Induction and detection of bystander effects after combined treatment of cells with 5-bromo-2'-deoxyurine, Hoechst 33 258 and ultraviolet A light.
OBJECTIVE
A combined treatment of cells with 5-bromo-2'-deoxyurine (BrdU), Hoechst 33 258 and ultraviolet A (UVA) light was used to introduce chromosomal aberrations in cells for the study of bystander effects in non-labelled cells.


METHODS
Mixtures of BrdU-labelled and non-labelled Chinese hamster cells (V79) in S phase were exposed to Hoechst 33 258 and/or UVA light. Metaphase cells were collected and analysed for chromosomal aberrations by Giemsa staining. BrdU immunostaining was performed to verify BrdU incorporation in the cells.


RESULTS
Combined treatment with BrdU, Hoechst dye and UVA light induced reduced cell survival and increased chromosomal aberrations, whereas treatment with Hoechst 33 258 and/or UVA light had no effect on cells. Elevated frequencies of chromosomal aberrations were found in non-labelled cells mixed with BrdU-labelled cells and exposed to Hoechst dye and UVA light, suggesting the induction of bystander effects by damaged BrdU-labelled cells. These bystander clastogenic effects were also observed in non-labelled cells mixed with dying cells, indicating a contribution of dying cells in the induction of the bystander effects.


CONCLUSIONS
The combined treatment with BrdU, Hoechst 33 258 and UVA light is a valid method for the study of bystander effects as it enables both induction of DNA damage and discrimination of targeted cells and bystander cells.
Xiao
Y
Y
Department of Cell Biology and Histology, Academic Medical Centre, M3-352, University of Amsterdam, PO Box 22700, NL-1100 DE Amsterdam, the Netherlands. y.xiao@amc.uva.nl
de Feyter
E
E
Van Oven
C H
CH
Stap
J
J
Hoebe
R
R
Havenith
S
S
Van Noorden
C J F
CJ
Aten
J A
JA
D000818:Animals; D006690:Bisbenzimidazole / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000494:pharmacology*; D024201:Bystander Effect; D002460:Cell Line; D002470:Cell Survival; D002869:Chromosome Aberrations; D006224:Cricetinae; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D005456:Fluorescent Dyes / Q000494:pharmacology; D008677:Metaphase / Q000528:radiation effects; D008856:Microscopy, Fluorescence; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D011838:Radiation-Sensitizing Agents; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole