An investigation of the role of tissue transglutaminase in programmed cell death. (Apoptosis)

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Thomas, G.L., 1998. An investigation of the role of tissue transglutaminase in programmed cell death. (Apoptosis). PhD, Nottingham Trent University.

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Abstract

The purpose of this study was to investigate the role of the tissue type II transglutaminase (tTGase) enzyme, (E.C. 2.3.2.13) in programmed cell death (apoptosis). tTGase is a member of a large family of enzymes which catalyse an irreversible crosslinking reaction between proteins, forming large insoluble polymeric complexes. Apoptosis is a form of cell death that is opposite to necrosis. Often referred to as "cell suicide", the cells shrink and break up forming small bodies which are the removed from healthy tissues by phagocytosis, with no resultant damaging immunological responses, as in necrosis. As homeostasis is maintained at all times during the apoptotic event it is thought to be a programmed or physiological form of cell death. Two, unique, polyclonal antisera, were raised against transglutaminase-mediated protein structures rich in the catalysis product of the s (γ-glutamyl) lysine isodipeptide : i) a high molecular weight sodium dodecyl sulphate (SDS) / insoluble polymer and ii) the SDS / insoluble "apoptotic envelope".

Hepatocellular carcinomas, formed by induction-feeding with 2-acetylaminofluorene (2-AAF) in the diet of rats, when examined were found to contain time-dependent increasing amounts of both of the tTGase-mediated antigens. Conversely, three metastatic variants of a Hamster fibrosarcoma cell-line when resected in vivo produced tumour masses which showed both a decrease in tTGase catalytic activity and amounts of both tTGase- mediated antigens. This difference was concluded to be due to a cell type dilution effect, caused by the heterogenous hypercellularity found in the hepatocellular carcinoma.

In vitro studies showed that tTGase activity and antigen could be induced in a time- and dose-dependent manner in two neoplastic cell-lines, WI38 VA13a (an SV40 transformed Human lung fibroblast) and HT29 (an endothelial cell isolated from an Human colon carcinoma) by treatment with sodium butyrate (NaB, 1-5 mM over 1-5 days) and all-trans retinoic acid (R.A., 10-50 μM over 1-5 days) respectively. Detectable levels of apoptosis, shown by SDS-isolation and counting ELISA quantitation of "apoptotic envelopes" in both in vitro models did not match the increase in tTGase enzymatic induction. Furthermore, it was shown that NaB altered the normal cell cycle of WI38 VA13a to produce an anueploid sub-population of cells which evaded both mitosis and apoptosis. Morphological examination by confocal laser microscopy, showed the presence of small "apoptotic" light-refi-active bodies (ARB's) in the HT29 / R.A. model. Characterisation of the ARB's by Normarski phase contrast microscopy showed them to be wholly intracellular fractions of the intact apoptotic cells. Numbers of apoptotic cells in both in vitro models was seen to be independent of the corresponding induction of tTGase. Finally, using a novel cell permeabilisation assay system, applied to the two tTGase induction models in WI38 VA13a and HT29, and to a third system on the Human umbilical vein cloned endothelial cell-line ECV 304, it was shown in situ that via the catalytic action of tTGase the biotinylated polyamine cadaverine can be specifically incorporated into a 42 kDa amine acceptor substrate. This acceptor protein was believed to be an essential extracellular matrix protein substrate of tTGase, crosslinked during the apoptotic event. Attempts were made to isolate and characterise this protein but amino acid sequence data could not be obtained.

The data in this thesis have shown that apoptosis can occur independently of tTGase activity; tTGase is therefore not an essential trigger for apoptosis initiation. However, tTGase has been shown to alter the biophysical nature of the apoptotic cell playing a fundamental role in formation of irreversible protein polymers in the apoptotic cell in vivo and stabilisation of the apoptotic envelope in vitro, via crosslinking of substrate proteins in the extracellular matrix.

Item Type:

Thesis

Creators:

Thomas, G.L.

Date:

1998

ISBN:

9781369323375

Identifiers: