An analysis of the molecular regulation of tumor necrosis factor-alpha expression in human mast cells
Date
2000-03
Authors
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Publisher
ORCID
Type
Degree Level
Masters
Abstract
The objective of this study was to analyze the molecular regulation of tumour
necrosis factor-alpha (TNF-α) gene expression in human cells. Allergic diseases (e.g.,
asthma) comprise one of the most prominent medical problems in Canada, and their
incidence and severity are on the increase here as in most industrialized countries.
Allergic responses are initiated by contact of allergens with specific IgE antibodies on
mast cells, leading to cellular activation. Such activation induces mast cells to release
very large amounts of the pro-inflammatory cytokine TNF-α which, by itself, mediates
much of the cellular recruitment associated with the allergic late phase response (LPR).
Most of the pathology of allergic diseases is directly attributable to the LPR. The
molecular mechanism regulating TNF-α expression in mast cells, as opposed to that in
macrophages (which can also produce an abundance of TNF-α) is poorly understood. My
hypothesis is that if TNF-α were differentially regulated in mast cells, it might be
possible to therapeutically manipulate its highly pathogenic contributions to allergic
diseases without affecting the other roles of this cytokine in the body. I examined the
kinetics of TNF-α gene transcription and protein expression in mast cell-differentiated
KU812 cells which I stimulated with phorbol-12, 13-myristate acetate (PMA) and
calcium ionophore A23187 (PMA and A23187 effectively mimic FcεRI stimulation of
mast cells). RT-PCR and ELISA methods were used to detect TNF-α mRNA and protein,
respectively. My results indicate that low levels of TNF-α is stored in the unstimulated
cells (but not in the supernatant fluids), high levels of TNF-α were secreted into the
culture supernatants within 2 hr after stimulation. The critical regulatory elements in the
human TNF-α promoter were studied. A human TNF -a gene promoter fragment (-625 to +19 bp relative to the transcription start site; TSS) was enzymatically digested from the
5' end inward and thereby I generated a series of promoter fragments of varying sizes.
Each of these promoter fragments were placed immediately 5' to a cDNA encoding the
marker protein luciferase. Then the constructs were transfected into differentiated KU812
cells and I assessed the TNF promoter-driven luciferase expression in the mast cells
following PMA/A23187 challenge. A negative (-331 to -205) and several positive (-82 to
+19, -131 to -82, -205 to -153) control regions were identified within the TNF-α
promoter.
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Degree
Master of Science (M.Sc.)
Department
Veterinary Microbiology
Program
Veterinary Microbiology