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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 3 1374-1383
Copyright © 2003 by The Endocrine Society

Abnormally High Expression of Proteasome Activator-{gamma} in Thyroid Neoplasm

Tomohisa Okamura, Shin-ichi Taniguchi, Tsuyoshi Ohkura, Akio Yoshida, Hideki Shimizu, Mitsue Sakai, Hiroyuki Maeta, Hiroko Fukui, Yoshihiko Ueta, Ichiro Hisatome and Chiaki Shigemasa

First Department of Internal Medicine (T.Ok., S.T., T.Oh., A.Y., H.S., H.F., Y.U., I.H., C.S.), Department of Bioscience (M.S.), and Second Department of Surgery (H.M.), Tottori University Faculty of Medicine, Yonaga 683-8504, Japan

Address all correspondence and requests for reprints to: Shin-ichi Taniguchi, M.D., Ph.D., First Department of Internal Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan. E-mail: stani{at}grape.med.tottori-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
PA28-{gamma} is the activator of 20S proteasome, the ATP-dependent proteolytic system that plays an important role in cell cycle progression in various cell types. In this paper, we show the abnormally high expression of PA28-{gamma} in various thyroid neoplasms. Thyroid samples were obtained from patients with normal thyroid (4 cases) and with the following diseases: papillary adenocarcinoma (13 cases), multinodular goiter (4 cases), and anaplastic carcinoma (1 case). PA28-{gamma} expression was estimated by immunohistochemical staining and Western blotting. In all of the papillary adenocarcinoma samples, PA28-{gamma} was abnormally overexpressed, especially in cancer cells existing at the peripheral region of the cancer mass or in cancer cells invading the capsular region surrounding the cancer mass. In cancer cells of these areas, PA28-{gamma} was predominantly distributed in nucleus rather than in the cytoplasm of cancer cells. On the other hand, no obvious PA28-{gamma} expression was observed in the adjacent normal thyroid follicular cells. In multinodular goiter, the expression of PA28-{gamma} was relatively low compared with papillary adenocarcinoma. In anaplastic carcinoma, PA28-{gamma} was expressed at the highest level, especially in poorly differentiated regions such as squamous metaplasia of anaplastic cancer tissue. Therefore, the PA28-{gamma} expression seems to be restricted to thyroid cancer cells, especially in the region where the growth rate of cancer cells is accelerated. This result is further confirmed by the fact that C2, {alpha}-subunit of 20S proteasome, and proliferating cell nuclear antigen are similarly overexpressed in this region. Thus, PA28-{gamma} might be involved in the regulatory system for the cell cycle. Moreover, the growth of thyroid cancer cell lines was affected by the proteasome inhibitor, clasto-lactacystin ß-lactone. These results demonstrate that PA28-{gamma} is overexpressed in thyroid cancer, especially in its growth-accelerated cells.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE 20S PROTEASOME is a 700-kDa macromolecule that is responsible for the ATP-dependent degradation of numerous cellular proteins, including regulatory enzymes, transcription factors, and cell cycle regulators (1, 2, 3, 4, 5, 6). Effective protein and peptide degradation by this macromolecule requires the assistance of regulatory protein (7). An assembly of regulatory proteins forms a 19S complex, known as PA700. This complex binds to both ends of the 20S proteasome to yield the 26S proteasome. The 26S proteasome is the macromolecular entity that recognizes and degrades ubiquitin-protein conjugates in an ATP-dependent manner. A second type of regulatory molecule is referred to as PA28 or 11S regulator. PA28 is composed of two homologous subunits ({alpha} and ß; Ref. 8) and a separate but related protein, PA28-{gamma} (9). PA28 binds to the outer rings of the 20S proteasome and greatly stimulates the small peptides but not proteins in an ATP-independent reaction. PA28-{alpha} and PA28-ß are markedly up-regulated by interferon-{gamma}, and they enhance the generation of antigenic peptides by the proteasome for presentation by the major histocompatibility complex class I pathway (10), but the function of PA28-{gamma} still remains elusive.

In eukaryotic cells, proteolysis is known to be involved in the cell cycle. Nuclear oncoproteins, such as c-myc and c-fos, and p53 antioncoprotein, which are required for regulation of cell growth, are degraded rapidly in an energy-dependent fashion (11, 12, 13). Cyclin, which is essential for mitosis, was demonstrated to be degraded by the ubiquitin pathway (14). With respect to cancer cells, several reports have demonstrated that the proteasome components are up-regulated in cancer tissue or cancer cell lines (15, 16, 17, 18, 19, 20). Proteasome expression has been found to be abnormally high in human leukemic cells (21). Kanayama et al. (17) demonstrated by Northern blot and immunohistochemical analyses that the expressions of the proteasomes, C2 and C9, and ubiquitin were abnormally high in most neoplastic regions of renal cell carcinomas and in renal cancer cell lines (17). These data suggest that proteasome is abnormally overexpressed and somehow contributes to the cell cycle progression of the cancer cells. Moreover, a recent study revealed that PA28-{gamma}-deficient mice were born without appreciable abnormalities, but their growth after birth was significantly retarded compared with that of PA28-{gamma}+/- or PA28-{gamma}+/+ mice. Moreover, the cultured embryonic fibroblast cells lacking PA28-{gamma} displayed a lower progression rate for S phase entry than their wild-type counterparts (22). This observation strongly suggests that PA28-{gamma} could positively contribute to cell cycle progression via its physiological role, activation of 20S proteasome.

