© The Author 2006. Published by Oxford University Press.
ARTICLE |
Effect of Chemotherapy-Induced DNA Repair on Oncolytic Herpes Simplex Viral Replication
Affiliation of authors: Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA
Correspondence to: Manish Aghi, MD, PhD, Brain Tumor Research Center, Massachusetts General Hospital–Simches Research Bldg., 185 Cambridge St., CPZN-3800, Boston, MA 02114 (e-mail: maghi{at}partners.org).
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ABSTRACT |
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Background: Gliomas treated with the alkylating agent temozolomide have incomplete responses in part because of tumoral repair of chemotherapy-induced DNA damage. Data from phase I trials suggest that G207, an oncolytic herpes simplex virus (HSV) with mutated ribonucleotide reductase (RR) and
34.5 genes, is safe but needs greater viral oncolysis to be effective. We hypothesized that temozolomide and G207 treatment limitations could be jointly addressed using temozolomide-induced tumor-protective DNA repair pathways to enhance viral replication. Methods: Human glioblastoma cells (U87, T98, and U373) and U87 cells transfected with the gene for the DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) were treated with G207 and/or temozolomide. Drug interactions, expression of the growth arrest DNA damage 34 (GADD34) and RR transcripts before and after their knockdown with short interfering RNAs, DNA strand breaks, and apoptosis were measured using Chou–Talalay analysis, real-time reverse transcription–polymerase chain reaction, the comet assay, and flow cytometry, respectively. Survival of mice (groups of ten) with intracranial U87 xenograft tumors treated with temozolomide and/or G207 was analyzed using Kaplan–Meier analysis. Results: Temozolomide exhibited strong synergy with G207 in both MGMT-negative and the MGMT inhibitor O6-benzylguanine–treated MGMT-expressing gliomas (Chou–Talalay combination indices = 0.005 to 0.39) and induced GADD34 expression primarily in nonapoptotic MGMT-negative U87 glioma cells (fold difference = 16, 95% confidence interval [CI] = 12.6 to 20.4, compared with untreated cells). MGMT-expressing T98 and U87/MGMT cells treated with temozolomide plus O6-benzylguanine had higher RR expression than untreated cells (fold difference =14.9, 95% CI = 10.1 to 22.0 [T98]; 9.9, 95% CI = 7.0 to 13.8 [U87/MGMT]). GADD34 and RR knockdown increased temozolomide-induced DNA damage and inhibited the synergy of G207 and temozolomide in U87 and O6-benzylguanine–treated U87/MGMT cells. Mice bearing intracranial U87 tumors survived longer after combination therapy (100% survival at 90 days) than after single-agent therapy (median survival = 46 and 48 days with G207 and temozolomide treatment, respectively). Conclusions: Temozolomide-induced DNA repair pathways vary with MGMT expression and enhance HSV-mediated oncolysis in glioma cells. These findings unveil the potential of HSV to target cells surviving temozolomide treatment.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Engineered oncolytic viruses take advantage of cancer cell mutations to induce tumor cell–selective destruction (1–3). G207, an oncolytic herpes simplex virus (HSV) (3), has deletions of both copies of neurovirulence gene
34.5 and an inactivating mutation of UL39, which encodes ICP6, the large subunit of HSV ribonucleotide reductase (RR). As a result of these mutations, G207 selectively replicates in and lyses dividing cells, possibly because dividing cells express mammalian RR and growth arrest DNA damage 34 (GADD34). These gene products have yet to be fully characterized, but they may regulate the cell cycle (4,5) and complement G207 mutations. In particular, mammalian RR generates deoxyribonucleotides in place of HSV RR, and the GADD34 carboxyl terminus substitutes for the homologous region of 34.5 (6). Glioblastomas, the first tumor type in which G207 was studied, are aggressive neoplasms that are resistant to current treatments; over the past decade, surgery, radiation, and chemotherapy have only minimally altered the median survival times for those with the disease (12–15 months) (7). Intratumoral G207 inoculation has efficacy in glioma animal models, and a phase I clinical trial demonstrated safety of intracranial G207 inoculation in glioma patients, albeit with only partial radiologic responses (8,9), suggesting a need for greater viral replication.
Temozolomide, an oral alkylating agent, was approved by the Food and Drug Administration for treatment of newly diagnosed glioblastoma in 2005, based on a phase III randomized multicenter trial in which patients with newly diagnosed glioblastoma who were treated with temozolomide plus radiotherapy survived for a median of 14.6 months, compared with 12.1 months among patients receiving radiotherapy alone (10). At nonacidic pH, temozolomide spontaneously converts into its active metabolite, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide, a DNA alkylating agent that methylates guanine at the O6 and N7 positions (11). O6-methylguanine is not itself lethal to cells, but when paired with thymine, it triggers mismatch DNA repair, a three-step process that involves 1) mismatched base removal by N-methylpurine-DNA glycosylase (12); 2) strand cleavage by apurinic/apyrimidic endonuclease; and 3) strand break recruitment of poly(ADP-ribose) polymerase, a nick sensor that targets the DNA repair synthetic machinery to damaged DNA (12). However, if repair fails to keep pace with DNA damage, repetitive futile rounds of mismatch repair create single-strand DNA breaks, which activate serine/threonine kinase ATR (ATM and Rad3-related) during S phase (13). If ATR activation fails to arrest the cell cycle, single-strand breaks are converted into double-strand breaks in subsequent S phases; these breaks activate serine/threonine kinase ATM (ataxia-telangiectasia mutated). Activated ATM promotes cell cycle arrest and apoptosis (11).
Unfortunately, many gliomas are resistant to temozolomide, primarily because they express the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). MGMT, which is expressed by 20% of gliomas, facilitates temozolomide resistance by removing the alkyl adduct from the O6 position of guanine before mismatch repair begins (14,15). MGMT-mediated temozolomide resistance can be partially overcome by simultaneous treatment with O6-benzylguanine, an MGMT inhibitor that is nontoxic and has been shown in phase 1 clinical trials to be capable of enhancing temozolomide responsiveness of MGMT-expressing gliomas (16).
Because each of these new pharmacologic and oncolytic viral therapies generates only partial responses, glioblastoma treatment may require multimodal therapy. We hypothesized that limitations in temozolomide and G207 glioma treatment could be jointly addressed by taking advantage of temozolomide induction of DNA repair genes to enhance HSV replication. Although some studies have demonstrated synergy between oncolytic HSV treatment and chemotherapy (17–21), the interaction was usually neither quantified nor mechanistically explained. We used Chou–Talalay multiple-drug effect analysis (22) to quantify the interaction between temozolomide and oncolytic HSV. We also examined the effect of glioma p53 and MGMT expression on this interaction and investigated whether interactions between temozolomide and HSV result from temozolomide-induced expression of DNA repair genes. Finally, we studied the combination of temozolomide and oncolytic HSV in vivo.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cell Lines
U87, U373, and T98 human glioblastoma and Vero (African green monkey kidney) cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Tumor cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin at 37 °C and 5% CO2. Vero cells were maintained in the same conditions, with calf serum substituting for fetal calf serum. Human astrocytes were obtained from ScienCell (San Diego, CA) and cultured in human astrocyte medium (ScienCell).
Plasmids
The MGMT cDNA was obtained from ATCC and subcloned into the pCDNA3 expression vector (Invitrogen, Carlsbad, CA). The pc53-SCX3 plasmid (kindly provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) contains a cDNA encoding a dominant p53 mutation in which a cytosine is changed to a thymidine, resulting in a substitution of alanine for valine at codon 143 (23). The 700-bp human GADD34 promoter was cloned using 30-cycle polymerase chain reaction (PCR; 20 seconds at 98 °C, 2 minutes at 68 °C per cycle) with the Takara LA Taq Polymerase (Panvera Corporation, Madison, WI) from template DNA isolated from U87 cells (Trizol per Invitrogen protocol) (forward primer, 5'-CAATTGGGGAGGCCAAGGCGGGAGGAT-3'; reverse primer, 5'-GAATTCTAAGAGCAACGAACACAATGGC-3'; Invitrogen). This generated a PCR product flanked by MfeI and EcoRI restriction sites that was then subcloned into the pCR2.1-TOPO vector (Invitrogen) per the manufacturer's protocol. The PCR product was sequenced and inserted in place of the cytomegalovirus (CMV) promoter into the plasmid pCMV-enhanced green fluorescent protein (EGFP; kindly provided by T. Kuroda, Massachusetts General Hospital, Charlestown, MA), generating the plasmid pGADD34-EGFP, in which expression of the EGFP reporter gene is driven by the human GADD34 promoter.
Transfection
The three plasmids pCDNA3-MGMT, pc53-SCX3, and pGADD34-EGFP were separately transfected into U87 cells using lipofectamine according to the manufacturer's protocol (Invitrogen). Clones were isolated in 1 mg/mL G418 (GIBCO, Carlsbad, CA). Clones from the pc53-SCX3 transfection were screened for mutant p53 expression using an enzyme-linked immunosorbent assay kit (Calbiochem; San Diego, CA), with the clone that expressed the highest level of mutant p53 designated as U87/mp53. Clones from the pGADD34-EGFP transfection were screened by flow cytometry for induction of fluorescence 48 hours after temozolomide treatment, with the clone that exhibited the highest level of fluorescence induction designated as U87/pGADD34-EGFP. Clones from the pCDNA3-MGMT transfection were screened for temozolomide sensitivity as described below. The clone with greatest resistance to both 300 and 1000 µM temozolomide was screened for MGMT mRNA levels by real time (RT) PCR and was designated U87/MGMT.
Viruses
Wild-type HSV-1 strain F (obtained from B. Roizman, University of Chicago, Chicago, IL), strain F–derived 34.5–ICP6–LacZ+ G207 (3), strain F–derived 34.5– R3616 (provided by B. Roizman) (24), wild-type HSV-1 strain KOS (obtained from D. Knipe, Harvard Medical School, Boston, MA), and KOS-derived ICP6–LacZ+ hrR3 (obtained from S. Weller, University of Connecticut, Farmington, CT) (25) were grown, purified, and titered by plaque assay on Vero cells, as described previously (3).
