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Loss of intracellular putrescine pool-size regulation induces apoptosis
1Department of Radiation Oncology/Cancer Biology Division, Arizona Health Sciences Center, The University of Arizona, Tucson 85724, USA.
Abstract:
Synthesis and uptake are two important regulated mechanisms by which eukaryotic cells maintain polyamine levels. The role that loss of synthesis and/or uptake regulation plays in mediating putrescine toxicity was investigated by comparing toxicity in an ornithine decarboxylase (ODC)-deficient Chinese hamster ovary cell line (C55.7) with a functional putrescine transport system and an ODC-overproducing rat hepatoma cell line (DH23b), which are transport regulation deficient. When C55.7 cells were transfected with either mouse ODC (M) or trypanosome ODC (Tb), intracellular putrescine content increased slightly in C55.7(Tb-ODC), compared to C55.7(M-ODC), due to the lack of response of Tb-ODC to polyamine regulation. The increase in putrescine content resulting from loss of ODC regulation had no impact on cell growth and viability. When the feedback repression of polyamine uptake was blocked with cycloheximide, C55.7 cells transfected with either ODC construct accumulated very high levels of putrescine from the medium, and underwent apoptosis in a putrescine dose-dependent manner. A similar correlation of deregulated putrescine uptake and increased apoptotic cells was observed in DH23b cells. These data demonstrate that loss of feedback regulation on the polyamine transport system, but not ODC activity, is sufficient to induce apoptosis. Thus, downregulation of the transport system is necessary to prevent accumulation of cytotoxic putrescine levels in rodent cells.
Insights
Loss of regulation in cellular polyamine uptake, not synthesis, triggers toxic putrescine accumulation and apoptosis. Downregulating transport prevents cytotoxic putrescine levels in cells.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Eukaryotic cells regulate polyamine levels through synthesis and uptake.
- Putrescine, a key polyamine, can be toxic if levels are not controlled.
Purpose of the Study:
- To investigate the role of synthesis and uptake regulation in mediating putrescine toxicity.
- To compare putrescine toxicity in cells with deficient synthesis versus deficient uptake regulation.
Main Methods:
- Utilized ornithine decarboxylase (ODC)-deficient Chinese hamster ovary cells (C55.7) and ODC-overproducing rat hepatoma cells (DH23b).
- Transfected C55.7 cells with mouse and trypanosome ODC constructs.
- Blocked feedback repression of polyamine uptake using cycloheximide.
- Assessed cell growth, viability, and apoptosis in response to varying putrescine levels.
Main Results:
- Loss of ODC regulation alone did not impact cell growth or viability.
- Blocking polyamine uptake regulation led to high intracellular putrescine accumulation and dose-dependent apoptosis.
- Deregulated putrescine uptake correlated with increased apoptotic cells in both cell lines.
Conclusions:
- Loss of feedback regulation on the polyamine transport system is sufficient to induce apoptosis.
- Downregulation of the polyamine transport system is crucial for preventing cytotoxic putrescine accumulation in rodent cells.