With regard to thyroid follicular cells, so far there have been no reports describing the possible involvement of 20S proteasome and PA28-{gamma} for cell growth or proliferation. We separately show that growth-stimulatory signals such as TSH or insulin could induce PA28-{gamma} expression and recruit it into the nucleus using a rat functional thyroid cell line, FRTL5 (23). Then, it is reasonable to assume that the proteasome compartment and its regulators are overexpressed in thyroid cancer cells, as observed in other cell types such as renal cell carcinoma or leukemic cells.

In this study, we estimated that the expression and distribution of PA28-{gamma} as well as C2, a component of {alpha}-subunit of proteasome, in v-{gamma} and C2 are overexpressed in various types of thyroid cancer tissues. Our observation implies that the proteasome proteolytic system is involved in thyroid cancer cell proliferation.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Human subjects

All human tissues were obtained by the University of Tottori Committee for the Protection of Human Subjects, in accordance with the Declaration of Helsinki. Thyroid samples were obtained at surgery from 18 patients with the following diseases: papillary adenocarcinoma (13 cases), anaplastic carcinoma (1 case), and multinodular goiter (4 cases). Normal thyroid tissues were obtained at autopsy from four patients without thyroid disease.

Western blot analysis

Human thyroid tissues were homogenized in 300 µl RIPA buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate in PBS with protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN)] and centrifuged at 15,000 rpm for 20 min at 4 C. The supernatant was used as the total cell lysate. Ten micrograms of each lysate were loaded to 10% SDS-PAGE and blotted onto membranes. Proteins were electrophoretically transferred to nitrocellulose membranes, blocked with Tris-buffered saline and 0.05% Tween-20 containing 5% nonfat dried milk, washed, and incubated with a polyclonal antibody against PA28-{gamma} (1:500), C2 (1:500; from Keiji Tanaka, Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), thyroid peroxidase (1:250; 8912-5059, Biogenesis, Poole, UK), proliferating cell nuclear antigen (PCNA; 1:500, M0879, DAKO Corp., Glostrup, Denmark), or actin (1:500; sc-1616, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). These membranes were exposed to the secondary antirabbit antibody (NA934V, Amersham Pharmacia Biotech, Little Chalfont, UK; diluted 1:3000) for PA28-{gamma} and C2, and antimouse antibody (NA931, Amersham Pharmacia Biotech; diluted 1:3000) for PCNA, respectively. After incubation with secondary antibody, the detection was performed using enhanced chemiluminescence.

Immunohistochemical evaluation of PA28-{gamma} and C2 expression

PA28-{gamma} and C2 expression were analyzed by immunocytochemical staining of thyroid tissues. Paraffin-embedded tissue sections, 4-µm thick, were deparaffinized in xylene and rehydrated through a graded alcohol series to deionized water. The endogenous peroxidase activity was blocked with H2O2. The incubation with the anti-PA28-{gamma} antibody (1:100) or the anti-C2 antibody (1:50) for 12 h at 4 C was followed by the incubation with biotinylated horse serum anti-IgG (1:3000) for 30 min at room temperature. The sections were immersed in a solution with the avidin-biotin complex (Vector Laboratories, Inc., Burlingame, CA) for 30 min, developed with diaminobenzidine, and counterstained with hematoxylin. The sections were scanned at magnification (x100) using light microscopy. All sections were evaluated by two authors (T.Ok. and S.T.) for determination of the interobserver variability. The immunoreactivity was evaluated according to the intensity and extent of the staining. The intensity of the immunoreaction was subjectively evaluated in four grades (0, 1+, 2+, and 3+). In cases showing variable intensity of the reaction, an average score was adopted. The extent of the reaction was considered as 1+ if the number of reactive cells was less than 25%, 2+ when the number of reactive cells was 25% to 75%, and 3+ if more than 75% of the cells stained. The final score was obtained in each case by multiplying the intensity by the extent score. The immunoreactivity of each case was then classified into five grades according to the combined score estimated by two examiners (T.Ok. and S.T.) as "expression score," as follows: 0, negative (scores 0 and 1+); 1, weak (scores 2+); 2, moderate (scores 3+); 3, strong (scores 4+); and 4, extremely strong (scores over 5+). Controls were obtained from the thyroid region adjacent to the primary tumors with proven histological absence of neoplasm.

Cell cultures

The NPA (24) and 8505C (25) clone was obtained from Dr. S. Kosugi (Department of Laboratory Medicine and Clinical Genetics Unit, Kyoto University School of Medicine, Kyoto, Japan). NPA cells and 8505C cells were grown in RPMI medium 1640 (31800-022, Invitrogen Corp., Carlsbad, CA), and DMEM (12800-017, Invitrogen Corp.), respectively, supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 50 µg/ml streptomycin. Culture medium was changed every 2 d, and cells were passaged every 5–6 d.

5-Bromo-2'-deoxy-uridine (BrdU) uptake assay

DNA synthesis was determined by measuring the incorporation of BrdU into the cellular DNA fraction. Incorporated BrdU was estimated by ELISA using peroxidase-conjugated anti-BrdU antibody (1-444-611, Roche Diagnostics GmbH, Ingelheim, Germany). To determine the function of proteasome, we applied proteasome-specific inhibitor such as the active form of lactacystin, clasto-lactacystin ß-lactone (BBI-100, Boston Biochem, Cambridge, MA), to NPA and 8505C cells in the presence of 10% fetal calf serum.

