Merlin-Deficient Schwann Cells Are More Susceptible to Radiation Injury than Normal Schwann Cells In Vitro

Abstract

Objectives  Vestibular schwannomas (VS) are intracranial tumors, which are caused by NF2 gene mutations that lead to loss of merlin protein. A treatment for VS is stereotactic radiosurgery, a form of radiation. To better understand the radiobiology of VS and radiation toxicity to adjacent structures, our main objectives were (1) investigate effects of single fraction (SF) radiation on viability, cytotoxicity, and apoptosis in normal Schwann cells (SCs) and merlin-deficient Schwann cells (MD-SCs) in vitro, and (2) analyze expression of double strand DNA breaks (γ-H2AX) and DNA repair protein Rad51 following irradiation.

Study Design  This is a basic science study.

Setting  This study is conducted in a research laboratory.

Participants  Patients did not participate in this study.

Main Outcome Measures  In irradiated normal SCs and MD-SCs (0–18 Gy), we measured (1) viability, cytotoxicity, and apoptosis using cell-based assays, and (2) percentage of cells with γ-H2AX and Rad51 on immunofluorescence.

Results  A high percentage of irradiated MD-SCs expressed γ-H2AX, which may explain the dose-dependent losses in viability in rodent and human cell lines. In comparison, the viabilities of normal SCs were only compromised at higher doses of radiation (>12 Gy, human SCs), which may be related to less Rad51 repair. There were no further reductions in viability in human MD-SCs beyond 9 Gy, suggesting that <9 Gy may be insufficient to initiate maximal tumor control.

Conclusion  The MD-SCs are more susceptible to radiation than normal SCs, in part through differential expression of γ-H2AX and Rad51. Understanding the radiobiology of MD-SCs and normal SCs is important for optimizing radiation protocols to maximize tumor control while limiting radiation toxicity in VS patients.

Introduction

Sporadic vestibular schwannoma (VS) are benign intracranial tumors originating from the Schwann cells (SC) of the cochleovestibular nerve. They are caused by inactivating mutations of the NF2 gene that encodes the merlin tumor suppressor protein. Due to the nature and location of these tumors, VS can cause progressive hearing loss and imbalance. The incidence of VS is rising, ¹ and it is estimated that 1 to 2 in 1,000 individuals will develop a VS in their lifetime. ^{2 3} In parallel, the use of stereotactic radiosurgery (SRS) for VS has risen from 5 to 24% in recent years. ^{4 5} Although the side-effect profile and tumor control rates with SRS appear to be favorable, up to 13% of tumors continue to progress in the long term. ^{6 7} Although we have a robust understanding of the molecular pathways leading to tumorigenesis, the radiobiology of VS and factors leading to radiation resistance are poorly understood. ^{8 9}

In general, ionizing radiation causes DNA damage by initiating base damage, single and double stranded breaks (DSB), and DNA crosslinking. In particular, radiation-induced DSBs are lethal and can initiate cell death via p53 activation, caspase-dependent apoptosis, and mitotic catastrophe in proliferating cell populations. ^{8 10 11} A molecular consequence of radiation-induced DSBs is the activation of ataxia-telangiectasia mutated (ATM) protein, ¹² which subsequently initiates the phosphorylation of histone H2AX (i.e., γ-H2AX or phospho-H2AX, an established marker for detection and resolution of DSBs). ^{13 14 15} The generation of γ-H2AX triggers the recruitment of proteins important for DNA repair. ¹⁶

Cells can avoid cell death by entering cell cycle arrest and activating DNA repair mechanisms through nonhomologous end joining (NHEJ) and homologous recombination (HR). ¹⁶ The primary mechanism of DNA repair depends on several factors including the type of insult and the phase of cell cycle that the DNA injury occurred. ^{17 18 19} When compared with NHEJ, HR repair is less prone to error and involves more protein complexes, including RPA, Rad50, Rad51, and BRCA1/2. ¹⁶ In HR, Rad51 protein is the major strand-transfer enzyme that enters the nucleus and repairs DSBs. ^{16 17 18 20} Specifically, Rad51 expression has been linked to radiation resistance in various malignances. ^{21 22}

