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J. Biol. Chem., Vol. 277, Issue 38, 34773-34784, September 20, 2002
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From the Department of Molecular, Cellular, and Developmental
Biology, University of Colorado, Boulder, Colorado 80309-0347 and
The mitochondrial respiratory chain is required
for the induction of some yeast hypoxic nuclear genes. Because the
respiratory chain produces reactive oxygen species (ROS), which can
mediate intracellular signal cascades, we addressed the
possibility that ROS are involved in hypoxic gene induction. Recent
studies with mammalian cells have produced conflicting results
concerning this question. These studies have relied almost exclusively
on fluorescent dyes to measure ROS levels. Insofar as ROS are very
reactive and inherently unstable, a more reliable method for measuring
changes in their intracellular levels is to measure their damage
(e.g. the accumulation of 8-hydroxy-2'-deoxyguanosine
(8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure
the expression of an oxidative stress-induced gene, e.g.
SOD1. Here we used these approaches as well as a
fluorescent dye, carboxy-H2-dichloro-dihydrofluorescein diacetate (carboxy-H2-DCFDA), to determine whether ROS
levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift
to anoxia. These studies also reveal that carboxy-H2-DCFDA
is an unreliable reporter of ROS levels in yeast cells shifted to
anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have
found that specific proteins become carbonylated during a shift to
anoxia and that some of these proteins are the same proteins that
become carbonylated during peroxidative stress. The mitochondrial
respiratory chain is responsible for much of this carbonylation.
Together, these findings indicate that yeast cells exposed to anoxia
experience transient oxidative stress and raise the possibility that
this initiates the induction of hypoxic genes.
Exposure of Yeast Cells to Anoxia Induces Transient Oxidative
Stress
IMPLICATIONS FOR THE INDUCTION OF HYPOXIC GENES*
, and AMC Cancer Research Center,
Denver, Colorado 80214
*
This work was supported by National Institutes of Health
Grant GM 30228 (to R. O. P.) and a National Institutes of Health postdoctoral fellowship (to K. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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