Immunostaining and microscopy

Cells were plated on cover slips and cultured in RPMI for NPA cells or DMEM for 8505C cells with 10% fetal calf serum, then washed twice with PBS, and fixed with 2% paraformaldehyde. Cells were permeabilized with 0.5% Triton X-100, incubated with anti PA28-{gamma} antibody (1:100), then visualized using FITC-conjugated antirabbit IgG antibody. To observe the fine localization of PA28-{gamma} within cells, we used a confocal microscopy system (Fluoview-Olympus, Olympus Corp., Tokyo, Japan).

Statistical analysis

The Mann-Whitney U test and Spearman correlation test were used to test for differences in the classified staining levels of PA28-{gamma} and C2 expression between groups. The values of Western blot analysis were estimated by densitometry and compared by ANOVA. BrdU-labeling index, which was the percentage of control, was analyzed for each treatment group derived from the same preparations. Differences between each group were statistically analyzed using ANOVA. Significant differences between groups were determined by Student’s t test. A P value of less than 0.01 was considered to indicate statistical significance. All statistical analyses were performed on a personal computer with the statistical package StatView 5.0 for Mackintosh (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Evaluation of PA28-{gamma} by Western blot analysis

We estimated PA28-{gamma} and PCNA expression by Western blotting in normal thyroid tissues of 4 patients (cases 14–17), papillary adenocarcinoma of 13 patients [cases 1–13; except for case 5, oxyphilic variant, the rest of the 12 samples were subclassified into classical (usual) type], and multinodular goiter of 4 patients (cases 18–21) by densitometry. To compare the expression of PA28-{gamma} between each membrane, we used PA28-{gamma} expression value, which is the densitometrically analyzed data of each membrane divided by the data of case 7, which was used as an internal control. In this study, most cases of papillary adenocarcinoma showed high expression of PA28-{gamma} (Fig. 1AGo). On the other hand, with regard to normal thyroid and multinodular goiter, the expression of PA28-{gamma} was relatively low compared with papillary adenocarcinoma. Faint expression of PA28-{gamma} was observed in two of the four samples of normal thyroid (cases 14 and 15); the other two samples of normal thyroid (cases 16 and 17) and all four of the multinodular goiter samples (cases 18–21) showed relatively lower expression of PA28-{gamma} than most samples of papillary adenocarcinoma (Fig. 1Go, A and B). Basically, PCNA expression of these samples appeared to be in parallel with PA28-{gamma} expression (Fig. 1Go). In PA28-{gamma} expression values, there were significant differences between normal thyroid and papillary adenocarcinoma (0.253 ± 0.100 vs. 4.219 ± 0.525; P < 0.01) or multinodular goiter and papillary adenocarcinoma (0.715 ± 0.116 vs. 4.219 ± 0.525; P < 0.01). But, there were no significant differences between normal thyroid and multinodular goiter (0.253 ± 0.100 vs. 0.715 ± 0.116; P = 0.6786). The same tendency with PA28-{gamma} expression value was observed in PCNA values. Therefore, we analyzed whole data regarding PA28-{gamma} and PCNA expression values in papillary adenocarcinoma, multinodular goiter, and normal thyroid. Using these samples, we found the positive correlation between PA28-{gamma} and PCNA expression values (y = 0.971 + 0.538 x; r = 0.779; P < 0.001; Fig. 2Go), indicating that PA28-{gamma} expression is significantly related to PCNA expression. These results suggest that PA28-{gamma} is somehow related to cancer cell growth and reflects cell growth activity, because PCNA is generally induced in early S phase during cell cycle progression and acts as a cofactor of DNA polymerase.



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  		Figure 1. Western blot analysis of PA28-{gamma} in papillary adenocarcinoma (A), multinodular goiter, and normal thyroid tissue (B). Case 7 was used as internal control in both panels. For immunoblotting, 10 µg of each lysate was loaded to 10% SDS-PAGE and blotted onto membranes. Membranes were then incubated with anti-PA28 antibody (1:500 dilution) and anti-PCNA antibody (1:500 dilution). The enhanced chemiluminescence was performed for the same time period for each experiment of papillary adenocarcinoma or normal thyroid and multinodular goiter. The expression of normal thyroid and multinodular goiter (B) was very low, so a longer exposure period was necessary to get enough enhancement of chemiluminescence to compare with papillary adenocarcinoma (A). To ensure that the total amount of protein in each lane was identical, membranes were incubated with antiactin antibody (1:500 dilution) in the same method mentioned above.

 


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  		Figure 2. The correlation between PA28-{gamma} and PCNA with Western blot analysis. We analyzed whole data regarding the intensity of PA28-{gamma} and PCNA, which was examined by densitometry in papillary adenocarcinoma (filled circles), multinodular goiter (open squares), and normal thyroid (open circles). To compare the expression of PA28-{gamma} between each membrane (Fig. 1Go, A and B), we used PA28-{gamma} expression value, which is the densitometrically analyzed data of each membrane divided by the data of case 7, which was used as an internal control. These samples show positive correlation between of PA28-{gamma} and PCNA (y = 0.971 + 0.538 x; r = 0.779; P < 0.001). The values of Western blots from each sample were analyzed densitometrically and divided by case 7 as the internal standard.