Cell Culture, Cytotoxicity, and Chou–Talalay Analysis
Temozolomide (Schering-Plough, Kenilworth, NJ) was dissolved in saline (20 mM, 4 mg/mL), cisplatin (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide (50 mM, 15 mg/mL), and MGMT inhibitor O6-benzylguanine (Sigma) was dissolved in distilled water (41.49 mM, 10 mg/mL). U87, T98, U87/MGMT, or human astrocytes were incubated overnight in 96-well plates (4000 cells/well). The next day, virus and/or chemotherapy were added to the cells. Temozolomide was sometimes supplemented with 100 µM O6-benzylguanine. Cells were incubated for 4 days, and survival was then assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma) per the manufacturer's protocol. Dose–response curves were fit to Chou–Talalay lines (22), which are derived from the law of mass action and are described by the equation log(fa/fu) = m log D – m log Dm, in which fa is the fraction affected (percent cell death), fu is the fraction unaffected (percent cell survival), D is the dose, Dm is the median-effect dose (the dose causing 50% of cells to be affected, i.e., 50% survival), and m is the coefficient signifying the shape of the dose–response curve. Chemotherapy and virus were then added in combinations in a ratio equaling the ratio of their median-effect doses, with each dose in each experiment plated in triplicate and each experiment performed three times. After fitting the combined dose–response curve from a single representative experiment to a Chou–Talalay line, Chou–Talalay combination indices (CIs) were calculated for each fa using the equation CI = (D1/Dx1) + (D2/Dx2) + (D1)(D2)/[(Dx1)(Dx2)], which defines CI for mutually nonexclusive treatment regimens (CIs reported in this article were derived assuming mutually nonexclusive treatments, although the interpretations of all CIs reported in this article did not change under the mutually exclusive assumption in which the third term in the CI equation is eliminated), in which Dx1 and Dx2 are the chemotherapy and virus doses required to achieve a particular fa, respectively, and D1 and D2 are the doses of the two combined required to achieve the same fa. Levels of interaction are defined as follows: CI greater than 1.3 indicates antagonism, CI between 1.1 and 1.3 indicates moderate antagonism, CI between 0.9 and 1.1 indicates additivity, CI between 0.8 and 0.9 indicates slight synergy, CI between 0.6 and 0.8 indicates moderate synergy, CI between 0.4 and 0.6 indicates synergy, and CI less than 0.4 indicates strong synergy (26). Per the protocol described by Chou and Talalay (26), CI values lack 95% confidence intervals, but the use of the above ranges to interpret CIs takes variability into account.
Single-Step Growth Curves
U87 cells (2.5 x 105) were plated into 12-well plates in DMEM–10% fetal calf serum containing no drug, 300 µM temozolomide, or 0.1 µM cisplatin and grown for 24 hours. These concentrations are nontoxic to U87 cells when treatment is for 72 hours (data not shown). Cells were then infected with G207 at a multiplicity of infection (MOI) of 1.5 in the presence or absence of the same temozolomide or cisplatin concentrations. At various time points up to 48 hours after infection, cells were scraped into the medium and subjected to three freeze–thaw cycles. Virus titers were determined by plaque assays on Vero cells. Each concentration in an experiment was plated in triplicate, and each experiment was performed three times.
Quantitative Real-Time RT-PCR
U87, T98, U87/MGMT, or human astrocyte cells were treated for 48 hours with temozolomide (10 µM–3 mM) or cisplatin (0.01–1 µM). These doses give 30%–90% survival after 4 days and 100% survival at 24–48 hours (data not shown). RNA was extracted from cells with Trizol (Invitrogen). A high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA) was used to generate cDNA. Real-time reverse transcription (RT)-PCR was performed on an ABI Prism 7000 (Applied Biosystems) machine using human GADD34 (forward, 5'-GGAGGAAGAGAATCAAGCCA-3'; reverse, 5'-TGGGGTCGGAGCCTGAAGAT-3'); MGMT (forward, 5'-CCTGGCTGAATGCCTATTTC-3'; reverse, 5'-GATGAGGATGGGGACAGGATT-3'); ATM (forward, 5'-CTTCAGTGGACCTTCATAATGC-3'; reverse, 5'-CCATACAAACTATCTGGCTCC-3'); ATR (forward, 5'-ACATTCCCTGATCCTACATCATG-3'; reverse, 5'-TTCAATAGATAACGGCAGTCCTG-3'); MPG (forward, 5'-GTCCTAGTCCGGCGACTTCC-3'; reverse, 5'-CTTGTCTGGGCAGGCCCTTTGC-3'); and PADPRP (forward, 5'-TCGCCCATGTTTGATGGAAA-3'; reverse, 5'-CTGTCACTCCTCCAGCTTC-3') primers (Invitrogen) combined with SYBR Green Master Mix (Applied Biosystems) or primer–probe combinations for RR subunits M1 and M2 (Part Nos. Hs00357247_g1 and Hs00168784_m1; Applied Biosystems) and 18S rRNA (Part No. 4308329; Applied Biosystems) combined with TaqMan Master Mix (Applied Biosystems). Relative quantification was performed using 18S rRNA as an endogenous control. All reactions began with 10 minutes at 95 °C for AmpliTaq Gold activation, followed by 40 cycles of 95 °C for 15 seconds for denaturation followed by 60 °C for 1 minute for annealing–extension.
Small Interfering RNA (siRNA)
Duplex RNA targeting human GADD34 (5'-GGACACUGCAAGGUUCUGA), the M2 subunit of human RR (5'-UGCUGUUCGGAUAGAACAG), and control small interfering (si) RNA with medium GC content (comparable to that of the other siRNAs used) that targets no known vertebrate sequences were synthesized with d(TT) at the 3' terminus of each strand (Invitrogen). siRNA was transfected into U87 or T98 cells using lipofectamine per the manufacturer's protocol (Invitrogen). Levels of GADD34 and RR M2 subunit mRNA in cells transfected with GADD34 or RR M2 siRNA, relative to levels in mock-transfected cells, were assessed at 24, 48, and 72 hours posttransfection using RT-PCR and were found to be reduced to 27%–30%, 3%–7%, and 25%–28% of baseline control cell values, respectively. Control siRNA maintained GADD34 and RR MR subunit mRNA levels at 95%–100% those of nontransfected cells 24–72 hours after transfection. Knockdown of protein levels by siRNA was confirmed by western blot analysis. Total protein from cultured cells was extracted in radioimmunoprecipitation assay buffer (150 mM NaCl, 1.0% IGEPAL CA-630; IPEGAL CA-630 = 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0), and 30 µg of protein was separated by sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes by electroblotting, and incubated with antibodies to GADD34 (goat polyclonal, 1 : 100; Imgenex Corp., San Diego, CA) or RR M2 subunit (chicken polyclonal, 1 : 200; GenWay Biotech, San Diego, CA) at 4 °C overnight. The next day, membranes were washed in Tris-buffered saline (1 M Tris pH 7.5, 5 M NaCl, 0.1% Tween 20) and incubated with peroxidase-conjugated secondary antibodies (donkey anti-goat polyclonal, 1 : 1000; Chemicon, Temecula, CA or donkey anti-chicken polyclonal, 1 : 1000; Affinity BioReagents, Golden, CO) for 40 minutes. Protein–antibody complexes were visualized using the Enhanced Chemiluminescence kit (Amersham Biosciences; Piscataway, NJ). Each treatment was repeated in triplicate.
