dc.contributor.author | Lowrie, Alastair Graeme | en |
dc.date.accessioned | 2019-02-15T14:33:27Z | |
dc.date.available | 2019-02-15T14:33:27Z | |
dc.date.issued | 2006 | en |
dc.identifier.uri | http://hdl.handle.net/1842/34972 | |
dc.description.abstract | | en |
dc.description.abstract | 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. | en |
dc.publisher | The University of Edinburgh | en |
dc.relation.ispartof | Annexe Thesis Digitisation Project 2019 Block 22 | en |
dc.relation.isreferencedby | Already catalogued | en |
dc.title | Neoplastic expression and cellular functions of proteolysis inducing factor/dermcidin | en |
dc.type | Thesis or Dissertation | en |
dc.type.qualificationlevel | Doctoral | en |
dc.type.qualificationname | MD Doctor of Medicine | en |