Characterization of neurotensin production by a line of rat medullary thyroid carcinoma cells.

Abstract

The rMTC 6-23 cell line was derived from a calcitonin-producing rat medullary thyroid carcinoma. During characterization of these cells we discovered that they also synthesize and secrete neurotensin (NT), a tridecapeptide originally isolated from the hypothalamus and not previously associated with C cells. Immunoreactive NT (iNT) Medullary thyroid carcinoma is a neoplasm of calcitonin (CT)-producing cells of the mammalian thyroid gland. Zeytino0lu et al. (1) and Gagel et al. (2) have established in culture a CT-secreting line (rMTC 6-23) derived from a transplantable WAG/Rij rat medullary thyftid carcinoma. These cells synthesize and secrete CT and respond with an increase in CT secretion during incubation with calcium, K+, and glucagon. In screening these cells for neuroendocrine peptides, some of which have previously been associated with medullary thyroid carcinoma (3-8), we found that the rat medullary thyroid carcinoma (rMTC) 6-23 line also produced and secreted neurotensin (NT), a tridecapeptide, which was originally isolated from bovine hypothalamus by Carraway and Leeman (9) and from human intestine by Hammer et al. (10). Although a possible neurohormonal or neurotransmitter role has been postulated for NT, it has no known effects on calcium metabolism or calcium-regulating hormones and has not previously been associated with C cells.MATERIALS AND METHODS rMTC Cell Line. The rMTC 6-23 cell line was established in culture from a transplantable rMTC (11) carried in the WAG/Rij strain. The tumor was passed between animal and culture five times over a 2-year period before a continuous cell line was successfully established. Primary cultures were prepared by a collagenase technique described elsewhere (1). The rMTC 6-23 cell line has been serially subcultured more than 25 times since February 1979. Cells are grown as monolayers in Dulbecco's modified Eagle's medium supplemented with 1.4% glutamine and heat-inactivated (600C, 1 hr) horse serum (15%) and fetal calf serum (2.5%). There has been no evidence of senescence, as shown by a decrease in growth rate or a decrease in the production of CT or NT over an 11-month period.Cell Growth. To determine growth, we plated cells at equal density in replicate 35-mm plastic culture dishes. Medium was changed every 2 days. At 48-hr intervals, medium was collected from six dishes selected at random and stored at -20°C for subsequent measurements of immunoreactive neurotensin (iNT). Three dishes at each time interval were washed with saline and frozen at -20°C for determination of total cell protein (12). Cell extracts for measurement of NT content were made by washing three dishes three times with saline and extracting with 1 ml of ice-cold 10 mM HC1 per dish. The cells were scraped with a rubber policeman and frozen and thawed three times. The resulting suspension was separated in a microfuge (Brinkman, model 3200) for 2 min, and the supernatant solution was frozen at -200C for subsequent radioimmunoassay of NT. An additional three dishes were incubated with ...

Cloning and sequence analysis of a cDNA encoding rat preprocholecystokinin.

“…Examples of such neuropeptides include neurotensin (2), somatostatin (3,4), cholecystokinin (CCK) (5), and proopiomelanocortin-derived peptides (6). Several of these peptides are synthesized initially as larger precursor proteins, which are in turn processed to biologically active peptide(s) (7,8).…”

“…Levels of immunoreactive CCK in 2 MTC lines (denoted here as WD or 1-1-4 and WE or 1-1-4A) were 20 to 40 times higher than the concentrations reported for porcine brain or gut CCK-8 (10). Poly(A) RNA was isolated from one of the MTC lines, and by using recombinant DNA techniques a cDNA library was prepared.…”

Neurotensin in human small cell lung carcinoma

“…Previously, NTLI has been shown to be present in endocrine pancreatic tumours (Blackburn et al, 1980;Feurle et al, 1981;Gutniak et al, 1980;Theodorsson-Norheim et al, 1983) and it also was found in human small cell lung carcinomas (Wood et al, 1981(Wood et al, , 1983. At present, the only cell lines known to produce NTLI have been derived from a rat medullary carcinoma of the thyroid and a rat phaeochromocytoma (Tischler et al, 1982;Zeytinoglu et al, 1980). Our findings constitute the first example of cell lines of human origin producing NTLI.…”

Neurotensin and Neuromedin N Are Differentially Processed from a Common Precursor by Prohormone Convertases in Tissues and Cell Lines

“…The endocrine rat medullary thyroid carcinoma (rMTC) 6-23 cell line was shown to express high levels of pro-NT/NN following dexamethasone induction (Zeytinoglu et al 1980;de Nadai et al 1993). Studies of precursor processing in this cell line showed that sites 2, 3, and 4 were readily cleaved to generate almost stoichiometric amounts of NT and NN and minor quantities of large NT and large NN ) (Table 1).…”