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The epidermal growth factor receptor (EGFR) is frequently dysregulated in malignant glioma that leads to increased resistance to cancer therapy. Upregulation of wild type or expression of mutant EGFR is associated with tumor radioresistance and poor clinical outcome. EGFR variant III (EGFRvIII) is the most common EGFR mutation in malignant glioma. Radioresistance is thought to be, at least in part, the result of a strong cytoprotective response fueled by signaling via AKT and ERK that is heightened by radiation in the clinical dose range. Several groups including ours have shown that this response may modulate DNA repair. Herein, we show that expression of EGFRvIII promoted γ-H2AX foci resolution, a surrogate for double-strand break (DSB) repair, and thus enhanced DNA repair. Conversely, small molecule inhibitors targeting EGFR, MEK, and the expression of dominant-negative EGFR (EGFR-CD533) significantly reduced the resolution of γ-H2AX foci. When homologous recombination repair (HRR) and non-homologous end joining (NHEJ) were specifically examined, we found that EGFRvIII stimulated and CD533 compromised HRR and NHEJ, respectively. Furthermore, NHEJ was blocked by inhibitors of AKT and ERK signaling pathways. Moreover, expression of EGFRvIII and CD533 increased and reduced, respectively, the formation of phospho-DNA-PKcs and -ATM repair foci, and RAD51 foci and expression levels, indicating that DSB repair is regulated at multiple levels. Altogether, signaling from EGFR and EGFRvIII promotes both HRR and NHEJ that is likely a contributing factor towards the radioresistance of malignant gliomas.
Glioblastoma multiforme (GBM) is the most deadly form of malignant glioma with a highly aggressive and radioresistant phenotype that results in grim patient prognosis. Epidermal growth factor receptor (EGFR) amplification or mutation is observed in a number of different cancers including GBM and is thought to be a major contributor to radioresistance.1,2 The most common EGFR mutation is the type III variant (EGFRvIII) accounting for almost 60% of all EGFR mutations.3 EGFRvIII is defined by a deletion of exons 2–7 resulting in a ligand-independent and constitutively active receptor that leads to the development of highly radioresistant tumors.4,5
Activation of EGFR by ligand (e.g., EGF, TGFα) binding triggers a cascade of cellular signaling events associated with increased cell proliferation, angiogenesis, invasion and metastasis.6 EGFR signaling is also activated in a transient, ligand-independent manner by radiation, initiating the same or similar signaling events as EGF (reviewed in ref. 7). This mechanism perhaps accounts for the radioresistant phenotype displayed by EGFR overexpressing tumors after repeated low-dose radiotherapy protocols. Over the last few years an increasing number of reports have suggested that one contributor towards this cytoprotective response initiated by EGFR signaling is enhanced double-strand break (DSB) repair.8–11
The repair of DSBs in mammalian cells primarily occurs by non-homologous end-joining (NHEJ) or homologous recombination repair (HRR), the dependence and utilization of each pathway is determined by a number of factors, including the cell cycle and the severity and type of the DNA damage. Both modes of DNA repair are vital to cell survival and maintenance of the genome.12 The two repair pathways are tightly regulated by members of the phosphatidyl-inositol-3' kinase-related (PIKK) family of kinases, including DNA-PKcs, ATM and A-T and RAD3-related (ATR), that serve as master regulators of the DNA damage response (DDR).13
EGFR has been linked to DNA-PKcs, ATM and DSB repair by several groups.8 The Nirodi group showed that EGFR kinase activity is important for the activation of DNA-PKcs and for DSB repair.14,15 Other studies have shown that EGFR signaling is important for complete resolution of repair foci.16,17 Previous work by our group demonstrated that expression of EGFR-CD533 (a kinase-dead mutant) is able to block signaling stemming from several members of the ErbB family and other receptor tyrosine kinases (RTKs), and radiosensitize human carcinoma and malignant glioma cells.18–21 This work also showed that expression of EGFR-CD533 reduced split-dose radiosurvival of breast carcinoma cells suggesting a role for RTKs in DSB repair.22 In line with the growth-promoting and radioresistant phenotype displayed by elevated EGFR signaling, we have also shown that forced expression of EGFRvIII from adenovirus in human glioma xenografts results in enhanced tumor growth and radioresistance that can be blocked by EGFR-CD533.18 Although several previous reports have shown global effects on survival and more general effects on DNA repair after modulation of EGFR expression or function, none have determined the mechanism involved or the type(s) of DSB repair being affected. Several studies have implied a role for EGFR in the regulation of the NHEJ pathway by modulating the cellular localization of DNA-PKcs, however, earlier studies did not determine the impact of EGFR mutants commonly found in GBMs, such as vIII, on DSB signaling and whether HRR is affected.
