Adenovirus E4orf4 Protein-Induced Death of p53 ^−/− H1299 Human Cancer Cells Follows a G ₁ Arrest of both Tetraploid and Diploid Cells due to a Failure To Initiate DNA Synthesis

Abstract

The adenovirus E4orf4 protein selectively kills human cancer cells independently of p53 and thus represents a potentially promising tool for the development of novel antitumor therapies. Previous studies suggested that E4orf4 induces an arrest or a delay in mitosis and that both this effect and subsequent cell death rely largely on an interaction with the B55 regulatory subunit of protein phosphatase 2A. In the present report, we show that the death of human H1299 lung carcinoma cells induced by expression of E4orf4 is typified not by an accumulation of cells arrested in mitosis but rather by the presence of both tetraploid and diploid cells that are arrested in G 1 because they are unable to initiate DNA synthesis. We believe that these E4orf4-expressing cells eventually die by various processes, including those resulting from mitotic catastrophe.

Analysis by Live Imaging of Effects of the Adenovirus E4orf4 Protein on Passage through Mitosis of H1299 Tumor Cells

“…We showed previously that E4orf4-expressing tumor cells with both 2n and 4n DNA content are arrested in G 1 and die (8). This arrest in G 1 seems due to an inability to initiate new rounds of DNA synthesis (8).…”

“…We showed previously that E4orf4-expressing tumor cells with both 2n and 4n DNA content are arrested in G 1 and die (8). This arrest in G 1 seems due to an inability to initiate new rounds of DNA synthesis (8). E4orf4's effects require an interaction with the B55 class of protein phosphatase 2A regulatory subunits (9,(14)(15)(16)(17), and we have found that at high levels, E4orf4 inhibits PP2A B55 (18) to induce effects on anaphase-promoting complex (APC) and perhaps other processes that may disrupt normal passage through mitosis (18,19).…”

“…4orf4 is a human adenovirus early protein that functions during infection to facilitate progression to the late phase (1-7); however, when overexpressed alone in cancer cells, it induces some accumulation of 4n (tetraploid) cells (8) and slow cell death, perhaps involving mitotic catastrophe (9)(10)(11)(12)(13)(14). We showed previously that E4orf4-expressing tumor cells with both 2n and 4n DNA content are arrested in G 1 and die (8).…”

“…Our earlier work by flow cytometry analysis of synchronized H1299 cells expressing high levels of E4orf4 indicated that, whereas cells arrested at G 1 /S with hydroxyurea (HU) were able to complete a round of DNA replication following removal of HU with those arrested at G 0 /G 1 , new rounds of DNA synthesis were not evident following addition of fresh serum (8). We reexamined such cells by live imaging.…”

“…We reexamined such cells by live imaging. Figure 2 shows results obtained with cells arrested in G 1/ S using HU (8) and then infected with the viral vectors; after 18 h, fresh medium containing 20% serum was added in the absence of HU. Figure 2A shows that the pattern with cells infected with the control rtTA vector resembled that shown in Fig.…”