The adenovirus E1A proteins differentially regulate AP‐1‐responsive genes. Collagenase and stromelysin are repressed by E1A, whereas the expression of c‐jun is elevated. Inhibition of collagenase has been found to be exerted through the consensus AP‐1 binding site TGAGTCA. Here we show that the distal AP‐1 binding site in the c‐jun promoter, the jun2TRE (TTACCTCA), is the decisive element of this promoter in mediating the positive response to the 243 amino acid E1A product. In vitro binding studies revealed that, in contrast to the consensus AP‐1 site which is preferentially targeted by dimers composed of the Jun and Fos families, the jun2TRE binds heterodimers composed of cJun and ATF‐2(‐like) proteins. Since stimulation of c‐jun transcription is a function of the transforming domain of E1A encoded by conserved region 1, cJun‐‐ATF‐2 may be one of the effector factors involved in transformation. The data further suggest that E1A can distinguish between cJun‐‐cJun and cJun‐‐ATF‐2 in imposing opposite states of activity.

Recent evidence suggests that vascular endothelial growth factor (VEGF) expression is up-regulated by oxidative stressors through activation of hypoxia-inducible Factor 1 (HIF-1). To investigate whether this is a general phenomenon, we studied the effects of the sulfhydryl reagent arsenite on VEGF expression in human ovarian cancer cells. Arsenite potently induces the production of reactive oxygen species (ROS) in several cell systems and directly interacts with sulfhydryl groups of cellular thiols. We report that arsenite induces VEGF mRNA and protein levels in normoxic H134 and OVCAR-3 cells. Arsenite also increases HIF-1␣ protein levels, suggesting a role for HIF-1 in the induction of VEGF expression. Pretreatment with the ROS inhibitors catalase and mannitol attenuated arsenite-induced ROS production, but did not affect induction of VEGF mRNA and HIF-1␣ protein. In contrast, pretreatment with the thiol antioxidants glutathione or N-acetylcysteine completely abrogated both effects, whereas a potentiation was observed by depletion of intracellular glutathione. These results demonstrate that arsenite-induced VEGF mRNA and HIF-1␣ protein expression is independent of increased ROS production but critically regulated by the cellular reduced glutathione content. In addition, these data suggest the involvement of a thiol-sensitive mechanism in the regulation of VEGF mRNA expression and HIF-1␣ protein in human ovarian cancer cells.

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1␣ (HIF-1␣) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1␣ and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1␣ protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1␣ ؊/؊ mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite.

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African trypanosomes evade the immune response of their host by periodically changing their variant surface glycoprotein (VSG) coat. Each coat is encoded by a separate VSG gene. Expressed genes are in a telomeric expression site (ES) and there are several sites in each trypanosome. To study the transcription control of VSG genes in Trypanosoma brucei we have analyzed an ES, called the dominant ES (DES), that readily switches off and on. The promoter area of the DES is very similar to that of the 221 ES (Zomerdijk et al., 1990). It can be switched off and on in vivo without detectable DNA alterations in the vicinity of the transcription start and it can drive high transient expression of a reporter gene in transfection experiments. However, there are also two major differences between the DES and the 221 ES. First, one version of the DES contains an additional upstream transcription unit overlapping the VSG gene ES promoter. The presence of this upstram transcription is dispensable, however, for the VSG gene ES promoter is active, even if transcription through this start from the upstream promoter is blocked using UV light. Moreover, a second version of the DES present in another trypanosome variant does not produce these upstream transcripts. Secondly, we find that the inactivation of DES transcription in one trypanosome variant is accompanied by DNA alterations in the DES upstream (greater than 2 kb) of the transcription start; reactivation of DES transcription is accompanied by another alteration far upstream. Although we cannot exclude that these DNA rearrangements are incidental, our results raise the possibility that the activity of ES promoters is negatively controlled in cis by far upstream sequences not included in transfection constructs and that alterations in these sequences may lead to (in)activation of the promoter.