cDNA‐directed expression of human thyroid peroxidase

Kimura, Shioko; Kotani, Takuya; Ohtaki, Sachiya; Aoyama, Toru

doi:10.1016/0014-5793(89)80759-8

Cited by 15 publications

(6 citation statements)

References 20 publications

(40 reference statements)

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“…Nevertheless, it could not be definitely excluded that this radioactivity represented hormone synthesized within the cell, and then rapidly secreted. The microsomal antigen, now known to be hTPO, can be detected by immunofluorescent techniques on the surface of thyroid cells (12,13), as well as on the surface of nonthyroidal cells transfected with hTPO cDNA (14,23). Ultrastructural, histochemical analysis shows TPO on the external surface of microvilli only in TSH-stimulated or autonomously active human thyroid tissue, and not in normal thyroid tissue (24).…”

Section: Discussionmentioning

confidence: 99%

Generation of a Biologically Active, Secreted Form of Human Thyroid Peroxidase by Site-Directed Mutagenesis

Foti¹,

Kaufman²,

Chazenbalk³

et al. 1990

Molecular Endocrinology

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Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.

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Section: Discussionmentioning

confidence: 99%

Generation of a Biologically Active, Secreted Form of Human Thyroid Peroxidase by Site-Directed Mutagenesis

Foti¹,

Kaufman²,

Chazenbalk³

et al. 1990

Molecular Endocrinology

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“…In most in vitro systems, the hTPO introduced by cDNAdirected gene expression was detected by means of immunohistology, immunoblotting or auto-antibodies of patients with thyroid auto-immune disease. However, the production of catalytically active hTPO has not been achieved so far, irrespective of the expression system and cell culture system employed for the experimental approach [24,25,26,27,28]. The function and activity of the hTPO are probably influenced by the multimerization of the protein as well as by additional factors, including the incorporation of heme, the glycosylation, the localization of the enzyme and the thyroglobulin content of the cells.…”

Section: Discussionmentioning

confidence: 99%

Iodide uptake in human anaplastic thyroid carcinoma cells after transfer of the human thyroid peroxidase gene

et al. 2001

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Human thyroperoxidase (hTPO) is critical for the accumulation of iodide in thyroid tissues. Poorly differentiated and anaplastic thyroid tumours which lack thyroid-specific gene expression fail to accumulate iodide and, therefore, do not respond to iodine-131 therapy. We consequently investigated whether transfer of the hTPO gene is sufficient to restore the iodide-trapping capacity in undifferentiated thyroid and non-thyroid tumour cells. The human anaplastic thyroid carcinoma cell lines C643 and SW1736, the rat Morris hepatoma cell line MH3924A and the rat papillary thyroid carcinoma cell line L2 were used as in vitro model systems. Employing a bicistronic retroviral vector based on the myeloproliferative sarcoma virus for the transfer of the hTPO and the neomycin resistance gene, the C643 cells and SW1736 cells were transfected while the L2 cells and MH3924A cells were infected with retroviral particles. Seven recombinant C643 and seven SW1736 cell lines as well as four recombinant L2 and four MH3924A cell lines were established by neomycin selection. They were studied for hTPO expression using an antibody-based luminescence kit, followed by determination of the enzyme activity in the guaiacol assay and of the iodide uptake capacity in the presence of Na125I. Genetically modified cell lines expressed up to 1,800 times more hTPO as compared to wild type tumour cells. The level of hTPO expression varied significantly between individual neomycin-resistant cell lines, suggesting that the recombinant retroviral DNA was integrated at different sites of the cellular genome. The accumulation of iodide, however, was not significantly enhanced in individual recombinant cell lines, irrespective of low or high hTPO expression. Moreover, there was no correlation between hTPO expression and enzyme activity in individual cell lines. The transduction of the hTPO gene per se is not sufficient to restore iodide trapping in non-iodide-concentrating tumour cells. Future studies will have to concentrate on the possible expression of enzymatically active proteins or the transfer of multiple genes involved in iodide trapping.

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“…In den meisten In-vitro-Systemen wurde nach Transfer des hTPO-Gens die Expression mittels Immunhistologie, Immuno blotting oder Autoantikörper von Patienten mit Autoimmun erkrankungen der Schilddrüse dokumentiert. Die Produktion eines katalytisch aktiven Enzyms wurde jedoch nicht erreicht -unabhängig vom Expressionssystem und verwendeten Zellen (10,15,18,19).…”

Section: Zusammenfassungunclassified

Gentechnisch modulierte Iodidanreicherung in malignen Tumoren

Haberkorn¹,

Askoxylakis²,

Markert³

et al. 2010

Nuklearmedizin

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SummaryAfter the cloning of the gene encoding the sodium-iodide symporter several trials were made to develop a radioiodine treatment for multiple tumour entities based on NIS gene transfer. These studies revealed in vitro as well as in vivo a tremendous enhancement of iodide accumulation, which was followed by a rapid efflux. Therapy effects were observed in vitro by clonogenic assays and in vivo by growth inhibition of the treated tumours. However, the interpretation of these results were largely different. Problems of radioiodine therapy after NIS transfer are low efficiency of gene transfer and the short exposure time for the tumours caused by the rapid efflux. Trials to enhance therapeutic efficiency by co-transfer of the gene encoding thyroperoxidase failed due to the low enzyme activity.

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cDNA‐directed expression of human thyroid peroxidase

Cited by 15 publications

References 20 publications

Generation of a Biologically Active, Secreted Form of Human Thyroid Peroxidase by Site-Directed Mutagenesis

Generation of a Biologically Active, Secreted Form of Human Thyroid Peroxidase by Site-Directed Mutagenesis

Iodide uptake in human anaplastic thyroid carcinoma cells after transfer of the human thyroid peroxidase gene

Gentechnisch modulierte Iodidanreicherung in malignen Tumoren

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