 
Overexpression of PA28-{gamma} in human thyroid carcinoma and its localization

Because PA28-{gamma} expression is especially high in thyroid papillary adenocarcinoma, we estimated the distribution of PA28-{gamma} by immunocytochemistry. Six samples of papillary adenocarcinoma (cases 1–7), four of normal thyroid (cases 14–17), four of multinodular goiter (cases 18–21), and one anaplastic carcinoma (case 23) were analyzed.

In all samples of papillary adenocarcinoma, the distribution of PA28-{gamma} showed a very peculiar property. All of the papillary cancer existed as a cancer mass accompanied by an adjacent capsular region. The highest intensity of PA28-{gamma} expression was observed in peripheral cells inside the cancer capsule or invading the capsular region (Fig. 3AGo). Moreover, if focused on intracellular distribution of PA28-{gamma} in these regions, the expression of PA28-{gamma} is especially high in the nucleus rather than in the cytosol (Fig. 3AGo, arrows). On the other hand, cancer cells localized in the central region of the cancer mass showed a relatively weak or moderate level of PA28-{gamma} expression (Fig. 3BGo). There was no obvious expression of PA28-{gamma} in the adjacent normal area (Fig. 3EGo). There were no significant differences about the expression of PA28-{gamma} between classical (usual) papillary thyroid carcinomas (cases 1–4, 6, and 7) and oxyphilic variant (case 5).



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  		Figure 3. Immunostaining of papillary carcinoma, anaplastic carcinoma, and normal thyroid using anti-PA28-{gamma} antibody. Immunostaining of papillary carcinoma (case 8) using anti-PA28-{gamma} antibody (A and B) and anti-C2 antibody (C and D). In A, arrows indicate the representative staining pattern of PA28-{gamma}. Adjacent normal follicular cells of the same tissue are shown in E (x600). A section of the area near the peripheral region of the cancer mass is shown in A (x600) and that of the central region of the mass is shown in B (x600). A section of almost the same area of A, which is the peripheral region of the mass, is shown in C (x600) which was stained with anti-C2 antibody. A section of almost the same area of B, which is the central region of the mass, is shown in D (x600). Immunostaining of anaplastic carcinoma (case 22; G and H) and normal thyroid (case 14; x600) using anti-PA28-{gamma} antibody.

 
To compare the expression of PA28-{gamma}, we classified the PA28-{gamma} expression into five categories (0, negative; 1, weak; 2, moderate; 3, strong; and 4, extremely strong) according to the intensity and extent of the staining.

We revealed that the intensity of PA28-{gamma} expression of cancer mass (classes 1–4) is higher than that of the adjacent normal region in both nucleus (class 0) and cytosol (class 0; Fig. 4AGo; {dagger}, P < 0.01). In the cancer mass region, the peripheral region (classes 2–4) showed higher expression of PA28-{gamma} than the central region (classes 1–2) with significant difference. Especially at the peripheral region of the cancer mass, the expression of PA28-{gamma} at nucleus (classes 3–4) was higher than at cytosol (classes 2–3) with significant difference (*, P < 0.01). On the other hand, there was no significant difference between nucleus and cytosol of cancer cells localizing the central region of the cancer mass and adjacent normal area.



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  		Figure 4. Comparison of PA28-{gamma} expression between nucleus and cytosol in the different groups. We performed the immunohistochemical analysis of multinodular goiter (n = 4) and papillary adenocarcinoma (n = 7). We classified the expression of PA28-{gamma} into five grades (0, negative; 1, weak; 2, moderate; 3, strong; and 4, extremely strong) according to the intensity and extent of the staining. A and B indicate the differences of the PA28-{gamma} expression between adjacent normal region, central region, and peripheral region of papillary adenocarcinoma and multinodular goiter, respectively. *, Significant difference (P < 0.01) compared with nucleus and cytosol; {dagger}, significant difference (P < 0.01) compared with adjacent normal area; {ddagger}, significant difference (P < 0.01) compared with multinodular goiter.

 
Such a peculiar property of PA28-{gamma} distribution in thyroid papillary cancer leads us to the idea that PA28-{gamma} expression may reflect the growth property of cancer cells. This result is quite compatible with our separate report showing that the PA28-{gamma} was mainly localized within the nucleus of growth-stimulated FRTL5 rat thyroid cells (23).

To estimate PA28-{gamma} expression in normal thyroid tissue without any neoplastic change, we analyzed four cases of normal human thyroid tissues obtained at autopsy (Fig. 3FGo). As expected, there was no obvious expression of PA28-{gamma} in all of the normal thyroid tissue, and all of the tissue was classified into class 0.

We next performed the immunohistochemical analysis of PA28-{gamma} expression in multinodular goiter. Multinodular goiter is characterized as a benign change of thyroid follicular cells, resulting in multifocal nodular region with capsule-like structure in the thyroid gland. We examined the nodular component that was completely separated from adjacent tissue by a capsule of multinodular goiters. The histological appearance of the nodule displayed a distinct morphologic pattern that clearly distinguished it from surrounding thyroid tissue.

