Biomedical subjects

P L Arscott

Publications and source records attributed to P L Arscott.

8 recordsLinked to original sources

PubMed2000

2-Methoxyestradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis.

The etiology of autoimmune thyroid diseases is unclear; however, the extreme female predominance suggests that sex hormones may have a pathogenic role. 2-Methoxyestradiol (2-ME) is present in the serum of women during the ovulatory and luteal phases of the menstrual cycle, and during pregnancy. We investigated the actions of 2-ME and estrogen on thyroid follicular cells. 2-ME induced dramatic changes in cell morphology and decreased the viability of the cells, as well as disrupted the structural integrity of cultured thyroid follicles. Flow cytometric analysis showed that 2-ME halted cell proliferation by arresting the cells in the G2/M cell-cycle compartment. Prolonged exposure to 2-ME led to apoptosis and to increased release of the autoantigen thyroid peroxidase (TPO). 17beta-estradiol failed to produce a similar effect even in 40-fold molar excess to 2-ME. Co-treatment with estrogen receptor antagonists did not alter the 2-ME effect, indicating that 2-ME was not operating through a classic nuclear estrogen receptor. In conclusion, this study indicates that 2-ME induces G2/M cycle arrest, apoptosis and the disruption of thyroid follicles. This process results in the release of thyroid antigens that may play a role in high incidence of thyroid autoantibodies and autoimmune thyroid disease in women.

2-Methoxyestradiol↗

PubMed1999

Inflammatory cytokine regulation of Fas-mediated apoptosis in thyroid follicular cells.

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of gamma-interferon and either tumor necrosis factor-alpha or interleukin 1beta allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.

Apoptosis↗

PubMed1999

TRAIL death pathway expression and induction in thyroid follicular cells.

To determine whether programmed cell death in thyroid follicular cells can be related to activation of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway, we examined the expression and function of this pathway in primary thyroid follicular cells and a papillary thyroid carcinoma cell line in vitro. Despite the expression of TRAIL receptors death receptor 4 and death receptor 5, purified TRAIL could not induce programmed cell death (PCD) in any of the thyroid follicular cells examined. However, pre-incubation with cycloheximide before TRAIL facilitated the induction of rapid and massive PCD. This suggested that despite the presence of a labile inhibitor of the TRAIL pathway, TRAIL could mediate PCD under appropriate conditions. To determine whether there were sources of TRAIL in the thyroid that could interact with thyroid follicular cell TRAIL receptors, RNase protection assays were used to determine TRAIL mRNA expression. TRAIL message was expressed in intrathyroidal lymphocytes isolated from a patient with thyroiditis, and unexpectedly, thyroid follicular cells themselves could be induced to express abundant TRAIL message in the presence of the inflammatory cytokines interferon gamma, tumor necrosis factor alpha, and interleukin 1beta. Furthermore, the papillary thyroid carcinoma cell line could be induced to kill the TRAIL-sensitive lymphoma cell line BJAB through a TRAIL-dependent mechanism.

Apoptosis↗

PubMed1999

Characterization of FAP-1 expression and function in thyroid follicular cells.

Human thyrocytes are resistant to Fas-mediated programmed cell death (PCD). It has been reported that a labile protein inhibitor is involved in the protection of thyrocytes from PCD, and its action can be reversed by incubation of thyrocytes with cycloheximide (CHX) during treatment with agonist anti-Fas Ab. Fas-associated phosphatase-1 (FAP-1) is a protein that has been shown to interact with the negative regulatory domain of Fas and block Fas-mediated apoptosis in FAP-1 transfected Jurkat cells. We investigated the possibility that FAP-1 might be involved in protection against Fas-mediated PCD in human thyrocytes. FAP-1 mRNA was detected in primary thyrocytes using a ribonuclease protection assay. The presence of FAP-1 protein was confirmed by immunohistochemical staining and flow cytometry using a polyclonal anti-FAP-1 Ab. FAP-1 protein also disappeared from thyroid cells in response to CHX. To determine whether FAP-1 is a functional inhibitor of PCD in thyrocytes, we incubated thyrocytes with synthetic SLV (Ac-SLV) tripeptide to compete with Fas for interaction with FAP-1. Thyrocytes treated with Ac-SLV tripeptide showed significantly increased cell death as compared to cells treated with control tripeptide. In addition, in the presence of a suboptimal concentration of CHX, the Ac-SLV tripeptide yielded a strong, synergistic increase in Fas-mediated PCD as compared to thyrocytes treated with control tripeptide. These results implicate FAP-1 as a regulator of Fas-induced PCD in thyrocytes.

