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Susana B Bravo,
Maria E R Garcia-Rendueles,
Angela R Garcia-Rendueles,
Joana S Rodrigues,
Sihara Perez-Romero,
Montserrat Garcia-Lavandeira,
Maria Suarez-Fariña,
Francisco Barreiro,
Barbara Czarnocka,
Ana Senra,
Maria V Lareu,
Javier Rodriguez-Garcia,
Jose Cameselle-Teijeiro,
Clara V Alvarez
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Susana B Bravo,
Maria E R Garcia-Rendueles,
Angela R Garcia-Rendueles,
Joana S Rodrigues,
Sihara Perez-Romero,
Montserrat Garcia-Lavandeira,
Maria Suarez-Fariña,
Francisco Barreiro,
Barbara Czarnocka,
Ana Senra,
Maria V Lareu,
Javier Rodriguez-Garcia,
Jose Cameselle-Teijeiro,
Clara V Alvarez
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Susana B Bravo,
Maria E R Garcia-Rendueles,
Angela R Garcia-Rendueles,
Joana S Rodrigues,
Sihara Perez-Romero,
Montserrat Garcia-Lavandeira,
Maria Suarez-Fariña,
Francisco Barreiro,
Barbara Czarnocka,
Ana Senra,
Maria V Lareu,
Javier Rodriguez-Garcia,
Jose Cameselle-Teijeiro,
Clara V Alvarez
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Susana B Bravo,
Maria E R Garcia-Rendueles,
Angela R Garcia-Rendueles,
Joana S Rodrigues,
Sihara Perez-Romero,
Montserrat Garcia-Lavandeira,
Maria Suarez-Fariña,
Francisco Barreiro,
Barbara Czarnocka,
Ana Senra,
Maria V Lareu,
Javier Rodriguez-Garcia,
Jose Cameselle-Teijeiro,
Clara V Alvarez
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ABSTRACT: 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 loss of thyroid phenotype could be related to culture conditions in which human cells are grown in medium optimized for rodent culture (5H), including hormones with marked differences in affinity for the relevant rodent/human receptor.Objective:To define conditions which allows 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 STRs. Human-rodent inter-species contamination was assessed.Results:We defined an 'h7H medium' enabling growth of human thyroid cultures for more than twenty passages maintaining thyrocyte phenotype. Thyrocytes proliferated and grouped as follicle-like structures (FLS); expressed Na+/I- symporter, pendrin, cytokeratins, thyroglobulin and thyroperoxidase, showed iodine-uptake and secreted thyroglobulin and FT3. Using these conditions, we generated a Bank of Thyroid Tumors in Culture (BANTTIC) from normal thyroids, Grave's hyperplasias, benign neoplasms (goiter, adenomas) and carcinomas.Conclusions:Using appropriate culture conditions is essential for phenotype maintenance in human thyrocytes. The BANTTIC generated under humanized h7H culture conditions will provide a much needed tool to compare similarly growing cells from normal versus pathological origins, and thus to elucidate molecular basis of thyroid disease.
The Journal of clinical endocrinology and metabolism 03/2013; · 6.50 Impact Factor
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Maria F Garces,
Elizabeth Sanchez,
Ariel I Ruíz-Parra,
Jorge Andrés Rubio-Romero,
Edith Angel-Müller,
Miguel A Suarez,
Luisa F Bohórquez, Susana B Bravo,
Rubén Nogueiras,
Carlos Diéguez,
Jorge E Caminos
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ABSTRACT: During gestation there are important changes in maternal metabolism and an increase in insulin resistance, coinciding with an increase in adiposity. Chemerin is an adipocytokine which is expressed and secreted in various tissues, including placenta, and may play an important role in metabolic regulation during pregnancy. The aim of this study was to determine serum levels of chemerin during gestation and compare them to other indicators of insulin resistance. A cross-sectional study was carried out analyzing serum chemerin levels of 20 pregnant women during three gestational periods, early, middle, and late (between the 10th-14th, the 23rd-26th, and the 34th-37th week) and 20 non-pregnant women were used as a control group. An analysis of chemerin levels during the menstrual cycle was performed in an eumenorrheic group (n=16) in the early follicular (cycle day 4±1) and the midluteal phase (cycle day 22±1), demonstrating that serum chemerin levels did not fluctuate significantly. Serum levels of chemerin were significantly elevated during late gestation when compared to early (p<0.001) and middle (p=0.001) gestation and a negative correlation between serum chemerin and adiponectin levels (r=-0.1643) became more significant when the non-pregnant group was included in the calculations (r=-0.2471). There was no significant association of triglycerides, total cholesterol, LDL, HDL, insulin, and HOMA levels with chemerin. Although chemerin rose significantly and is negatively associated with adiponectin levels, it is not correlated with other markers of insulin sensitivity, suggesting that more study is needed to determine whether chemerin is useful in predicting insulin resistance during gestation.