In multinodular goiter, the expression of PA28-{gamma} was relatively lower than papillary adenocarcinoma (Fig. 3Go, A and B), but higher than normal thyroid (Fig. 3FGo). Although the nodular component of multinodular goiter showed morphological heterogeneity (i.e. follicular size, colloid content, and follicular morphology), there were no significant differences in the expression of PA28-{gamma} between these cells inside the capsule. The expression of PA28-{gamma} was clearly higher than the adjacent surrounding morphologically normal thyroid tissue. But there was no significant difference between nucleus and cytosol in the peripheral tissue and the region of the mass.

Next, we classified the PA28-{gamma} expression of multinodular goiter into five categories (0, negative; 1, weak; 2, moderate; 3, strong; and 4, extremely strong) according to their staining level. In multinodular goiter, the intensity of PA28-{gamma} expression was moderate (classes 0–2), although the expression of PA28-{gamma} was observed to be higher in multinodular goiter than in the adjacent normal thyroid region (class 0). In multinodular goiter, there were no significant differences in PA28-{gamma} distribution between nucleus and cytosol in both the peripheral region (classes 0–2) and the central region (classes 0–2; Fig. 4BGo, multinodular goiter, nucleus vs. cytosol in peripheral region, P = 0.5050; nucleus vs. cytosol in central region, P = 0.3496).

Finally, the most malignant converted form of thyroid follicular cells, thyroid anaplastic carcinoma, was analyzed (Fig. 3Go, G and H). In anaplastic carcinoma, several transformation steps are usually observed. Basically, PA28-{gamma} was extremely expressed in the whole area of anaplastic cancer mass. Interestingly, PA28-{gamma} is expressed at its highest level (class 4), especially in the most poorly differentiated cancer cells, such as squamous metaplasia (Fig. 3GGo). In such a poorly differentiated region, PA28-{gamma} was predominantly distributed in the nucleus rather than cytoplasm of cancer cells, as observed in papillary carcinoma (Fig. 3AGo). This result repeatedly supports the idea that the expression and subcellular distribution of PA28-{gamma} may reflect cancer cell growth.

Expression of C2 in human thyroid neoplasm and its relationship to the expression of PA28-{gamma}

PA28-{gamma} works as an activating molecule of 20S proteasome. We estimated the relationship between PA28-{gamma} and 20S proteasome in thyroid neoplasm. Because 20S proteasomes are unusually large polysubunit complexes that are assumed to have several distinct enzyme activities in a single structure (26, 27), we estimated one of the primary structures of proteasome {alpha}-subunits, named C2 (28), by immunohistochemistry. Seven samples of papillary adenocarcinoma (cases 1–7), four of normal thyroid (cases 14–17), four of multinodular goiter (case 18–21), and one of anaplastic carcinoma (case 22) were analyzed.

Interestingly, C2 expression showed a similar property with PA28-{gamma} in terms of its intensity. In papillary carcinoma, C2 was highly expressed in cancer cells localizing at the peripheral region of the cancer mass (Fig. 3CGo) and the central region (Fig. 3DGo). On the other hand, in adjacent normal follicular thyroid cells, there was no obvious expression of C2 in either cytosol or nucleus. To compare the expression of each region, we classified regions into five categories (0, negative; 1, weak; 2, moderate; 3, strong; and 4, extremely strong) according to the intensity and extent of the staining (Fig. 5AGo). Both the central and peripheral regions of cancer mass showed significantly higher expression of C2 than the adjacent normal region (Fig. 5AGo; P < 0.01).



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  		Figure 5. The comparison of C2 expression between nucleus and cytosol in the different groups. We performed the immunohistochemical analysis of multinodular goiter (n = 4) and papillary adenocarcinoma (n = 7). We classified the expression of C2 into five categories (0, negative; 1, weak; 2, moderate; 3, strong; 4, extremely strong) according to the intensity and extent of the staining. A and B indicate the differences of the C2 expression between adjacent normal region, central region, and peripheral region of papillary adenocarcinoma and multinodular goiter, respectively. {dagger}, Significant difference (P < 0.01) compared with adjacent normal area; {ddagger}, significant difference (P < 0.01) compared with multinodular goiter.

 
However, the distribution of C2 appeared to be distinct from that of PA28-{gamma}. Although PA28-{gamma} was predominantly expressed in the nucleus of cancer cells (Fig. 4AGo), C2 expression showed no significant difference between nucleus and cytosol of cancer cells localizing at the peripheral and central regions of the cancer mass of papillary carcinoma (Fig. 5AGo; papillary adenocarcinoma, nucleus vs. cytosol in peripheral region, P = 0.2445; nucleus vs. cytosol in central region, P = 0.1736).

In multinodular goiter, significant difference in C2 expression was observed between papillary adenocarcinoma and multinodular goiter (Fig. 5Go, A and B; {ddagger}, P < 0.01). But there was no significant difference in C2 expression between nucleus and cytosol of thyroid follicular cells (Fig. 5BGo; multinodular goiter, nucleus vs. cytosol in peripheral region, P > 0.9999; nucleus vs. cytosol in central region, P = 0.3688).

To estimate C2 expression in normal thyroid tissue without any neoplastic change, we analyzed four cases of normal human thyroid tissues obtained at autopsy. As expected, there was no obvious expression of C2 in any normal thyroid tissue (class 0; data not shown).

Then, we presumed PA28-{gamma} expression might be related to lymph node metastasis, because PA28-{gamma} expression seems to reflect cancer cell growth. However, in this study, we could not find any significant relationship between the PA28-{gamma}/C2 expression and lymph node metastasis (data not shown).

