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Published on: February 16, 2015
Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines
Shruti Nagar1, Leslie E Smith, William F Morgan
1Radiation Oncology Research Laboratory, BRB-6010, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA. snag001@umaryland.edu
Abstract:
We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells. We termed this the death-inducing effect (DIE). The goal of the present study was to determine how DIE killed cells. Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells. First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays. Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (approximately 2%) level of apoptosis. The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis. We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones. Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis. Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE. These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells. These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE.
Insights
The death-inducing effect (DIE) involves cytotoxic factors in radiation-induced unstable cell medium that cause DNA damage and cell death in healthy cells. These factors lead to apoptosis or mitotic cell death, explaining the observed cytotoxicity.
Area of Science:
- Cell Biology
- Radiation Biology
- Genetics
Background:
- Ionizing radiation can induce chromosomal instability in cells.
- A unique non-targeted effect, termed the death-inducing effect (DIE), was observed where medium from unstable clones was cytotoxic to parental cells.
Purpose of the Study:
- To elucidate the mechanism by which the death-inducing effect (DIE) causes cell death.
- To investigate whether DIE is mediated by secreted factors or products from dead/dying cells.
Main Methods:
- Assessed apoptosis in parental and unstable clones using Annexin V binding and TUNEL assays.
- Exposed parental cells to medium from unstable clones and analyzed DNA double-strand breaks, micronucleus formation, and apoptosis induction.
Main Results:
- Unstable clones exhibited varying spontaneous apoptosis levels.
- Medium from unstable clones induced DNA double-strand breaks and increased apoptosis in recipient cells.
- Increased micronucleus frequencies indicated a role for mitotic-linked cell death in DIE.
Conclusions:
- The death-inducing effect (DIE) is likely mediated by cytotoxic factors secreted into the culture medium.
- These secreted factors induce DNA double-strand breaks in recipient cells, leading to either apoptosis or mitotic cell death.
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