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TRAIL preferentially induces apoptosis in tumor cells and virus-infected cells. Unlike other tumor necrosis factor family members, TRAIL does not kill cells from most normal tissues and has thus been proposed as a promising new cancer treatment. Our study demonstrated that IFNgamma combined with TRAIL can trigger apoptosis in vitro in several resistant thyroid tumor cell lines, such as thyroid anaplastic carcinoma cells (ARO cells), while either agent alone exerts only a minimal effect. We further tested this effect on a mouse thyroid tumor model, when in vivo tumor growth was also significantly inhibited by this combination. The mechanism of how IFNgamma sensitized thyroid carcinoma cells to TRAIL-induced apoptosis was investigated by screening global gene alterations in ARO cells treated with IFNgamma. Microarray data revealed that a proapoptotic gene, Bak, is markedly upregulated by IFNgamma, and this was confirmed by RNase protection assay. Western blot analysis also showed a significant increase in Bak at the protein level. Upregulation of Bak and sensitization for apoptosis by IFNgamma was blocked by overexpression of antisense Bak in ARO cells. Furthermore, overexpression of Bak sensitized ARO cell to TRAIL-induced apoptosis without the need for IFNgamma pretreatment. This suggests that Bak is a regulatory molecule involved in IFNgamma-facilitated TRAIL-mediated apoptosis in thyroid cancer cells.

Exosomes are 50-90 nm vesicles with antigen presenting ability carrying major histocompatibility complex (MHC) class I, class II, abundant co-stimulatory molecules and some tetraspan proteins. Although dendritic cells (DCs) are one of the professional antigen presenting cells capable of presenting exogenous antigens in MHC class I-mediated antigen specific manner (cross-presentation), the cross-presentation ability by exosomes from immature or mature DCs are unknown. Here we show that exosomes released from ovalbumin (OVA) protein-pulsed bone marrow derived dendritic cells (BM-DCs) weakly present the peptide determinants to OVA specific MHC class I-restricted CD8(+) T cell hybridomas. The exosomes secreted by OVA(257-264) peptide- or OVA protein-pulsed mature BM-DCs activated OVA specific MHC class I-restricted T cell hybridomas more efficiently than those from immature BM-DCs. Transporters associated with antigen processing (TAP) deficient mice-derived BM-DCs were also used to examine whether functional TAP activity was required for cross-presentation by exosomes. The exosomes obtained from OVA(257-264) peptide-pulsed BM-DCs derived from TAP(-/-) mice showed a significant antigen presenting ability to OVA specific MHC class I-restricted T cell hybridomas. Altogether, our data indicate that BM-DCs secrete exosomes with weak cross-presentation ability.

The specific pathogenesis of nodular goiter and the role of apoptosis in goitrogenesis are not known. We sought to examine the regulation of the TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL)-induced apoptosis pathways in primary thyroid cells from 17 patients with nodular goiter, using 10 normal thyroids as controls. Both goitrous and normal thyroid cells were resistant to recombinant human TRAIL and an agonist anti-Fas antibody under basal conditions. However, all normal thyrocytes could be sensitized by TNF/IL-1 or interferon /IL-1 to undergo apoptosis in response to TRAIL or FasL, respectively. In contrast, the majority of goiter-derived cells remained resistant to TRAIL (12 of 17 samples) or FasL (9 of 17 samples) after cytokine pretreatment; 14 of 17 goiter nodules were resistant to at least one death ligand. Goiter size was inversely correlated with the sensitivity to TRAIL-mediated apoptosis. The resistance of goiter cells to TRAIL did not appear to be due to transcriptional regulation or cell surface expression of death and decoy receptors. However, increased proteasome activity was found in a subset of goiter cells resistant to both death ligands, and proteasome inhibitors could sensitize these goiter cells to TRAIL-mediated apoptosis. In conclusion, goiter-derived thyroid cells are resistant to TRAIL and/or Fas-induced apoptosis in vitro, and this may represent a new aspect of aberrant growth regulation in goiter nodules. The increased proteasome activity associated with this resistance suggests that the proteasome may be an important regulator of apoptosis in nodular goiter. (J Clin Endocrinol Metab 87: 4264 – 4272, 2002)

