| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
BRIEF REPORT |
Functional Genomic Unit (G.C., M.M., E.V., R.P., C.A., L.P.), National Cancer Institute, Fondazione G. Pascale, 80131 Naples, Italy; Department of Surgical and Oncological Sciences (M.Z., M.T., G.S.), University of Palermo, 90100 Palermo, Italy; and Department of Pharmaceutical Sciences (M.A., A.B., A.R., M.F., A.G., A.T., M.P., L.M., M.A.B., M.C.T., A.L.), University of Salerno, 84084 Fisciano, Italy
Address all correspondence and requests for reprints to: Maria Caterina Turco, M.D., Ph.D., Dipartimento di Scienze Farmaceutiche (DiFarma), University of Salerno, Via ponte don Melillo, 84084 Fisciano, Italy. E-mail: mcturco{at}unisa.it.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Objective: The objective of this study was to analyze BAG3 expression in thyroid neoplastic cells and investigate its influence in cell apoptotic response to TNF-related apoptosis-inducing ligand (TRAIL).
Design, Setting, and Patients: We investigated BAG3 expression in human thyroid carcinoma cell lines, including NPA, and the effect of BAG3-specific small interfering RNA on TRAIL-induced apoptosis in NPA cells. Subsequently, we analyzed BAG3 expression in 30 benign lesions and 56 carcinomas from patients of the Naples Tumor Institute Fondazione Senatore Pascale.
Main Outcome Measures: The main outcome measures were: analysis of BAG3 protein in NPA cells by Western blot and immunocytochemistry; analysis of apoptosis in TRAIL-stimulated NPA cells by flow cytometry; and evaluation of BAG3 expression in specimens from thyroid lesions by immunohistochemistry.
Results: BAG3 was expressed in human thyroid carcinoma cell lines; small interfering RNA-mediated downmodulation of its levels significantly (P < 0.0195) enhanced NPA cell apoptotic response to TRAIL. The protein was not detectable in 19 of 20 specimens of normal thyroid or goiters, whereas 54 of 56 analyzed carcinomas (15 follicular, 28 papillary, and 13 anaplastic) were clearly positive for BAG3 expression.
Conclusions: BAG3 downmodulates the apoptotic response to TRAIL in human neoplastic thyroid cells. The protein is specifically expressed in thyroid carcinomas and not in normal thyroid tissue or goiter.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Proteins that share the BAG (Bcl-2-associated athanogene) domain bind the ATPase domain of the heat shock proteins (Hsp) and modulate their chaperone activity in protein folding (10). The first identified member of this family, BAG1, is expressed in some tumors, particularly breast carcinomas, where it is implicated in maintaining cell growth (10). The BAG family includes BAG3 protein, also known as CAIR-1 or Bis, expressed in muscle, heart, brain, and other tissues (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). In some cell types, BAG3 expression can be induced by heat shock (17) or cell transformation (13). We recently showed that BAG3 expression is induced in human leukocytes by oxidative stress and the increase of its levels results in reducing stress-induced apoptosis (20). Furthermore, we found that BAG3 was expressed in B chronic lymphocytic (18) and acute lymphoblastic (19) leukemia cells and antagonized cell response to chemotherapeutic compounds (18, 19). BAG3 therefore appeared to exert an antiapoptotic activity in some cell types. The mechanism of apoptosis regulation relies on BAG3 interaction with Hsp70 (18, 19, 20), a chaperone able to interfere with more than one apoptotic event, including apoptosome assembly, caspase-mediated deoxyribonuclease activation, and degradation of the antiapoptotic kinase Akt (24). The expression of BAG3 in thyroid tumors and its influence on thyroid cell apoptosis had not been investigated so far. Furthermore, BAG3 ability to modulate TRAIL-induced apoptosis was not previously verified in any cell type.
We analyzed whether BAG3 was detectable in human thyroid tumor cell lines and influenced TRAIL-induced apoptosis. Results prompted us to investigate BAG3 expression in thyroid malignant vs. benign lesions.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
ARO, FRO, WRO and NPA cells (American Type Culture Collection, Manassas, VA) were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UK) in 5% CO2 humid atmosphere.