Alkaline Comet Assay
Temozolomide-induced DNA damage was quantified using the alkaline comet assay. Cells were treated with various temozolomide concentrations for 6 hours, suspended in low-melting agarose (Trevigen, Inc., Gaithersburg, MD), and placed onto comet slides (Trevigen). Once the agarose solidified, cells were lysed and electrophoresed per the manufacturer's protocol for the alkaline comet assay. Nuclei were labeled with SYBR green dye (Trevigen) per the manufacturer's protocol, cells were photographed using confocal microscopy, and tail moments of 50 cells per slide were calculated using CometScore software (TriTek Corporation, Sumerduck, VA).
Flow Cytometry
U87/pGADD34-EGFP cells were treated for 48 hours with various temozolomide concentrations. Apoptosis was assessed using the Annexin V-PE Apoptosis Detection kit (BD Biosciences, San Jose, CA). A BD FACScalibur flow cytometer (BD Biosciences) was used to sort cells on the basis of phycoerythrin, EGFP, and 7-amino-actinomycin D (7-AAD) positivity, with 7-AAD–positive (i.e., dead) cells gated out of any subsequent analysis. To assess viral replication in GADD34-expressing cells, temozolomide-treated U87/pGADD34-EGFP cells were sorted on the basis of EGFP expression using a BD FACS Vantage SE flow cytometer (BD Biosciences). EGFP-positive and -negative cells were then infected with G207 (MOI = 1.5), and viral yield was determined by plaque assays in Vero cells as described above. Three replicates were performed for each experiment.
Immunohistochemistry
U87/pGADD34-EGFP cells were treated for 48 hours with 100 µM temozolomide and then infected with G207 (MOI = 0.1) for 10 hours. Cells were then fixed in 4% paraformaldehyde, stained with mouse anti–
-galactosidase (1 : 100; Promega, Madison, WI), stained with Texas Red–conjugated sheep anti-mouse antibody (1 : 100; Amersham), and counterstained with 1.5 µg/mL 4'-6-diamidino-2-phenylindole (DAPI) in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Three replicates were performed for each experiment.
In Vivo Experiments
Athymic mice at 6–8 weeks of age with median weight of 20 grams (National Cancer Institute, Bethesda, MD) were subcutaneously inoculated with 106 U87 cells. Two weeks later, mice with tumors of 12–36 mm3 were placed into four treatment groups (five mice per treatment group), each with the same mean tumor volume. Mice were treated by intraperitoneal injection with 100 mg of temozolomide per kilogram of body weight, the maximum tolerated temozolomide dose, every day for 14 days and/or treated intratumorally with 5 x 106 plaque-forming units (pfu) G207 in 30 µL on treatment days 2 and 5. Mock-treated mice received equivalent intraperitoneal or intratumoral volumes of saline. Tumor length, height, and width were measured two times per week using calipers, with the measurer blinded to each animal's treatment group. Tumor volume was the product of these dimensions, and fold-growth was relative to tumor volume on treatment day 1. Measurement of a mouse's tumor continued until tumors reached a maximum size of 2.1 cm in one dimension, when the mouse had to be killed by anesthesia due to excessive tumor burden.
To assess viral replication in subcutaneous tumors, 20 days after subcutaneous inoculation of 106 U87 cells into athymic mice (n = 40), when tumors had achieved volumes of 80–120 mm3, mice were treated intraperitoneally with saline (n = 20) or with 100 mg/kg/day of temozolomide (n = 20). On the third treatment day, 5 x 106 pfu of G207 were inoculated into each tumor. Temozolomide treatment continued, and mice were killed and tumors excised at 2, 4, 6, and 8 days post–G207 inoculation (five mice per group per time point). Tumors were weighed, cut in small pieces with a razor blade, suspended in a volume of phosphate-buffered saline that was twice the tumor volume, homogenized manually, sonicated, and centrifuged at 16 110g for 5 minutes at 4 °C. The supernatant was isolated and freeze–thawed three times, and viral yield was determined by plaque assays on Vero cells as described above.
For intracranial studies, athymic mice (n = 10 per treatment group) were anesthetized with ketamine/xylazine (75/15 mg/kg intraperitoneally) and immobilized in a stereotactic apparatus. A midline sagittal incision was made, and a 1-mm burr hole was drilled in the skull 1 mm anterior to and 2.5 mm lateral to the bregma on the right side. U87 cells (2 x 105) in 2 µL of DMEM were then injected 4.5 mm below the dura using a Hamilton syringe. On days 7, 8, and 9 after injecting tumor cells, mice were treated with 100 mg/kg of temozolomide or saline by intraperitoneal injection. On day 10, mice were treated by intratumoral injection of 7 x 105 pfu of G207 in 2 µL of virus buffer (150 mM NaCl, 20 mM Tris, pH 7.5) or virus buffer lacking virus using the same burr hole and coordinates at which tumor cells were injected. Mice were then observed until they became moribund, lethargic, anorexic, dehydrated, or distressed, at which point they were killed. The presence of intracranial tumors was macroscopically confirmed postmortem in all mice. The Massachusetts General Hospital Subcommittee on Research Animal Care approved all animal protocols.
