Immediate and long-term consequences of vascular toxicity during zebrafish development.

Proper formation of the vascular system is necessary for embryogenesis, and chemical disruption of vascular development may be a key event driving developmental toxicity. In order to test the effect of environmental chemicals on this critical process, we evaluated a quantitative assay in transgenic zebrafish using angiogenesis inhibitors that target VEGFR2 (PTK787) or EGFR (AG1478). Both PTK787 and AG1478 exposure impaired intersegmental vessel (ISV) sprouting, while AG1478 also produced caudal and pectoral fin defects at concentrations below those necessary to blunt ISV morphogenesis. The functional consequences of vessel toxicity during early development included decreased body length and survival in juvenile cohorts developmentally exposed to inhibitor concentrations sufficient to completely block ISV sprouting angiogenesis. These data show that concentration-dependent disruption of the presumed targets for these inhibitors produce adverse outcomes at advanced life stages.

Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter.

Here we show that the adenovirus early region 4 (E4) open reading frame 4 (ORF4) protein autoregulates its own transcription by inhibiting adenovirus E1A-induced activation of E4 transcription both in transient transfection experiments and during lytic virus growth. The inhibitory activity of E4-ORF4 was selective for E1A-CR3-dependent transactivation and had no effect on CR1 transactivation. The inhibitory activity of E4-ORF4 was relieved by okadaic acid treatment, which inhibits the cellular protein phosphatase 2A (PP2A), suggesting that E4-ORF4 controls the phosphorylated status of transcription factors important for E4 promoter activity. This conclusion agrees with previous demonstrations that E4-ORF4 associates with PP2A and causes a partial dephosphorylation of certain transcription factors, including E1A (U. Müller, T. Kleinberger, and T. Shenk, J. Virol. 66:5869-5878, 1992; T. Kleinberger and T. Shenk, J. Virol. 67:7556-7560, 1993). However, our results indicate that dephosphorylation of E1A itself might not be the primary target for E4-ORF4. Instead, the E4-ORF4-PP2A complex appears to work by dephosphorylation of multiple cellular transcription factors that are involved in E1A transactivation of the E4 promoter.

An adenovirus E1A transcriptional repressor domain functions as an activator when tethered to a promoter.

The adenovirus E1A protein contains three well conserved regions, designated conserved region (CR) 1, 2 and 3, which are important for the multiple activities ascribed to E1A. The CR3 domain constitutes a prototypic transcription activator, consisting of a promoter targeting region and a transactivating region. Here we demonstrate the existence of a second transactivating region located within amino acids 28 to 90 (essentially the CR1 domain) of the E1A protein. A fusion protein, containing the Gal4 DNA binding domain linked to CR1, was as efficient as the classical CR3 transactivator in activating transcription from a reporter plasmid containing Gal4 binding sites. However, competition experiments suggest that Gal/CR1 and Gal/CR3 work through different cellular targets. The E1A-243R protein has previously been extensively characterized as a repressor of transcription. Here we show that a Gal4 fusion protein expressing the CR1 domain is indeed sufficient for repression of SV40 enhancer activity. Collectively, our results suggest that CR1 functions as an activator if tethered to a promoter and as a repressor in the absence of promoter association.

The carboxy-terminal exon of the adenovirus E1A protein is required for E4F-dependent transcription activation.

The adenovirus-2 E1A 289R transcription activator protein contains a 49 amino acid sequence (designated CR3) that has been suggested to represent the minimal domain required for E1A-induced activation of viral early transcription. We show here that the non-conserved carboxy-terminal E1A exon contains two interchangeable elements that are required for efficient CR3-dependent transactivation of the adenovirus E4 promoter in HeLa cells. These two elements do not encode independent transactivation functions and have been designated auxiliary regions (ARs) 1 and 2. The effects of AR1 and AR2 are not additive, suggesting that they contribute a mechanistically analogous function in transcription. Previous studies have suggested that two cellular transcription factors, ATF-2 and E4F, can function together with E1A to induce transcription of the E4 promoter. The importance of respective factors for E4 transcription has not been resolved. We find that E1A activation of E4F, but not ATF-2 (or other ATF factors), is AR1- and AR2-dependent. This result suggests that E1A induction of the E4 promoter in HeLa cells is primarily mediated by E4F.

Enhanced invasive properties of rat embryo fibroblasts transformed by adenovirus E1A mutants with deletions in the carboxy-terminal exon.

E1A genes deficient in the carboxy-terminal exon can cooperate with activated ras oncogenes to induce transformation of rat embryo fibroblasts. However, the resulting transformed foci show a distinct appearance characterized by a decreased adhesion of the cells to the substrate. Here, we demonstrate that cell lines derived from foci showing the variant morphology are defective in down-regulation of stromelysin 1 metalloprotease expression and show an increased invasive propensity compared with cells transformed by wild-type E1A. The altered focus morphology, the high invasive propensity and the elevated stromelysin 1 expression were abrogated by glucocorticoid treatment. Our results show that E1A functions necessary for transformation and inhibition of invasive properties may be separated, and indicate that a 23 amino acid serine/threonine-rich region within the E1A carboxy-terminal exon is required for efficient repression of metalloprotease expression in transformed cells.

Independent transformation activity by adenovirus-5 E1A-conserved regions 1 or 2 mutants.

Two conserved regions (CR1 and CR2) on the adenovirus E1A proteins have previously been shown to be required for cooperation with the ras oncogene in the transformation of primary rodent cells. Sequences within these regions are essential for the ability of E1A to associate with the 105K product of the retinoblastoma susceptibility gene, p105-RB, as well as with other cellular proteins, including a 107K (p107) and a 300K (p300) species. In this paper, we show that CR1 mutants deficient in p300 binding and CR2 mutants with lost or reduced binding of p105-RB and/or p107 have a low, but not abolished focus formation activity. In contrast, CR1/CR2 double mutants were deficient in focus formation, suggesting that the transformation activities displayed by the single CR1 or CR2 mutants were due to an independent transformation activity by both CR1 and CR2. No strict correlation between p105-RB binding and E1A-mediated transformation was observed. The E1A enhancer repression function was found to correlate with the binding of p300 but not with E1A-mediated transformation. Complex formation between E1A and p107, similar to the p105-RB binding, required sequences within both CR1 and CR2. The CR2 sequences required for binding of p107K or p105-RB were overlapping, but not identical. Finally, a larger segment of CR2 was required for stable complex formation between E1A and phosphorylated forms of p105-RB or p107 compared to corresponding unphosphorylated species.