Unfortunately, the role of γ-H2AX and Rad51 in VS have not been thoroughly elucidated. With a better understanding of VS radiobiology, we can optimize SRS radiation protocols to maximize tumor toxicity and reduce radiation resistance while minimizing injury to surrounding normal structures. In this study, we investigate the effects of single-fraction (SF) radiation on normal SCs and merlin-deficient Schwann cells (MD-SC) on caspase-3/7 activity, cytotoxicity, and cell viability in vitro. We also analyzed the expression of DSBs in DNA (as measured by γ-H2AX) and Rad51 DNA repair proteins following irradiation.

Cell Lines and Culture

Primary SCs from adult rat and adult human nerve tissues were prepared by the Monje Research Laboratory according to established protocols. ^{23 24} Mouse MD-SCs (FV-MNMSC-032910 aka MTC-10 + luciferase) ²⁵ and human MD-SCs (HSC7228-45 aka MD-HSC-45) ²⁶ were developed by the Fernandez-Valle Research Laboratory as previously published. Primary SCs were expanded at 37°C and 5% CO ₂ on 0.01% poly-L-ornithine (Sigma–Aldrich) and laminin (5 µg/mL)-coated flasks, and in mitogenic media consisting of Dulbecco's modified eagle medium (DMEM), 10% heat inactivated fetal bovine serum (FBS; Seradigm), forskolin (2 μM; Sigma–Aldrich), heregulin (2.25 μM; Sigma–Aldrich), and 1% penicillin-streptomycin. The MD-SCs were cultured at 37°C and 5% CO ₂ in N2 media, which is comprised of DMEM/F12 (Thermo Fisher), 1% N2 supplement (Thermo Fisher), and 1% penicillin-streptomycin (Sigma–Aldrich) on CellBIND plates (Corning).

Radiation Protocol

An RS 2000 biological cabinet X-Irradiator (Rad Source Technologies, Suwannee, Georgia, United States) was used for irradiations. Dosimetry was confirmed by using a standard ion chamber sensor (10 × 6–0.6 high dose rate chamber, Radcal, Inc.) attached to an Accu-dose Meter Dose (Radcal, Inc.). Cells were irradiated at room temperature with SF radiation (0, 3, 6, 9, 12, 15, and 18 Gy) at 160 kV and 25 mA, using a dose rate of 550c Gy/min.

Caspase-3/7, Cytotoxicity, and Viability Assays

For cell-based assays, 10,000 cells were seeded on 96-well plates precoated with 0.01% poly-L-ornithine and laminin (5 μg/mL) and cultured in their respective media at 37°C and 5% CO ₂ ( n  = six per condition and time-point). After 24 hours in vitro , the media was changed to maintenance medium consisting of DMEM, 10% FBS, and 1% penicillin-streptomycin. Subsequently, they were treated with SF radiation (0, 3, 6, 9, 12, 15, and 18 Gy). A cell-based assay that simultaneously detects caspase-3/7 activity, cytotoxicity (“membrane integrity”) and cell viability (ApoTox-Glo Triplex Assay, Promega) was performed by using the manufacturer's recommended protocol at 24 and 96 hours postirradiation. Fluorescence and luminescence were measured with the Glomax luminometer and fluorometer (Promega).

