Induction of neuronal and tumor-related genes by adenovirus type 12 E1A.

Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region between the second and third conserved regions. Substitution of a single amino acid in the spacer is able to abrogate Ad12 tumorigenesis. To investigate the function of the spacer, microarray analysis was performed on cells transformed by tumorigenic and nontumorigenic Ad12s that differ only by one amino acid in the spacer. Fewer than 0.8% of approximately 8,000 genes in the microarray exhibited differential expression of threefold and higher. Of these, more than half of the known genes with higher expression in the wild-type Ad12-transformed cells have neuronal-specific functions. Some of the other differentially expressed genes are involved in the regulation of the cell cycle, transcription, cell structure, and tumor invasiveness. Northern blot analyses of a subset of the neuronal genes, including Robo1, N-MYC, and alpha-internexin, confirmed their strong expression in multiple Ad12 tumorigenic cell lines. In contrast, these neuronal genes displayed only minor or negligible expression in cells transformed by spacer-mutated Ad12. Significantly, stable introduction of E1A-12 into nontumorigenic Ad5-transformed cells induced neuronal gene expression. We found that the neuron-restrictive silencer factor, which serves as a master repressor of neuronal genes, was inactivated in both Ad12- and Ad5-transformed cells via cytoplasmic retention, though only Ad12-transformed cells exhibited neuronal gene induction. Mutational analyses of the alpha-internexin promoter demonstrated that E1A-12-mediated neuronal gene induction further required the activation of neuronal promoter E-box elements. These results indicate that the spacer is involved in mediating neuronal and tumor-related genes.