Transition in survival from low-dose hyper-radiosensitivity to increased radioresistance is independent of activation of ATM Ser1981 activity.
OBJECTIVE
The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G(2)-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance.


METHODS
Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 microg/mL chloroquine, 15 microM genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eight cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G(2)-phase cells entering mitosis, using histone H3 phosphorylation analysis.


RESULTS
The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G(2)-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range.


CONCLUSIONS
Overcoming HRS is reliant on the function of the early G(2)-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.
Krueger
Sarah A
SA
Department of Radiation Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA.
Collis
Spencer J
SJ
Joiner
Michael C
MC
Wilson
George D
GD
Marples
Brian
B
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D016195:G2 Phase / Q000502:physiology / Q000528:radiation effects; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D034741:RNA, Small Interfering; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000502:physiology*; D051381:Rats; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases