The Radioresistance to Killing of A1–5 Cells Derives from Activation of the Chk1 Pathway

Baocheng Hu,Xiang-Yang Zhou,Xiang Wang,Zhao-Chong Zeng,George Iliakis,Ya Wang

doi:10.1074/jbc.m009340200

Baocheng Hu, Xiang-Yang Zhou + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m009340200

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Abstract

Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

Highlights

A1–5 and B4 are two independently isolated cell lines that were derived in the same laboratory from primary rat embryo fibroblasts transformed with activated Ras (T24) and mutant p53val-135 [1]
After caffeine or UCN-01 treatment, the survival of irradiated A1–5 cells decreased to levels similar to that of B4 cells
The activation of DNA damage checkpoints arrests the normal progression through the cycle and facilitates repair, which in turn increases the survival probability

Summary

Introduction

A1–5 and B4 are two independently isolated cell lines that were derived in the same laboratory from primary rat embryo fibroblasts transformed with activated Ras (T24) and mutant p53val-135 [1]. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G2 arrest and at the same time reduces the phosphorylation of Cdc2 in A1–5 cells. Caffeine, which sensitizes cells to ionizing radiation and other genotoxic agents by abrogating DNA damage checkpoints, has been shown to be an effective inhibitor of ATM1 and ATR [25].

Results

Conclusion