EGFR primarily transmits signals through the RAS/RAF/MEK/ ERK and PI3K/AKT pathways,6 with EGFRvIII showing a preference for PI3K/AKT signaling, that results in increased radioresistance.19,23 However, cross-talk between ErbB receptors and other RTKs complicates the response to radiation.7 Recently, DNA-PKcs was shown to phosphorylate AKT on S473,24,25 a critical step in the activation of AKT. Furthermore, siRNA knockdown of AKT1 resulted in the inhibition of DNA-PKcs in NSCLC cell lines,26 suggesting that AKT and DNA-PKcs are tightly co-regulated. In addition, our recent work demonstrated that the MEK/ERK signaling pathway is critical for efficient HRR and complete activation of ATM.27 In this report we directly address the role of EGFR signaling in modulating HRR and/or NHEJ and show that the common EGFRvIII mutant stimulates both types of DSB repair and the dominant-negative EGFR (CD533) inhibits, supporting the idea that EGFR signaling promotes both major types of DSB repair. Expression of CD533 inhibited radiation-induced phosphorylation of ATM in addition to DNA-PKcs, suggesting that blocking EGFR signaling results in a more general attenuation of the DDR than previously thought. Furthermore, inhibiting either AKT or MEK/ERK resulted in significant inhibition of NHEJ suggesting that both AKT and MEK/ERK are critical effectors downstream of EGFR signaling that modulate NHEJ. Our findings suggest that enhanced DSB repair may contribute significantly to the increased radioresistance seen in GBM with derailed EGFR signaling. Therefore, DSB repair may be a suitable target for therapeutic intervention in GBM.
The phosphorylation of histone H2AX at S139 (γ-H2AX) after exposure to ionizing radiation (IR), and the formation of γ-H2AX IR-induced foci (IRIF) at the site of DNA damage is considered a molecular marker for DSBs.28 The kinetics of γ-H2AX foci removal or resolution is well-accepted as a surrogate indicator of DSB repair. We first assessed the effects of specific EGFR (AG1478) and MEK1/2 (PD184352) inhibitors on the resolution of γ-H2AX foci in human U87 glioma cells. Inhibition of either EGFR or MEK resulted in delayed resolution of γ-H2AX foci. Thirty minutes after exposure to IR cells pre-treated with PD184352 or AG1478 had 30% or 50% more γ-H2AX foci, respectively, compared to untreated control (Fig. 1A). This trend continued and 6 h post-IR approximately twice as many foci remained in PD184352 (two-fold above control), and AG1478 (2.3-fold above control) treated cells. These data suggest a role for EGFR and MEK/ERK in radiation-induced signaling that mediates the kinetics of formation and resolution of γ-H2AX foci.
