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the E1A protein and not the association of p53 with the E1B 55-kDa protein (6).

Since the E1B and E4orf6 proteins bind at different sites on p53, the E1B 55-kDa and the E4orf6 proteins might interact simultaneously with the residual low level of p53 that accumulates in the presence of the E4orf6 protein. The combination of adenovirus proteins might more completely inactivate p53 than either viral gene product alone. It is also possible that the altered localization of p53 in the presence of E4orf6 (Fig. 6) modifies the oncogenic properties of adenovirus-transformed cells. It has been shown that E1A/E1B-transformed 3Y1 rat cells containing a low steady-state level of the E1B 55-kDa protein form tumors more rapidly in nude mice than transformants with high levels of the transforming protein (36). 3Y1 transformants with relatively low levels of the E1B 55-kDa protein do not accumulate p53 in cytoplasmic bodies; rather, they contain nuclear p53 (36), just as we have observed for transformants containing the E4orf6 protein (Fig. 6).

It seems contradictory that the E4orf6 protein can cooperate with the E1A protein to transform cells (Fig. 2), presumably by blocking E1A-induced apoptosis (Fig. 1), while cells infected with mutant viruses lacking only the E1B 19-kDa protein undergo extensive apoptosis (4547). The mutant viruses contain wild-type E4 genes and should express the E4orf6 protein in the cells undergoing apoptosis. However, the E1A protein has been shown to induce apoptosis through a p53-independent pathway (48, 49) in addition to the p53-dependent pathway. The p53-independent apoptosis might be induced indirectly by the E1A protein; that is, it might be caused by another adenovirus gene product whose expression is activated by the E1A protein. The E1B 19-kDa protein can block both p53-dependent and p53-independent apoptosis induced by the E1A protein. In contrast, the E4orf6 protein can prevent p53-induced apoptosis, but not apoptosis mediated by TNF-α in the absence of p53 (Fig. 1). So, if the p53-independent apoptosis seen in infected cells is due to a viral protein whose expression is induced by E1A protein, then one can propose an explanation for the inability of the E4orf6 protein to prevent apoptosis in virus-infected cells. Presumably, the E4orf6 protein antagonizes p53-dependent apoptosis induced directly by E1A, but it does not prevent p53-independent apoptosis induced indirectly by the E1A protein when it activates another apoptosis-promoting viral gene in adenovirus-infected cells.

Many adenovirus vectors that are being considered for gene delivery in humans contain the E4orf6 coding region. Given the ability of this protein to alter p53 function (13) and its oncogenic potential demonstrated here, it would be prudent to remove this coding region from gene transfer vectors.

We thank D.A.Haber for AT6 cells, A.Teresky for instruction and help in mouse injections, J.Goodhouse and H.Zhu for assistance with confocal microscopy, and Y.Shen for helpful discussions on transformation assays. This work was supported by grants from the National Cancer Institute (CA41086) and the Cystic Fibrosis Foundation (Z998). T.S. is an American Cancer Society Professor and an Investigator of the Howard Hughes Medical Institute.

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