Serum deprivation and protein synthesis inhibition induce two different apoptotic processes in N18 neuroblastoma
1Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Av. Rovira Roure, 44, Lleida, Catalonia, 25198, Spain.
Abstract:
N18 are murine neuroblastoma cells that underwent cell death upon serum deprivation or inhibition of protein synthesis by means of cycloheximide (CHX). In both cases, an ultrastructural morphology and an internucleosomal pattern of DNA fragmentation typical of apoptosis were found. However, electron microscopy revealed abundant lipid vesicles in the cytoplasm of CHX-treated cells that were not found in their serum-deprived counterparts. In addition, when both types of apoptotic cells were compared by means of flow cytometry and chromatin staining with propidium iodide, the former showed consistently less fluorescence than the latter. Therefore, in N18 cells, both apoptotic processes seemed to differ at a structural level. At a functional level, we found that apoptosis was blocked by the protease inhibitor TLCK in CHX-treated but not in serum-deprived cells. On the other hand, we generated N18 clones that overexpressed Bcl-2 protein. After a period of 48 h we found that identical levels of Bcl-2 protein were able to block apoptosis in serum-deprived but not in CHX-treated cells. In conclusion, two different biochemical pathways leading to apoptosis seem to coexist in N18 neuroblastoma cells.
Insights
N18 neuroblastoma cells exhibit apoptosis via two distinct pathways. These pathways differ structurally and functionally, with one sensitive to protease inhibitors and Bcl-2 overexpression, and the other not.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Biology
Background:
- Neuroblastoma is a pediatric cancer with complex cell death mechanisms.
- Understanding apoptosis pathways is crucial for developing targeted therapies.
- N18 murine neuroblastoma cells provide a model to study distinct apoptotic processes.
Purpose of the Study:
- To investigate the differences between apoptosis induced by serum deprivation and cycloheximide (CHX) in N18 cells.
- To elucidate the distinct structural and functional characteristics of these two apoptotic pathways.
- To determine the role of protease activity and Bcl-2 in modulating these distinct apoptotic processes.
Main Methods:
- Induction of apoptosis via serum deprivation and CHX treatment in N18 cells.
- Ultrastructural analysis using electron microscopy.
- Flow cytometry and propidium iodide staining for DNA fragmentation assessment.
- Functional assays using protease inhibitors (TLCK) and Bcl-2 overexpression.
Main Results:
- Both serum deprivation and CHX induced apoptosis with DNA fragmentation, but CHX-treated cells showed unique cytoplasmic lipid vesicles.
- CHX-induced apoptosis exhibited reduced DNA content (fluorescence) compared to serum-deprived apoptosis.
- Apoptosis was blocked by TLCK in CHX-treated cells but not serum-deprived cells.
- Bcl-2 overexpression inhibited serum-deprived apoptosis but not CHX-induced apoptosis.
Conclusions:
- N18 neuroblastoma cells possess at least two distinct biochemical pathways leading to apoptosis.
- These pathways differ in their ultrastructural morphology, DNA content, and sensitivity to protease inhibitors and Bcl-2.
- This suggests context-dependent regulation of apoptosis in neuroblastoma cells.
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