Regulation Mechanisms of Peptide Hormones in Neuroendocrine Cancers

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 autocrine feedback loop was operating in DMS53, and based on the hCTR2 isoform, may be mediated by signal transduction through the cAMP- and Ca2+- dependent signalling pathways. To assess which signalling enzymes are activated by hCTR, signal transduction pathways were investigated using small molecule enzymes inhibitors, and their effects on the levels of CT-related species observed. It was observed that treatment of DMS53 cultures with the protein kinase C inhibitor, GF109203X had an effect on the levels of CT-related species in the medium. Again, the relative levels of the intracellular CT-related species were not changed by treatment with this inhibitor. This implicated PKC as a component of the hCTR signal transduction pathway. It was concluded that DMS53 cultures have mechanisms to maintain the intracellular and extracellular concentrations of CT-related species. The concentration of extracellular CT is regulated by a positive feedback mechanism, mediated by hCTR activation, and subsequent signalling involving PKC and AC. Treatment with biosynthetic and signalling inhibitors had no significant effect on the intracellular levels of CT-related species, demonstrating that DMS53 cultures prioritise tight control of intracellular concentrations over extracellular concentrations. With the methodology to detect and quantify peptide hormones in cell culture medium and lysate in hand, the generality of CT glycosylation was explored. Preliminary experiments successfully characterised the presence of glycosylated CT and CT-G in the medullary thyroid carcinoma cell line, TT. To broaden the range of detected hormones, HPLC-fluorescence methodology was developed to detect and quantify oxytocin (OT) and its precursors, and this methodology was used to investigate the presence of OT in the DMS79 SCLC cell line. Show more