Induction of endogenous genes following infection of human endothelial cells with an E1(-) E4(+) adenovirus gene transfer vector

Abstract

Recombinant adenovirus (Ad) gene transfer vectors are effective at transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. However, in the process of gene transfer, the Ad vectors induce the expression of target cell genes, some of which may modify the function of the target cell and/or alter the local milieu. To develop a broader understanding of Ad vector-mediated induction of endogenous gene expression, genes induced by first-generation E1(-) E4(+) Ad vectors in primary human umbilical vein endothelial cells were identified by cDNA subtraction cloning. The identified cDNAs included signaling molecules (lymphoid blast crisis [LBC], guanine nucleotide binding protein alpha type S [Galpha-S], and mitogen kinase [MEK5]), calcium-regulated/cytoskeletal proteins (calpactin p11 and p36 subunits, vinculin, and spinocerebellar ataxia [SCA1]), growth factors (insulin-like growth factor binding protein 4 and transforming growth factor beta2), glyceraldehyde-6-phosphate dehydrogenase, an expressed sequence tag, and a novel cDNA showing homology to a LIM domain sequence. Two- to sevenfold induction of the endogenous gene expression was observed at 24 h postinfection, and induction continued up to 72 h, although the timing of gene expression varied among the identified genes. In contrast to that observed in endothelial cells, the Ad vector-mediated induction of gene expression was not found following Ad vector infection of primary human dermal fibroblasts or human alveolar macrophages. Empty Ad capsids did not induce endogenous gene expression in endothelial cells. Interestingly, additional deletion of the E4 gene obviated the upregulation of genes in endothelial cells by the E1(-) E3(-) Ad vector, suggesting that genes carried by the E4 region play a central role in modifying target cell gene expression. These findings are consistent with the notion that efficient transfer of exogenous genes to endothelial cells by first-generation Ad vectors comes with the price that these vectors also induce the expression of a variety of cellular genes.

FIG. 1

RT-PCR analysis of endothelial-cell gene expression identified by a subtraction library as being upregulated by an E1⁻ E4⁺ Ad gene transfer vector. Total RNA (200 ng/reaction) extracted from uninfected control cells (C) or cells infected with E1⁻ E4⁺ Ad vector (Ad) was used as templates for RT-PCR analysis with gene-specific primers. GAPDH primers were used to confirm RNA integrity and use of equal amounts of RNA. Note that in most cases, there appears to be significant upregulation of expression of the genes identified by the subtraction library.

FIG. 2

Northern analysis of kinetics of expression of endogenous genes of human endothelial cells following infection by an E1⁻ E4⁺ Ad vector. (A) Control, uninfected cells. Total RNA (10 μg/lane) was isolated from uninfected control cells over time (6 to 72 h). Each lane was then hybridized to a specific probe as indicated. Note that, other than the 4.0-kb transcript of TGF-β2, there is little change in the transcripts. (B) Cells infected with an E1⁻ E4⁺ Ad vector. Total RNA was assessed as in panel A. In contrast to panel A, there are marked changes in all transcripts other than GAPDH. The GAPDH probe was used to confirm the integrity and equal loading of RNA.

FIG. 3

RT-PCR analysis of endogenous gene expression of human endothelial cells following E1⁻ E4⁻ Ad vector infection. (A) Expression of Ad E4 genes in human endothelial cells following infection with an E1⁻ E4⁻ Ad gene transfer vector compared to that with an E1⁻ E4⁺ vector. Total RNA (200 ng/reaction) extracted from uninfected control cells (C) or cells infected with an E1⁻ E4⁺ (E4⁺) or E1⁻ E4⁻ (E4⁻) Ad gene transfer vectors were used for RT-PCR analysis with AdE4 gene-specific primers. Note that with the E1⁻ E4⁺ vector but not with the E1⁻ E4⁻ vector, E4 transcripts are observed. (B) Expression of endogenous endothelial genes. The analysis was carried out as in Fig. 1, with total RNA (200 ng/reaction) extracted from uninfected control cells (C) or cells exposed to E1⁻ E4⁻ Ad vector (Ad) used as templates for RT-PCR analysis with gene-specific primers. For both panels A and B, GAPDH primers were used to confirm RNA integrity and use of equal amounts of RNA. (C) Northern analysis of expression of TGF-β2, calpactin p36, and Gα-S expression in endothelial cells infected with E1⁻ E4⁺ compared to E1⁻ E4⁻ Ad vectors. Total RNA (10 μg) extracted from uninfected control cells or cells infected with either E1⁻ E4⁺ or E1⁻ E4⁻ Ad gene transfer vectors were used for Northern analysis and probed with TGF-β2, calpactin p36, and Gα-S cDNA probes. Note that there is little change in transcript levels with the E1⁻ E4⁻ vector but there is upregulation with the E1⁻ E4⁺ vector.