To strengthen these findings, we then modulated EGFR function genetically expressing either EGFRvIII or CD533 that would stimulate or block EGFR signaling, respectively.18 EGFRvIII and EGFR-CD533 were expressed from adenovirus at a multiplicity of infection known to result in radioprotection or radiosensitization, respectively.29 As early as 30 min after exposure to IR cells expressing EGFRvIII had 45% less γ-H2AX foci compared to LacZ-infected cells (Fig. 1B and C). This trend continued and at 6 h post-IR there were 50% fewer foci in EGFRvIII expressing cells compared to LacZ-infected. In line with this finding, cells expressing EGFR-CD533 had 35% more foci 1 h post-IR, which increased to 50% more foci than vector control at 6 h. Under these conditions the extent of adenovirus infection was verified by immunohistochemistry using anti-EGFR antibody and found to be >95% (Fig. 1C, right). Altogether, EGFRvIII stimulates γ-H2AX foci resolution (DSB repair), whereas EGFR-CD533 blocks resolution. These data support a role for EGFR in IR-induced DSB repair in line with previous reports.8–10 However, whether DSB repair is carried out by NHEJ, HRR or both cannot be concluded from this experiment.
In order to specifically examine the quality of DSB repair we employed two different I-Sce-I-based repair assays—one specific for HRR and the other specific for NHEJ30 (Suppl. Fig. S1). Growth-arrested U87/HRR-GFP cells or U87/NHEJ-DsRed cells were infected with Ad-SceI and treated with increasing doses of EGF. EGF induced a dose-dependent increase in HRR (1.7-fold) peaking at physiological concentrations of EGF, which was reduced to 50% of basal levels in the presence of AG1478 (Suppl. Fig. S2A). Similarly, NHEJ increased ~2-fold (Suppl. Fig. S2B). To confirm that EGF stimulated EGFR signaling, a western blot of extracts from cells treated with EGF or IR showed the expected increase in ERK phosphorylation, and the ability of AG1478 to block these responses (Suppl. Fig. S2C). Collectively, these results provide evidence that ligand-induced EGFR signaling stimulates both HRR and NHEJ.
The impact of manipulating EGFR signaling on HRR and NHEJ was then determined using our fluorescence-based assays and transient expression of EGFRvIII or CD533 from adenovirus. Cells were first serum-starved to reduce indirect cell cycle effects on DSB repair as we have done previously.27 Using the U87/HRR-GFP cells, EGFRvIII expression led to significant 1.4–1.8-fold increases in HRR, whereas a 2.9-fold increase in NHEJ was seen with the U87/NHEJ-DsRed cells (Fig. 2A and B, and data not shown). Consistent with a role for EGFR signaling in both HRR and NHEJ, EGFR-CD533 expression on the other hand resulted in a 70% reduction in HRR and a 20% reduction in NHEJ. EGFRvIII and EGFR-CD533 stimulated and blocked ERK phosphorylation (Fig. 2C), respectively, in line with our previous results.22 Importantly, neither of the EGFR mutants had significant effects on the cell cycle profile (Fig. 2D). These results are consistent with the stimulating effects of EGF on HRR and NHEJ (Suppl. Fig. S2).
I-SceI-based repair assays are now frequently utilized for the analysis of mammalian DSB repair. However, these assays depend on the transcription and translation of reporter or drug resistance genes as endpoints after the I-SceI-generated DSB break is sealed and thus take several days to carry out. Our novel I-SceI NHEJ assay offers a more rapid repair assay. By determining DNA resealing using quantitative PCR (qPCR) we are able to detect repair significantly earlier than by monitoring protein fluorescence. This more direct approach eliminates the possibility that repair measurements could be influenced by indirect DsRed transcriptional or translational effects.
Thus, by using PCR conditions that favor the amplification of sealed DSBs (A. Hawkins, unpublished observations), we then determined the kinetics of NHEJ at the dual I-SceI sites (Fig. 3). A time-course experiment showed that repair can be detected at 8 h and later but not at 4 h after Ad-SceI infection (Fig. 3A). This result is consistent with our previous work showing I-SceI expression within hours of Ad-SceI infection.30 Repair levels increased 6.4-fold by 16 h (over those at 8 h) and plateaued at 8.6-fold 24 h post-infection (Fig. 3A). This result shows that NHEJ can be detected as early as 8 h after infection with Ad-SceI, eliminating the need for detecting repair by reporter assays at later times.
