Francisco Barreiro

TGF-β-induced apoptosis in human thyrocytes is mediated by p27kip1 reduction and is overridden in neoplastic thyrocytes by NF-κB activation

Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low proliferative index, low apoptotic index and a high incidence of metastasis. TGF-β is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-β in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Graves disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-β induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-β induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apoptosis in the absence of TGF-β. By contrast, in cells in which p27 was overexpressed, TGF-β had a survival effect. In growth medium, a net survival effect occurs in neoplastic thyrocytes only, not normal thyrocytes, due to activation of the NF-κB survival program. Together, these findings suggest that (a) thyroid neoplasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-β induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-κB program.

GSM radiation triggers seizures and increases cerebral c-Fos positivity in rats pretreated with subconvulsive doses of picrotoxin

This study investigated the effects of mobile-phone-type radiation on the cerebral activity of seizure-prone animals. When rats transformed into an experimental model of seizure-proneness by acute subconvulsive doses of picrotoxin were exposed to 2 h GSM-modulated 900 MHz radiation at an intensity similar to that emitted by mobile phones, they suffered seizures and the levels of the neuronal activity marker c-Fos in neocortex, paleocortex, hippocampus and thalamus increased markedly. Non-irradiated picrotoxin-treated rats did not suffer seizures, and their cerebral c-Fos counts were significantly lower. Radiation caused no such differences in rats that had not been pretreated with picrotoxin. We conclude that GSM-type radiation can induce seizures in rats following their facilitation by subconvulsive doses of picrotoxin, and that research should be pursued into the possibility that this kind of radiation may similarly affect brain function in human subjects with epileptic disorders.

Plitidepsin Has a Cytostatic Effect in Human Undifferentiated (Anaplastic) Thyroid Carcinoma

Undifferentiated (anaplastic) thyroid carcinoma is a highly aggressive human cancer with very poor prognosis. Although there have been a few studies of candidate treatments, the fact that it is an infrequent tumor makes it very difficult to design clinical trials. A strong association has been observed between undifferentiated thyroid carcinoma and TP53 mutations in numerous molecular genetic and expression studies. Plitidepsin (Aplidin, PharmaMar, Madrid, Spain) is a novel anticancer compound obtained from a sea tunicate. This compound has been reported to induce apoptosis independently of TP53 status. We investigated the actions of plitidepsin in human thyroid cancer cells. In initial experiments using primary cultured cells from a differentiated (papillary) carcinoma, we found that 100 nmol/L plitidepsin induced apoptosis, whereas lower doses were cytostatic. Because our aim was to study the effects of plitidepsin at clinically relevant concentrations, subsequent experiments were done with a dosage regimen reflecting plasma concentrations observed in previously reported clinical trials: 100 nmol/L for 4 hours, followed by 10 nmol/L for 20 hours (4100/2010 plitidepsin). This plitidepsin dosage regimen blocked the proliferation of a primary undifferentiated/anaplastic thyroid carcinoma culture obtained in our laboratory and of a commercial cell line (8305C) obtained from an undifferentiated thyroid carcinoma; however, it did not induce apoptosis. The proportion of cells in the G1 phase of the cell cycle was greatly increased and the proportion in the S/G2-M phases greatly reduced, suggesting that plitidepsin blocks G1-to-S transition. Levels of the cyclin D1/cyclin-dependent kinase 4/p21 complex proteins were decreased and, in line with this, the levels of unphosphorylated Rb1 increased. The decrease in cell cycle proteins correlated with hypoacetylation of histone H3. Finally, we did experiments to assess how rapidly tumor cells return to their initial pretreatment proliferative behavior after 4100/2010 plitidepsin treatment. Cells from undifferentiated tumors needed more than 3 days to recover logarithmic growth, and after 7 days, cell number was still significantly lower than in control cultures. 4100/2010 plitidepsin inhibited the growth in soft agar. Together, our data show that plitidepsin is able to block in vitro cell cycle progression at concentrations similar to serum concentrations observed in vivo, and that this effect is persistent for several days after plitidepsin removal. Whether plitidepsin will prove to be clinically useful in the treatment of undifferentiated thyroid cancers remains to be established. However, our results raise the possibility that plitidepsin might be effective alone or in combination with radiotherapy and/or other drug treatments.

Electromagnetic fields at 2.45 GHz trigger changes in heat shock proteins 90 and 70 without altering apoptotic activity in rat thyroid gland

Summary Non-ionizing radiation at 2.45 GHz may modify the expression of genes that codify heat shock proteins (HSP) in the thyroid gland. Using the enzyme-linked immunosorbent assay (ELISA) technique, we studied levels of HSP-90 and HSP-70. We also used hematoxilin eosin to look for evidence of lesions in the gland and applied the DAPI technique of fluorescence to search for evidence of chromatin condensation and nuclear fragmentation in the thyroid cells of adult female Sprague-Dawley rats. Fifty-four rats were individually exposed for 30 min to 2.45 GHz radiation in a Gigahertz transverse electromagnetic (GTEM) cell at different levels of non-thermal specific absorption rate (SAR), which was calculated using the finite difference time domain (FDTD) technique. Ninety minutes after radiation, HSP-90 and HSP-70 had decreased significantly (P<0.01) after applying a SAR of 0.046±1.10 W/Kg or 0.104±5.10−3 W/Kg. Twenty-four hours after radiation, HSP-90 had partially recovered and HSP-70 had recovered completely. There were few indications of lesions in the glandular structure and signs of apoptosis were negative in all radiated animals. The results suggest that acute sub-thermal radiation at 2.45 GHz may alter levels of cellular stress in rat thyroid gland without initially altering their anti-apoptotic capacity.

