• Corpus ID: 53463523

ISOLATION OF TSH-RESPONSIVE HUMAN THYROCYTES\x=req-\ ANALYSIS OF VARIOUS THYROID TUMOURS

@inproceedings{Nolde2008ISOLATIONOT,
  title={ISOLATION OF TSH-RESPONSIVE HUMAN THYROCYTES\x=req-\ ANALYSIS OF VARIOUS THYROID TUMOURS},
  author={Siegmar Nolde and Matthias Schumacher and Helmuth Hilz},
  year={2008}
}

• S. Nolde, M. Schumacher, H. Hilz
• Published 2008
• Biology, Medicine

A method for the isolation of hormone-responsive thyrocyte suspensions from human tumours is described. Cells from normal and adenomatous thyroid tissue responded to thyroid stimulating hormone (TSH) by a concentration-dependent increase in cAMP. Bovine TSH usually produced half-maximal elevation of cAMP levels at concentrations between 2 and 4 mU/ml. Human TSH caused half\x=req-\ maximal stimulation at 3\p=n-\8 times lower concentrations indicating considerable species specificity of the human TSH receptor. Other hormones were without influence except prostaglandin E1 (PGE1) which evoked moderate elevations of the cAMP levels. Kinetic analyses of various thyroid tumours revealed marked differences in TSH induced increases in the intracellular cAMP levels with respect to the time course as well as to the extent of the maximal response. Theophylline, which by itself had a rather small effect on basal cAMP levels, acted synergistically with TSH in most cases, leading to values up to 20 times above the control values. The kinetics also indicated the existence in stimulated cells of two cAMP pools exhibiting different susceptibility to phosphodiesterase. Cells from adenomatous goitres with hyperplasia exhibited significantly lower basal cAMP levels than thyrocytes from the other goitres and from normal tissue. Other classes of thyroid tumours also showed characteristic differences with regard to basal cAMP and degree of stimulation by TSH and theophylline. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 34). II. Medizinische Klinik und Institut für Physiologische Chemie, Universität Hamburg, FRG Downloaded from Bioscientifica.com at 10/31/2018 01:58:54AM via free access i umour cells often retain their capacity for the special metabolic functions oí the tissue from which they originate. However, partial or complete deletions in regulatory abilities is one of the first signs of cancerous deviation (cf. Pitot et al. 1961; Schorr et al. 1972; Anderson et al. 1974). Such deletions have also been described in thyroid tumours of the rat (cf. Macchia et al. 1972; D'Armiento 8c Monaco 1974) as well as in humans (Schorr et al. 1972; DemeesterMirkine et al. 1976). The aim of our analyses is to localize regulatory deletions in human thyroid tumours. Since most of the physiological effects of the thyroid stimulating hormone (TSH) are mediated by the cAMP system (see Schell-Frederick 8c Dumont 1970; Dunwnt 1971; Field 1975 for a review), we have analyzed as a first step of the regulatory sequence: Hormone bindingshormone response, the ability of human thyroid tumours to respond to TSH by an increase in intracellular cAMP. Few human thyroid tumours have been analyzed previously for their ability to increase cAMP in response to TSH. In two analyses (cf. Schorr et al. 1972; Sand et al. 1976), only adenylate cyclase activity was measured in homogenates, while the other determinations were carried out with tissue slices (Burke 8c Szabo 1972; DeRubertis et al. 1971; Dumont et al. 1975). In slices, slow pene¬ tration of peptide hormones to the target cells and their possible destruction by liberated proteases usually leads to an unfavourable response with respect to time and hormone concentrations (cf. Swillens et al. 1976). Furthermore, because of the heterogeneous structure of the tumourous tissue along with the drastic differences in the content of connective tissue, blood constituents, and the size of the follicles, it is difficult to obtain true aliquots for comparative studies. We therefore decided to develop a technique for the preparation of hcrmone-responsive thyroid cell suspensions, which is described in this paper. Its efficiency is documented by the analysis of various forms of thyroid tumours. Some of these results have been reported in preliminary form (Breustedt et. al. 1975; Nolde et al. 1975). MATERIAL AND METHODS Preparation of cell suspension. Thyroid tissue removed by surgery was immediately put into cold buffered Earles's salt solution (Flow Laboratories, Nr. 