Immunofluorescence

For immunofluorescence studies, 10,000 to 20,000 cells were seeded in their respective culture media onto precoated 16-well culture slides. After 24 hours, respective media were changed to maintenance media, and plates were irradiated with 0, 6, 12, or 18 Gy. After incubation for 0, 15, 60, and/or 360 minutes in maintenance media, cells were fixed, permeabilized, and blocked. Subsequently, the slides were treated with 1:500 mouse phospho-histone H2AX (Ser139) antibody (GT2311, Invitrogen) or 1:2000 rabbit Rad51 antibody (ab63801, Abcam) overnight at 4°C, donkey anti-rabbit secondary antibody conjugated to Alexa-594 (Thermo Fisher Scientific) for 2 hours, and 4′,6-diamidino-2-phenylindole dihydrochloride nuclear stain (DAPI, D8542, Sigma–Aldrich) for 15 minutes at room temperature. Subsequently, antifade medium was applied, and slides were cover-slipped. Representative images were obtained by using a confocal microscope (×40, Carl Zeiss LSM 700, Germany). A blinded reviewer counted the number of cells that demonstrated nuclear foci for γ-H2AX (Ser139) and Rad51 on images measuring 320 × 320 μm ( n  = 4–6 per condition). Subsequently, the percentage of cells expressing nuclear foci were calculated per high power field.

Statistical Analysis

Data were analyzed with two-way analysis of variance with least square means for multiple comparisons and Tukey–Kramer adjustment, and Wilcoxon rank-sum test with Bonferroni correction. Significance was set at p -value less than 0.05.

Results

Viability, Caspase-3/7, and Cytotoxicity by Radiation Dosage and Cell-Type

Normal SCs and MD-SCs (rodent and human) were irradiated with 0, 3, 6, 9, 12, 15, and 18 Gy of SF radiation. Caspase-3/7, cytotoxicity, and viability assays were performed at 24 and/or 96 hours. At 24 hours, when caspase-3/7 activity is expected, no significant changes in caspase-3/7 activity were detected in all cell lines ( Fig. 1A and B ). At the same time point, no significant increases in cytotoxicity were noted in our rodent SCs and MD-SCs ( Fig. 1C ). Conversely, radiation initiated significant increases in cytotoxicity (measure of loss of membrane integrity) in the human cell lines, beginning at 6 Gy for normal SCs and 15 Gy for MD-SCs ( Fig. 1D ); these results suggest that radiation affects membrane integrity differently in human SCs and MD-SCs. Furthermore, there were no significant changes in cell viability at 24 hours ( Fig. 1E and F ); however, at 96 hours, rodent and human MD-SCs demonstrated dose-dependent losses in viabilities at all radiation doses tested, starting at 3 Gy ( Fig. 1G and H ). In contrast, normal SCs were more resistant to radiation injury at all doses, exhibiting reductions in viabilities only at higher doses of radiation (15 and 18 Gy) in human SCs ( Fig. 1H ).

Fig. 1.

Apoptosis, cytotoxicity, and viability assays following single fraction radiation of rodent and human normal SC and MD-SC. Mean fold changes of relative luminescence or fluorescence units were calculated. ( A, B ) At 24 hours, normal SCs and MD-SCs in both rodent and human cell lines had similar levels of caspase 3/7 activation at all doses of radiation. ( C, D ) No significant differences in cytotoxicity were found in rodent normal SCs and MD-SCs; however, cytotoxicity was observed in human normal SCs at lower doses than MD-SCs. ( E, F ) No radiation-induced loss of cell viability was observed in either rodent or human normal SCs and MD-SCs at 24 hours. ( G, H ) MD-SCs demonstrated dose-dependent reductions in viability with 3 to 18 Gy, while human normal SCs showed loss of viability only at higher radiation doses (i.e., 15 and 18 Gy). There were significant differences in viability between MD-SCs and normal SCs at 3 to 18 Gy in both rodent and human cell lines. There were no additional losses in viability after 6 and 9 Gy for MD-SCs of rodent and human origin, respectively. Boxplot = 25–50–75th percentiles. Circles = mean. Error bar represent minimum and maximum values. * p  < 0.05; ** p  < 0.01. n  = six replicates each. MD-SC, merlin-deficient Schwann cell; NS, not significant.

