivities to death
induced by treatment with anti-Fas mAb. We examined the expression of
one such inhibitor, FAP-1 (18), which has been reported to be present
in the thyroid (19). The level of mRNA for FAP-1 was analyzed by
ribonuclease protection assay in paired normal and cancer thyrocytes
from four patients (Fig. 5).
Interestingly, levels of FAP-1 did not differ substantially between the
normal and cancer thyrocytes, even in the two sets where Fas mRNA was
increased in cancer thyrocytes (Fig. 3, sets 2 and 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In thyroid cells associated with thyroiditis, Fas is constitutively expressed and apoptosis is also evident, especially in regions proximal to lymphocytic centers (15, 26). The Fas expressed in Hashimoto’s thyroiditis is, therefore, thought to contribute to increased apoptosis in the thyroid (15, 21). PTC is often accompanied by lymphocytic infiltrates (4), and evidence of increased levels of apoptosis in PTC has also been reported (15, 27). Thus, the Fas expressed on PTC may provide a possible mechanism for the initiation of apoptosis in neoplastic thyroid cells.
We then considered whether the increased expression of Fas in PTC is functional and, therefore, a potential target for immune control. By examining thyrocytes isolated from normal and cancerous tissue from the same patient, we were able to directly compare the expression of Fas, and the ability of these cells to respond to Fas-mediated cell death. Expression of Fas mRNA and protein were documented in both normal and cancer-derived thyrocytes. Whereas Fas mRNA levels did not differ between normal and cancerous tissue for all patients, the increased levels found in the cancer thyrocytes from two patients suggest possible transcriptional control in these patients. Importantly, it is the expression of Fas protein that more directly relates to the susceptibility of PTC thyrocytes to Fas-mediated apoptosis. As quantified by flow cytometry, Fas protein expression on cancer-derived thyrocytes was increased nearly 3-fold compared with normal thyrocytes. Correspondingly, susceptibility to Fas-mediated death was also significantly enhanced in these PTC cells.
We have previously shown that thyrocytes were resistant to death
initiated by anti-Fas mAb alone, but susceptible in the presence of
CHX, which reduced levels of a labile inhibitor (16). In our present
study, the cancer-derived thyrocytes, as well as the normal thyrocytes,
also demonstrated the presence of a labile inhibitor of Fas-mediated
apoptosis and required the presence of CHX to initiate death. In the
case of PTC, once inhibitor levels were decreased by CHX it is likely
that the up-regulation of Fas on cancer thyrocytes was then a
significant factor contributing to increased sensitivity to
Fas-mediated cell death. After IFN- pretreatment, anti-Fas mAb in
the presence of CHX induced significant death, in both normal and
cancer thyrocytes, demonstrating that both cell populations are capable
of responding with maximal death.
The presence of inhibitors of Fas-mediated apoptosis may contribute to the overall sensitivity of thyrocytes to death signaled through this pathway. It is probable that activation of the Fas death pathway in thyrocytes depends on the relative concentrations of Fas and Fas inhibitor present. Inhibitors that specifically block Fas signaling include the FLICE (FADD-like ICE)-inhibitory protein (28) and FAP-1 (18), which binds to the negative regulatory domain of Fas. We have detected FLICE-inhibitory protein in thyrocytes, however, its expression was not regulated by CHX treatment (data not shown) and, therefore, is unlikely to be directly involved in thyrocyte resistance to death induced by Fas signaling. On the other hand, FAP-1 is present in normal thyrocytes, and protein expression decreases after CHX treatment (19), so it may contribute to resistance to Fas-mediated apoptosis observed in thyrocytes. We have provided evidence that FAP-1 is also expressed by thyrocytes in PTC. Although, Fas mRNA is present at higher levels in some cancer thyrocytes, little difference was seen in the levels of inhibitor mRNA. Thus, the differential sensitivity of cancer and normal thyrocytes to Fas-mediated cell death is less likely the result of depressed inhibitor concentrations than the level of Fas expressed on these cells.
The Fas/FasL system has been proposed as a mechanism for tumor cell
defense (29), although there is controversy regarding the importance of
FasL in this role (30). Interestingly, using ribonuclease protection
assay to detect specific FasL mRNA, we found no expression of FasL in
either normal or cancer thyrocytes (31) (data not shown), whereas mRNA
for Fas was present in all cases. Unfortunately, controversy over the
specificity of several antibodies to FasL make the study of FasL
protein expression difficult (31, 32, 33). Conversely, the Fas expressed on
PTC may allow for the immune control of tumor growth and provide a
possible target for treatment. Death of tumor cells has been
demonstrated after inducing sensitivity to Fas-mediated apoptosis with
anticancer drugs, such as doxorubicin (34). In another study, treatment
with IFN-
induced expression of functional CD95 in basal cell
carcinomas, eventually leading to tumor regression (35). Thus, similar
treatments or factors that enhance Fas-mediated apoptosis could effect
PTC tumor lysis.
In summary, our results indicate that Fas is up-regulated in some PTC cases, and these cells demonstrate greater sensitivity to apoptosis through this death pathway. This, along with the improved outcome in the presence of immune infiltration, suggests that the Fas death pathway could be involved in the immune control of PTC. Furthermore, our results suggest that the increased Fas expressed on some PTC may provide a possible target for treatment.
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Acknowledgments |
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Footnotes |
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Received June 16, 1999.
Accepted August 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß lo/-CD4-CD8- cells, in autoimmune thyroid
disease. Clin Exp Immunol. 93:430–436.[Medline]
treatment is mediated by CD95 (Apo-1/Fas)-CD95
ligand-induced suicide. J Clin Invest. 100:2691–2696.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) and cancer
(
) thyrocytes from two different PTC patients.
) in the presence or absence of a
suboptimal concentration of CHX. Cell viability was measured using MTT.
Each value is the average and SE from three wells
(*P < 0.005 vs. IgM control). B,
Thyrocytes treated with the same combinations of antibodies and CHX
after pretreatment with IFN-