Regulation Mechanisms of Peptide Hormones in Neuroendocrine Cancers
type: CreativeWork
description: In the work described in this thesis, analytical methods for the detection and quantification of peptide hormones featuring on-line analyte concentration, post-separation tagging and HPLC-fluorescence detection were presented. These methods were used to detect and quantify calcitonin (CT) and its prohormones glycyllysyllysine-extended CT (CTGKK), glycyllysine-extended CT (CTGK) and glycine-extended CT (CT-G) for the first time, in DMS53 small cell lung carcinoma (SCLC) cell culture medium and lysate. Additionally, novel glycosylated versions of each species were also identified, suggesting the presence of a parallel biosynthetic pathway in DMS53. Extracellular but not intracellular levels of CT were reduced as a result of treatment with biosynthesis inhibitors, and it was suggested CT precursor flux through the glycosylated pathway acts as a bypass mechanism to maintain intracellular CT levels. Moreover, the up-regulation of extracellular levels of CT-related species in response to increased medium volume provided evidence of a homeostatic feedback loop maintaining extracellular CT concentrations. To interrogate the mechanism of this feedback, DMS53 cultures were treated with a specific human calcitonin receptor (hCTR) agonist, SUNB8155, to determine if the hCTR is involved in the regulation of CT. It was observed that the relative levels of extracellular CT increased with SUNB8155 treatment, but that the relative levels of the intracellular CT-related species were unchanged. This suggested that hCTR is expressed in DMS53, and that activation of the receptor influences the expression and biosynthetic processing of CT-related species. To investigate this hypothesis, hCTR was identified in DMS53 cells using reverse transcription PCR and Western blot analyses. Specifically, transcriptional and translational evidence of the isoform hCTR2 was identified. Thus, for the first time,hCTR activation was implicated in the up-regulation of CT. This suggested that a positive aut…
abstract: In the work described in this thesis, analytical methods for the detection and quantification of peptide hormones featuring on-line analyte concentration, post-separation tagging and HPLC-fluorescence detection were presented. These methods were used to detect and quantify calcitonin (CT) and its prohormones glycyllysyllysine-extended CT (CTGKK), glycyllysine-extended CT (CTGK) and glycine-extended CT (CT-G) for the first time, in DMS53 small cell lung carcinoma (SCLC) cell culture medium and lysate. Additionally, novel glycosylated versions of each species were also identified, suggesting the presence of a parallel biosynthetic pathway in DMS53. Extracellular but not intracellular levels of CT were reduced as a result of treatment with biosynthesis inhibitors, and it was suggested CT precursor flux through the glycosylated pathway acts as a bypass mechanism to maintain intracellular CT levels. Moreover, the up-regulation of extracellular levels of CT-related species in response to increased medium volume provided evidence of a homeostatic feedback loop maintaining extracellular CT concentrations. To interrogate the mechanism of this feedback, DMS53 cultures were treated with a specific human calcitonin receptor (hCTR) agonist, SUNB8155, to determine if the hCTR is involved in the regulation of CT. It was observed that the relative levels of extracellular CT increased with SUNB8155 treatment, but that the relative levels of the intracellular CT-related species were unchanged. This suggested that hCTR is expressed in DMS53, and that activation of the receptor influences the expression and biosynthetic processing of CT-related species. To investigate this hypothesis, hCTR was identified in DMS53 cells using reverse transcription PCR and Western blot analyses. Specifically, transcriptional and translational evidence of the isoform hCTR2 was identified. Thus, for the first time,hCTR activation was implicated in the up-regulation of CT. This suggested that a positive aut…
author: Morgan, Jeremy Andrew McInnes
language: en
keywords: calcitonin, hCTR, calcitonin receptor, intracellular signalling, GPCR, peptide hormone, cancer, lung carcinoma, signalling pathway, feedback, TT, DMS53, HPLC, post-column, labeling, mass spectrometry
date published: 2016
date modified: 2026-07-24T00:54:54Z
provider: Australian National University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Australian National University

PropertyValue
property ID: Handle
value: http://hdl.handle.net/1885/144624