Using this assay, we then determined the effect of EGFR signaling on NHEJ after infection with EGFR-CD533 virus. We found that EGFR-CD533 inhibited NHEJ 45–70% between 10 and 24 h after infection with Ad-SceI (Fig. 3B, and data not shown). This result shows that NHEJ is indeed affected by EGFR signaling and more so than initially determined by DsRed fluorescence (see Fig. 2B). We believe the PCR data is more accurate and reliable than the DsRed data because it examines repair directly and much sooner after I-SceI expression than when DsRed is used as an endpoint for repair. We attribute this difference to transcriptional effects on DsRed expression and the longer time (>48 h) it takes to obtain quantifiable levels of DsRed. Regardless, all combined, these data suggest that EGFR signaling is important for both HRR and NHEJ.
Earlier work using cell fractionation and subsequent western blotting analyses demonstrated that EGFR is important for the nuclear localization of DNA-PKcs, a factor critically important for NHEJ.17 In an extension of this work we examined the effect of EGFRvIII or EGFR-CD533 expression on radiation-induced phospho-DNA-PKcs (T2609) foci formation (Fig. 4). We show that EGFR-CD533 blocked radiation-induced accumulation of nuclear phospho-DNA-PKcs foci by 70% (Fig. 4A). In addition, expression of EGFRvIII increased the formation of nuclear phospho-DNA-PKcs foci between 20–60% (Fig. 4A and data not shown). Our previous work demonstrated that MEK/ERK signaling is important for the formation of phospho-(S1981) ATM foci,27 and ATM has been reported to phosphorylate DNA-PKcs at T2609 in response to radiation which is believed to regulate DNA-PKcs activity.31 Interestingly, we found that EGFR-CD533 also blocked the formation of radiation-induced phospho-ATM foci by 70% (Fig. 4B). We saw little to no effect of EGFRvIII on phospho-ATM foci formation under these conditions. Combined, our data shows that blocking EGFR signaling impairs the formation of both p-(T2609) DNA-PKcs and p-(S1981) ATM foci. At this point it is not clear whether EGFR-CD533 inhibits the activation of ATM, that in turn blocks the activation of DNA-PKcs, or whether both PIKKs are directly affected. Alternatively, EGFR could modulate the proper localization of DNA-PKcs and ATM. Regardless, blocking EGFR signaling with EGFR-CD533 results in impaired repair foci formation in line with a model by which EGFR signaling modulates DNA damage processing and DSB repair via both DNA-PKcs and ATM.
RAD51 expression was shown previously to decrease in the presence of the EGFR inhibitor Gefitinib.32,33 Thus, we examined the effect of EGFRvIII and EGFR-CD533 on RAD51 foci formation and expression (Fig. 5). In several independent experiments EGFRvIII expression increased the number of cells positive for RAD51 foci by 20–30%, whereas the expression of EGFR-CD533 compromised RAD51 foci formation by 20–30% (Fig. 5A, and data not shown). When we examined RAD51 expression by western blotting EGFRvIII increased RAD51 levels about 1.5-fold whereas EGFR-CD533 decreased levels by 10% (Fig. 5B). Collectively, our data show that EGFRvIII upregulates and CD533 downregulates both major DSB repair pathways perhaps by modulating the function, localization and expression of vital repair proteins such as DNA-PKcs, ATM and RAD51.
Since EGFRvIII preferentially signals to the PI3K/AKT pathway,19,23 it is possible that NHEJ is modulated by PI3K/AKT.24,34 Thus, we examined the effect of a pan-AKT inhibitor (SH-5) on NHEJ using the qPCR repair assay (Fig. 6). SH-5 is a phosphatidylinositol analog that prevents PIP3 formation and binding to AKT, thus blocking AKT activation.35 We also examined the effect of inhibiting EGFR with AG1478 and MEK/ERK signaling with PD184352. We found that inhibiting AKT reduced the NHEJ levels at 10 h by 25%, whereas inhibiting either EGFR or MEK/ERK resulted in a 50% reduction. In parallel, we examined by western blotting the effects of blocking AKT and MEK/ERK signaling on radiation-induced phospho-(S473) AKT and p-ERK levels, respectively. The pan-AKT inhibitor SH-5 reduced basal AKT phosphorylation by 60%, and completely inhibited the radiation-induced increase (Fig. 6B). PD184352 completely blocked ERK phosphorylation (data not shown), as expected.30 Altogether, these results show that signaling through the PI3K/AKT and MEK/ERK pathways are both critical for efficient NHEJ.
