Zabaglo, Lila; (2000) The relationship between hypoxia, proliferation and apoptosis in murine CaNT tumours and human HT29 xenografts. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tumour hypoxia and cell proliferation are two factors which greatly influence cancer therapy. In this thesis the cell biology of tumours whose internal micro-environment was perturbed by therapeutically-relevant radiation doses or an oxygenation modifier (carbogen) was investigated. A well-defined murine experimental tumour, CaNT, and a widely used human tumour xenograft, HT29 were used in vivo and in vitro to study the relationship between hypoxia, proliferation and apoptosis. Hypoxia was assessed by measuring the incorporation of 7-(4'-(2-nitroimidazole-1-yl)-butyl)-theophylline (NITP), an immunologically identifiable hypoxia marker that binds bioreductively to cells under low-oxygen conditions. Experiments investigating the influence of various oxygen levels on NITP binding in separate phases of the cell cycle showed that aneuploid G2 cells disaggregated from CaNT tumours bind more NITP at all oxygen tensions than cells in other phases. The NITP binding in G1 and S phase cells was similar which could suggest that this phenomenon may be independent of cell size. HT29 xenografts showed a similar pattern of NITP binding, but with a lower overall nitroreductase activity compared with that seen in the murine tumours. CaNT and HT29 cells grown in vitro for several cell generations showed a different pattern of NITP binding. Simultaneous fluorescent staining of cells for hypoxia (NITP), proliferation (BrdUrd) and DNA content showed that treatment with a single fraction of carbogen induced a G2 phase delay in CaNT tumours. A 2-hour exposure of CaNT tumours to carbogen induced apoptosis, measured with the TdT assay, which could reflect re-perfusion injury to previously hypoxic cells. The experiments exploring the influence of carbogen breathing on proliferation in HT29 xenografts showed marked changes to the cell cycle. In HT29 xenografts carbogen treatment did not alter the cell cycle progression of oxic cells, whereas hypoxic cells completely halted progression through the cell cycle. The apoptotic response of CaNT and HT29 cell lines to radiation-induced DNA damage was investigated both in vivo and in vitro. CaNT and HT29 cell lines showed little or no radiation-induced apoptosis in vitro respectively. In contrast, in vivo doses as small as 0.5 Gy caused marked apoptosis. In CaNT tumours, the number of apoptotic cells increased from 10% in the controls to 33% 2 hours post treatment with 0.5 Gy. The percentage of apoptosis remained at an elevated level (22% even 24 hours after irradiation. HT29 xenografts treated with 0.5 and 5 Gy X-rays showed 24 and 26% apoptosis respectively. The influence of micro-environmental factors such as hypoxia, nutrient deprivation and cell density on the induction of apoptosis was tested in vitro. However none of these factors could explain the differences between the apoptotic response to ionising radiation in vivo and in vitro.

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