A variety of proteases have been shown to promote the progression of cancer by virtue of their ability to degrade extracellular proteinaceous barriers, such as
basement membrane and interstitial stroma. At the outset of this study available
evidence strongly implicated cathepsin D in breast cancer metastasis. It was
envisaged that an antibody inhibitory to the activity of this enzyme might retard
invasion, and restrain a tumour from spreading. To this end anti-peptide
antibodies were generated against a peptide sequence derived from the substrate
capturing "flap" of the enzyme. Inhibition of enzyme activity by these antibodies
could not be demonstrated, probably due to the lack of a suitably sensitive
enzyme assay. However, the rationale of this study and the expertise gained from
it could be applied, in the future, to enzymes that have since been found to be
more relevant to tumour invasion.
A feature of many transformed cells is an anomalous lysosomal enzyme
trafficking system, and concomitant hyper-secretion of some enzymes. The
distribution of low pH compartments and lysosomal enzyme-containing
compartments was investigated in human breast epithelial cells, and their c-Ha-ras-
transformed counterparts. Immunofluorescence and immunoelectron
microscopy showed that these compartments have a more peripheral cellular
distribution with respect to normal cells, and cathepsins B and D were cell
surface-associated.
Studies were undertaken to reveal the extracellular matrix degrading ability of c-Ha-
ras-transformed cells. Transformed cells exhibited increased degradation of
fluorescein-labelled extracellular matrix in serum free medium, and increased motility, and degradation and disruption of extracellular matrix in serum-containing
medium. In vitro invasion through artificial basement membrane by
transformed cells was investigated using scanning electron microscopy, and was
further used to preliminarily identify the proteases involved in invasion by
specific inhibition. By this means, greatest inhibition of in vitro invasion was
obtained using a specific metalloproteinase inhibitor. Overexpression by
transformed cells of a metalloproteinase was detected by gelatin zymography.
Together these results suggest that the increased invasive capacity of ras-transformed
breast epithelial cells may be largely due to increased
metalloproteinase activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg , 1996.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9728 |
| Date | 11 October 2013 |
| Creators | Fortgens, Philip Hendrik. |
| Contributors | Dennison, Clive. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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