EGFR has become an important molecular target for cancer therapy with the development of tyrosine kinase inhibitors (TKIs) and monoclonal antibodies directed towards blocking the EGFR signaling pathway.36 The notion that EGFR signaling affects DNA repair has existed for some time,37 yet little is known about the mechanism. EGF has been shown to modulate the radiosensitivity of human cells,38 and depletion of EGFR or inhibition of EGFR signaling led to decreased cell growth and activation of DNA repair factors.39,40 A direct effect on HRR was suggested when blunted expression of RAD51 was found in cells treated with the EGFR inhibitor Tarceva,41 and our own work implicated signaling from the EGFR downstream effectors RAF/MEK/ERK in the regulation of HRR.27 In the last two years studies have focused on the physical interaction between EGFR and DNA-PKcs, nuclear transport, and the resulting impact on DSB repair.8,9 Increased survival in split-dose radiation experiments is considered to be the result of DSB repair. Our group previously demonstrated that expression of EGFR-CD533 radiosensitizes breast carcinoma cells in split-dose radiosurvival assays,22 suggesting a role for RTKs in DSB repair.
In order to solidify these earlier findings at the mechanistic level we used γ-H2AX foci resolution as a surrogate for DSB repair. We show that repair in human glioma cells is enhanced by EGFRvIII expression and compromised by EGFR-CD533. These findings are in agreement with those of others using this same approach (γ-H2AX foci resolution) with NSCLC cell lines harboring naturally occurring mutations in EGFR, or using the monoclonal EGFR antibody Cetuximab.14,16,17 The first direct molecular evidence for EGFR’s influence on DSB repair was reported by Das et al.15 who showed that the EGFR status affected micro-homology end-joining. Herein, we examined DSB repair at unique I-SceI sites of integrated DNA repair substrates that are expected to more closely reflect physiological conditions than repair assays based on transient transfections. However, since I-SceI only creates a single DSB insufficient for triggering a global DDR as IR does, these differences need to be taken into account when results are interpreted. For example, radiation activates signaling initiated from the cytoplasm/membrane compartments, such as EGFR, that could influence IRIF resolution whereas this may not occur in response to a single I-SceI break. Nevertheless, our findings using the resolution of γ-H2AX IRIF as a surrogate for DSB repair and the fluorescence-based repair assays are consistent. Thus, our findings strongly support the conclusion that EGFR signaling is important for both types of DSB repair.
Interestingly, in some cases we saw more effects on DSB repair and foci formation by blocking signaling with EGFR-CD533 than with AG1478. Cross-talk and trans-activation between the RTK receptors, such as EGFR, IGF-1R, PDGFR and c-Met, is now an accepted mechanism of growth control in GBM and other solid cancers.4,7,42 In addition to blocking EGFR signaling, EGFR-CD533 also blocks the oligomerization of EGFR with ErbB2, ErbB4, IGF-1R and potentially other RTKs,7 which would be expected to broaden the effect on signaling and DSB repair. Both ErbB2 and IGF-1R have been implicated in the regulation of ATM and DNA repair.43–47 Thus, EGFR-CD533 is likely impacting more signaling pathways than most small molecule TKIs or monoclonal antibodies specific for EGFR explaining the more robust effects seen with CD533 on DSB repair.