Peptides 01/2013; · 2.43 Impact Factor
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Susana B Bravo,
Jorge E Caminos,
Carmen R González,
María J Vázquez,
María F Garcés,
Libia A Cepeda,
María E R García-Rendueles,
Antonio Iglesias-Gamarra,
Consuelo Gómez-Díaz,
Miguel Lopez,
Justo P Castaño,
Carlos Diéguez,
Rubén Nogueiras
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ABSTRACT: The stomach secretes a wide range of peptides with essential metabolic functions, and thereby plays an important role in the regulation of energy homeostasis. Disulfide isomerase glucose-regulated protein 58 (GRp58) is a molecular chaperone member of the endoplasmic reticulum (ER) stress signaling pathway, which is a marker for human gastric cancer. Since GRp58 seems to be regulated by a phosphorylation/dephosphorylation pattern shift, we used the 2DE gel methodology and peptide mass fingerprinting-protein identification by means of MALDI-TOF mass spectrometry. We show that gastric mucosa GRp58 is dephosphorylated by fasting, and this effect is blunted when fasted rats are treated with leptin. Furthermore, we assessed the gene expression of GRp58 under different physiological settings known to be associated with energy homeostasis (fasting, leptin treatment and leptin deficiency). We found that intraperitoneal administration of leptin increases whereas leptin deficiency decreases GRp58 mRNA levels. However, GRp58 expression remains unchanged after fasting, indicating that leptin actions on GRp58 are no direct sensitivity to fasting. Dissection of the molecular pathways mediating the interactions between ER stress-related factors and nutrient availability, as well as their target genes, may open a new avenue for the study of obesity and other metabolic disorders.
International Journal of Peptides 01/2011; 2011:969818.
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[hide abstract]
ABSTRACT: 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.
Analytical Biochemistry 05/2010; 400(2):219-28. · 3.00 Impact Factor
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ABSTRACT: The RET receptor is a tyrosine kinase receptor implicated in kidney and neural development. In the adenopituitary RET and the co-receptor GFRa1 are expressed exclusively in the somatotrophs secreting GH. RET is implicated in a clever pathway to maintain at physiological levels the number of somatotrophs and the GH production. Thus, in absence of its ligand GDNF, RET induces apoptosis through massive expression of Pit-1 leading to p53 accumulation. In the presence of the ligand GDNF, RET activates its tyrosine kinase and promotes survival at the expense of reducing Pit-1 expression and downregulating GH. Recent data suggest that RET can also have a second role in pituitary plasticity through a second co-receptor GFRa2.
Frontiers of hormone research 01/2010; 38:127-38. · 1.71 Impact Factor
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Jorge E Caminos, Susana B Bravo,
Maria F Garcés,
C Ruth González,
Libia A Cepeda,
Adriana C González,
Rubén Nogueiras,
Rosalía Gallego,
Tomás García-Caballero,
Fernando Cordido,
Miguel López,
Carlos Diéguez
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ABSTRACT: Amylin (islet amyloid polypeptide) and vaspin (visceral adipose tissue specific serpin) are gut and adipocyte hormones involved in the regulation of body weight homeostasis. The aim of this study was to examine whether amylin and vaspin are expressed in human and rat placenta, as well as their regulation by nutritional status. Our results demonstrate that amylin and vaspin are localized in both human and rat placenta. In the rat term placenta vaspin was demonstrated in the trophoblast of the fetal villi, the labyrinth. Vaspin immunostaining in human placenta was localized in cytotrophoblast and syncytiotrophoblast in the first trimester placentas while in the third trimester vaspin was localized in the syncytiotrophoblast. Regarding amylin, rat placenta of 16 days of gestational age showed an intense immunostaining, mainly localized in the labyrinth. On the other hand, in the human third trimester placenta amylin immunoreactivity was intense in the syncytiotrophoblast of the chorionic villi and in decidual cells. Furthermore, placental amylin and vaspin showed an opposite pattern of expression during pregnancy, with vaspin showing the highest expression level at the end and amylin at the beginning of pregnancy. Finally, food restriction also has contrary effects on their expression, increasing vaspin but decreasing amylin placental mRNA and protein levels. Taken together, our results demonstrate that vaspin and amylin are modulated by energy status in the placenta, which suggests that these proteins may be involved in the regulation of placental metabolic functions.