Immunofluorescence Images for Double-Strand DNA Breaks and DNA Repair Following Irradiation

To detect levels of DSBs and DNA repair at 0, 6, 12, and 18 Gy, immunofluorescence was performed at 15, 60, and 360 minutes postirradiation for γ-H2AX and Rad51, respectively ( Figs. 2–3 ). At 60 minutes, rodent MD-SCs demonstrated evident nuclear γ-H2AX with 6, 12, and 18 Gy, while rodent SCs demonstrated low levels of γ-H2AX even at 18 Gy ( Fig. 2A and B ). This is consistent with viability assays showing that rodent MD-SCs were more susceptible to radiation injury than normal SCs. Furthermore, at 360 minutes, very few rodent MD-SCs expressed nuclear Rad51 at all radiation dosages, suggesting that lack of Rad51 DNA repair may contribute to their radiation susceptibility. In addition, rodent SCs demonstrated obvious Rad51 predominantly at 6 Gy, suggesting that this dose may be an important threshold for the initiation of DNA repair mechanisms in normal SCs ( Fig. 3A and B ).

Fig. 2.

γ-H2AX Expression in normal SC and MD-SC. γ-H2AX immunofluorescence was performed 60 minutes after cells were exposed to radiation. Intense nuclear γ-H2AX expression was seen in varying degrees at 6, 12, and 18 Gy in all cell lines: ( A ) rodent normal SCs, ( B ) rodent MD-SCs, ( C ) human normal SCs, and ( D ) human MD-SCs. DAPI, nuclear stain. White bar = 40 μm. MD-SC, merlin-deficient Schwann cell.

Fig. 3.

Rad51 Expression in normal SC and MD-SC. To evaluate the activation of DNA repair, immunofluorescence for Rad51 was performed 360 minutes after cells were exposed radiation. ( A ) Rodent normal SCs demonstrated Rad51 expression at 6 Gy alone. ( B ) No obvious Rad51 expression was seen in rodent MD-SCs at all radiation doses. ( C ) Several human normal SCs showed weak-to-moderate expression of Rad51 after 6, 12, and 18 Gy. ( D ) Rad51 expression was seen at baseline in human MD-SCs, as well as 6, 12, and 18 Gy. DAPI, nuclear stain. White bar = 40 μm. MD-SC, merlin-deficient Schwann cell.

In contrast to our rodent cell lines, both human SCs and MD-SCs demonstrated high levels of γ-H2AX at 60 minutes ( Fig. 2C and D ), suggesting that both cell lines are susceptible to developing radiation-induced DSBs. This is inconsistent with our viability assays that showed human SCs were more resistant than MD-SCs to radiation injury, which may reflect dynamic γ-H2AX resolution through the activation of DNA repair in human SCs. At 360 minutes, some normal SCs demonstrated nuclear Rad51 at all radiation doses, suggesting that normal SCs are capable of repairing DSBs by rejoining DNA. On the other hand, human MD-SCs exhibited Rad51 at 6 Gy, but not 12 or 18 Gy, suggesting the absence of Rad51 at higher radiation doses ( Fig. 3C and D ) may also explain the dose-dependent losses of MD-SCs in our viability assays. All together, these results suggest that MD-SCs may mount insufficient DNA repair to address the high levels of DNA damage seen with large doses of radiation.

In addition, when evaluating DNA repair mechanisms, we found that irradiated cells demonstrated both cytoplasmic and nuclear staining of Rad51 protein ( Figs. 2–3 ). These findings may represent cytoplasmic to nuclear transport for DNA repair, further evidence highlighting the dynamic processes that occur following radiation injury. ²⁷

Percentage of Cells with Double-Strand DNA Breaks and DNA Repair Following Irradiation

The percentages of cells with γ-H2AX and Rad51 were calculated for each radiation dose and cell-type at 15, 60, and 360 minutes ( Fig. 4A–L ). At various time points, a majority of rodent MD-SCs demonstrated γ-H2AX at 6, 12, and 18 Gy, but only a small portion of MD-SCs had transient Rad51 at 6 Gy (15 minutes) ( Fig. 4A–F ). These results suggest that insufficient DNA repair in MD-SCs could contribute to the dose-dependent losses seen on viability assays ( Fig. 1G ). Only a small percentage of normal SCs demonstrated γ-H2AX foci overall, except at 6 Gy (360 minutes), when a small but signficant increase in normal SCs with Rad51 was observed ( Fig. 4A–F ). These findings suggest that rodent SCs are less susceptible to radiation injury overall and those that are susceptible at 6 Gy may be upregulating Rad51 in an attempt to maintain cell survival.

