Apoptosis is initiated in human keratinocytes exposed to signalling factors from microbeam irradiated cells.
OBJECTIVE
There is now no doubt that bystander signalling from irradiated cells occurs and causes a variety of responses in cells not targeted by the ionizing track. However, the mechanisms underlying these processes are unknown and the relevance to radiotherapy and risk assessment remains controversial. Previous research by our laboratory has shown bystander effects in a human keratinocyte cell line, HPV-G cells, exposed to medium from gamma irradiated HPV-G cells. The aim of this work was to investigate if similar mechanisms to those identified in medium transfer experiments occurred in these HPV-G cells when they are in the vicinity of microbeam irradiated cells. Demonstration of a commonality of mechanisms would support the idea that the process is not artifactual.


METHODS
HPV-G cells were plated as two separate populations on mylar dishes. One population was directly irradiated using a charged particle microbeam (1 - 10 protons). The other population was not irradiated. Bystander factor-induced apoptosis was investigated in both populations following treatment by monitoring the levels of reactive oxygen species and mitochondrial membrane potential using fluorescent probes. Expression of the anti-apoptotic protein, bcl-2, and cytochrome c were determined, as well as apoptosis levels.


RESULTS
Microbeam irradiation induced increases in reactive oxygen species and decreases in mitochondrial membrane potential at 6 h post-exposure, increased expression of bcl-2 and cytochrome c release at 6.5 h and increased apoptosis at 24 h.


CONCLUSIONS
This study shows that similar bystander signalling pathways leading to apoptosis are induced following microbeam irradiation and following medium transfer. This demonstrates that the mechanisms involved are common across different radiation qualities and conditions and indicates that they may be relevant in vivo.
Lyng
Fiona M
FM
Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin, Ireland. fiona.lyng@dit.ie
Maguire
Paula
P
Kilmurray
Niamh
N
Mothersill
Carmel
C
Shao
Chunlin
C
Folkard
Melvyn
M
Prise
Kevin M
KM
D017209:Apoptosis / Q000502:physiology / Q000528:radiation effects*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D005453:Fluorescence; D005720:Gamma Rays; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D008564:Membrane Potentials / Q000502:physiology / Q000528:radiation effects; D008928:Mitochondria / Q000378:metabolism / Q000528:radiation effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011836:Radiation Tolerance; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000528:radiation effects; D015398:Signal Transduction / Q000502:physiology / Q000528:radiation effects*; D013997:Time Factors
D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D045304:Cytochromes c