Lowrie, Alastair Graeme

2019-02-15T14:33:27Z

2019-02-15T14:33:27Z

2006

Proteolysis inducing factor (PIF) is a pro-cachectic glycopeptide purified from the urines of mice innoculated with the MAC 16 tumour and from the urines of weight losing patients with pancreatic carcinoma. It arises from the dermcidin gene which produces 2 other peptides, Y-P30, an unglycosylated neuronal survival factor with homology to the PIF peptide core and DCD-1, an antibiotic peptide secreted by eccrine sweat glands which has no homology with PIF. We sought to investigate PIF / dermcidin expression in cell lines and pancreatic carcinoma tissue, the role of proteolysis inducing factor as a growth and survival factor in tumour cells and the influence on these effects of the structural features known to be important to the induction of cachexia by PIF and the functions of Y-P30 and dermcidin. In vitro expression was assessed by PCR, Western blotting, in vitro translation and immunocytochemistry. In vivo expression was assessed using laser capture microdissection, PCR, real-time PCR, Western blotting, immunoprecipitation and the analysis of urine by mass spectrometry. Growth and survival functions were assessed in the HuH7 cell line with immunocytochemistry and flow cytometry. Cells were treated with a synthetic PIF peptide or stably transfected with PIF / dermcidin expression vectors. Site-directed mutagenesis of these vectors was used to assess glycosylation and the role of different sequences within the polypeptide. Dermcidin was expressed by pancreatic carcinoma cell lines and primary human hepatocytes but not by the HuH7 cell line. In HuH7 cells induced expression promoted cell growth and improved survival following oxidative stress. The YP-30 / PIF core peptide sequence was sufficient to induce cell growth. Survival promotion did not require glycosylation but was prevented by mutagenesis of the asparagine residues of the PIF core peptide. Analysis of mRNA expression suggested dermcidin was expressed in a low number ofpancreatic carcinomas. Non-specific antibody binding prompted the development of mass spectrometry for detection of PIF in urine samples of patients with these tumours. This did not reveal PIF but did demonstrate other proteomic changes. We have demonstrated growth and survival functions of PIF/dermcidin which may be relevant in a range of physiological and pathological processes including cachexia and neoplasia. The PIF core peptide sequence appears to be important for these effects but its glycosylation does not appear to be required. However, differential glycosylation may account for PIF's cachectic affects and the difficulty in its immunological detection. Further investigation using techniques such as mass spectrometry may cast further light on the structure of PIF and its relationship to disease processes.

http://hdl.handle.net/1842/34972

The University of Edinburgh

Annexe Thesis Digitisation Project 2019 Block 22

Already catalogued

Neoplastic expression and cellular functions of proteolysis inducing factor/dermcidin

Thesis or Dissertation

Doctoral

MD Doctor of Medicine