Involvement of 20S proteasome in thyroid cancer cell proliferation

Our data indicate that PA28-{gamma} may play a key role in thyroid cancer cell proliferation with 20S proteasome. To test this hypothesis, we performed an inhibition study using the proteasome inhibitor ß-lactone (29), and we checked the fine localization of PA28-{gamma} within cancer cells by a confocal microscopy system using two types of thyroid cancer cell lines, i.e. NPA, derived from papillary adenocarcinoma, and 8505C, derived from anaplastic carcinoma. In the presence of 10% fetal calf serum, the maximal proliferation was observed in both NPA and 8505C cell lines (doubling time, about 22 h and 18 h, respectively).

To evaluate DNA synthesis of PA28-{gamma} in the thyroid cancer cell lines NPA and 8505C, we measured the incorporation of BrdU into the cellular DNA fraction. We applied a proteasome-specific inhibitor, clasto-lactacystin ß-lactone (29), which is the active form of lactacystin, to NPA and 8505C cells in the presence of 10% fetal calf serum. Lactacystin and its ß-lactone derivative inhibit protein breakdown by proteasomes but do not affect other types of proteases; they appeared very useful to test the involvement of proteasome for cell growth (30). Generally, 50% inhibition was obtained at 1–10 µM after 2-h exposure of ß-lactone. Maximal inhibition was achieved in these assays at concentrations above 20 µM. In a variety of mammalian cells, these inhibitors blocked degradation of both long-lived and short-lived proteins similarly. Inhibition of 75–95% of protein degradation was achieved within 1-h exposure to 20 µM inhibitors. Nevertheless, these reagents did not reduce cell viability or protein synthesis for at least several hours (30).

As shown in Fig. 6Go, the treatment of ß-lactone markedly reduced BrdU incorporation of both NPA (Fig. 6AGo) and 8505C (Fig. 6BGo) cells in a dose-dependent manner. At maximal concentration of ß-lactone (25 µM), BrdU uptake was suppressed to under 50% in both cell lines. Cells treated with these inhibitors remained fully viable for the duration of the experiment. These results are compatible with other reports of inhibition study using ß-lactone (30), suggesting 20S proteasome is involved in cell growth machinery of the thyroid cancer cell lines NPA and 8505C.



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  		Figure 6. Inhibition of thyroid cancer cell lines, NPA and 8505C proliferation by proteasome-specific inhibitor ß-lactone. To determine the involvement of proteasome in thyroid cancer cell growth, we applied a proteasome-specific inhibitor such as clasto-lactacystin ß-lactone to NPA (A), derived from a papillary adenocarcinoma, and 8505C (B), derived from an anaplastic carcinoma. NPA and 8505C cells that had been cultured in 10% fetal calf serum were treated with ß-lactone (0 µM, 2.5 µM, 25 µM) for 40 h and were labeled with BrdU for 24 h before the end of treatment. The treatment of ß-lactone markedly reduced BrdU uptake of both NPA (A) and 8505C cells (B) in a dose-dependent manner. At maximal concentration (25 µM), ß-lactone significantly reduced BrdU incorporation (*, P < 0.01) compared with the control (0 µM). Data are the mean ± SEM of triplicate determinations from a representative experiment that was replicated twice with separate batches of experiments.

 
To observe the fine localization of PA28-{gamma} within these thyroid cancer cells, we used a confocal microscopy system. The cytoplasm showed a weak, fine granular staining, whereas the nucleus showed a brighter fluorescence with a coarse granular staining pattern in both NPA and 8505C cells (Fig. 7Go). This indicates that PA28-{gamma} was highly expressed, especially in the nucleus of proliferating thyroid cancer cell lines. These staining patterns resemble the PA28-{gamma} staining patterns observed in anaplastic carcinoma and papillary adenocarcinoma.



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  		Figure 7. Cytolocalization of PA28-{gamma} in NPA and 8505C cells. Differential interference contrast image of NPA (A) and 8505C (C), and immunofluorescence microscopy of NPA (B) and 8505C (D). Scale bar, 10 µm.

 
Therefore, our observations indicate that PA28-{gamma} is localized especially in the nucleus of proliferating thyroid cancer cells, and 20S proteasome appears to play a critical role in thyroid cancer cell growth.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Proteasomes are cylindrical multisubunit proteases that degrade many cellular proteins in ATP-dependent fashion. Two distinct protein complexes have been found to bind the ends of the proteasome and activate proteasome activity. One of the activators is the 19S regulatory complex of the 26S proteasome; the other activator is a protein termed PA28, also referred to as 11S regulator (31, 32). Two homologous subunits of PA28 (PA28-{alpha} and -ß) were first identified, and they are responsible for the enhancement of proteasomal processing of intracellular antigens (22). In contrast, PA28-{gamma} was originally isolated as Ki antigen, which is the target of the autoantibody appearing in patients’ serum of systemic lupus erythematosus. PA28-{alpha} and PA28-ß are located mainly in the cytoplasm and present diffusely in the nucleus, whereas PA28-{gamma} is predominantly located in the nucleus (32), indicating that PA28-{gamma} plays a distinct role from PA28-{alpha} and PA28-ß. So far, the exact physiological role of PA28-{gamma} still remains elusive, but several lines of evidence suggest that PA28-{gamma} may play a key role in cell proliferation. First, PA28-{gamma}-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was significantly retarded compared with that of PA28-{gamma}+/- or PA28-{gamma}+/+ mice. Moreover, the embryonic fibroblasts derived from PA28-{gamma} deficient mice were larger and displayed a lower saturation density than their wild-type counterparts, and flow cytometric analysis of embryonic fibroblasts revealed that the proportion of cells that entered S phase was significantly lower in PA28-{gamma}-/- cells than in wild-type cells (22). Second, we have already confirmed that growth stimulatory signals such as TSH or insulin could induce PA28-{gamma} and recruit it into the nucleus in FRTL5 thyroid cells (23). On the basis of these observations, we presumed that PA28-{gamma} could be highly expressed in various thyroid neoplasms.