3F-041D, without Ca+" + , + 20 niM Hepes pH 7.4), freed from fat and connective tissue and cut into small pieces. The chopped tissue was washed four times with 5-10 vols, of buffer by slow stirring for 8 min at 4°C and removing the supernatant by suction. The tissue was finally sedimented (580 x g, 8 min), weighed and incubated in 6 vols, of the above mentioned Hepes-salt buffer containing 0.25 °/o collagenase (Worthington, Freehold; CLS 45K148), 0.1 °/o hyaluronidase (E. Merck; Nr. 4508) (cf. Vale et al. 1972) and 3 °/o inactivated calf serum (GIBCo, USA). During incubation at 37°C the mixture was slowly stirred (120 r. p. m.) and every 10 min the mixture was sucked 6 times into a Downloaded from Bioscientifica.com at 10/31/2018 01:58:54AM via free access 70 ml syringe provided with silicone tubing (inner diameter 5 mm). After 60 min the tissue fragments were allowed to settle and the cloudy supernatant was sucked off and discarded. The residue was washed once with salt buffer and the pellet suspended in 6 vols, of warmed (37°C) salt buffer containing 0.25 %> Viocase (GIBCo, USA) followed by an incubation for 10-15 min at 37°C with slow stirring (120 r. p. m.). At times 10 min and 15 min of incubation the mixture was passed through a 70 ml syringe as described above and finally poured through a plastic sieve (1 mm2) into a polycarbonate tube containing inactivated bovine serum to give a final serum concentration of 10 °/o. The cells were sedimented by centrifugation (8 min, 2000 r. p. m., 4°C) and washed 3 times in cold MEM medium containing 20 dim Hepes pH 7.4 (Flow Laboratories, Nr. 1F-121C) and 3 °/o inactivated serum. The cells were finally re-suspended in 3-5 ml of the same medium, filtered through four layers of perlón stocking and diluted to a concentration of 1 x 107 cells/ml. Cell number was determined by counting in a haemocytometer. Incubation of cells with TSH and determination of cAMP levels. Aliquots of the cell suspension (0.5-1 x 10'' cells) were routinely incubated in Hepes-buffered MEM pH 7.2 (37°C) for 20 min ± 2 mM theophyllinc. Then, 200 mU/ml of bovine TSH was usually added where indicated to a final volume of 200 ¡A (shaking water bath, 37°C). The reaction was stopped by the addition of 50 fil 25 °/o cold trichloroacetic acid. After centrifugation, 200 til of the supernatant was extracted five times with 1 ml cold water-saturated ether. Aliquots were analyzed for cAMP by the method of Gilman (1970), or Brown et al. (1972), or by a modification (Schumacher, unpublished experiments) of the radioimmunoassay (RIA) (Steiner et al. 1969). All methods gave identical values. Morphological analyses. — Before disaggregation, parts of the tumour tissues were fixed in 5 %> phosphate buffered formaldehyde for routine histological examination. The morphological integrity of the dispersed cells was analyzed by phase contrast microscopy. Dye exclusion tests were routinely performed in aliquots of the cell sus¬ pension by staining with 5 °/o trypan blue in phosphate buffered saline. For electron microscopy cells were sedimented at 1200 g for 7 min and the pellets fixed at 4°C for 90 min in 2.3 °/o glutaraldehyde in 0.2 m cacodylate buffer pH 7.4. The fixative was removed and the pellet rinsed for 24 h at 4°C with cacodylate buffer. Post-fixation was performed for 1 h at 0°C in 2 °/o osmium tetroxide in phosphate buffer followed by dehydration in cold alcohol. The pellets were then embedded in Epon 800. Semi-thin sections were cut with a Reichert OmU 2 microtome and the sections stained with toluidine blue for light microscopic examination. Ultra-thin sections were obtained from light microscopically selected cell clusters. The sections were counterstained in lead hydroxide solution. Electron micrographs were obtained with a Siemens EM 300 microscope. Clinical diagnosis was based in most cases on the analysis of iodide uptake, T3/T4 levels in serum, TSH determination, and scintiscans. DNA was determined according to Burton (1968). Human TSH was kindly supplied by the hormone distribution officer (NIH) and the WHO Intern. Laboratory for Biological Standards (London), which also supplied a bovine TSH standard. Most experiments were performed with commercial bovine TSH (Thyrostimulin®, Organon GmbH, München), which exhibited activities similar to the WHO standard preparation. Downloaded from Bioscientifica.com at 10/31/2018 01:58:54AM via free access