Establishment of a Calcitonin-Producing Rat Medullary Thyroid Carcinoma Cell Line. II. Secretory Studies of the Tumor and Cells in Culture*

Abstract

In the preceding paper we described the establishment of a calcitonin(CT)-producing cell line from the WAG/Rij transplantable rat medullary thyroid carcinoma (rMTC). In this paper we present the results of studies on CT secretion by both the parent tumor in vivo and the rMTC 6–23 cells in culture. CT was measured by a heterologous RIA using antibody against human CT (hCT), hCT tracer, and hCT standard. Complete and quantitative cross-reactivity with synthetic rat CT (rCT) was shown. A progressive increase in plasma CT concentration was observed in rats after transplantation of the rMTC. Tumor CT content was 42 ±5.7 μ/g wet wt tissue (mean ±SE). Intravenous injection of calcium gluconate (10 mg/kg) produced a prompt rise in the plasma CT concentration; no consistent change in plasma CT was seen after iv pentagastrin (5 μxg/kg). Gel filtration studies indicated that the predominant form of immunoreactive CT eluted with [^l25I]hCT in both tumor extracts and plasma, although a small peak of higher molecular weight CT was detected in tumor extracts. CT secretion by rMTC 6–23 cells in culture was stimulated by calcium chloride (1.7 mM), glucagon (2.8 x 10^-6M), and KC1 (50 mM);no response was seen after incubation with pentagastrin (1.3 × 10^-6M), hypothalamic extract, or ethanol (0.1%). Half-maximal enhancement of CT secretion in the presence of calcium chloride occurred at 1.9 mM calcium, and maximal secretion occurred between 3–4 mM. KC1- induced stimulation of CT release was dependent on the presence of calcium in the medium. The average CT content of the cultured cells was 2.0 ng/mg cell protein, and basal secretion ranged from 3.5–6.6 ng/mg cell protein/48 h. Gel filtration studies of cell extracts and medium indicated that the predominant form of immunoreactive CT eluted with [¹²⁵I]rCT, although a small void volume peak of immunoreactive CT was noted. We conclude that the WAG/Rij transplantable rMTC and a cell line derived from this tumor are useful models for studying rCT synthesis and secretion.