Humanized Medium (h7H) Allows Long-Term Primary Follicular Thyroid Cultures From Human Normal Thyroid, Benign Neoplasm, and Cancer

CONTEXT Mechanisms of thyroid physiology and cancer are principally studied in follicular cell lines. However, human thyroid cancer lines were found to be heavily contaminated by other sources, and only one supposedly normal-thyroid cell line, immortalized with SV40 antigen, is available. In primary culture, human follicular cultures lose their phenotype after passage. We hypothesized that the loss of the thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture, including hormones with marked differences in its affinity for the relevant rodent/human receptor. OBJECTIVE The objective of the study was to define conditions that allow the proliferation of primary human follicular thyrocytes for many passages without losing phenotype. METHODS Concentrations of hormones, transferrin, iodine, oligoelements, antioxidants, metabolites, and ethanol were adjusted within normal homeostatic human serum ranges. Single cultures were identified by short tandem repeats. Human-rodent interspecies contamination was assessed. RESULTS We defined an humanized 7 homeostatic additives medium enabling growth of human thyroid cultures for more than 20 passages maintaining thyrocyte phenotype. Thyrocytes proliferated and were grouped as follicle-like structures; expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin, and thyroperoxidase showed iodine-uptake and secreted thyroglobulin and free T3. Using these conditions, we generated a bank of thyroid tumors in culture from normal thyroids, Graves hyperplasias, benign neoplasms (goiter, adenomas), and carcinomas. CONCLUSIONS Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The bank of thyroid tumors in culture generated under humanized humanized 7 homeostatic additives culture conditions will provide a much-needed tool to compare similarly growing cells from normal vs pathological origins and thus to elucidate the molecular basis of thyroid disease.

Expression of exogenous proteins and short hairpin RNAs in human primary thyrocytes

Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma, or melanoma. Others have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumors from patients. Thus, it is important to use several primary cells with a common pathological origin to achieve reproducible results, and it is necessary to find common methods for manipulation of protein expression in such various cultures. We have standardized a transfection method for efficient expression of exogenous proteins in human primary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Electroporator PulseAgile [PA]-4000, Microporator MP-100, and Nucleofector II). Nucleofection was unquestionably the most efficient even for promoter regulation studies, and it was effective in cultures from different origins as normal thyroid, papillary carcinoma, or lymphoid node metastasis. We also standardized, through lentiviral infection, the short hairpin RNA downregulation of protein expression generating human thyrocytes with low levels of p27KIP1 as a model system.

Rewiring of the apoptotic TGF-β-SMAD/NFκB pathway through an oncogenic function of p27 in human papillary thyroid cancer

Papillary thyroid carcinoma (PTC), the most frequent thyroid cancer, is characterized by low proliferation but no apoptosis, presenting frequent lymph-node metastasis. Papillary thyroid carcinoma overexpress transforming growth factor-beta (TGF-β). In human cells, TGF-β has two opposing actions: antitumoral through pro-apoptotic and cytostatic activities, and pro-tumoral promoting growth and metastasis. The switch converting TGF-β from a tumor-suppressor to tumor-promoter has not been identified. In the current study, we have quantified a parallel upregulation of TGF-β and nuclear p27, a CDK2 inhibitor, in samples from PTC. We established primary cultures from follicular epithelium in human homeostatic conditions (h7H medium). TGF-β-dependent cytostasis occurred in normal and cancer cells through p15/CDKN2B induction. However, TGF-β induced apoptosis in normal and benign but not in carcinoma cultures. In normal thyroid cells, TGF-β/SMAD repressed the p27/CDKN1B gene, activating CDK2-dependent SMAD3 phosphorylation to induce p50 NFκB-dependent BAX upregulation and apoptosis. In thyroid cancer cells, oncogene activation prevented TGF-β/SMAD-dependent p27 repression, and CDK2/SMAD3 phosphorylation, leading to p65 NFκB upregulation which repressed BAX, induced cyclin D1 and promoted TGF-β-dependent growth. In PTC samples from patients, upregulation of TGF-β, p27, p65 and cyclin D1 mRNA were significantly correlated, while the expression of the isoform BAX-β, exclusively transcribed in apoptotic cells, was negatively correlated. Additionally, combined ERK and p65 NFκB inhibitors reduced p27 expression and potentiated apoptosis in thyroid cancer cells while not affecting survival in normal thyroid cells. Our results therefore suggest that the oncoprotein p27 reorganizes the effects of TGF-β in thyroid cancer, explaining the slow proliferation but lack of apoptosis and metastatic behavior of PTC.