Statistical Analysis
Comparisons of variables (in vitro and in vivo viral yield, mean tail moment, percentage of cells that underwent apoptosis, percentage of cells that were -galactosidase positive, and fold-growth of subcutaneous tumors) were made using a two-sided Student's t test. Comparisons of Kaplan–Meier curves were made using the log-rank test. P<.05 was considered statistically significant.
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RESULTS |
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Treatment of p53-Intact MGMT-Negative Glioma Cells With Oncolytic HSV and Chemotherapy
We first investigated the interaction between temozolomide and G207. For cultured U87 human glioma cells, which are MGMT negative and p53 wild type (27), median-effect doses (Dm = dose causing 50% survival) of temozolomide (i.e., 390 µM) or of G207 (i.e., MOI of 0.08) were reduced 100-fold by combined treatment (Dm = 3.9 µM temozolomide + 0.0008 MOI G207). All Chou–Talalay combination indices in U87 cells were less than 0.009, which is well below the 0.4 cutoff for strong synergy (Fig. 1, A; Table 1).
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To determine if the strong synergism in U87 cells was unique to the temozolomide–G207 combination, temozolomide was replaced with cisplatin, another chemotherapeutic agent that is commonly used to treat glioma. Cisplatin is an intercalating agent that disrupts DNA replication and/or transcription, a different mechanism for affecting tumor cells than temozolomide. The synergism of the combination of G207 and cisplatin in U87 cells was less (CI = 0.43 to 0.59; Table 1) than that of the temozolomide–G207 combination.
To determine if the mutations of 34.5 and RR in G207 HSV contributed to the strong synergy between temozolomide and G207, we treated U87 cells with temozolomide and with HSVs R3616 (from which the neurovirulence gene 34.5 is deleted) or hrR3 (which is mutated in the nucleotide metabolism gene ICP6), or their respective wild-type parental HSV-1 strains, F and KOS. In U87 cells, there was strong synergy between R3616 and temozolomide (CI = 0.21 to 0.32) but virtually none between hrR3 and temozolomide (CI = 0.56 to 1.01) (Fig. 1, A; Table 1). Also, there was no synergy in U87 cells between temozolomide and the wild-type strain F (CI = 0.72 to 1.19) or KOS (CI = 0.81 to 0.95) (Fig. 1, A; Table 1). Lack of synergy of temozolomide and viruses other than G207 is unlikely to reflect greater replication proficiency of the other viruses because Chou–Talalay analysis adjusts doses to achieve comparable effects. In the absence of temozolomide, the Dm of G207 (0.08 MOI) on U87 cells was higher than that of strain F (0.04 MOI), but in the presence of temozolomide, the Dm of G207 became considerably lower than that of strain F (G207 combined with temozolomide, Dm = 0.0008 MOI; strain F combined with temozolomide, Dm = 0.02 MOI; Fig. 1, B).
In contrast to U87 glioma cells, cultured human astrocytes exhibited minimal sensitivity to temozolomide (Dm = 4784 µM) or G207 (Dm = 0.99 MOI). Temozolomide and G207 had an antagonistic interaction in astrocytes (Table 1; Fig. 1, B).
Effect of Glioma p53 Mutations and MGMT Expression on the Interaction of Temozolomide With Oncolytic HSV
In contrast to U87 cells, T98 human glioma cells express mutated p53 and are temozolomide resistant, presumably because they express MGMT (16,27,28). Treatment of cultured T98 cells with G207, R3616, or hrR3 combined with temozolomide resulted in additive (R3616 and G207) or antagonistic (hrR3) interactions (CIs = 0.62 to 5.66, Table 1; Fig. 2, A). However, after adding MGMT inhibitor O6-benzylguanine, interactions between temozolomide and G207 or hrR3 were synergistic (CI = 0.06 to 0.38), whereas those between temozolomide and R3616 were not (CI = 0.74 to 4.80; Table 1; Fig. 2, B).
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The role of p53 mutations in the interaction between temozolomide and G207 was explored using U373, a p53-mutant MGMT-negative human glioma cell line (27). Temozolomide and G207 were strongly synergistic in U373 cells (Table 1). Thus, p53 was not necessary for the synergy between temozolomide and G207.
We then studied U87/mp53, a transfectant expressing mutant p53. The temozolomide Dm in U87/mp53 cells was approximately twice that observed in untransfected U87 cells (Table 1). Real-time RT–PCR revealed that all pc53-SCX3–transfected U87 clones, including U87/mp53 cells, had twice as much MGMT mRNA as untransfected U87 cells, suggesting that the U87/mp53 p53 mutation increased MGMT expression, which reduced sensitivity to temozolomide. The interactions observed between temozolomide and the various oncolytic HSVs in U87/mp53 cells were similar to the interactions observed in T98 cells (Table 1).
To distinguish the roles of MGMT and p53 status in the synergistic interaction between temozolomide and HSV, we used U87/MGMT, a transfectant cell line that retains wild-type p53 but expresses eightfold more MGMT mRNA than U87 (i.e., an MGMT mRNA level similar to that expressed by T98 cells). Compared with the temozolomide Dm of U87 cells, that of U87/MGMT cells was 4.6-fold higher (Table 1). The interactions between temozolomide and oncolytic HSV were similar in U87/MGMT and T98 cells: Virtually no synergy between temozolomide and any HSV (CI = 0.54 to 1.48; Table 1; Fig. 2, C), strong synergy between temozolomide and G207 or hrR3 in O6-benzylguanine–treated cells (CI = 0.15 to 0.39), and additivity/antagonism between temozolomide and R3616 in O6-benzylguanine–treated cells (CI = 0.84 to 1.43; Table 1; Fig. 2, D).
