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Isolation of DAP3, a novel mediator of interferon-gamma-induced cell death
J L Kissil1, L P Deiss, M Bayewitch
1Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Interaction of certain cytokines with their corresponding cell-surface receptors induces programmed cell death. Interferon-gamma induces in HeLa cells a type of cell death with features characteristic of programmed cell death. Here, we report the isolation of a novel gene, DAP3 (death-associated protein-3), involved in mediating interferon-gamma-induced cell death. The rescue of this gene was performed by a functional selection approach of gene cloning that is based on transfection with an antisense cDNA expression library. The antisense RNA-mediated inactivation of the DAP3 gene protected the cells from interferon-gamma-induced cell death. This property endowed the cells expressing it with a growth advantage in an environment restrictive due to the continuous presence of interferon-gamma and thus provided the basis of its selection. The gene is transcribed into a single 1.7-kilobase mRNA, which is ubiquitously expressed in different tissues and codes for a 46-kDa protein carrying a potential P-loop motif. Ectopic expression of DAP3 in HeLa cells was not compatible with cell growth, resulting in a 16-fold reduction in the number of drug-resistant stable clones. The data presented suggest that DAP3 is a positive mediator of cell death induced by interferon-gamma.
Insights
Researchers identified a new gene, death-associated protein-3 (DAP3), crucial for interferon-gamma-induced programmed cell death in HeLa cells. Inactivating DAP3 protected cells, highlighting its role in mediating this cell death pathway.
Area of Science:
- Molecular Biology
- Cell Biology
- Immunology
Background:
- Cytokine-receptor interactions can trigger programmed cell death.
- Interferon-gamma (IFN-γ) induces cell death in HeLa cells with programmed cell death characteristics.
Purpose of the Study:
- To isolate and characterize novel genes involved in IFN-γ-induced cell death.
- To identify the specific gene mediating IFN-γ-induced apoptosis in HeLa cells.
Main Methods:
- Functional selection using an antisense cDNA expression library for gene cloning.
- Transfection and antisense RNA-mediated gene inactivation.
- Analysis of gene expression and protein product characteristics.
Main Results:
- A novel gene, death-associated protein-3 (DAP3), was identified as being involved in IFN-γ-induced cell death.
- Antisense RNA inactivation of DAP3 conferred resistance to IFN-γ-induced cell death.
- DAP3 is transcribed into a 1.7-kb mRNA, coding for a 46-kDa protein; ectopic expression inhibited cell growth.
Conclusions:
- DAP3 is a positive mediator of programmed cell death induced by interferon-gamma.
- The identification of DAP3 provides new insights into the molecular mechanisms of cytokine-mediated apoptosis.