We have previously shown that AKT signaling was enhanced in cells expressing EGFRvIII relative to wild type EGFR,19 a finding which was confirmed by another group.23 Herein, we follow up on this interesting observation and show that blocking AKT signaling inhibits NHEJ, thus identifying AKT as a downstream effector of EGFR signaling during DSB repair. In addition, signaling through the MEK/ERK pathway is also critical for efficient NHEJ. We believe the relative difference in response between blocking AKT vs. MEK seen here might be misleading since PD184352 is particularly effective and treatment results in a complete block of ERK phosphorylation as we showed previously.27 In contrast, we see a relatively less effect of SH-5 on AKT phosphorylation, which may account for the less pronounced effects on repair. Future experiments will examine the role of AKT and its different isoforms on NHEJ in glioma cells. It should be noted that EGFR downstream effectors are not limited to AKT and ERK and further investigation of other molecules such as STAT3 should be performed.42,48
There are several mechanisms by which EGFR/ERK/AKT signaling could affect DSB repair; the activity and assembly of repair factors at the DSB, cell cycle regulation and transcription of repair genes. Our results suggest that all three levels are affected to some degree by EGFR manipulation that would modulate repair. Our work supports the idea that EGFR regulates DNA-PKcs nuclear transport and phosphorylation,8 and goes one step further by showing that the same appears to be the case with ATM perhaps via cross-regulation with DNA-PKcs. One point of note is that the anti-phospho-(S1981) ATM antibody may recognize other repair proteins with similar phospho-motifs gathering at the DSB,49 therefore it could be a conglomerate of repair proteins that fail to assemble at the DSB. In addition a dependence on ERK signaling for the activation, nuclear localization and foci formation of ATR was recently suggested.50 Therefore, taken together, these findings give credence to the notion that growth factor signaling is important for IRIF formation of several members of the PIKK family, although to put this notion to the test would require a more thorough investigation.
EGFR signaling has also been associated with the activation of various transcription factors.7 EGFR has been implicated in the transcriptional regulation of ATM and base excision repair factor XRCC1.51,52 In seeking a cause for the positive effects of EGFR on HRR, we observed increases in RAD51 foci formation which could perhaps be explained by changes in RAD51 expression in line with a number of recent reports.12,32,33,41 Thus, our results support the idea that DSB repair is regulated at several levels by growth factor signaling. It would make sense for the cell if growth-promoting and apoptosis-triggering signals are coordinated at the level of the DSB. In a situation when repair fails, EGFR (and ERK/AKT) pro-survival signaling would be blunted at the expense of apoptosis, whereas after completion of successful repair, cell growth would resume.
EGFR and growth factor signaling in general has long been known to modulate cell cycle regulation.7 Although not directly addressed in this work, one cannot rule out such indirect effects on DSB repair. It is well-accepted that HRR is most active in late S-phase and G2.12 Therefore, cell cycle effects could to some extent account for increases or decreases observed in HRR when EGFR function was manipulated even though we were unable to see any correlation. However, this could not account for the large changes observed in NHEJ which is considered to be most active in G0/ G1.12 Furthermore, using our novel qPCR-based repair assay shows that these effects can be detected as early as 6–8 h after infection with I-SceI adenovirus, suggesting that there is insufficient time for cell cycle effects to have real impact on repair. Thus, cell cycle effects cannot explain the effects on NHEJ we see after manipulating EGFR signaling.
