Taylor, Samantha; (1993) The expression of mature human calcitonin in Caco-2 cells. Masters thesis (M. Phil.), UCL (University College London).

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Abstract

The aim of this work was to transfect genetic material encoding the peptide, human calcitonin (hCT) into Caco-2 cells (an intestinal epithelial cell model, Hidalgo et aL, 1989) and to examine secretion, in particular from the basolateral surface. It was expected that information on the intracellular sorting of hCT by the Caco-2 cell model would give valuable insight into peptide processing by GI epithelial cells and have implications for the oral delivery of the peptide. cDNA encoding hCT and a small genomic fragment of the hCT gene, CALC-I were successfully transfected into Caco-2 cells. This was confirmed by resistance of transfected cells to G418, a neomycin analogue. The neomycin gene was a marker for the plasmids with which the cells were transfected, G418 being found to be cytotoxic to parent Caco-2 cells within 14 days. All mammalian expression constructs were also transfected into COS cells. Electroporation of COS cells was validated by transfection of the cells with a plasmid encoding Esherichia coli β-galactosidase. hCT secretion could not be detected in transfected COS cells or Caco-2 cells by immunoprecipitation. However, rat calcitonin (rCT) secretion from the rat medullary thyroid carcinoma cell line, rMTC 6-23 could not be detected by immunoprecipitation using the same method. Radioimmunoassay (RIA) of cell medium and cell lysates subjected to concentration on a Sep-Pak Cig cartridge confirmed secretion of rCT by 6-23 cells. This was found to be 0.32 ± 0.03 ng rCT.mg-1 total cellular protein medium and 0.10 ± 0.01 ng rCT.mg-1 total cell protein for cell lysates. No detectable hCT was found in or secreted from transfected Caco-2 or COS cells. Results are discussed in relation to calcitonin gene expression and suggestions are made as to how one might proceed with the project by examination of gene transcription and subsequent expression of hCT or its precursors in Caco-2 cells.

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