Effect of Bcl-2 on the cellular response to oxidative stress
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Exposure of cells to hydrogen peroxide can cause oxidative damage to cellular constituents including lipids, protein, and DNA. At elevated concentrations, hydrogen peroxide can trigger cell death by apoptosis or necrosis. Apoptotic cell death can be prevented by overexpression of the oncoprotein Bcl-2. The exact mechanism by which Bcl-2 blocks cell death is controversial. Some researchers believe that Bcl-2 possesses antioxidant properties that protect cells from apoptosis. The purpose of this thesis was to assess oxidative stress and apoptosis following hydrogen peroxide exposure in Jurkat T cells overexpressing Bcl-2. One of the major objectives was to ascertain whether or not Bcl-2 overexpression elevated the antioxidant capacity of Jurkat T cells to provide protection from oxidant-induced cell death. Hydrogen peroxide treated Jurkat cells became apoptotic at moderate levels of oxidant (25-100 uM H2O2), and necrotic at higher doses (greater than 200 uM H2O2). Bcl-2 overexpression prevented caspase activation and cell death at the apoptotic doses of H2O2, but not the necrotic doses. Caspase inhibition studies demonstrated that Bcl-2 overexpression provided a greater level of resistance from H2O2-induced cell death than the broad-spectrum caspase inhibitor z-VAD.fmk. A systematic study was carried out examining the antioxidant status of Jurkat cells overexpressing Bcl-2. Several Bcl-2 transfectants were utilised for the study, so that any differences seen could be correlated to the level of Bcl-2 expression. Surprisingly, there were no statistically significant differences among the Bcl-2 transfectants for any of the antioxidant enzymes. Jurkat cells overexpressing Bcl-2 exhibited the same level of oxidative damage to lipids and protein in response to H2O2 exposure as the parental Jurkat cells. Interestingly, Jurkat cells overexpressing Bcl-2 continued to grow in culture after H2O2 exposure, despite harboring damage to cellular constituents. Consistent with these results, H2O2 treated Jurkat cells overexpressing Bcl-2, which failed to undergo apoptosis, were more prone to genomic instability. Together, these findings suggest that Bcl-2 overexpression protects Jurkat cells from H2O2-induced cell death by blocking apoptosis. Jurkat cells overexpressing Bcl-2 were no better at detoxifying oxidants and showed the same level of oxidative damage following H2O2 exposure. As a result, the overexpression of Bcl-2 considerably enhanced the mutagenicity of H2O2.