Western blotting
For Western blot analysis, 50 µg of protein was run on 12% SDS-PAGE gels and transferred to nitrocellulose. Nitrocellulose blots were blocked with 5% BSA in Tris buffer saline Tween-20 buffer [20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 0.01% Tween 20] and incubated with primary anti-BAG3 (10, 11, 12) (Alexis Biochemicals, San Diego, CA) or anti-
-tubulin (Sigma, St. Louis, MO) antibody in Tris buffer saline Tween-20 5% BSA overnight at 4 C. Immunoreactivity was detected by sequential incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Pittsburgh, PA) and enhanced chemiluminescence reagents (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL) following standard protocols.
BAG3 small interfering RNA (siRNA) construction
siRNAs with two thymidine residues (dTdT) at the 3' end of the sequence were designed for bag3 gene (5'-AAGGUUCAGACCAUCUUGGAAdTdT-3') or unspecific, scrambled (5'-CAGUCGCGUUUGCGACUGGdTdT-3') sequences (Dharmacon Research Inc., Lafayette, CO). RNAs were dissolved in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) as 200 µM solutions and annealed at room temperature after heating to 95 C in buffer (30 mM HEPES-KOH, pH 7.9, 100 mM potassium acetate, 2 mM magnesium acetate). NPA cells were transfected with bag3-specific or control-scrambled siRNA. For cell transfection, approximately 1 x 106 cells were plated in six-well plates, in media containing 10% fetal bovine serum, to give 30–50% confluency. The cells were transfected with a final siRNA concentration of 20 nM using oligofectamine (Invitrogen, Carlsbad, CA). After 72 h, cell total protein lysates were prepared in sample buffer (2% sodium dodecyl sulfate, 10% glycerol, 2% mercaptoethanol, and 60 mM Tris-HCl, pH 6.8, in demineralized water) on ice. Efficiency of transfection was evaluated by Western blot analysis.
Apoptosis evaluation
Cells transfected with bag3-specific or control scrambled siRNA were incubated with or without recombinant TRAIL (human recombinant Superkiller TRAIL; Alexis Biochemicals). At the indicated time intervals, apoptosis was evaluated by analyzing Annexin V/fluorescein isothiocyanate (Bender Medsystem, Vienna, Austria) binding and propidium iodide incorporation (25) using a Becton Dickinson FACScan flow cytometer. Moreover, caspase 8 activity was evaluated by flow cytometry using a carboxyfluorescein FLICA Assay Kit (B-Bridge International, San Jose, CA). Statistical analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA; www.graphpad.com).
Immunohistochemistry
Specimens from normal and pathological human thyroid tissues were analyzed as previously described (26). Briefly, the specimens were isolated, rinsed with PBS, fixed in 4% buffered neutral formalin, and embedded in paraffin. Five- to 6-µm-thick paraffin sections were then deparaffinized, placed in a solution of absolute methanol and 0.3% hydrogen peroxide for 30 min, and then washed in PBS before immunoperoxidase staining. Slides were then incubated overnight at 4 C in a humidified chamber with saturating amounts of rabbit polyclonal anti-BAG3 antibody (10, 11, 12) (Alexis Biochemicals) and subsequently with biotinylated goat antirabbit IgG (Dako A/S, Glostrup, Denmark) for 20 min, followed by streptavidin horseradish peroxidase for 20 min (LSAB2 System; Dako). Negative controls were performed by omitting the primary antibody or substituting with rabbit control Ig (data not shown). After incubation in a solution containing 0.06 mM diaminobenzidine (Dako) and 2 mM hydrogen peroxide in 0.05% PBS (pH 7.6) for 5 min, slides were washed, dehydrated with alcohol and xylene, and mounted with coverslips using a permanent mounting medium (Permount-Proscitech, Kirwan, Australia).
