Human recombinant anti-thyroperoxidase autoantibodies: in vitro cytotoxic activity on papillary thyroid cancer expressing TPO

International audienceBACKGROUND: Thyroid cancers are difficult to treat due to their limited responsiveness to chemo- and radiotherapy. There is thus a great interest in and a need for alternative therapeutic approaches. RESULTS: We studied the cytotoxic activity of anti-thyroperoxidase autoantibodies (anti-TPO aAbs, expressed in baculovirus/insect cell (B4) and CHO cells (B4') or purified from patients' sera) against a papillary thyroid cancer (NPA) cell line. Anti-TPO aAbs from patients' sera led to a partial destruction of NPA cell line by complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) and exhibited an anti-proliferative activity. Comparison of the cytotoxic activity of anti-TPO aAbs shows that B4' induced an anti-proliferative effect and a better ADCC than B4, but a lower one than anti-TPO aAbs from patients' sera. Antibody-dependent cell-mediated cytotoxicity was increased when human peripheral blood mononuclear cells were used as effector cells, suggesting that FcgammaRs, CD64, CD32 and CD16 are involved. Indeed, anti-TPO aAbs from patients' sera, but not B4 and B4', exhibited CDC activity. CONCLUSIONS: These data indicate that anti-TPO aAbs display moderate ADCC and anti-proliferative activities on NPA cells; IgG glycosylation appears to be important for cytotoxic activity and ADCC efficiency depends on FcgammaR-bearing cells. Finally, recombinant human anti-TPO aAbs cannot yet be considered as an optimal tool for the development of a novel therapeutic approach for thyroid cancer

Chromatin Immunoprecipitation to Analyze DNA Binding Sites of HMGA2

BACKGROUND: HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP) experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. CONCLUSION: After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp) of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences

UbcH10 overexpression may represent a marker of anaplastic thyroid carcinomas

The hybridisation of an Affymetrix HG_U95Av2 oligonucleotide array with RNAs extracted from six human thyroid carcinoma cell lines and a normal human thyroid primary cell culture led us to the identification of the UbcH10 gene that was upregulated by 150-fold in all of the carcinoma cell lines in comparison to the primary culture cells of human normal thyroid origin. Immunohistochemical studies performed on paraffin-embedded tissue sections showed abundant UbcH10 levels in thyroid anaplastic carcinoma samples, whereas no detectable UbcH10 expression was observed in normal thyroid tissues, in adenomas and goiters. Papillary and follicular carcinomas were only weakly positive. These results were further confirmed by RT–PCR and Western blot analyses. The block of UbcH10 protein synthesis induced by RNA interference significantly reduced the growth rate of thyroid carcinoma cell lines. Taken together, these results would indicate that UbcH10 overexpression is involved in thyroid cell proliferation, and may represent a marker of thyroid anaplastic carcinomas

Les catalyseurs porphyriniques : un outil pour la préparation de modèles de métabolites

International audienc

Ghrelin acylation by ghrelin-O-acyltransferase can occur in healthy part of oncologic liver in humans

INTRODUCTION: Activation of ghrelin is controlled by the enzyme Ghrelin-O-Acyl Transferase (GOAT). In humans, localization of this acylation is poorly understood. The aim of that study was to explore GOAT localization and activation in human Liver by evaluating both bioactive and non-bioactive ghrelin in the blood stream entering and leaving liver and to simultaneously evaluate GOAT mRNA expression in Liver. METHODS: Healthy part of oncologic hepatic tissue collected from nine patients undergoing hepatectomy was used to evaluate GOAT mRNA expression by quantitative real time polymerase chain reaction (RTqPCR). Simultaneously blood from portal vein, supra hepatic vein, sub clavicular vein and radial artery was also sampled to assay total and acylated ghrelin. RESULTS: Acylated ghrelin level was significantly increased in supra hepatic vein compared to portal vein level (385±42 ng/ml vs. 268±24 ng/ml, p=0.04). Supra hepatic vein to portal vein ratio for acylated ghrelin (acylation ratio) is at 1.4±0.1. Mean expression of GOAT mRNA in liver, expressed as 2-∆Ct/µg total RNA/1µl of liver tissue was at 0.042±0.021 arbitrary units. GOAT mRNA expression in liver was correlated with acylated to total ghrelin ratio in supra hepatic vein (p=0.016, R=0.75) and with acylation liver ratio (p=0.05, R=0.61). CONCLUSIONS: Blood concentration of acylated ghrelin was found significantly increased after its passage through liver suggesting acylation can occur in the liver. RTqPCR data confirmed the presence of GOAT in liver, with positive correlation between GOAT expression and acylated ghrelin liver ratio. This study strongly suggests that liver is a site of ghrelin acylation in human