Transformation-defective adenovirus 5 E1A mutants exhibit antioncogenic properties in human BLM melanoma cells

Abstract

Adenoviral E1A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1A delCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1A CR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1A WT ) or only the exon 2 sequences (E1A Ex2 ) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1A Ex2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1A delCR2 -or E1A CR3Ex2 -expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1A WT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.

Tumor-specific activation of hTERT-derived promoters by tumor suppressive E1A-mutants involves recruitment of p300/CBP/HAT and suppression of HDAC-1 and defines a combined tumor targeting and suppression system

“…The 13S instead of the 12S cDNA was chosen due to its dominant transformation suppressing effect when expressed together with the 12S mRNA species, suggesting an intinsic antioncogenic activity within the CR3 (Haley et al, 1984). This suggestion is supported by our previous results where we have shown that Ad5 E1A-CR3Ex2 mutant is an excellent tumor suppressor, whereas Ad5 E1A-Ex2 alone does not suppress malignant tumor growth (Dickopp et al, 2000).…”

“…Therefore, we had constructed transformation-defective Ad5 and Ad12 13S-E1A mutants with similar or even improved tumorsuppressive activity as compared with E1A-WT (Dickopp et al, 2000;and manuscript in preparation). The 13S instead of the 12S cDNA was chosen due to its dominant transformation suppressing effect when expressed together with the 12S mRNA species, suggesting an intinsic antioncogenic activity within the CR3 (Haley et al, 1984).…”

“…However, this antitumor effect of exon 2 functions does not seem to be sufficient for tumor suppression, since we have recently shown that Ad5 exon 2 alone has no antioncogenic activity in BLM melanoma cells. Thus, additional functions mediated by the CR3 for suppression of BLM-induced tumor growth seem to be required (Dickopp et al, 2000).…”

“…Cloning of the Ad12 E1A-Spm2 and all Ad5 E1A mutants into the pRcRSV vector Dickopp et al, 2000) and construction of the hTERT promoter constructs (Horikawa et al, 1999) was described previously. Ad12 E1A-Spm2, Ad5 E1A-CR3NLS or Ad12 E1A-SpEx were cloned into the pVP22-Topo vector (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions using PCR-amplified inserts derived from pRcRSV-based plasmids excluding the AUG start codons.…”

“…Unfortunately, E1A proteins are able to transform at least rodent cells in cooperation with other oncoproteins. Therefore, as the therapeutic use of wild type E1A may harbor the risk of enhancing tumor growth, we previously constructed antioncogenic and transformation-defective E1A 13S cDNA-derived mutants from Ad 12 and Ad5 (Dickopp et al, 2000). Among these, the Rb-binding-deficient Ad12 E1A-Spm2 mutant lacking six consecutive alanine residues in the Ad12 E1A unique 'spacer', the Ad5 E1A-delCR2 and the Ad5 E1A-CR3Ex2 mutant (Figure 1) turned out to be efficient tumor suppressors (Dickopp et al, 2000;and manuscript in preparation).…”