Abstract
Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200-400 ppm O(2) or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.
Highlights
DNA replication in mammalian cells is subject to a fast acting regulation that depends on the O2 tension in the cellular environment
As inhibition of mammalian replicon initiation and elongation under hypoxia is relieved by addition of deoxycytidine even without reoxygenation, it is possible that ribonucleotide reductase acts as a sensor of O2-dependent replication control transmitting its signal through alterations in the deoxynucleoside triphosphate pool
As glucose can mimic or modulate diverse cellular responses induced by hypoxia [11, 13, 14, 19, 20], we asked whether Simian virus 40 (SV40) DNA replication is influenced by variations of the glucose concentration in the cell culture medium of virus-infected CV1 cells
Summary
DNA replication in mammalian cells is subject to a fast acting regulation that depends on the O2 tension in the cellular environment. We examined the incorporation of [methyl-3H]deoxythymidine into SV40 DNA at the two glucose concentrations by pulse labeling of SV40-infected cells at different times after start of hypoxic or normoxic incubation.
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