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N E Heldin

Publications and source records attributed to N E Heldin.

At least 37 records · Page 2Linked to original sources
PubMed1996

EGF-receptors in human normal and pathological thyroid tissue.

Expression of the epidermal growth factor receptor (EGFR) was studied in cryosections from human thyroid tissues. Normal tissue (4 cases), nodular goitre (12), toxic goitre (9), adenoma (9), follicular carcinoma (1), papillary carcinoma (7) and poorly differentiated carcinoma (1) were used for immunohistochemistry. Northern blot analysis was performed in two nodular goitres, three adenomas, two papillary carcinomas, one follicular carcinoma and the adjacent normal tissue in five cases as well as in two cell lines from anaplastic carcinomas. Epidermal growth factor receptor immunoreactivity was detected in all tissues examined. The amount of EGFR mRNA did not differ between normal and abnormal tissues. However, the EGFR staining was weaker in normal thyroid tissue compared to the adjacent neoplastic areas suggesting an upregulation at the posttranslational level in the latter. A strong staining was also seen in hyperfunctioning thyroid glands. The EGFR location was mainly basal or basolateral in all thyroid tissues with normal histology and in toxic diffuse goitre. Pericellular and sometimes cytoplasmatic staining was seen in neoplastic tissues. In nodular goitre the staining was both basal, lateral and apical and varied in intensity. Our data suggest that a non-polarized location of EGFR probably indicates a loss of the normal epithelial cell polarity and could be interpreted as an early sign of dedifferentiation. Furthermore, a role for the EGFR is proposed, not only in the development of thyroid neoplasias but also in goitre formation.

Adenocarcinoma, Follicular↗
PubMed1995

Functional analysis of a variant of the thyrotropin receptor gene in a family with Graves' disease.

Nucleotide sequence analysis of PCR fragments corresponding to the TSH-receptor (TSHR) amplified from genomic DNA collected from the four members of a family, two of which had Graves' thyrotoxicosis, revealed a nucleotide substitution in the first position of codon 36 of the TSH-receptor gene in the two patients. The nucleotide substitution was from G to C, leading to a 36D-->36H change (D36H) in the predicted amino acid sequence of the receptor. The altered sequence was also found in DNA obtained from their mother, but not in DNA from their father. We stably expressed the two receptor variants in NIH 3T3 cells, by transfection of cDNA encoding the wildtype (WT) and D36H variants of the TSHR. Neither the binding of 125I-TSH nor the responsiveness to TSH measured as cAMP formation, appeared to be different in the TSHR-D36H compared to the TSHR-WT. Furthermore, the D36H-receptor also became desensitized when exposed to TSH as did the WT-receptor.

3T3 Cells↗
PubMed1994

Point mutations of the thyrotropin receptor determining structural requirements for its ability to bind thyrotropin and to stimulate adenylate cyclase activity.

The two cysteines C494 and C569, located in the first and second extracellular loop, respectively, of the thyrotropin (TSH) receptor, were mutated to serines to test the functional significance of the putative disulfide bond between these two cysteines. Single (C494S and C569S) and double (C494/569S) mutant receptors were generated, transiently expressed in COS cells, and compared with regard to the ability to bind ligand and to mediate stimulation of adenylate cyclase activity. The double mutant retained ligand binding capacity, in contrast to the single cysteine mutants that were essentially devoid of binding capacity. The ability of the mutated receptor variants to stimulate adenylate cyclase activity was lost or greatly reduced.

Adenylyl Cyclases↗
PubMed1994

Thyrotropin (TSH)-induced receptor internalization in nonthyroidal cells transfected with a human TSH-receptor complementary deoxyribonucleic acid.

TSH-induced desensitization was studied in nonthyroidal cells expressing functionally active TSH receptors (TSHR). Chinese hamster ovary (CHO) cells and mouse NIH 3T3 cells were stably transfected with a human TSHR cDNA. Stimulation of the CHO-TSHR and NIH-TSHR cells with 10 mU/ml TSH resulted in a decreased sensitivity to a second TSH stimulation only in the NIH-TSHR cells. A decrease in TSH-induced cAMP was present within 1 h and coincided with a decreased binding of [125I]TSH. The half-maximal effect was observed after a 3- to 4-h stimulation with TSH, and exposure of cells to TSH for 20 h led to a 70-80% inhibition of cAMP formation. After withdrawal of TSH, cells regained full responsiveness to TSH after 6 h. Moreover, the desensitization effect observed in NIH-TSHR cells was not mimicked by forskolin and, therefore, was not mediated by cAMP. Stimulation of the CHO-TSHR cells with TSH did not result in a desensitization toward a second TSH stimulation, nor did it reduce the binding of [125I]TSH. This difference between the two cell lines might be explained by a higher turnover rate of receptors in the CHO cells. Indeed, incubation of cells with [125I]TSH showed a more efficient internalization of ligand in the CHO-TSHR cells compared to the NIH-TSHR cells. In summary, the homologous desensitization observed in TSHR-transfected NIH 3T3 cells appears to be the result of ligand-induced receptor down-regulation.

3T3 Cells↗
PubMed1994

Thyrotropin receptor and leukocyte adhesion molecules in autoimmune thyroid disease: a study of their gene expression by northern blot analysis and in situ hybridization.

In order to characterize the role of leukocyte-activating antigens and other immunological parameters in autoimmune thyroid disease, mRNA levels of intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1, E-selectin), invariant chain (Ii) and the thymic hormone thymosin beta (T beta 4) were investigated in 18 human thyroid glands, including eight Graves' thyroids, two Hashimoto's thyroids, two endemic goiters and six healthy controls. Northern blot analysis showed that in autoimmune thyroid disease, expression of ICAM-1 and T beta 4 was correlated to transcript levels of Ii, whereas in the healthy controls, expression of T beta 4, ICAM-1 and ELAM-1 was low or nearly absent. ELAM-1 and TSH receptor (TSH-R) expression, the latter serving as a thyroid specific marker, was increased in some diseased gland but showed no relation to the immunological parameters mentioned above. Localization of the specific mRNAs by in situ hybridization demonstrated a cell-specific expression of TSH-R (thyrocytes), ELAM-1 (vascular endothelial cells) and T beta 4 (cells of hematopoietic origin). In contrast, transcripts of Ii and ICAM-1 were found in thyrocytes, leukocytes and endothelial cells. Our results implicate a coordinate expression of ICAM-1, T beta 4 and Ii in autoimmune thyroid disease, yielding distinct cellular expression patterns. Differential expression of ICAM-1, Ii and the TSH-R in thyroid epithelial cells indicates active regulatory events within the thyrocyte.

Autoimmune Diseases↗
PubMed1993

Differential expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human lung carcinoma cell lines.

In the present investigation we have studied the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) in ten different human lung carcinoma cell lines, four small cell carcinomas and six non-small cell carcinomas. None of the small-cell lung carcinoma cell lines demonstrated expression of PD-ECGF/TP mRNA. However, four of six of the non-small cell carcinoma cell lines expressed the 1.8 kb PD-ECGF/TP transcript. The cell lines derived from the single squamous cell carcinoma and the two adenocarcinomas expressed the PD-ECGF/TP mRNA, and were found to have the corresponding protein both in cell lysates and conditioned media as determined both by immunoblotting and measurement of thymidine phosphorylase activity. Only one of three studied large cell carcinoma cell lines expressed low levels of PD-ECGF/TP mRNA, but the corresponding PD-ECGF/TP protein was not demonstrated by immunoblotting.

Carcinoma, Non-Small-Cell Lung↗
PubMed1993

Expression levels of the thyrotropin receptor gene in autoimmune thyroid disease: coregulation with parameters of thyroid function and inverse relation to major histocompatibility complex classes I and II.

