Abstract
Oxygen radicals generated during hypoxia cause oxidative base modifications in the VEGF promoter that may be linked to induction of VEGF mRNA expression (FASEB J. 19: –394, 2005). To explore this idea, we searched for oxidative modifications in functionally relevant sequences of genes that are up-, down-, or non-regulated by hypoxia. PCR was used to amplify selected DNA sequences before and after treatment with formamidopyrimidine-DNA glycosylase (Fpg), an N-glycosylase and AP-lyase that releases damaged bases from DNA, under the premise that if oxidation products are present, then Fpg will reduce PCR amplification efficiency. Hypoxic, but not normoxic, PAECs displayed Fpg-induced reductions in PCR amplification of the hypoxic response elements of the VEGF and HO-1 promoters but not in functionally insignificant promoter sequences or in intronic or exonic coding sequences. Fpg-detectable modifications were related in time to changes in VEGF and HO-1 mRNA expression. Neither promoter nor coding sequences of the hypoxia down-regulated ODC gene displayed Fpg sensitive modifications. Similarly, neither promoter nor coding sequences of the constitutively active actin gene were modified in hypoxia. These observations suggest that hypoxia-induced oxidative base modifications are targeted to functionally significant sequences of hypoxia-inducible genes.
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