NPA cells were fixed in 4% buffered neutral formalin, washed in PBS, and incubated overnight at 4 C in a humidified chamber with preimmune serum or polyclonal anti-BAG3 antibody (10, 11, 12) (Alexis Biochemicals) (1:100 in PBS). Slides were then incubated with biotinylated goat antirabbit IgG for 20 min and subsequently with streptavidin horseradish peroxidase for 20 min. After incubation in a solution containing 0.06 mM diaminobenzidine (Dako) and 2 mM hydrogen peroxide in 0.05% PBS (pH 7.6) for 5 min, slides were washed, counterstained with hematoxylin for 1 min, and incubated in water for 10 min. Slides were then washed, dehydrated with alcohol and xylene, and mounted with coverslips using a permanent mounting medium.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
). To investigate the antiapoptotic property of BAG3 in these cells, we analyzed the effect of BAG3-specific siRNAs in cells of the human papillary carcinoma NPA line. Cells transfected with BAG3-specific, but not a scrambled, siRNA displayed reduced levels of BAG3 protein in immunoblotting of total cell protein lysates. A siRNA concentration of 200 nM appeared optimal (Fig. 1A) and was used in the subsequent experiments. The cells were incubated with medium either with or without recombinant TRAIL and, at the indicated time intervals, cell apoptosis was evaluated. Cell apoptotic response to TRAIL was significantly (P < 0.0195) enhanced in cultures treated with BAG3-specific, but not control scrambled, siRNA (Fig. 1Ba). Accordingly, caspase 8 activation, an event involved in TRAIL-triggered apoptosis pathway (3), was amplified in bag3 siRNA-treated cells (Fig. 1Bb). These findings prompted us to investigate the expression of BAG3 in thyroid tumors.
|
). Then, we analyzed a number of normal and pathological human thyroid specimens in immunohistochemistry. The proportion of positively stained cells was scored from 0 to 4 (0, no positive cells; 1+, <10% of positive cells; 2+, 11–50% of positive cells; 3+, 51–75% of positive cells; 4+, 75–100% of positive cells). To evaluate the statistical significance of the difference between benign and malignant tissues, values of 0.1, 1, 2, 3, and 4 were applied to lesions scored with 0, 1+, 2+, 3+, and 4+, respectively; mean values and SE values were calculated for the two groups, and the Student’s t test was applied. Normal thyroid and nodular goiter specimens were negative for BAG3 expression (only 1 of 15 nodular goiters showed a very weak staining with anti-BAG3 antibody); 8 of 10 analyzed follicular adenomas showed a weak positivity (<10% positive cells). However, 54 of 56 analyzed carcinoma specimens were clearly positive for BAG3 expression (Fig. 2B). Furthermore, although immunohistochemistry did not allow a quantitative evaluation, a stronger intensity of staining was generally observed in the undifferentiated (anaplastic) thyroid tumors, compared with well-differentiated carcinomas. Representative images of the analyzed cases are shown in Fig. 2C.
|
Investigation of BAG3 expression and activity in thyroid carcinomas could contribute to our understanding of the pathogenesis and sensitivity to therapy in these tumors. More in general, the involvement of BAG3 in the phenotypic and functional assets of thyroid carcinomas lends support to a valuation of BAG co-chaperone proteins as elements involved in neoplastic processes (10, 13, 18, 19).
|
Footnotes |
|---|
Abbreviations: BAG, Bcl-2-associated athanogene; Hsp, heat shock protein; siRNA, small interfering RNA; TRAIL, TNF-related apoptosis-inducing ligand.
Received August 8, 2006.
Accepted December 4, 2006.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
sensitize human thyroid epithelial cells to TNF-related apoptosis-inducing ligand-induced apoptosis through increases in procaspase-7 and bid, and the down-regulation of p44/42 mitogen-activated protein kinase activity. J Clin Endocrinol Metab 89:250–257
sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression. Oncogene 23:928–935[CrossRef][Medline] and Hsp70/Hsc70. Oncogene 19:4385–4395[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