Together, these results indicate that O6-benzylguanine–treated MGMT-expressing cells exhibited synergy between temozolomide and RR-negative HSVs regardless of p53 status. Although p53 mutations in T98 (codon 237) and U87/mp53 (codon 143) were associated with MGMT expression and the U87/mp53 p53 mutation induced MGMT expression in multiple clones, the U373 p53 mutation (codon 273) was not associated with MGMT expression.
G207 Yield in Temozolomide-Treated Infected Cells In Vitro and In Vivo
To determine if synergy resulted from temozolomide enhancement of G207 replication in U87 cells, the effect of chemotherapy on yield of infectious G207 was analyzed using single-step growth experiments. Forty-eight hours after infection, temozolomide-treated cells produced at least fivefold more G207 than untreated or cisplatin-treated cells (3 x 106 pfu in temozolomide-treated versus 6 x 105 pfu in untreated and 3 x 105 pfu in cisplatin-treated, P = .005; Fig. 3, A). Similarly, 2–8 days after G207 infection of subcutaneous U87 xenograft tumors in athymic mice, tumors from mice pretreated with temozolomide produced two- to sixfold more G207 than those treated with saline (P<.001–.005; Fig. 3, B).
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, P = .05 (12 hours); 
, P = .006 (24 hours); 
, P = .002 (36 hours); and ||, P = .005 (48 hours). B) Titers of G207 harvested from excised subcutaneous U87 tumors in athymic mice at 2, 4, 6, and 8 days post–G207 inoculation in mice pretreated for 3 days with 100 mg/kg of body weight/day temozolomide (closed circle) or saline (open circle). Two-sided Student's t test was used to compare G207 recovery from temozolomide-treated tumors versus that from saline-treated tumors: ¶, P<.001 (2 days); #, P<.001 (4 days); **, P = .005 (6 days); and Temozolomide-Induced DNA Repair Gene Expression and MGMT Expression
Next, we investigated the mechanism by which temozolomide treatment may enhance G207 replication in U87 cells—that is, whether it involved increased DNA repair gene expression—and whether the interactions between temozolomide and HSV mutants observed in MGMT-expressing cells reflected induction of different DNA repair genes. For these analyses we used real-time RT-PCR to investigate the expression of genes contributing to repair of temozolomide-induced DNA damage, some of which might enhance HSV replication: MPG, PADPRP, ATR, and ATM (12,13,29). We also investigated the expression of GADD34 and RR, genes that complement specific HSV mutations, and which may assist the cellular DNA damage response. U87 cells that were treated with 1 mM temozolomide for 48 hours had higher GADD34 expression than untreated cells (fold difference = 16, 95% confidence interval [CI] = 12.6 to 20.4). This increase was also higher than that of the other genes that were assessed before and after temozolomide treatment (Fig. 4, A; Table 2). Cisplatin treatment of U87 cells did not induce the expression of any of the genes. Also, temozolomide treatment did not increase expression of the assessed genes in human astrocytes or in T98, U87/MGMT, or U87/mp53 cells (Fig. 4, A; Table 2). However, treating T98, U87/MGMT, or U87/mp53 cells with 1 mM temozolomide and 100 µM O6-benzylguanine did increase RR M2 subunit expression compared with that in untreated cells (fold difference = 14.9, 95% CI = 10.1 to 22.0 [T98]; 9.9, 95% CI = 7.0 to 13.8 [U87/MGMT]; 9.2, 95% CI = 6.8 to 12.4 [U87/mp53]) and that of the other assessed transcripts (Fig. 4, A; Table 2). Western blot analysis confirmed that temozolomide induced expression of GADD34 and RR M2 subunit protein expression in U87 and O6-benzylguanine–treated T98 cells and that siRNA inhibited protein expression (Fig. 4, B).
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GADD34 and RR in DNA Repair and Drug Synergy
Because GADD34 and RR were the most temozolomide-induced of the assessed transcripts in MGMT-negative and O6-benzylguanine–treated MGMT-positive cells, respectively, we investigated whether GADD34 and RR assist repair of temozolomide-induced DNA damage. The alkaline comet assay was used to measure temozolomide-induced single strand breaks. The mean tail moment, reflecting cumulative DNA damage, of U87 cells treated for 6 hours with temozolomide varied with temozolomide concentration, whereas DNA damage was minimal in U87/MGMT cells regardless of temozolomide dose (Fig. 4, C). O6-benzylguanine–treated U87/MGMT cells had the same increase in tail moment with temozolomide concentration that was observed in U87 cells, as did U87 cells treated with RR siRNA, which targets RR M2 subunit mRNA, and O6-benzylguanine–treated U87/MGMT cells treated with GADD34 siRNA (Fig. 4, C). Adding GADD34 siRNA to U87 cells or RR siRNA to O6-benzylguanine–treated U87/MGMT cells increased the tail moment at each temozolomide concentration (Fig. 4, C). Also, GADD34 and RR siRNA inhibited the synergy of G207 and temozolomide in U87 and O6-benzylguanine–treated U87/MGMT cells, respectively (Table 1).
GADD34 Expression After Temozolomide Treatment
GADD34 has been speculated to function in DNA repair and apoptosis (4). Our hypothesis that temozolomide-induced GADD34 expression in MGMT-negative gliomas enhances G207 replication could explain the observed synergy only if GADD34 expression was not limited to cells that undergo apoptosis after temozolomide treatment, because enhanced viral replication in such cells would not augment cytotoxicity. Therefore, we investigated which cells expressed GADD34 after temozolomide treatment using U87 cells transfected with pGADD34-EGFP, in which the human GADD34 promoter drives EGFP expression, causing green fluorescence after temozolomide treatment (Fig. 5, A). More EGFP-negative live cells underwent apoptosis than did EGFP-positive live cells (52.7% versus 19.0% at 300 µM temozolomide; 56.7% versus 11.1% at 900 µM temozolomide; P<.001), suggesting that cells that would survive temozolomide treatment expressed GADD34 more frequently than cells that would not (Fig. 5, B–C).
