Importance of Nuclear Localization of Apoptin for Tumor-specific Induction of Apoptosis

Abstract

The chicken anemia virus-derived protein Apoptin induces apoptosis specifically in human tumor and transformed cells and not in normal, untransformed cells. The cell killing activity correlates with a predominantly nuclear localization of Apoptin in tumor cells, whereas in normal cells, it is detected mainly in cytoplasmic structures. To explore the role of nuclear localization for Apoptin-induced cell death in tumor cells, we employed a mutagenesis strategy. First, we demonstrated that the C terminus of Apoptin contains a bipartite-type nuclear localization signal. Strikingly, further investigation showed that Apoptin contains two different domains that induce apoptosis independently, and for both domains, we found a strong correlation between localization and killing activity. Using inhibitors, we ruled out the involvement of de novo gene transcription and translation and further showed that Apoptin itself does not have any significant transcriptional repression activity, suggesting that Apoptin exerts its effects in the nucleus by some other method. To determine whether nuclear localization is sufficient to enable Apoptin to kill normal, untransformed cells, we expressed fulllength Apoptin fused to a heterologous nuclear localization signal in these cells. However, despite its nuclear localization, no apoptosis was induced, which suggests that nuclear localization per se is not sufficient for Apoptin to become active. These studies increase our understanding of the molecular pathway of Apoptin and may also shed light on the mechanism of cellular transformation.

Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing

“…Mutation of a specific part of Apoptin resulted in a strongly reduced binding and delayed apoptosis. Recently, we have shown that Apoptin contains two different domains that induce apoptosis independently, 13 which might explain why we only observed a delayed apoptosis induction when the DEDAF/Apoptin interaction was prevented. DEDAF was isolated earlier in a yeast twohybrid screen, using the DED of pro-caspases 8 and 10, 16 and was also found by others as a binding partner for the transcription factor E4TF1/hGABP and named YEAF1.…”

“…Recently, we have shown that inhibitors of transcription or translation do not affect Apoptin's activity; however, this does not exclude a function in transcriptional repression. 13 Overexpression of DEDAF induced apoptosis in three different tumor cell lines, but not in normal fibroblasts or mesenchymal stem cells. In contrast, FADD overexpression did induce apoptosis in these normal cells, indicating that the cells were competent to execute apoptosis.…”

“…8 Mutational analyses indicate that nuclear localization is required, but not sufficient, for the pro-apoptotic activity of Apoptin in tumor cells. 12,13 These data suggest that the tumor-specific activity of Apoptin is not caused by nuclear localization per se. Rather, differential phosphorylation was found to be involved; 14 furthermore, binding to factors that are expressed at higher levels in transformed cells than in normal diploid cells may also play a role.…”

“…In transformed cells, however, it assumes a predominantly nuclear localization, which is likely required for its pro-apoptotic activity. 8,12,13 We examined whether the subcellular distribution of overexpressed DEDAF was consistent with a functional interaction with Apoptin in intact cells. When expressed alone in Saos-2 osteosarcoma cells, DEDAF was to some extent cytoplasmic, but the great majority of staining was found in the nucleus, distributed evenly in a fine granular pattern, as seen by fluorescent and confocal microscopy ( Figure 4j-k).…”

Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin

“…The subcellular localization of apoptin appears to be important for the selective toxicity towards transformed and cancer cells. In cells resistant to apoptin-mediated cell death, the protein has a cytoplasmic localization, whereas in sensitive cells, apoptin is found in the nucleus (Danen-Van Oorschot et al, 2003). Apoptin was described to be specifically phosphorylated in transformed cells by an unknown kinase, which was proposed to be important for the nuclear localization and apoptotic activity of apoptin (Rohn et al, 2002).…”