Fig. 4.

Percentage of normal SC and MD-SC with γ-H2AX and RAD51 expression. Immunofluorescence for γ-H2AX (marker of DNA damage) and Rad51 (DNA repair enzyme) was performed on irradiated normal and MD-SCs. ( A–C ) More rodent MD-SCs demonstrated γ-H2AX expression when compared with normal SCs at 6, 12, and 18 Gy (particularly at 60 minutes postirradiation). ( D–F ) When exposed to 6 Gy of radiation, more normal rodent SCs demonstrated Rad51 expression when compared at all time points postirradiation. ( G–I ) At higher doses (i.e., 12 and 18 Gy), there were more normal human SCs expressing γ-H2AX at all time points when compared with lower doses. There were higher percentages of human MD-SCs with notable γ-H2AX expression at all doses at 360 minutes and at 18 Gy at 60 minutes. ( J–L ) Except at 6 Gy (60 minutes) and 18 Gy (360 minutes), there were no significant differences in the percentage of normal human SCs expressing Rad51. The percentage of human MD-SCs expressing Rad51 increased from baseline only at 6 Gy (360 minutes). Boxplot = 25–50 to 75th percentiles. Circles = mean. Error bar represent minimum and maximum values. * p  < 0.05, ** p  < 0.01. n  = 4–6 replicates. MD-SC, merlin-deficient Schwann cell.

Overall, a majority of human SCs expressed nuclear γ-H2AX, particularly evident at higher doses (i.e., 12 and 18 Gy) ( Fig. 4G–I ). In parallel, the mean percentage of human SCs with Rad51 also increased at various time points with all radiation dosages, obtaining significance particularly at 6 Gy (60 minutes) and 18 Gy (360 minutes) ( Fig. 4J–L ), which could explain why human SCs were highly resistant to radiation on our viability assays ( Fig. 1H ). On the other hand, a majority of human MD-SCs expressed γ-H2AX, particularly at 360 minutes; however, the percentage of irradiated cells expressing Rad51 did not increase above 0 Gy levels, except at 6 Gy (360 minutes) ( Fig. 4K ), suggesting that human MD-SCs may be more susceptible to radiation-induced losses in viability due to insufficient Rad51 DNA repair. Furthermore, there were significant differences between the percentages of human SCs and MD-SCs with γ-H2AX and Rad51 expression at 6 Gy, suggesting 6 Gy of radiation may initiate differential responses to radiation between the two cell lines.

Discussion

The SRS is a common method for delivery of SF radiation in patients with VS. ²⁸ With modern SRS dosing, the progression-free survival at 15 years is between 87 and 89% in patients with sporadic VS. ^{6 7} In general, several factors can contribute to radiation resistance, including (1) radiation dosage and fractionation, (2) expression of DSBs and DNA repair proteins, (3) focal population of radiation-resistant cells, (4) proliferative index and cell cycle checkpoint, (5) activation of various cell death pathways, (6) hypoxic state, and (7) differential expression of non-NF2 oncogenes and tumor suppressor genes. ^{26 27 29 30 31 32 33 34} However, the exact mechanisms responsible for radiation resistance in VS tumors are not well elucidated. Existing studies with VS suggest a possible link between radiation resistance and the expression levels of phosphatase and tensin homolog (PTEN) and ErbB2 (receptor tyrosine-protein kinase erbB-2). ^{35 36} Although PTEN is important for maintaining genomic stability and repairing DSBs, ³⁷ how PTEN and ErbB2 contribute to DNA repair, cell survival, and ultimately radiation resistance in VS is not very clear. ³⁸ Thus, the aim of our study was to assess the impact of radiation dose on DSBs, DNA repair, and cell death of MD-SCs in comparison to normal SCs.

