ATM-dependent ERK signaling via AKT in response to DNA double-strand breaks.
Ionizing radiation (IR) triggers many signaling pathways primarily originating from either damaged DNA or non-nuclear sources such as growth factor receptors. Thus, to study the DNA damage-induced signaling component alone by irradiation would be a challenge. To generate DNA double-strand breaks (DSBs) and minimize non-nuclear signaling, human cancer cells having bromodeoxyuridine (BrdU) - substituted DNA were treated with the photosensitizer Hoechst 33258 followed by long wavelength UV (UV-A) treatment (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose- dependent generation of DSBs equivalent to radiation doses between 0.2 - 20 Gy, as determined by pulsed-field gel electrophoresis, and accompanied by dose-dependent ATM (ser-1981), H2AX (ser-139), Chk2 (thr-68), and p53 (ser-15) phosphorylation. Interestingly, low levels (≤ 2 Gy equivalents) of BrdU photolysis stimulated ERK phosphorylation whereas higher (> 2 Gy eq.) resulted in ERK dephosphorylation. ERK phosphorylation was ATM-dependent whereas dephosphorylation was ATM-independent. The ATM-dependent increase in ERK phosphorylation was also seen when DSBs were generated by transfection of cells with an EcoRI expression plasmid or by electroporation of EcoRI enzyme. Furthermore, AKT was critical for transmitting the DSB signal to ERK. Altogether, our results show that low levels of DSBs trigger ATM- and AKT-dependent ERK pro-survival signaling and increased cell proliferation whereas higher levels result in ERK dephosphorylation consistent with a dose-dependent switch from pro-survival to anti-survival signaling.
Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA, USA.
Ashraf
A
Khalil
Rhiannon N
RN
Morgan
Bret R
BR
Adams
Sarah E
SE
Golding
Seth M
SM
Dever
Elizabeth
E
Rosenberg
Lawrence F
LF
Povirk
Kristoffer
K
Valerie
D064007:Ataxia Telangiectasia Mutated Proteins; D001973:Bromodeoxyuridine / Q000737:chemistry; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D010782:Photolysis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology*; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D001973:Bromodeoxyuridine
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