Effect of octreotide on proliferation of in vitro cultured thyroid medullary carcinoma cells

In TT cells, originating from medullary carcinoma of the human thyroid, the presence of receptors for somatostatin was demonstrated at the ultrastructural level. Inhibitory effect of octreotide (a somatostatin analogue) was observed on proliferation of in vitro cultured TT cells and confirmed by evaluating levels of PCNA and Ki-67 proliferation-associated antigens and examining the extent of DNA damage using the comet assay. Our studies indicate a potential for application of somatostatin analogues to diagnosis and adjunct treatment in thyroid medullary carcinomas.     

Hybridocytochemical and immuno-ultrastructural study of calcitonin gene expression in cultured medullary carcinoma cells

The study was aimed at a morphological demonstration of calcitonin (CT) gene expression in cultured TT cells, or, more specifically, hybridocytochemical detection of CT mRNA and calcitonin gene-related peptide (CGRP) mRNA and ultrastructural localization of the two hormones. The TT cells originated from medullary carcinoma of human thyroid gland. Ultrastructural studies of TT cells demonstrated a well-developed rough endoplasmic reticulum, large Golgi apparatus and low number of secretory granules. Hybridocytochemical studies showed the presence of mRNAs for CT and CGRP in all TT cells. At the ultrastructural level, double immunolabelling demonstrated that the two hormones were always expressed together in the same secretory granules. Our results provide a significant addition to the biochemical studies performed up to now and indicate that all TT cells produce both mRNAs and both hormones in parallel.     

Quantification of calcitonin gene expression at the cellular level in medullary carcinoma of the thyroid

Calcitonin mRNA was detected in human medullary carcinoma using an in situ hybridization technique. This specific and reproducible method could lead to a better functional characterization of medullary carcinoma and should allow the definition of new prognosis factors in medullary thyroid cancer.     

Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.     

Oncogene expression in a medullary thyroid carcinoma

We report the expression of Ha-ras, fos, c-myc and N-myc mRNA in a human medullary carcinoma of the thyroid gland, both in primary tumor and lymph node metastasis, as demonstrated by in situ hybridization and Northern blot analysis. A significant difference in the oncogene expression in the primary tumor and the metastasis was not observed. Tumor tissue revealed a significant overexpression of Ha-ras, c-myc and N-myc mRNA as compared to the normal thyroid gland. The amount of fos mRNA expression in non tumorous thyroid gland did not significantly differ from tumor tissue, sis, fms and abl mRNA expression was not detectable in tumor tissue and non tumorous thyroid gland. We conclude, that the (over)expression of the oncogenes Ha-ras, c-myc and N-myc may be associated with initiation and progression of medullary thyroid carcinoma. Similar studies on additional cases of human medullary thyroid carcinoma will be necessary to reveal further information.     

Biosynthesis of calcitonin by a rat medullary thyroid carcinoma cell line

The 32-amino acid form of the peptide hormone calcitonin is the product of a series of post-translational processing steps of a 13,400-dalton precursor, procalcitonin. We have now identified the steps involved in proteolytic paring of the precursor to the mature secretory form. Cultures of the CA-77 cell line were radiolabeled and the various forms of calcitonin were isolated by specific immunoprecipitation followed by fractionation on gel filtration and reversed-phase high performance liquid chromatography. Pulse-chase kinetics showed that procalcitonin was cleaved to a 6,500-dalton biosynthetic intermediate which was subsequently processed to the size of mature calcitonin (3,400 daltons). Partial microsequencing of the [35S] methionine-labeled intermediate indicated that the sequence consisted of the COOH-terminal 52 residues of procalcitonin. Partial microsequencing of the [35S]methionine- or [3H]proline-labeled 3,400-dalton species revealed that it was indistinguishable from naturally occurring, amidated calcitonin. These data define the major pathway for calcitonin biosynthesis in this neoplastic cell line and presumably in normal cells.     

Simultaneous localization of calcitonin mRNA and peptide in a medullary thyroid carcinoma

We report the visualization of calcitonin gene expression products at the mRNA and peptide levels on the same section of a medullary thyroid carcinoma by combined in situ hybridization and immunohistochemistry. mRNA detection was accomplished by hybridization with radioactively labeled antisense RNA probes followed by autoradiography and immunohistochemically using the avidin-biotin complex method. Best results were obtained when in situ hybridization preceded immunohistochemistry, as determined by quantitative analysis of the autoradiographs. When immunohistochemistry was performed prior to in situ hybridization, the RNase inhibitor heparin had to be added to the antibodies to retain hybridizable mRNA. The intensity of the two reactions varied in individual cells, indicating a functional heterogeneity of tumor cells with regard to calcitonin mRNA content and storage of the related immunoreactive peptide. These results, in combination with elevated serum calcitonin levels, suggest significant differences in the rate of secretion of individual tumor cells. Simultaneous localization of mRNA and its peptide within the same cell may, therefore, provide further insight into gene expression and secretory activity at the single cell level.     

Release of calcitonin by cultures of medullary carcinoma of the thyroid.

Three biopsies of medullary carcinoma of the thyroid were grown in monolayer culture. All three cultures initially released high levels of calcitonin into the medium, but the conretion from the culture cells was not stimulated when the medium calcium concentration was increased from 1.8 to 3.6 mEq/L. Four peaks of calcitonin immunoreactivity were found when the culture medium of one cell line was fractionated by gel filtration on Bio-Gel P-10. This closely corresponded to the heterogeneous molecular profile of calcitonin in the serum of this patient and other patients with medullary carcinoma of the thyroid.