Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro.
G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures.
Masunaga
S
S
Radiation Oncology Research Laboratory, Kyoto University, Osaka, Japan.
Keng
P C
PC
D000818:Animals; D016466:CHO Cells; D002110:Caffeine / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects / Q000528:radiation effects*; D002469:Cell Separation; D006224:Cricetinae; D004260:DNA Repair / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects / Q000528:radiation effects; D016195:G2 Phase / Q000187:drug effects / Q000528:radiation effects*; D005720:Gamma Rays; D006918:Hydroxyurea / Q000494:pharmacology; D066298:In Vitro Techniques; D016196:S Phase / Q000187:drug effects / Q000528:radiation effects
D002110:Caffeine; D006918:Hydroxyurea