Baseline Expression of γ-H2AX and Rad51

Most cells express H2AX protein at baseline, but ordinarily H2AX is degraded through poly-ubiquitination. However, when radiation causes DSBs, H2AX poly-ubiquitination stops temporarily; H2AX becomes rapidly phosphorylated at Ser139 to become γ-H2AX, which is then incorporated into chromatin where DSBs occur. ^{14 15 39} Subsequently, several DNA repair mechanisms are activated, including the recruitment of the DNA repair protein, Rad51. ⁴⁰ Because proliferative cells are more often in the S and G2 phases of the cell cycle, these cells express more phosphorylated γ-H2AX and Rad51 proteins at baseline. ^{41 42} Thus, it is understood that more proliferative cells, such as tumor and transformed cell lines, are more prone to radiation-induced cell death because they progress through mitosis with uncorrected DNA breaks from inefficient repair mechanisms. The findings of our study are consistent with this theory and demonstrate that baseline expression of γ-H2AX and Rad51 is relatively higher in our MD-SCs.

Radiation Dose and Cell Death

When examining radiation doses, it is anticipated that higher doses will lead to more radiation injury and DSBs. ³⁹ When DSBs overwhelm the DNA repair mechanisms, cells become irreparably damaged, leading to the onset of cell death via various pathways, for example, caspase-dependent apoptosis, caspase-independent cell death, and autophagy. ^{10 11 27} Our results suggest that the radiation-induced cell death is not related to caspase-3/7 cleavage (a hallmark of apoptosis) in both human and rodent cell lines ( Fig. 1A and B ). When we look at cytotoxicity (a marker of dead cell protease leakage) ( Fig. 1C and D ), we find that normal human SCs and human MD-SCs had significant increases to suggest that radiation-induced damage may affect membrane integrity that could contribute to caspase-independent cell death ( Fig. 1G and H ). However, rodent cell lines did not express cytotoxicity at all radiation doses tested, but this phenomenon may potential occur at a later time point. Some examples of caspase-independent cell death that may occur in irradiated normal SCs and MD-SCs are autophagy, mitotic catastrophe, and apoptosis-inducing factor. ^{10 11 27} Investigation into these signaling cascades is a direction of future research.

Radiation Response in Merlin-Deficient Schwann Cells

When we look at viabilities after irradiation, we found that MD-SCs were more radiosensitive than normal SCs ( Fig. 4G and H ). The MD-SCs were more susceptible possibly because they expressed higher levels of radiation-induced DSBs than normal SCs. This may be due to the inability of MD-SCs to adequately correct radiation-induced DNA breaks, as suggested by insufficient expression of Rad51 DNA repair ( Fig. 4A-C and G-I ).

In particular, human MD-SCs showed dose-dependent losses in viability from 0 to 9 Gy of SF radiation, but no further reductions from 9 to 18 Gy ( Fig. 1H ). These results suggest that perhaps radiation doses less than 9 Gy are not sufficient to initiate maximal reduction in the viability of MD-SCs. A reason why 9 Gy may be an important threshold for radiation injury in MD-SCs is that lower doses of radiation (e.g., 6 Gy) can increase Rad51 DNA repair ( Fig. 4L ). At lower doses (i.e., 6 Gy), human MD-SCs may express more Rad51 in an attempt to evade radiation-induced cell death; at higher doses, these cells may potentially initiate cell death through activation of robust cell cycle checkpoints. In rodent MD-SCs, the threshold for radiation-induced cell death is even lower (i.e., 3 Gy), Fig. 1G potentially because cells did not express obvious Rad51 protein at the radiation doses and time points tested ( Fig. 4D–F ).

Radiation Response in Normal Schwann Cells

In contrast to MD-SCs, normal SCs demonstrated less radiation-induced losses in viability ( Fig. 1G and H ). Specifically, normal rodent SCs had no reductions in viability at all radiation doses ( Fig. 1G ), while normal human SCs showed only mild reductions (∼30%) at 15 and 18 Gy of radiation ( Fig. 1H ). These findings may be partly explained by comparing the dose- and time-dependent shifts in γ-H2AX and Rad51 between normal SCs and MD-SCs. In rodent cell-lines, MD-SCs demonstrated γ-H2AX as early as 15 minutes, while normal SCs demonstrated γ-H2AX at later time points ( Fig. 4A–C ). In human cell lines, MD-SCs demonstrated γ-H2AX at all radiation doses, while normal SCs expressed γ-H2AX only at 12 and 18 Gy ( Fig. 4G–I ). Moreover, normal SCs of both rodent and human origin may be less susceptible to radiation at 6 Gy because the percentages of cells with Rad51 DNA repair increased significantly at this dose ( Fig. 4D–F and K ).

Potential Clinical Significance

The optimal radiation threshold maximizes radiation injury to tumor cells while reducing toxicity to normal surrounding structures. Overall, higher doses of radiation result in higher levels of γ-H2AX and lower levels of Rad51. ^{43 44 45} Based on our viability studies, we observe a radiation threshold at approximately 9 to 12 Gy that is optimal for tumor control without significant injury to normal SCs. This determination also takes into consideration patterns in γ-H2AX and Rad51 expression. Because Rad51 DNA repair is seen in MD-SCs exposed to 6 Gy, we suspect that that radiation doses <9 Gy would be insufficient for tumor control. In addition, radiation doses ≥12 Gy are detrimental to normal human SCs because they express more γ-H2AX at these higher doses.

Limitations and Follow-Up Studies

Our study is important because it highlights differences in DNA damage and repair following SF radiation in normal and MD-SCs. However, there are many aspects of tumor control that need further investigation. Our conclusions are significant but are limited by various factors related to the in vitro models used (1) MD-SCs are transformed cell lines that are deficient in merlin through viral knockout of exon 2 of the NF2 gene, (2) rat normal SCs were utilized as controls for mouse MD-SCs because primary mouse SCs do not expand well in vitro, and (3) human normal SCs were isolated from adult peripheral nerves and were not the isogenic controls of the human MD-SCs. Investigating other forms of DNA damage (e.g., nonlethal single-strand breaks) and DNA repair would be important in future investigations of the radiation response. Additional studies using neuronal cells, primary VS cultures, and functional animal models are warranted to better understand radiation biology of VS and normal nerve.

Conclusion

Understanding the radiobiology of MD-SCs and normal SCs is important for optimizing radiation protocols to maximize tumor control while limiting radiation toxicity in patients with VS. We found differences in DNA damage and repair proteins that help explain why MD-SCs are more susceptible to radiation than normal SCs. In addition, we identified radiation thresholds that could potentially help us reduce radiation toxicity while controlling tumor growth. Furthermore, understanding how radiation affects DNA damage and repair mechanisms in MD-SCs will help establish a foundation to investigate other factors contributing to radiation resistance in patients suffering from VS.

Funding Statement

Funding American Hearing Research Foundation Bernard & Lottie Drazin Memorial Grant to Dr. Erin Cohen and the North American Skull Base Society Research Grant to Drs. Telischi, Dinh, and Ivan. Normal SC cultures were prepared in the laboratory of Paula Monje, in part funded by the Craig H. Neilsen Foundation, U.S. Department of Health and Human Services, National Institutes of Health (grant 339576). The merlin-deficient Schwann cells were prepared in the laboratory of Cristina Fernandez-Valle, National Institute on Deafness and Other Communication Disorders and in part funded by the NIH/NIDCD 1R01-DC010189-06.