Using a human TSH receptor (TSH-R) cDNA probe, we investigated TSH-R transcript levels in 13 human thyroid fragments by Northern blot analysis; 7 Graves' disease, 2 Hashimoto's disease, 3 endemic goiter, and 1 healthy thyroid gland were studied. TSH-R expression levels were variable, but displayed a close correlation to the expression of thyroid peroxidase (r = 0.703; P < 0.05), thyroglobulin (r = 0.817; P < 0.01), and the nuclear oncogene c-fos (r = 0.935; P < 0.001), but not c-myc. Overall, TSH-R transcript levels were low or absent in those thyroids in which expression of the major histocompatibility complex class I or II (MHC I or II) was high, thus establishing an inverse relation (MHC I, r = -0.791; P < 0.01; MHC II, r = -0.784; P < 0.01). In situ hybridization showed that apart from lymphocytes, thyroid cells themselves were the source of MHC II transcripts. gamma-Interferon expression was only detectable in 1 Hashimoto's goiter. Our findings suggest that next to lymphocyte infiltration, active regulatory events in the thyrocyte are responsible for the inverse relation between functional parameters (TSH-R, thyroid peroxidase, thyroglobulin, and c-fos) and immunological markers (MHC I and II).

Adolescent↗
PubMed1991

Characterisation of a cell line (LCC-18) from a cultured human neuroendocrine-differentiated colonic carcinoma.

A cell line (LCC-18) from a neuroendocrine colonic tumour was established. The tumour cells retained their endocrine characteristics through more than 100 passages and showed positive immunocytochemistry for synaptophysin, vasoactive intestinal polypeptide (VIP) and glucagon. The culture medium also contained VIP and glucagon, which indicates that mechanisms for release of some of the active peptides were preserved. Transplantation of LCC-18 tumour cells into nude rats resulted in tumour formation with similar endocrine characteristics. The c-myc gene was amplified which might have been a prerequisite for establishment of the cell line. The chromosomes in LCC-18 were studied by G-banding and C-banding. The cell line had a distinctive mode in the hypotriploid region, at S = 61. The double minute (Dms) positive stemline karyotype showed numerical and structural aberrations more similar to findings in ordinary colonic adenocarcinomas than to observations in previously studied, pure intestinal neuroendocrine tumours. The Dms may be correlated with amplification of c-myc. LCC-18 may become valuable for studies of neuroendocrine differentiation, regulation of growth and production and release of hormones and for studies of drug effect.

Adult↗
PubMed1991

Coexpression of functionally active receptors for thyrotropin and platelet-derived growth factor in human thyroid carcinoma cells.

In the present study we show the simultaneous expression of functionally active receptors for TSH and platelet-derived growth factor (PDGF) in a newly established human anaplastic thyroid carcinoma cell line, HTh 74. In Northern blot analysis of RNA extracted from HTh 74 cells a low expression of both TSH and PDGF receptor messenger RNA was found. These observations in conjunction with the fact that the cells contain cytokeratin clearly demonstrate that the cells are bona fide epithelial thyroid cells. Stimulation of HTh 74 cells with TSH led to a concentration-dependent increase in cAMP formation, showing a functional activity of the TSH receptors. Northern blot analysis, immunoprecipitation, immunofluorescence staining, and binding experiments showed the presence of both alpha- and beta-type PDGF receptors in the HTh 74 cells. The functional activity of the PDGF receptors was demonstrated by ligand-induced internalization of the receptors and PDGF-induced growth of the HTh 74 cells. The significance of the expression of PDGF receptors on thyroid epithelial cells is not clear. However, it might reflect the gain of a new growth stimulatory pathway participating in the transformation of the epithelial thyroid cells. Alternatively, the PDGF receptors may be remnants from an immature progenitor cell from which the undifferentiated carcinoma has evolved.

Carcinoma↗
PubMed1991

A somatic point mutation in a putative ligand binding domain of the TSH receptor in a patient with autoimmune hyperthyroidism.

Nucleotide sequence analysis of PCR fragments of TSH receptor cDNA synthesized from thyroid RNA of a patient with autoimmune hyperthyroidism, revealed two different sequences in the first position of codon 36. In one of the sequences, there was a C for G substitution leading to the D36----H substitution in the predicted peptide. Both variants were also found in genomic DNA of thyroid tissue. However, only the germ line sequence was found in other tissues representing all three different germ layers. The novel sequence is therefore likely to represent a somatic mutation in the thyroid tissue, of possible relevance for the pathogenesis of the patient's thyroid disorder.

