The mechanisms by which altered processing, distribution and secretion of proteolytic
enzymes occur, facilitating degradation of the extracellular matrix in invasive and
metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell
line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown
that the ras-transfected cell line has a more alkaline pH. The objective of this study was to
determine which organelles of the endosome-lysosome route were alkalinized and shifted
to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl
(DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin
(DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated
to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In
rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein
(OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a
sustained anti-OVA (anti-self) response (implying a potential strategy for cancer
immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent
microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake,
immunolabelling for DNP (a component of DAMP) and unique markers for the early
endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent
mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense
morphology and lysosome-associated membrane protein-2, LAMP-2) and electron
mlcroscopy was performed. The pH of late endosomes and lysosomes in the
ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and
Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required
to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR),
process the precursors to active proteases and to allow receptor recycling. The more
alkaline pH observed potentially explains the altered processing of proteases in rastransfected
cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for,
among other things, the positioning and trafficking of various organelles, causing
relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may
enable fusion of Iysosomes with the plasma membrane and the release of proteolytic
enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/5511 |
| Date | January 2005 |
| Creators | Jackson, Jennifer Gouws. |
| Contributors | Elliott, Edith. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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