Approaches to Define the Involvement of Reactive Oxygen Species and Iron in Ultraviolet-A Inducible Gene Expression

  • Charareh Pourzand
  • Olivier Reelfs
  • Rex M. Tyrrell
Part of the Methods in Molecular Biology™ book series (MIMB, volume 99)

Abstract

Oxidative stress is implicated in a wide range of human diseases as well as0 the ageing process and major efforts have been concentrated on development of markers of this state in cells. The discovery that the expression of the gene which encodes the heme catabolic enzyme, heme-oxygenase-1 (HO-1), is strongly induced by oxidizing agents such as ultraviolet-A (UVA, 320–400nm) radiation, has provided a powerful indicator of cellular redox state (see also  Chapter 23). This phenomenon, which was originally observed in primary human cultured skin fibroblasts, was later shown to occur in most human cell types, although not in keratinocytes (1). The expression of the HO-1 gene is clearly dependent on cellular reducing equivalents. It is induced by several oxidants, including hydrogen peroxide (H2O2), and glutathione (GSH) depletion strongly enhances both basal levels and oxidant-induced expression of the gene (2,3). The enhanced expression of the HO-1 gene by UVA and other agents can be entirely accounted for by a very strong enhancement in transcription rate (4) and given the fact that the mRNA for human HO-1 is relatively stable (half-life 2.7 h, ref. [4]), the measurement of mRNA accumulation by the Northern blot technique is evidently the simplest way of monitoring expression. This approach is currently used in several laboratories as a positive control when testing other genes suspected of being oxidant inducible. A second redox-regulated gene is CL-100, whose transcription is also strongly activated by UVA and oxidants (5). CL-100 is interesting since it encodes a dualspecificity (tyrosine/threonine) protein phosphatase that specifically inactivates cellular mitogen-activated protein (MAP) kinases. It is therefore implicated in the modulation of signal transduction events involved in the cellular stress response (3,5).

Keywords

DMSO NADPH Thiol Porphyrin Mannitol 

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Charareh Pourzand
    • 1
  • Olivier Reelfs
    • 1
  • Rex M. Tyrrell
    • 1
  1. 1.Department of Pharmacy and PharmacologyUniversity of BathBath

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