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Title: Tissue transglutaminase & tumour progression : cell death & extracellular matrix stabilisation
Author: Parry, James
ISNI:       0000 0001 3473 2830
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2002
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The importance of tissue transglutaminase (tTGase, type 2) in tumour progression has been a matter of some debate for a number of years. The role of the enzyme in cell death was investigated following a study carried out some years earlier suggesting that treatment of Met B cells with dexamethasone caused an increase in tTGase within the cells and this was followed by an increase in apoptosis, as measured by the formation of SDS-insoluble bodies. Dexamethasone treatment was shown to induce a powerful, dose-dependent, mRNA synthesis-dependent increase in tTGase activity. The number of SDS-insoluble bodies was found to be comparable with increases in tTGase activity, however, no increase in apoptosis was observed when DNA fragmentation, induction of Caspase-3 and ATP:ADP ratios indicators of classical apoptosis were used. Incubation of cells with the labelled primary amine substrate fluorescein-cadaverine led to its incorporation into cellular proteins following damage resulting from exposure to trypsin during cell passage. The formation of these bodies was shown to be via the activation of tTGase following loss of Ca²⁺ homeostasis rather than induction of classical apoptosis, since Met B cells expressing the bcl-2 cDNA were not protected from SDS-insoluble body formation. It was concluded that a novel type of cell death was taking place which was related to the intracellular activation of tTGase. Met B cells were further treated with a variety of apoptosis inducing agents both in the presence and absence of dexamethasone. Treatment of the cells with dexamethasone showed no increase in apoptosis, inferring a type of protection. This new form of cell death was shown to be evident in other cell types (e.g. the P8 rat osteosarcoma), and occurring naturally in organs of normal animals. It was also demonstrated that this type of death could be induced in tumours, following systemic injections of dexamethasone and intra-tumour injections of ionomycin. Given the increasing evidence for the involvement of tTGase in cell attachment, migration and extracellular matrix stabilisation, work was continued using a partially characterised transplantable rat osteosarcoma (P8). A reduction in tTGase expression in the primary tumour was observed during tumour growth and metastasis. In contrast, secondary tumours showed high levels of expression of tTGase. Immunohistochemical techniques, in situ hybridisation and in situ activity experiments showed fibrillar patterns of staining of extracellular tTGase, which become reduced as the tumour grows and as tTGase expression drops. Also observed was an increase in the deposition of matrix proteins tibronectin and general collagens, although this may be an accumulation of matrix as the tumour grows, rather than an increase in expression. The limited amount of angiogenesis within the P8 osteosarcoma was also noted. Cells isolated from the P8 primary tumour were transfected with tTGase cDNA under the control of a constitutive viral promoter. These clones showed increased cell attachment and matrix processing when compared to transfected controls and wild type P8 cells. This is in agreement with current research on tTGase localised at the cell surface. These clones did not form tumours when re-injected back into animals, supporting previous data obtained using other tumour models.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Apoptosis