The mechanism behind the increased radioresistance of mutant EGFR, especially EGFRvIII, expressing tumors is as yet undefined. Our results suggest that a major, but certainly not only, contributor to this phenotype is enhanced DSB repair. Other factors such as the existence of cancer stem cells, hypoxia and repopulation should not be ignored but were beyond the scope of this study.1,4 However, targeting of EGFR by TKIs or monoclonal antibodies has given mixed results in the clinic with a reported acquired resistance to treatment.4,36 This acquired radioresistance is thought to be the result of the upregulation of compensatory signaling via other ErbB family members or indeed other RTKs.4 Furthermore, co-expression of EGFRvIII with mutant or deleted PTEN resulting in increased AKT signaling correlates with such resistance.36 The findings reported herein extend our current understanding of EGFR signaling and its influence on DSB repair. We show that EGFR signaling modulates DSB repair in a multi-tiered manner. We are the first to describe EGFRvIII-mediated stimulation of both HRR and NHEJ a finding that may account, in part, for the increased radioresistance of gliomas harboring this important EGFR mutation. Furthermore, we identify AKT and MEK/ERK, important downstream effectors in the EGFRvIII signaling pathway, as critical modulators of NHEJ. We further consolidate the role of DNA-PKcs in EGFR stimulated DSB repair, and additionally, we identify ATM as a participant in this response. Our findings show that increased EGFR signaling has a global and multi-faceted effect on the DDR to influence DSB repair. Therefore, targeting DSB repair in conjunction with radiotherapy either alone or in combination with a TKI may be an attractive approach for therapeutic intervention in GBM.
Anti-phospho-ERK1/2 and ERK2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-EGFR (R19/48) antibody was from Biosource (Invitrogen, Carlsbad, CA). Anti-phospho-ATM (S1981), -phospho-AKT (S473), and AKT antibodies were from Cell Signaling Technology, Inc., (Danvers, MA), γ-H2AX (S139 clone JBW301) was from Upstate/Millipore, MA. Anti-phospho-DNA-PKcs (T2609) antibody was from Biolegend (San Diego, CA), and anti-RAD51 antibody was purchased from EMD Biosciences (Gibbstown, NJ). AG1478 was purchased from AG Scientific (San Diego, CA). PD184352 has been described.27 EGF was purchased from Sigma-Aldrich (St Louis, MO). SH-5 was purchased from EMD Biosciences (Gibbstown, NJ). All drugs were dissolved in DMSO with the exception of EGF which was dissolved in ethanol.
Human malignant glioma U87 (p53+) cells, were cultured as described.30 The adenoviruses Ad-SceI, Ad-LacZ, Ad-EGFR-CD533 and Ad-EGFRvIII have been described.18,30 Ad-SceI was added to the culture medium at 30 MOI, whereas Ad-LacZ, Ad-EFGRvIII and Ad-EGFR-CD533 were used at MOIs of 3–10. Cells were incubated with virus for 4 h at 37°C. Inhibitors were added to the cell culture medium to the indicated final concentrations 1 h before treatment and left in the medium throughout the experiment. Irradiations were performed using an MDS Nordion Gammacell 40 (ON, Canada) research irradiator with a Cs-137 source delivering a dose of 1.05 Gy/min.
Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were exposed to antibodies at 1:500–2,000 dilutions. Specific proteins bands were detected and quantified using infrared emitting dye-conjugated secondary antibodies, anti-mouse IgG 680 Alexa (Invitrogen), or anti-rabbit IgG IRDYE 800 (Rockland Immunochemicals, Gilbertsville, PA) using the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE). Densitometry of immuno-reactive bands was carried out using the Odyssey Application software (version 1.2) from Li-Cor Biosciences.
The HRR-GFP assay has been described.30 The NHEJ-DsRed assay is based on a DNA cassette with two I-SceI recognition sequences flanking an ATG codon acting as a decoy upstream of the DsRed reporter gene. Upon cleavage with I-SceI the decoy is removed, the DNA sealed and DsRed expressed (see Suppl. Fig. S1 and Suppl. Methods for more details). The IRIF assay has been described previously,27 see Supplemental Methods.
Unpaired two-tailed t tests were performed on triplicate or more data sets using GraphPad Prism 3.0 (GraphPad Software, Inc.).
The Massey Cancer Center Flow Cytometry and Imaging Facility are supported in part by NIH grant P30 CA16059.
Supported by NIH P01CA72955 (K.V.), R01CA40615 (L.F.P.) and T32CA085159 (S.E.G.).
Supplementary materials can be found at: www.landesbioscience.com/supplement/GoldingCBT8-8-Sup.pdf