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Inhibition of c-Jun N-Terminal Kinase Activity Enhances Vestibular Schwannoma Cell Sensitivity to Gamma Irradiation

Yue, Wei Ying PhD*§,§; Clark, J. Jason MS*; Telisak, Michael MD*,¶; Hansen, Marlan R. MD*,‡

doi: 10.1227/01.neu.0000431483.10031.89

BACKGROUND: Radiosurgery is increasingly used to treat vestibular schwannomas (VSs). Increasing the sensitivity of VS cells to irradiation (IR) could allow for lower and/or more effective doses of IR, improving safety and efficacy. Persistent c-Jun N-terminal kinase (JNK) activity in VS cells reduces cell death by suppressing the accumulation of reactive oxygen species (ROS), raising the possibility that JNK activity protects against IR-induced VS cell death, which is mediated by ROS.

OBJECTIVE: To determine the extent to which JNK signaling contributes to VS cell radiosensitivity.

METHODS: Primary human VS cultures, derived from acutely resected tumors, received single doses (5-40 Gy) of gamma irradiation. Histone 2AX phosphorylation, a marker of IR-induced DNA damage, was assayed by Western blot and immunostaining. ROS levels were quantified by measuring 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. Cell apoptosis was determined by terminal deoxynucleotidyl transferase 2′-deoxyuridine, 5′-triphosphate nick end labeling.

RESULTS: The JNK inhibitors SP6000125 and I-JIP reduced histone 2AX phosphorylation after IR. They also increased H2DCFDA fluorescence in nonirradiated cultures and significantly increased IR-induced (5-10 Gy) H2DCFDA fluorescence 72 hours, but not 2 hours, after IR. Finally, I-JIP (50 μmol/L) significantly increased VS cell apoptosis in cultures treated with 20 to 40 Gy. I-JIP (20 μmol/L), SP600125 (20 μmol/L), and JNK1/2 short interfering RNA knockdown each increased VS cell apoptosis in cultures treated with 30 to 40 Gy, but not lower doses, of IR.

CONCLUSION: Inhibition of JNK signaling decreases histone 2AX phosphorylation and increases ROS and apoptosis in VS cells after gamma irradiation. These results raise the possibility of using JNK inhibitors to increase the effectiveness of radiosurgery for treatment of VSs.

ABBREVIATIONS: ATM, ataxia telangiectasia mutated

DAPI, 4′,6-diamidino-2-phenylindole

FRS, fractionated stereotactic radiotherapy

H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate

H2AX, histone 2AX

IR, irradiation

JNK, c-Jun N-terminal kinase

NF2, neurofibromatosis type 2

PBS, phosphate-buffered saline

ROS, reactive oxygen species

siRNA, small interfering RNA

SRS, stereotactic radiosurgery

TUNEL, deoxynucleotidyl transferase 2′-deoxyuridine, 5′-triphosphate nick end labeling

VS, vestibular schwannoma

Departments of *Otolaryngology-Head and Neck Surgery and

Neurosurgery, University of Iowa, Iowa City, Iowa;

§Department of Otolaryngology-Head and Neck Surgery, Mayo Clinic, Rochester, Minnesota;

Aurora Wilkinson Medical Clinic, Summit, Wisconsin

Correspondence: Marlan R. Hansen, MD, Department of Otolaryngology-Head and Neck Surgery, University of Iowa, Iowa City, IA 52242. E-mail:

Received May 8, 2012

Accepted May 21, 2013

Copyright © by the Congress of Neurological Surgeons