In this study, we showed that PA28-{gamma} was expressed at quite high levels in papillary cancer of the thyroid by Western blot analysis (Fig. 1Go). In contrast, normal thyroid cells express PA28-{gamma} at very weak levels. And in multinodular goiter, the expression of PA28-{gamma} was lower than papillary adenocarcinoma but higher than normal thyroid. This result is compatible with previous reports showing that the key transition factors for cell cycle progression, such as PCNA, Ki-67, or cyclin, are overexpressed in various thyroid neoplasms (33, 34, 35). In particular, PCNA is mainly expressed in nucleoli and closely related to proliferative activity in papillary thyroid carcinoma (33, 36, 37). And we proved a significant correlation between PA28-{gamma} and PCNA expression by Western blot analysis (Fig. 2Go), suggesting that PA28-{gamma} may be involved in the regulatory machinery of thyroid cancer cell growth like PCNA.

Next, we analyzed the localization of PA28-{gamma} by immunohistochemistry. We observed that PA28-{gamma} was basically overexpressed in thyroid neoplasm such as papillary carcinoma and anaplastic carcinoma. And the expression of PA28-{gamma} was especially high in peripheral cells inside the cancer capsule or invading the capsular region (Fig. 3AGo). More importantly, the immunocytochemical examination revealed that PA28-{gamma} was predominantly distributed in the nucleus of cancer cells. In this region, the expression of PA28-{gamma} is especially high in the nucleus rather than in the cytoplasm with significant difference (P < 0.01; Fig. 4AGo).

The recruitment of PA28-{gamma} into the nucleus is partially driven by its nuclear localization signal (38), although the intracellular signal that translocates PA28-{gamma} into the nucleus remains unknown. The clear shift of PA28-{gamma} distribution in cancer cells led us to the idea that PA28-{gamma} may be a pivotal regulator of nuclear proteasome activity. But we still don’t have enough evidence to show that the recruitment of PA28-{gamma} up-regulates intranuclear proteasome activity and thereby contributes to the rapid degradation of a cell cycle regulator like cyclin. Indeed, Wilk et al. (39) reported that recombinant PA28-{gamma} stimulates the proteasome-mediated hydrolysis of systemic substrates and may facilitate the later stages of protein metabolism in the nucleus and/or have a more specialized role in controlling the levels of biologically active peptides in the nucleus. Moreover, in the lung cancer cell lines, Machiels et al. (16) reported that the subcellular distribution of proteasome epitopes depends on the culture condition of the cancer cells. They revealed that in cells growing under favorable conditions (1 or 2 d in fresh medium), proteasomes are detected mainly in the nuclei, and the cytoplasm is only slightly stained. In cells growing under unfavorable conditions (4- or 5-d-old medium), the staining pattern changes with a much less pronounced nuclear staining than under favorable conditions, whereas the cytoplasm remains slightly stained. This observation is quite compatible with our result showing that in papillary carcinoma, PA28-{gamma} expression was especially high in the nucleus of the cancer cells localizing at the peripheral region of cancer mass (Fig. 4Go). In general, the proliferation of cancer cells appears to be accelerated at the peripheral region of the cancer mass (40, 41, 42, 43).

Furthermore, we examined the localization of PA28-{gamma} in the two different types of thyroid cancer cell lines, NPA, derived from papillary adenocarcinoma, and 8505C, derived from anaplastic carcinoma using immunofluorescence microscopy. It clearly showed that NPA and 8505C, growing under optimal conditions, give the bright staining pattern with most of the PA28-{gamma} fluorescence localized in the nuclei, whereas the low staining pattern is localized in the cytoplasm (Fig. 7Go). These data are quite compatible with our observations about thyroid neoplasm tissue.

We also examined the expression of C2 because PA28-{gamma} requires the {alpha}-/ß-subunit components of 20S proteasome to fulfill its functional activity (44). The high expression of C2 was observed in papillary adenocarcinoma at the peripheral region of cancer mass. The tendency of the expression of PA28-{gamma} and C2 was quite similar (Fig. 6AGo), supporting our hypothesis that PA28-{gamma} could be a pivotal regulator of nuclear proteasome activity and contribute to cancer cell growth. Unlike PA28-{gamma}, the subcellular distribution of C2 in papillary cancer cells seems to be diffuse in both the nucleus and the cytoplasm of cancer cells (Fig. 5AGo, papillary adenocarcinoma). This finding is not surprising, because 20S proteasome is also responsible for the degradation of many cytoplasmic proteins (45).

In thyroid neoplasm, we could not find any reports examining proteasome expression or positive involvement of proteasome for thyroid cancer cell growth. Thus, this is the first report describing the expression of a proteasome component in thyroid carcinoma. Proteasome plays important roles in many physiological processes, such as degradation of numerous cellular proteins including regulatory enzymes and transcription factors (1, 2, 3, 4, 5, 6). Most importantly, extensive works revealed that proteasome is one of the critical regulators for cell cycle progression (46). In human cells, proteasome plays an important role in the nucleus of cells to regulate the rapid degradation of ubiquitinated cyclin-dependent kinase inhibitors such as p21cip1 and p27kip1, the degradation of which is required for G1 to S transition in the cell cycle (47). In tumors developed from other tissues, some reports have pointed out the importance of proteasome for the development and proliferation of cancer cells (17, 21). The mRNAs for two of the multiple subunits of proteasomes were expressed at abnormally high levels in most neoplastic regions of patients with various primary renal cell carcinomas and in all renal cancer cell lines, and they correlated with the proteasomal activities of these cells (17). Consistent with the increased expressions of proteasomal mRNAs, the expressions of three ubiquitin genes were found to be greatly increased in these renal cancer cells. The levels of both proteins appeared to be considerably increased in the nuclei of carcinoma cells of kidney (17). Thus, the acceleration of proteasome seems to be indispensable for cancer cell growth in other tissues. Our result of the PA28-{gamma} expression pattern is quite compatible with these observations.

The nuclear distribution of proteasome and its functional acceleration seem to be critical for cell cycle progression. So far, it is impossible to selectively disrupt the nuclear proteasome activity. Proteasome inhibitors such as MG132 or lactacystin are able to interrupt the proteasome activity of the whole cell, not the nucleus itself. There is a report that proteasome inhibitor blocked the proliferation of gastric cancer cell lines (18). We showed that proteasome inhibitor, ß-lactone, could inhibit the proliferation of human thyroid cancer cell lines such as NPA and 8505C at their physiological concentration, interrupting the proteasome activity (Fig. 6Go).

These results indicate that proteasomes within the nucleus could help to regulate thyroid cancer cell growth, although it remains unclear whether proteasomes directly affect the regulation of cell cycle regulators such as cyclin in thyroid cancer cell lines. If the proteasome within the nucleus somehow relates to cell cycle progression, the recruitment of PA28-{gamma} into the nucleus is a reasonable way to regulate the proteasome activity of preexisting proteasome within nucleus. Further study using the dominant negative form of PA28-{gamma} is necessary to isolate the PA28-{gamma} roles in thyroid cancer cell growth.

In contrast to thyroid malignant neoplasm, PA28-{gamma} was also expressed in well differentiated tumors, such as multinodular goiter. The expression of PA28-{gamma} in multinodular goiter was higher than normal thyroid, but significantly lower than papillary adenocarcinoma (Fig. 4Go, A and B). But the increased PA28-{gamma} staining of the periphery was not observed in nodules of multinodular goiter. We speculate that cells existing at the periphery of multinodular goiter may be less proliferative than those of papillary carcinoma. These results indicate that the expression of PA28-{gamma} is not cancer specific, but rather it reflects the activity of the thyroid cell proliferation.

To define new prognostic indicators in several human malignancies, including thyroid tumors, the evaluation of oncogene-encoded proteins has been used (48). The significant correlation between PCNA and PA28-{gamma} expression in this study suggests that PA28-{gamma} could be a novel prognostic marker for thyroid carcinoma. It will be valuable to predict the prognosis of patients with thyroid cancer by estimating the PA28-{gamma} expression in cancer tissue.

In summary, we found that PA28-{gamma} was highly expressed in thyroid cancer tissue by Western blot analysis. Moreover, significant correlation was observed between PCNA and PA28-{gamma} expression. These data suggest that PA28-{gamma} may be involved in the machinery of thyroid cancer cell growth. By immunohistochemistry, the expression of PA28-{gamma} was especially high in the nucleus of cancer cells localizing at the peripheral region of the cancer mass or in cancer cells invading the capsular region. The expression of C2 showed the quite similar staining pattern with PA28-{gamma}. Moreover, PA28-{gamma} is highly expressed with the nucleus of growing human thyroid cancer cell lines. The proliferation of these cells was significantly inhibited by the addition of proteasome inhibitor, ß-lactone. And the immunofluorescence studies clearly showed that NPA and 8505C cells, growing under optimal condition, give the bright staining pattern with PA28-{gamma} in the nuclei but the low staining pattern in the cytoplasm. The proteasome inhibitor ß-lactone, which is the effective inhibitor of thyroid cancer cell growth, interfered with the BrdU uptake of NPA (Fig. 6AGo) and 8505C (Fig. 6BGo) cell lines. These data support our idea that proteasomes within the nucleus could help to regulate thyroid cancer cell growth.

Our data indicate that, in thyroid cancer, PA28-{gamma} is overexpressed and distributed in the nucleus of cancer cells. We presume that PA28-{gamma} might be involved in the regulatory system for cell cycle progression of cancer cells via regulating nuclear proteasome activity. Further study should be directed toward identifying the mechanism of PA28-{gamma} overexpression in thyroid carcinoma and estimating the proteolytic activity of the nuclear proteasome assembled with PA28-{gamma}.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, and Culture (no. 12671088).

Abbreviations: BrdU, 5-Bromo-2'-deoxy-uridine; C2, {alpha}-subunit of the 20S proteasome; PCNA, proliferating cell nuclear antigen.

Received September 9, 2002.

Accepted December 11, 2002.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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