Adult↗
PubMed1991

The molecular biology of the human anaplastic thyroid carcinoma cell.

In the present investigation we show data from our studies of anaplastic human thyroid carcinoma cell lines. The cell lines employed in the study were HTh 7, HTh 74, C 643 and SW 1736, all derived from tumours diagnosed as anaplastic thyroid carcinomas. Northern blot analysis with four different thyroid specific cDNA probes showed a varying pattern of expression. Thyroglobulin mRNA was found in three of the carcinoma cell lines, although the signal was very weak compared to the expression in tissue from a toxic goitre, used as positive control. Interestingly, two of the cell lines expressed the receptor for thyrotropin, but none of them contained thyroperoxidase mRNA. Three of the cell lines expressed mRNA for receptors platelet-derived growth factor, PDGFR-alpha and/or PDGFR-beta type. Messenger RNA of a thyroid specific transcription factor, TTF-1, known to regulate the normal function of thyrocytes, was found in the toxic goitre but not in the anaplastic thyroid carcinoma cell lines. Lack of expression of TTF-1 might the immediate cause of the anaplastic phenotype, considering the possibility that TTF-1 functions as a master regulatory gene in thyroid cell differentiation.

Blotting, Northern↗
PubMed1990

Stromal influences on transformation of human mammary epithelial cells overexpressing c-myc and SV40T.

The proto-oncogene c-myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c-myc (A1N4-myc) or SV40T antigen (A1N4-T). Neither the A1N4, A1N4-myc, or A1N4-T cells will grow in soft agar or form tumors in nude mice. However, A1N4-T or A1N4-myc cells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or basic fibroblast growth factor (bFGF). Like EGF and TGF alpha, bFGF is moderately mitogenic for the anchorage-dependent growth (ADG) of all three cell lines. Further, co-cultivation of A1N4-T or A1N4-myc cells with primary diploid mammary fibroblasts can also induce the anchorage-independent growth (AIG) and stimulate the ADG of A1N4-T or A1N4-myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4-T and A1N4-myc cells and was found to contain immunoreactive TGF alpha and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFG alpha or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.

Agar↗
PubMed1989

Induction of cyclic AMP synthesis by forskolin is followed by a reduction in the expression of c-myc messenger RNA and inhibition of 3H-thymidine incorporation in human fibroblasts.

We have studied the effect of increased intracellular levels of cyclic AMP on the growth response to platelet-derived growth factor (PDGF) of human foreskin fibroblasts in culture. It was found that forskolin, a potent stimulator of adenylate cyclase activity, inhibits the stimulatory effect of PDGF on 3H-thymidine incorporation with a dose dependence similar to that observed with regard to cyclic AMP formation. A time-course study indicated that forskolin has no effect on ongoing DNA synthesis but affects events in the prereplicative phase. The cell-cycle block induced by forskolin was found to be reversible; after removal of the drug, DNA synthesis was initiated after a lag period, similar to that of the prereplicative phase of control cells. Forskolin had no effect on PDGF binding, receptor autophosphorylation, or c-fos mRNA expression. However, a reduction in PDGF-induced c-myc mRNA expression was observed in cultures given forskolin. Forskolin was also found to have a marked stimulatory effect on the expression of interferon-beta 2 mRNA expression. However, we were unable to demonstrate that the growth-inhibitory effect of forskolin is mediated by interferon-beta. In conclusion, an increase in cAMP levels leads to a reversible inhibition of PDGF-induced DNA synthesis in human fibroblasts, which may be related to an inhibition of c-myc mRNA expression.

Cell Line↗
PubMed1989

Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.

Animals↗
PubMed1988

Aberrant expression of receptors for platelet-derived growth factor in an anaplastic thyroid carcinoma cell line.