Apoptosis↗

PubMed1999

Fas (CD95) expression is up-regulated on papillary thyroid carcinoma.

Thyrocyte apoptosis signaled through the Fas receptor has been proposed as a mechanism for the cytotoxicity observed in thyroiditis, but the role the Fas pathway plays in thyroid cancer is not known. We examined Fas expression in thyroid tissue derived from patients with papillary carcinoma and follicular cancer. More intense immunohistological staining for the Fas protein was observed on papillary cancer cells as compared with adjacent normal follicles. To further characterize the expression of Fas in papillary cancer, paired normal and cancerous thyroid tissues were obtained at thyroidectomy from several donors, digested, and placed into cell culture. Messenger RNA was analyzed by ribonuclease protection assays, and protein was identified by flow cytometry. Fas expression was detected at levels up to 3-fold higher in cancerous thyrocytes compared with paired normal cells. To determine whether the expressed Fas antigen was functional, thyrocytes were treated with a monoclonal IgM anti-Fas antibody (clone CH11; Upstate Biotechnology, Inc., Lake Placid, NY) in the presence of interferon-gamma 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.

Adenocarcinoma, Follicular↗

PubMed1998

Apoptosis and thyroiditis.

The origin of the various forms of autoimmune thyroiditis remains unclear. Most investigations into the pathogenesis of these disorders have focused on immune abnormalities that might lead to an autoimmune response. However, no unique immune response to thyroid autoantigens has been identified that either is limited to patients with thyroiditis or is absolutely correlated with clinical disease expression. CD8 T-cell-mediated cytotoxicity is thought to be a major cause of thyroid follicular cell damage in thyroiditis. This damage is produced in part through the induction of apoptosis in thyroid cells. Recent studies have demonstrated that programmed cell death is regulated in thyroid cells and that a major pathway for immune-mediated apoptosis, the Fas pathway, is blocked by labile inhibitors in a manner that could prevent cytotoxicity. This review also examines several other types of regulation of apoptotic pathways in thyrocytes. We hypothesize that the regulation of programmed cell death pathways in the thyroid may alter the expression of autoimmune thyroid diseases by modifying the susceptibility of thyroid cells to immune-mediated apoptosis.

Apoptosis↗

PubMed1997

Fas (APO-1, CD95)-mediated apoptosis in thyroid cells is regulated by a labile protein inhibitor.

To determine whether thyroid cell apoptosis observed in autoimmune thyroid disease could be related to activation of the Fas pathway, we examined the expression and function of Fas on thyroid follicular cells in vitro. Fas messenger RNA was found to be present using two different techniques and was expressed at equal levels in thyrocytes cultured either in the presence or absence of TSH. Fas antigen protein expression was demonstrated by Western blot of thyroid cell lysates and by immunohistochemical staining of thyrocytes, and the amount of Fas protein present did not appear to vary regardless of culture conditions. Despite expressing substantial amounts of Fas protein, thyrocytes treated with anti-Fas monoclonal antibody failed to undergo apoptosis. The addition of either interferon-gamma or interleukin-1beta to the anti-Fas-treated cell cultures also did not promote apoptotic signaling through this pathway. In contrast, the concomitant administration of cycloheximide allowed the induction of apoptosis through the activation of Fas in thyrocytes. These results suggest that Fas is constitutively expressed in thyrocytes, but that the induction of apoptosis through the Fas pathway is blocked by a labile protein inhibitor.

Antibodies, Monoclonal↗

PubMed1996

Unique autoantibody epitopes in an immunodominant region of thyroid peroxidase.

To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.

Adolescent↗