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G207 Protein Expression and Replication in GADD34-Expressing Tumor Cells
To determine whether G207 protein expression was enhanced in tumor cells that express GADD34 after temozolomide treatment, U87/pGADD34-EGFP cells were treated with 100 µM temozolomide for 48 hours, infected with G207 (MOI = 0.1) for 10 hours, and then stained with an antibody to the G207-expressed marker protein -galactosidase (Fig. 5, D). -Galactosidase expression was fourfold more frequent in cells expressing EGFP than in those not expressing EGFP (15.8% versus 3.9%, difference = 11.9%, 95% CI on the difference = 2.3% to 21.7%; P = .001; Fig. 5, E), suggesting that cells expressing GADD34 after temozolomide treatment expressed higher levels of G207 protein than cells not expressing GADD34. To confirm whether G207 replication was higher in tumor cells expressing GADD34 after temozolomide treatment than in cells not expressing GADD34, U87/pGADD34-EGFP cells treated with 100 µM temozolomide for 48 hours were fluorescence-activated cell sorted by EGFP expression. EGFP-positive and -negative cells were then infected with G207 (MOI = 1.5) for 48 hours. The G207 yield was higher in EGFP-positive than in -negative cells (7.4 x 106 pfu versus 1.8 x 106 pfu, difference = 5.6 x 106 pfu, 95% CI on the difference = 4.3 x 106 pfu to 6.9 x 106 pfu; P = .002; Fig. 5, F).
Combined Treatment In Vivo
Because of their strong synergy in U87 cells, G207 and temozolomide were combined to treat subcutaneous U87 tumors in athymic mice at doses that alone inhibited tumor growth by less than 50% (Fig. 6, A). Mice were treated with intraperitoneal temozolomide for 14 consecutive days and intratumoral G207 on treatment days 2 and 5. Fifteen days after treatment began, tumors receiving combined treatment had reached statistically significantly smaller fold-growth than saline- or single agent–treated tumors (saline: 11.5-fold growth, 95% CI = 6.9- to 16.1-fold; temozolomide: 9-fold, 95% CI = 6.2- to 11.8-fold; G207: 9.5-fold, 95% CI = 6.2- to 12.8-fold; temozolomide + G207: 5.6-fold, 95% CI = 5.0- to 6.2-fold; Fig. 6, A), a difference that remained statistically significant thereafter. Tumor growth in single agent– versus saline-treated mice was not statistically significantly different until 26 days after treatment began, the last day of comparison, at which point tumors had enlarged by 62.9-fold (saline; 95% CI = 47- to 78.8-fold), 34.9-fold (G207; 95% CI = 24.8- to 45-fold), 35.5-fold (temozolomide; 95% CI = 30.7- to 40.3-fold), and 9.7-fold (G207 + temozolomide; 95% CI = 9.4- to 10-fold). Excessive tumor burden, defined as largest dimension greater than or equal to 2.1 cm, occurred after medians of 29 days (saline treatment), 40 days (single treatment), and 54 days (combined treatment) (Fig. 6, B). Athymic mice bearing orthotopic intracranial U87 tumors treated with intraperitoneal temozolomide or intratumoral G207 achieved median survival of 30.5 days (saline), 46 days (G207), or 48 days (temozolomide). In contrast, 100% of mice treated with temozolomide before G207 treatment began were still alive after 90 days (Fig. 6, C).
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DISCUSSION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We hypothesized that mutations enabling cancer cells to express DNA repair genes after chemotherapy treatment could be used to improve oncolytic viral therapy. We therefore studied the effect of glioma p53 and MGMT expression on temozolomide-induced DNA repair genes and whether these gene products could increase the replication of appropriately engineered oncolytic HSVs. We observed that, in cultured MGMT-negative U87 cells, the synergy of
34.5-deleted HSV with temozolomide was strong, whereas combining G207 with cisplatin caused lesser interactions. In contrast, in cultured MGMT-expressing temozolomide-resistant cells, there was no synergy between temozolomide and any HSV; however, synergy between temozolomide and RR-mutated HSV was observed after adding the MGMT inhibitor O6-benzylguanine. Also, the DNA repair genes GADD34 and the RR M2 subunit were the most highly expressed, compared with the other transcripts analyzed, after temozolomide treatment in MGMT-negative and O6-benzylguanine–treated MGMT-positive gliomas, respectively, and synergy did not occur when these transcripts were selectively targeted. Synergy between temozolomide and G207 occurred independent of whether glioma cells expressed wild-type or mutant p53. Furthermore, the benefits of combined treatment were confirmed in vivo, in that all athymic mice with intracranial U87 xenograft tumors that were treated with G207 and temozolomide exhibited 100% 90-day survival, compared with virtually none (10% G207, 0% temozolomide) treated with either agent alone.
Our findings suggest that mutations that enable gliomas to express DNA repair genes after temozolomide treatment can be used to improve viral oncolysis. In particular, we found that increased tumor cell expression of GADD34 and RR after temozolomide treatment reduced temozolomide-induced DNA damage in MGMT-negative and MGMT-positive glioma cell lines, respectively, and enhanced the replication of 34.5- and RR-mutated HSV viruses, respectively. Cisplatin, part of procarbazine–cisplatin–vincristine (PCV) glioma chemotherapy (30), only minimally increased expression of the DNA repair genes assessed; this finding may explain the lesser interactions we observed between cisplatin and G207.