Treatment of cultured primary human thyroid cells with IFN-γ and TNF-α uniquely allows the induction of Fas-mediated apoptosis. To investigate the role of this cytokine combination in vivo, CBA/J mice were immunized with thyroglobulin and then injected with IFN-γ and TNF-α. Compared with control animals, mice treated with IFN-γ and TNF-α showed significantly sustained lymphocytic infiltration in the thyroid, which was associated with the destruction of portions of the follicular architecture at wk 6 after initial immunization. Furthermore, the number of apoptotic thyroid follicular cells was increased only in the thyroids from mice treated with the IFN-γ and TNF-α. We also analyzed the function of the Fas pathway in vivo in cytokine-treated mice by using an agonist anti-Fas Ab injected directly into the thyroid. Minimal apoptosis of thyroid epithelial cells was observed unless the mice were pretreated with IFN-γ and TNF-α. These data demonstrate that this unique combination of inflammatory cytokines facilitates the apoptotic destruction of thyroid follicular cells in experimental autoimmune thyroiditis, in a manner similar to what is observed in Hashimoto’s thyroiditis in humans.

7-Hydroxystaurosporine (UCN-01) is a selective protein kinase C (PKC) inhibitor and is being developed as a novel anticancer agent. Because of reports that PKC may be involved in the pathogenesis of some forms of thyroid cancers, we examined four thyroid carcinoma lines (FRO, KAT5, NPA, and WRO). These cells were found to have different susceptibility to UCN-01 treatment, and there appeared to be a correlation between UCN-01-induced death and expression levels of endogenous Bcl-2. KAT5 cells, which normally express a low amount of Bcl-2, exhibited significantly higher sensitivity to UCN-01-induced death than the other cell lines. Of interest, susceptibility did not relate to PKC activity or its inhibition by UCN-01. In order to investigate the role of Bcl-2 in UCN-01-induced death, KAT5 cells were transfected to overexpress Bcl-2. KAT5/Bcl-2 cells were capable of conferring resistance to UCN-01-induced death. Furthermore, upregulating of Bcl-2 by 1alpha,25-dihydroxyvitamin D3 (VD3) could protect primary thyroid cell from death induced by UCN-01. Both in situ TUNEL staining and the flow cytometric analysis of cytokeratin-18 (CK18) cleavage confirmed that UCN-01 was indeed inducing apoptosis, and that this effect was inhibited by increased expression of Bcl-2. These results suggest that the Bcl-2 can block the UCN-01-activated cell death pathway and that the expression of Bcl-2 is inversely related to thyroid carcinoma cell susceptibility to UCN-01. Therefore, the analysis of the expression of apoptosis suppressors provides a basis for the use of UCN-01 in the treatment of thyroid cancer.

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto’s thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-γ and IL-1β cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-γ/IL-1β pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.

Primary thyroid cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL). Previously we showed that the combination of IL-1beta and TNFalpha facilitated TRAIL-mediated apoptosis in these cells and enhanced cell surface expression of TRAIL receptors. The aim of this study was to further characterize the mechanism by which these cytokines sensitized primary thyroid cells to TRAIL-mediated apoptosis. IL-1beta and TNFalpha increased the concentrations of procaspase-7 and Bid. In contrast, the p44/42 MAPK (Erk) pathway was active in thyroid cells and this activity was significantly decreased after exposure to IL-1beta/TNFalpha. A MAPK kinase inhibitor (U0126) could enhance the cytokine-induced sensitization of thyroid cells to TRAIL, reinforcing the inhibitory role of Erk on TRAIL signaling. In conclusion, IL-1beta/TNFalpha treatment sensitizes human thyroid cells to TRAIL-mediated apoptosis through increased surface expression of TRAIL receptors, increased expression of procaspase-7 and Bid, and the inhibition of p44/42 MAPK (Erk) pathway.