Ionizing radiation induces DNA double-strand breaks in bystander primary human fibroblasts.
That irradiated cells affect their unirradiated 'bystander' neighbors is evidenced by reports of increased clonogenic mortality, genomic instability, and expression of DNA-repair genes in the bystander cell populations. The mechanisms underlying the bystander effect are obscure, but genomic instability suggests DNA double-strand breaks (DSBs) may be involved. Formation of DSBs induces the phosphorylation of the tumor suppressor protein, histone H2AX and this phosphorylated form, named gamma-H2AX, forms foci at DSB sites. Here we report that irradiation of target cells induces gamma-H2AX focus formation in bystander cell populations. The effect is manifested by increases in the fraction of cells in a population that contains multiple gamma-H2AX foci. After 18 h coculture with cells irradiated with 20 alpha-particles, the fraction of bystander cells with multiple foci increased 3.7-fold. Similar changes occurred in bystander populations mixed and grown with cells irradiated with gamma-rays, and in cultures containing media conditioned on gamma-irradiated cells. DNA DSB repair proteins accumulated at gamma-H2AX foci, indicating that they are sites of DNA DSB repair. Lindane, which blocks gap-junctions, prevented the bystander effect in mixing but not in media transfer protocols, while c-PTIO and aminoguanidine, which lower nitric oxide levels, prevented the bystander effect in both protocols. Thus, multiple mechanisms may be involved in transmitting bystander effects. These studies show that H2AX phosphorylation is an early step in the bystander effect and that the DNA DSBs underlying gamma-H2AX focus formation may be responsible for its downstream manifestations.
Sokolov
Mykyta V
MV
Department of Nuclear Medicine, Clinical Center, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Smilenov
Lubomir B
LB
Hall
Eric J
EJ
Panyutin
Igor G
IG
Bonner
William M
WM
Sedelnikova
Olga A
OA
D024201:Bystander Effect / Q000528:radiation effects*; D018920:Coculture Techniques; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage / Q000528:radiation effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000528:radiation effects*; D006146:Guanidines / Q000494:pharmacology; D001556:Hexachlorocyclohexane / Q000494:pharmacology; D006657:Histones / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007306:Insecticides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011839:Radiation, Ionizing
D003497:Cyclic N-Oxides; D004791:Enzyme Inhibitors; D006146:Guanidines; C468783:H2AX protein, human; D006657:Histones; D007093:Imidazoles; D007306:Insecticides; C079391:2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; D009569:Nitric Oxide; D001556:Hexachlorocyclohexane; D004247:DNA; D019001:Nitric Oxide Synthase; C004479:pimagedine