<p>Mammalian cells have the ability to endogenously generate and metabolize hydrogen peroxide (H₂O₂). This H₂O₂ can interact with protein and lipid components of intracellular signal transduction pathways to regulate cell behaviour. It has been demonstrated in our laboratory and by others that many types of cells, including transformed cells, require H₂O₂ for efficient growth in culture. Excess H₂O₂ has long been identified as a source of oxidative stress that can mediate cell growth arrest and death. However, the addition of the H₂O₂ scavenger catalase to culture media or the overexpression of intracellular catalase can block proliferation. This effect is reversed by inactivation of the enzyme or upon co-incubation of cells with H₂O₂ and H₂O₂-generating sources such as glucose oxidase. Thus, at non-toxic levels, H₂O₂ appears to act as a cellular growth factor. The purpose of this work is to help elucidate molecular mechanisms underlying this H₂O₂-regulated aspect of cell physiology. The effects of extracellular H₂O₂ level manipulation upon the activities of the HER-2/Neu receptor tyrosine kinase, mitogen-activated protein kinases (MAPKs), and stress-activated protein kinases (SAPKs) are discussed. The c-Jun-NH₂terminal kinase 1 (JNK1), a member of the SAPK family, is involved in several diverse aspects of cellular functioning including apoptosis and transformation. The JNK1 signal has also been implicated as a cell sensor of redox (reducing/oxidizing) stress. We have observed that the growth-inhibitory effect of both high level H₂O₂ treatment and H₂O₂-scavenging catalase treatment is accompanied by a transient increase in JNK1 activity. To determine the importance of this response in growth regulation, the JKN1 signal wa stably altered in SK-OV-3 human ovarian adenocarcinoma cells by the expression of ectopic JNK1 (HA-JNK1). Levels of HA-JNK1 protein expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase activity, however with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H₂O₂. Transient HA-JNK1 expression also reduced H₂O₂ generation, and this effect was reversed by the JNK inhibitor SP600125. These resultts indicate that the JNK1 stress response contributes to the inhibition of proliferation by catalase treatment, possibly via additional reductions in environmental H₂O₂ caused by a lowered endogenous production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon basal H₂O₂ levels for efficient growth. These data contribute to the understanding of the mechanisms involved with the overexpression and/or hyperactivity of JNKs observed in certain cancers.</p> / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/6033 |
| Date | 02 1900 |
| Creators | Preston, Thomas J. |
| Contributors | Singh, Gurmit, Medical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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