Histology and histopathology 09/2009; 24(8):979-90. · 2.48 Impact Factor
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ABSTRACT: Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.
Regulatory Peptides 03/2008; 146(1-3):106-11. · 2.11 Impact Factor
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Fernando Palos,
María E R García-Rendueles,
David Araujo-Vilar,
Maria Jesús Obregon,
Rosa Maria Calvo,
Jose Cameselle-Teijeiro, Susana B Bravo,
Oscar Perez-Guerra,
Lourdes Loidi,
Barbara Czarnocka,
Paula Alvarez,
Samuel Refetoff,
Lourdes Dominguez-Gerpe,
Clara V Alvarez,
Joaquin Lado-Abeal
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ABSTRACT: We studied two families from Galicia (northwest Spain) with Pendred syndrome (PS) and unusual thyroid phenotypes. In family A, the proposita had a large goiter and hypothyroxinemia but normal TSH and free T3 (FT3). In family B, some affected members showed deafness but not goiter.
Our objective was to identify the mutations causing PS and molecular mechanisms underlying the thyroid phenotypes.
Interventions included extraction of DNA and of thyroid tissue.
Propositi and 10 members of the two families participated in the study.
Main outcome measures included SLC26A4 gene analysis, deiodinase activities in thyroid tissue, and c.416-1G-->A effects on SLC26A4 splicing. In addition, a primary PS thyrocyte culture, T-PS2, was obtained from propositus B and compared with another culture of normal human thyrocytes, NT, by Western blotting, confocal microscopy, and iodine uptake kinetics.
Proposita A was heterozygous for c.578C-->T and c.279delT, presented with goiter, and had normal TSH and FT3 but low FT4 attributable to high type 1 and type 2 iodothyronine deiodinase activities in the goiter. Propositus B bore c.279delT and a novel mutation c.416-1G-->A; some deaf relatives were homozygous for c.416-1G-->A but did not present goiter. The c.279delT mutation was associated with identical haplotype in the two families. T-PS2 showed truncated pendrin retained intracellularly and high iodine uptake with low efflux leading to iodine retention.
c.279delT is a founder mutation in Galicia. Proposita A adapted to poor organification by increasing deiodinase activities in the goiter, avoiding hypothyroidism. Lack of goiter in subjects homozygous for c.416-1G-->A was due to incomplete penetrance allowing synthesis of some wild-type pendrin. Intracellular iodine retention, as seen in T-PS2, could play a role in thyroid alterations in PS.
Journal of Clinical Endocrinology & Metabolism 02/2008; 93(1):267-77. · 6.50 Impact Factor
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ABSTRACT: Neuropeptide Y (NPY), agouti related peptide (AgRP), cocaine and amphetamine-regulated transcript (CART) and melanocortins, the products of the proopiomelanocortin (POMC), are hypothalamic peptides involved in feeding regulation and energy homeostasis. Recent evidence has demonstrated their expression in rat and human placenta.
In the current study, we have investigated the expression of those neuropeptides in the rat placenta by real-time PCR using a model of maternal food restriction.
Our results showed that placental-derived neuropeptides were regulated through pregnancy and following food restriction.
These data could indicate that placental-derived neuropeptides represent a local regulatory circuit that may fine-tune control of energy balance during pregnancy.
Reproductive Biology and Endocrinology 02/2008; 6:14. · 2.05 Impact Factor
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Cesar A. Vélez,
Martha R. García,
Radoslav Omelka,
Mario F. Guerrero,
Miriam A. Wilches,
Jesús E. Rojas,
María F. Garóes,
Rita M. Baldrich, Susana B. Bravo,
Libia A. Cepeda,
Miguel A. Suarez,
Iván H. Moreno,
Jorge E. Caminos
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ABSTRACT: Objective : The purpose of the presera study was to devélop a molecular genotyping method test by using a real time PCR hybridization probé and applying it to the analysis of C1843T mutations of the Sus scrofa RYR1 gene. Animáis population Three PSS-susceptible and PSS non-susceptible crossbred swine races were used for the experiments: Pietrain X Landrace Belga, Pietrain X Large White and Pietrain X Duroc. Methods: We have devéloped a genotyping method by using a hybridization probé and applied it to the analysis of C1843T mutations of the RYR1 gene, associated with PSS susceptibility. Genotyping results obtained by hybridization probé strategies were confirmed by restriction analysis and sequencing. In addi-tion, phenotype/genotype correlation analyses were devéloped by using the in vitro contracture test and confirmed the in vivo hálothane-succinylcholine challenge. Results: The real-time PCR with fluorescent hybridization probé methodology was designed to identify ho-mozygous PSS-resistant, PSS-susceptible animáis as well as heterozygous carriers. All cases genotyped by fluorescent hybridization probes were in agreement with PCR restriction enzyme digestión and sequencing and showed a 100% concordance between the in vivo and in vitro porcine stress syndrome (PSS) susceptibility results. Conclusions and clinical relevance: The real-time PCR with fluorescent hybridization probé method described here provides a rapid, easily interpretable and réliáble tool for genotyping the C1843T (Arg615-Cys) polymorphism of the RYR1 gene. This new methodology may be useful in the wide-scale genotyping of PSS-susceptibility and genetic selection.
