Absence of an immediate G1/S checkpoint in primary MEFs following gamma-irradiation identifies a novel checkpoint switch.
DNA double-strand breaks caused by ionizing radiation have been shown to induce G(1)/S, intra-S-phase, and G(2)/M cell cycle checkpoints. However, analysis of the immediate induction of G(1)/S checkpoint at a cellular level has been hampered by the inability to distinguish cells that were already replicating DNA at the time of damage from cells that entered S phase following the DNA damage. We have developed a novel strategy for assessing the initiation of the G(1)/S checkpoint following gamma-irradiation within asynchronous, low passage, primary mouse embryonic fibroblast cultures (MEFs) using a staggered CldU/IdU double-labeling protocol. Contrary to the current model of the G(1)/S checkpoint, we found that 65% of late- G(1) primary MEFs still proceeded into S phase after a gamma-irradiation dose of 5 Gy. The delayed p53-dependent G(1)/S checkpoint was intact in these cells, and a G2/M checkpoint that was over 90% effective was induced within one hour and maintained through six hours post-irradiation. Furthermore, these cells also exhibited an intra-S-phase replication slow-down, as there was a decrease in the S/ G(2) transition frequency of primary MEFs following gamma-irradiation. The absence of an immediate G(1)/S checkpoint in primary MEFs suggests that in late G(1) these cells may predominantly respond to DNA damage at the level of individual replication origins, rather than by inducing a complete shut-down of S-phase entry.
Cann
Kendra L
KL
Manitoba Institute of Cell Biology and the University of Manitoba, Winnipeg, Manitoba, Canada.
Hicks
Geoffrey G
GG
D000818:Animals; D002453:Cell Cycle; D002455:Cell Division; D049109:Cell Proliferation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis / Q000235:genetics; D042522:DNA Replication Timing; D003857:Deoxyuridine / Q000031:analogs & derivatives; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D016193:G1 Phase; D016195:G2 Phase; D005720:Gamma Rays; D006367:HeLa Cells / Q000528:radiation effects; D006801:Humans; D007065:Idoxuridine; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis; D016196:S Phase; D013194:Staining and Labeling / Q000379:methods
D050759:Cyclin-Dependent Kinase Inhibitor p21; D012333:RNA, Messenger; C012244:5-chloro-2'-deoxyuridine; D007065:Idoxuridine; D003857:Deoxyuridine