This study forms part of an investigation into the possible relevance of the
lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this
study, the main technique adopted was the Tokuyasu "cryo" method, in which the
tissues were fixed, frozen and sectioned and labelled using the relevant antibodies,
which were detected with protein A gold probes.
In order to implement the Tokuyasu technique, it was necessary to rebuild a knife
maker, for the production of adequately sharp glass knives, and to modify a sputter-coater
into a glow-discharger, for rendering carbon-coated grids hydrophilic, to
promote adhesion of hydrated sections.
This study was directed towards human tissues and peptide antibodies were
investigated as a means of avoiding isolation of proteins from scarce human tissue,
and as a means of obtaining antibodies that will target specific regions of proteins of
interest. Peptide antibodies were also considered promising for studies of
proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer
therapy. Various prediction programmes were investigated for their effectiveness
in predicting whether a given peptide sequence will elicit antibodies that will react
with the native protein. Successful prediction would increase the success rate of
peptide antibody production and thus lower the cost.
Leucocytes were studied as a model of an invasive cell, since they are more readily
available than tumour cells and serve the purpose during the development of
methods. In the course of these studies, an optimal protocol for the fixation of
PMNs was developed, involving lateral fixation of cut sections, that should be
useful for future studies on these cells. Elastase and cathepsins D and G were found
on the surface of activated PMNs and could thus play a role in the invasive
properties of these cells.
Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed
Neo-T counterparts revealed that upon transformation, lysosomes shift from a
perinuclear position, to a more peripheral position. None of the cathepsins studied
was found on the cell surface of either the normal or ras-transfected cells,
suggesting that surface distribution of these enzymes may not be a requirement for
invasiveness. These studies suggest that immunocytochemical investigation of
cells, in the process of invading through a barrier membrane, might be profitable in
elucidating the role of proteinases in invasive cancer. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9526 |
| Date | January 1993 |
| Creators | Elliott, Edith. |
| Contributors | Dennison, Clive. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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