Revista Colombiana de Anestesiologia 01/2008; 36(1):11-18.
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ABSTRACT: 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 (4(100)/20(10) 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 G(1) phase of the cell cycle was greatly increased and the proportion in the S/G(2)-M phases greatly reduced, suggesting that plitidepsin blocks G(1)-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 4(100)/20(10) 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. 4(100)/20(10) 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.
Clinical Cancer Research 12/2005; 11(21):7664-73. · 7.74 Impact Factor
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ABSTRACT: 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-beta is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-beta in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-beta induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-beta 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-beta. By contrast, in cells in which p27 was overexpressed, TGF-beta 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-kappaB 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-beta induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-kappaB program.
Oncogene 11/2003; 22(49):7819-30. · 6.37 Impact Factor
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ABSTRACT: 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- is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF- in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF- induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF- 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-. By contrast, in cells in which p27 was overexpressed, TGF- 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-B 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- induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-B program.Keywords: TGF-, thyroid cancer, p27, apoptosis, NF-B
Oncogene 10/2003; 22(49):7819-7830. · 6.37 Impact Factor
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ABSTRACT: Millions of people worldwide suffer goiter, a proliferative disease of the follicular cells of the thyroid that may become neoplastic. Thyroid neoplasms have low prolif-erative index, low apoptotic index and a high incidence of metastasis. TGF-b is overexpressed in thyroid follicular tumor cells. To investigate the role of TGF-b in thyroid tumor progression, we established cultures of human thyrocytes from different proliferative pathologies (Grave's disease, multinodular goiter, follicular adenoma, papillary carcinoma), lymph node metastasis, and a normal thyroid sample. All cultures maintained the thyrocyte phenotype. TGF-b induced cell-cycle arrest in all cultures, in contrast with results reported for other epithelial tumors. In deprived medium, TGF-b induced apoptosis in normal thyrocyte cultures and all neoplastic cultures except the metastatic cultures. This apoptosis was mediated by a reduction in p27 kip1 levels, inducing cell-cycle initiation. Antisense p27 expression induced apop-tosis in the absence of TGF-b. By contrast, in cells in which p27 was overexpressed, TGF-b 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-jB survival program. Together, these findings suggest that (a) thyroid neo-plasms are due to reduced apoptosis, not increased division, in line with the low proliferative index of these pathologies, and (b) TGF-b induces apoptosis in normal thyrocytes via p27 reduction, but that in neoplastic thyrocytes this effect is overridden by activation of the NF-jB program. Oncogene (2003) 22, 7819–7830. doi:10.1038/sj.onc.1207029
Oncogene 01/2003; · 6.37 Impact Factor
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ABSTRACT: Brush borders (microvilli) are cell membrane specialized structures that function mainly as high-throughput absortive/secretory areas. It has been well-established that brush borders are particularly rich in membrane lipids characteristic to lipid rafts. Here, we report 57 proteins identified from microvillous membranes (MVM) isolated from human syncytiotrophoblast cells using an experimental method that avoids the use of nonionic detergents. About 60% of the proteins reported here have been described previously as lipid-raft specific. Well-known lipid raft-markers such as Annexin A2 and alkaline phosphatase were identified. Cytoskeleton structural constituents and proteins related with the control and modulation of the cytoskeletal architecture as well as the regulation of the interaction of cytoskeletal constituents with the cell membrane and particularly with lipid raft domains were found (Ezrin, IQGAP1 and 2, EBP50). Other proteins identified include signal transduction molecules, such as Ras-related protein Rab-1B and Rab-7, and ADP-ribosylation factor 1. Several proteins harbor putative post-translational modifications that favor its localization in the lipid-raft environment, such as GPI (alkaline phosphatase and 5'-nucleotidase) and myristoylation (BASP1 and MARCKS). On the whole, this extensive description demonstrates from the protein composition point of view that brush border membranes are indeed highly enriched in lipid raft microdomains.
Journal of Proteome Research 4(6):2435-41. · 5.11 Impact Factor
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ABSTRACT: 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.
Analytical Biochemistry.