Receptors for platelet-derived growth factor (PDGF) have previously only been found on cells of mesenchymal and glial origin. This study shows PDGF receptors on an anaplastic thyroid carcinoma cell line, C 643, that was found to express thyroglobulin mRNA, confirming its origin from thyroid epithelium. Northern blot analysis of poly(A)+ RNA hybridized with a human PDGF B-type receptor cDNA probe revealed a 5.4-kilobase transcript in the C 643 cells. The existence of receptor protein on the cell surface was shown by immunofluorescence microscopy with a PDGF receptor monoclonal antibody. Binding experiments with 125I-labeled dimeric forms of PDGF indicated that the cells contain B-, but not A-, type PDGF receptors. The addition of PDGF to C 643 membranes in the presence of [32P]ATP induced phosphorylation of the receptor. A polyclonal PDGF B-type receptor peptide antiserum was used to immunoprecipitate a receptor protein from metabolically labeled C 643 cells; the receptor was found to be 5-10 kDa larger than that in normal human fibroblasts. Removal of N-linked carbohydrates using endoglycosidase H resulted in deglycosylated receptor proteins of similar size in C 643 cells and fibroblasts, indicating differences in glycosylation patterns of the two receptor proteins. The aberrant expression of receptors might be crucial in tumor development by conferring a selective growth advantage to the cancer cells.

Blotting, Northern↗
PubMed1988

Epidermal growth factor, but not thyrotropin, stimulates the expression of c-fos and c-myc messenger ribonucleic acid in porcine thyroid follicle cells in primary culture.

Previous studies have shown that TSH stimulates functional parameters in cultures of porcine follicle cells, but has no stimulatory effect on their growth. Epidermal growth factor (EGF), on the other hand, is a potent mitogen in such cells. The aim of the present investigation was to study the effect of EGF and TSH on the expression of proto-oncogene mRNA in porcine thyroid follicle cells. The addition of EGF caused a marked increase in the c-fos and c-myc mRNA levels. Stimulation of cells with 10 mU/ml TSH, however, failed to stimulate expression of the proto-oncogenes. The increase in c-fos mRNA caused by EGF was transient, with a maximum after 30 min. The c-myc transcript level increased after 30-60 min; the maximum level was at 90 min, and it decreased thereafter but remained at an elevated level throughout the 24 h of investigation. Half-maximal effects on proto-oncogene expression were obtained using 0.5-1 ng (c-fos) and 0.2-0.5 ng (c-myc) EGF/ml. Our data clearly demonstrate that increased expression of c-fos and c-myc mRNA is an early event in the prereplicative phase of EGF-stimulated porcine thyroid follicle cells. Since c-fos and c-myc expression may be necessary events in the prereplicative phase, the lack of effect of TSH on these functions may be closely related to its inability in stimulating the growth of porcine thyrocytes.

Animals↗
PubMed1987

A growth stimulatory effect of iodide is suggested by its effects on c-myc messenger ribonucleic acid levels, [3H]thymidine incorporation, and mitotic activity of porcine follicle cells in suspension culture.

In the present study the effect of iodide on thyroid cell growth was investigated in primary suspension cultures of porcine thyroid cells capable of organifying iodide. The addition of a high dose of iodide (10(-4) M) to such cultures caused a marked increase in c-myc mRNA levels, [3H]thymidine incorporation, and mitotic activity. The incorporation of [3H]thymidine started 30-36 h after the addition of iodide. The stimulatory effect was abolished by a simultaneous incubation with methimazole. The concentration dependence of the iodide-induced stimulation of [3H]thymidine incorporation was similar to that of an inhibitory effect on adenylate cyclase activity. W-7, an inhibitor of calmodulin activity, as well as epinephrine, agents that reduce cAMP levels, also stimulated [3H]thymidine incorporation. Moreover, the stimulatory effect of iodide was reduced in the presence of forskolin. The results suggest that an organic form of iodine stimulates thyroid cell growth by reducing cAMP levels and demonstrate the presence of a growth stimulatory pathway in porcine thyroid cells that is independent of exogenous polypeptide growth factors or hormones.

Adenylyl Cyclase Inhibitors↗