"Hit-and-run" transformation by adenovirus oncogenes

Abstract

According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the "hit-and-run" hypothesis claims that viruses can mediate cellular transformation through an initial "hit," while maintenance of the transformed state is compatible with the loss ("run") of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.

FIG. 1

Cells transformed by Ad5 E1A and Ad5 E4orf6-neo or E4orf3-neo are G418 sensitive. Shown are representative plates from transfections of primary rat cells with empty vector-neo or combinations of pCMV-E1A with empty vector-neo, pCMV-E1B-55 kDa-neo, pCMV-E4orf6-neo, or pCMV-E4orf3-neo, which contain transformed colonies obtained in the absence or presence of the selective drug G418.

FIG. 2

Absence of E1A and E4 proteins and genes in the majority of cell lines transformed by E1A plus E4orf6 or E4orf3. (a to c) Analysis of viral oncogene expression in 10 different AB (a), AS (b), and AT (c) cell lines by immunoblotting with M73 (E1A) or 2A6 (E1B-55 kDa) antibodies or by combined immunoprecipitation and immunoblotting with RSA3 (E4orf6) or 6A11 (E4orf3) antibodies. (d to f) PCR screening for the presence of E1A-, E1B-, E4orf6-, or E4orf3-specific DNA in 10 different AB (d), AS (e), and AT (f) cell lines. IgG, immunoglobulin G.

FIG. 3

Transient coexpression of Ad5 E4orf6 or E4orf3 with Ad5 E1A increases the mutation frequency at the hprt locus. For mutagenesis assays, 10⁵ CHO-D422 cells were transfected with either empty vector or plasmids encoding the indicated viral genes. After 4 days, cells were trypsinized and replated at a density of 10⁵ cells per plate in selective growth medium containing 6-thioguanine. Drug-resistant colonies from two of these plates corresponding to a total of 2 × 10⁵ cells plated in selective medium were counted 7 to 8 days thereafter, and numbers were corrected for the plating efficiency. The mean and standard deviation for at least three independent experiments are presented. After correction for the plating efficiency, the average number of 6-thioguanine-resistant colonies was 7 per plate for the vector control. The same results were obtained in two separate experiments in which we plated a total of 10⁶ cells (10 plates with 10⁵ cells/plate) in selective growth medium (data not shown).