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Caspase-independent apoptosis induced by differentiation-inducing factor of Dicytostelium discoideum in INS-1 cells
S Fujimaki1, Y Kubohara, I Kobayashi
1Department of Laboratory Medicine, Gunma University School of Medicine, 371-8512, Maebashi, Japan.
Abstract:
Differentiation-inducing factor (DIF) is a lipophilic hormone of Dicytostelium discoideum and has been shown to exert diverse effects in mammalian cells. We investigated the effect of DIF on cell viability in insulin-secreting INS-1 cells. DIF induced cell death in a dose-dependent manner. In DIF-treated cells, nuclear condensation and shrinkage of the cell body were observed. After 6 h of DIF treatment, cells became Tdt-mediated dUTP-biotin nick end-labeling-positive, and DNA ladder formation was detected, indicating that DIF induced apoptosis in these cells. DIF did not activate caspase-3, a key enzyme mediating apoptotic signals generated by various agents. Furthermore, DIF-induced cell death was not affected by Z-asp-2, 6-dichlorobenzoyloxymethylketone, a broad inhibitor of the caspases. As is the case in other types of cells, DIF increased cytoplasmic free calcium concentration in INS-1 cells. However, DIF-induced cell death was not affected by chelating intracellular free calcium by 1, 2-bis(2-aminoophenoxy)ethane-N, N, N, N-tetra acetic acid (BAPTA). These results indicate that DIF induces apoptosis in INS-1 cells by a mechanism independent of caspase-3. DIF-induced elevation of cytoplasmic calcium does not mediate the effect of DIF on cell death.
Insights
Differentiation-inducing factor (DIF) induces apoptosis in insulin-secreting INS-1 cells. This cell death occurs independently of caspase-3 activation and cytoplasmic calcium levels, suggesting a novel apoptotic pathway.
Area of Science:
- Cell Biology
- Biochemistry
- Endocrinology
Background:
- Differentiation-inducing factor (DIF), a lipophilic hormone from Dictyostelium discoideum, exhibits varied effects on mammalian cells.
- Insulin-secreting INS-1 cells are a key model for studying pancreatic beta-cell function and dysfunction.
Purpose of the Study:
- To investigate the impact of DIF on the viability of insulin-secreting INS-1 cells.
- To elucidate the molecular mechanisms underlying DIF-induced cell death in these cells.
Main Methods:
- DIF treatment of INS-1 cells.
- Assessment of cell viability and apoptosis using morphological analysis, Tdt-mediated dUTP-biotin nick end-labeling (TUNEL) assay, and DNA laddering.
- Enzyme activity assays for caspase-3.
- Measurement of intracellular calcium levels and assessment of effects using calcium chelators (BAPTA) and caspase inhibitors (Z-asp-2, 6-dichlorobenzoyloxymethylketone).
Main Results:
- DIF induced apoptosis in INS-1 cells in a dose-dependent manner, evidenced by nuclear condensation, cell shrinkage, TUNEL positivity, and DNA ladder formation.
- DIF-induced cell death was not inhibited by Z-asp-2, 6-dichlorobenzoyloxymethylketone, indicating caspase-3 independence.
- While DIF increased cytoplasmic calcium, chelating intracellular calcium with BAPTA did not prevent DIF-induced cell death.
Conclusions:
- DIF triggers apoptosis in INS-1 cells through a caspase-3-independent pathway.
- Elevated cytoplasmic calcium levels do not mediate DIF-induced cell death in this context, suggesting a novel apoptotic signaling mechanism.
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