DNA damage causes cells to initiate DNA repair and to stop dividing. Although the exact functions of the five known GADD proteins remain unconfirmed, they are associated with apoptosis and cell cycle arrest (4) and are increased by some chemotherapies (20,21,31). RR synthesizes nucleotide precursors using homodimeric large (M1) and small (M2) subunits. M2 expression is increased during S phase and by certain chemotherapies (32), whereas M1 expression is constant during the cell cycle, diminishing only during G0 arrest. This report builds on previous findings that certain chemotherapies induce GADD34 and RR (20,21,31,32) by demonstrating that both proteins prevent accumulation of chemotherapy-induced DNA damage, possibly through enhancement of DNA repair. Also, our observation of increased GADD34 expression primarily in nonapoptotic temozolomide-resistant cells suggests that increased G207 replication occurs in cells that survive temozolomide treatment due to increased DNA repair and underscores the complementary tumoricidal effects of these two treatments. Taken together, our results show that oncolytic HSV can target tumor cells that evade chemotherapy through DNA repair.
Our finding that chemotherapy-induced DNA repair genes enhanced the replication of specific HSV mutants is consistent with reports that DNA repair enhances HSV replication (29). In contrast, DNA repair inhibits replication of adenovirus, another engineered oncolytic virus that has been studied in clinical trials (29,33).
Although previous studies reported that restoring wild-type p53 expression in p53-mutated gliomas reduces MGMT expression (34), our finding that only certain p53 mutations increased MGMT expression suggests that many factors may regulate MGMT and that MGMT expression may not be increased in all of the 20% of glioblastomas with p53 mutations (35). We found that MGMT expression determines which DNA repair genes are induced by temozolomide. Although one would expect O6-benzylguanine–treated MGMT-expressing cells to accumulate and repair temozolomide-induced DNA damage using a similar mechanism as that of MGMT-negative cells, previous reports have already highlighted differences between the two scenarios, including that temozolomide induces apoptotic cell death in MGMT-negative cells, whereas it induces autophagic cell death in O6-benzylguanine–treated MGMT-positive cells (36). The ability of temozolomide to induce the expression of different DNA repair genes in different contexts suggests that these contexts differ not just in cell death mechanisms but also in DNA repair mechanisms.
MGMT-mediated resistance to temozolomide is an important issue in the treatment of gliomas. Of the 20% of gliomas that express MGMT, 90% fail to respond to temozolomide; of the remaining 80% of gliomas that do not express MGMT, 40% fail to respond (14,15). In phase I clinical trials, the MGMT inhibitor O6-benzylguanine enhanced the response of MGMT-expressing gliomas to temozolomide (16). Temozolomide induction of GADD34 expression in MGMT-negative cells and of RR in O6-benzylguanine-treated MGMT-positive cells suggests that constructs such as G207 may be ideal to combine with temozolomide because of synergy in both scenarios through the induction of different complementary mammalian genes.
Dual mutations in G207 may increase the safety of this HSV in patients relative to HSVs with single mutations, but sometimes dual mutations reduce in vivo oncolysis compared with single-mutation viruses (3,37). Because temozolomide strongly synergizes with specific HSVs through tumoral GADD34 or RR induction but antagonizes G207 replication and does not enhance the expression of GADD34 or RR in astrocytes, temozolomide increases G207's potency and widens the therapeutic window. In fact, in this study, temozolomide increased G207 potency beyond that of its wild-type parental virus.
Glioma treatment with G207 and temozolomide also enhanced temozolomide's potency, which might enable reduction of patient dosage and potential toxicity, such as the myelosuppression seen in 14% of glioma patients receiving temozolomide in a phase III clinical trial (10). Indeed, the temozolomide dose we used was the maximum tolerated murine dose (rodents exhibit slightly more temozolomide sensitivity than humans) and was minimally effective alone, confirming the need for a wider temozolomide therapeutic window in the animal model. G207 also has a maximal in vivo dose, due not to toxicity but to the large number of cells in a postsurgical glioma cavity, inefficient delivery and distribution, and viral titer limitations reflecting viral biology and production constraints (8,9). The synergy described here increases the efficacy of the lower viral MOIs that are observed in vivo.
This study has several limitations. One is potential differences in chemotherapy-induced DNA repair genes in glioma cell lines compared with glioblastoma in vivo. Differences in DNA repair gene expression have not yet been found in solid tumors compared with tumor cell lines. However, differences in expression of genes controlling the extracellular matrix, cell-to-cell communication, and the protein biosynthesis system have already been identified in solid tumors compared with tumor cell lines (38), underscoring the caution with which data from tumor cell lines must be interpreted. A second limitation is the potential of an immune response to HSV to influence the interaction of temozolomide and HSV. This factor would not be seen in the athymic mice used in this study but could be important in humans. A final limitation is the limited invasiveness of the cell lines used in this study, which would make them more treatable than human glioblastoma.
Nevertheless, two suggestions emerge from the strong synergy found between specific engineered oncolytic HSVs and temozolomide in this study. First, the concept of generating a drug-induced viral oncolysis–enhancing response selectively in tumor cells with specific mutations warrants further investigation. Second, glioma treatment with temozolomide and G207, possibly giving temozolomide before inoculating virus during surgery to take advantage of temozolomide-induced DNA repair in residual glioma cells, warrants a clinical trial.
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NOTES |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We thank Drs. Roizman, Knipe, and Weller for providing virus.
Supported in part by National Institutes of Health grants NS32677 (to R. L. Martuza) and P30 NS045776 (to S. Rabkin) for the real-time RT-PCR core.
Robert Martuza and Samuel Rabkin are consultants to MediGene AG, which has a license from Georgetown University for G207. The funding agencies were not involved in the data collection, experimental design, or analysis of this study.
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