Mutagenesis vol. 18 no. 6 pp. 549-560,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines
1Radiation Oncology Research Laboratory, BRB-6010, 2Graduate Program in Human Genetics and 3Marlene and Stewart Greenebaum Cancer Center, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA
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 (
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.
4To whom correspondence should be addressed at: Radiation Oncology Research Laboratory, BRB-6010, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA. Tel: +1 410 706 0254; Fax: +1 410 706 6138; Email: snaga001{at}umaryland.edu
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