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DNA inclusions within autolytic cytoplasmic vacuoles of hemopoietic stem cell line FDCP-Mix
S Reipert1, J A Hickman, T D Allen
1CRC Department of Structural Cell Biology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom.
Abstract:
FDCP-Mix, a pluripotent routine hemopoietic stem cell line undergoes internucleosomal cleavage of DNA when induced to apoptosis either by drugs or by withdrawal of growth factor (IL-3), and also displays a pattern of nuclear morphology that is typical for apoptosis. However, increased autolytic activity in the cytoplasm precedes the nuclear changes. For etoposide-treated FDCP-Mix cells, mitochondria were identified as a target for autolytic digestion in large autolytic vacuoles, but during this period an increase in the number of mitochondria was observed. The autolytic vacuoles displayed variations in their content. Large, electron-dense inclusions resembling "condensed chromatin" could regularly be found in FDCP-Mix cells treated with low concentrations of etoposide (<4 microM). Confocal fluorescence microscopy and DNAse-gold labeling were employed to demonstrate the presence of DNA in the formation of the electron-dense inclusions within autolytic vacuoles. The identification of mitochondrial macroautophagy, the evidence for an etoposide-induced proliferation of mitochondria, and the fact that electron-dense inclusions are formed at a stage when the morphology of the nucleus is still not effected, suggests that the DNA within the autolytic vacuoles may be of mitochondrial origin.
Insights
Hematopoietic stem cells undergoing apoptosis show cytoplasmic digestion of mitochondria before nuclear changes. DNA in autolytic vacuoles may originate from these proliferating mitochondria.
Area of Science:
- Cell Biology
- Molecular Biology
- Hematopoiesis
Background:
- Pluripotent hemopoietic stem cells (FDCP-Mix) undergo internucleosomal DNA cleavage during apoptosis.
- Apoptosis induction can occur via drugs or growth factor withdrawal (e.g., IL-3).
- Cytoplasmic autolytic activity precedes nuclear morphological changes in apoptosis.
Purpose of the Study:
- To investigate the source of DNA found in autolytic vacuoles during etoposide-induced apoptosis in FDCP-Mix cells.
- To elucidate the role of mitochondria in the early stages of apoptosis.
Main Methods:
- Confocal fluorescence microscopy
- DNAse-gold labeling
- Analysis of mitochondrial morphology and proliferation during apoptosis
Main Results:
- Etoposide treatment induced increased mitochondrial numbers and their autophagic digestion within vacuoles.
- Electron-dense inclusions, resembling condensed chromatin, were observed in autolytic vacuoles.
- DNA was detected within these inclusions, suggesting a non-nuclear origin.
- Mitochondrial macroautophagy was identified as a key process.
Conclusions:
- The DNA within autolytic vacuoles during etoposide-induced apoptosis in FDCP-Mix cells likely originates from mitochondria.
- Mitochondrial proliferation and subsequent autophagy precede nuclear DNA fragmentation in this model.
- This suggests a novel pathway for DNA involvement in early apoptotic events.