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Induction of p53-independent apoptosis by simian virus 40 small t antigen
O Gjoerup1, D Zaveri, T M Roberts
1Department of Cancer Biology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
Simian virus 40 small t antigen (st) is required for optimal transformation and replication properties of the virus. We find that in certain cell types, such as the human osteosarcoma cell line U2OS, st is capable of inducing apoptosis, as evidenced by a fragmented nuclear morphology and positive terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining of transfected cells. The cell death can be p53 independent, since it also occurs in p53-deficient H1299 cells. Genetic analysis indicates that two specific mutants affect apoptosis induction. One of these (C103S) has been frequently used as a PP2A binding mutant. The second mutant (TR4) lacks the final four amino acids of st, which have been reported to be unimportant for PP2A binding in vitro. However, TR4 unexpectedly fails to bind PP2A in vivo. Furthermore, a long-term colony assay reveals a potent colony inhibition upon st expression, and the behavior of st mutants in this assay reflects the relative frequency of nuclear fragmentation observed in transfections using the same mutants. Notably, either Bcl-2 coexpression or broad caspase inhibitor treatment could restore normal nuclear morphology. Finally, fluorescence-activated cell sorting analysis suggests a correlation between the ability of st to modulate cell cycle progression and apoptosis. Taken together, these observations underscore that st does not always promote proliferation but may, depending on conditions and cell type, effect a cell death response.
Insights
Simian virus 40 small t antigen (st) can induce apoptosis in some cells, independent of p53. This cell death response is linked to st
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Simian virus 40 small t antigen (st) is crucial for viral replication and cell transformation.
- The precise role of st in cellular processes, particularly its potential to induce cell death, remains incompletely understood.
Purpose of the Study:
- To investigate the capacity of Simian virus 40 small t antigen (st) to induce apoptosis in specific cell types.
- To elucidate the mechanisms underlying st-induced apoptosis, including its relationship with p53 and protein phosphatase 2A (PP2A) binding.
Main Methods:
- Transfection of human osteosarcoma (U2OS) and p53-deficient (H1299) cells with wild-type st and mutant forms.
- Apoptosis assessment via nuclear morphology, TUNEL staining, and colony formation assays.
- Analysis of st's interaction with PP2A in vivo and in vitro.
- Evaluation of cell cycle progression using fluorescence-activated cell sorting (FACS).
Main Results:
- Simian virus 40 small t antigen (st) induces apoptosis in U2OS and H1299 cells, characterized by nuclear fragmentation and DNA fragmentation.
- Apoptosis induction by st is p53-independent.
- Mutant st proteins (C103S and TR4) show altered apoptosis induction, with TR4 exhibiting impaired in vivo PP2A binding.
- st expression inhibits colony formation, and this inhibition correlates with apoptosis levels.
- Bcl-2 coexpression or caspase inhibition rescues cells from st-induced apoptosis.
- st modulates cell cycle progression, correlating with its apoptotic effects.
Conclusions:
- Simian virus 40 small t antigen (st) can induce p53-independent apoptosis in certain cell types.
- The interaction of st with PP2A, particularly in vivo, is critical for its apoptotic function.
- st's role is context-dependent, capable of promoting proliferation or inducing cell death based on cellular environment and specific viral protein interactions.