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10.1245/s10434-006-9127-4
Annals of Surgical Oncology 14:258-269 (2007)
© 2007 Society of Surgical Oncology
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Original Article

Radiation-Induced Cellular DNA Damage Repair Response Enhances Viral Gene Therapy Efficacy in the Treatment of Malignant Pleural Mesothelioma

Prasad S. Adusumilli, MD, Mei-Ki Chan, BS, Michael Hezel, BS, Zhenkun Yu, MD, PhD, Brendon M. Stiles, MD, Ting-Chao Chou, PhD, Valerie W. Rusch, MD and Yuman Fong, MD

Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

Correspondence: Address correspondence and reprint requests to: Yuman Fong, MD; E-mail: fongy{at}mskcc.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Malignant pleural mesothelioma (MPM) treated with radiotherapy (RT) has incomplete responses as a result of radiation-induced tumoral stress response that repairs DNA damage. Such stress response is beneficial for oncolytic viral therapy. We hypothesized that a combination of RT and NV1066, an oncolytic herpes virus, might exert an additive or synergistic effect in the treatment of MPM.

Methods: JMN, a MPM cell line, was infected with NV1066 at multiplicities of infection of .05 to .25 in vitro with and without radiation (1 to 5 Gy). Virus replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 (growth arrest and DNA damage repair 34, a DNA damage-repair protein) by real-time reverse transcriptase–polymerase chain reaction and Western blot test. Synergistic cytotoxicity dependence on GADD34 upregulation was confirmed by GADD34 small inhibitory RNA (siRNA).

Results: Synergism was demonstrated between RT and NV1066 across a wide range of doses. As a result of such synergism, a dose-reduction for each agent (up to 5500-fold) can be accomplished over a wide range of therapeutic-effect levels without sacrificing tumor cell kill. This effect is correlated with increased GADD34 expression and inhibited by transfection of siRNA directed against GADD34.

Conclusions: RT can be combined with oncolytic herpes simplex virus therapy in the treatment of malignant pleural mesothelioma to achieve synergistic efficacy while minimizing dosage and toxicity.

Key Words: Ionizing radiation • Gene therapy • Viruses • Herpes simplex virus • Combination therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Malignant pleural mesothelioma (MPM) is an aggressive, treatment-resistant cancer that results in a median survival of 12 months from the time of diagnosis.1 The rising worldwide incidence of MPM is not expected to peak for another 10 to 20 years2,3 as a result of increased use of asbestos in developing countries.4 Because of patterns of occupational asbestos exposure and the long latency period, the annual incidence of new cases in the United States is expected to increase by >50% in the coming decade.5,6 Pleural mesothelioma is a diffuse disease and is resistant to currently available therapeutic modalities.7 Even with combined surgery, chemotherapy, and radiotherapy (RT), only a few patients experience prolonged disease-free survival. Local control is a patient’s best chance for long-term survival. RT provided to reduce the tumor burden must include the entire hemithorax, and the dose of radiation required to produce local control of the disease is high.8,9 Higher doses of radiation needed to achieve local control are associated with toxicity.10 Thus, therapies are also sought that may synergize with RT to increase the tumor response and to decrease toxicities.

Replication-competent herpes simplex viruses (HSV) are novel oncolytic agents with potent activity against a wide range of human cancer cell lines.11–17 NV1066 is one such multimutated replication-competent oncolytic HSV attenuated by deletion of virulent viral growth genes infectious cell protein (ICP) 0, ICP34.5, and ICP4.12,18 Tumor cells, unlike normal cells, support virus replication as a result of their higher replicative nature and are specifically targeted for virus-induced cell lysis. Large numbers of progeny virus are released from relatively few initially infected cells to subsequently infect neighboring cancer cells, and this life cycle continues. Previously published studies demonstrated the efficacy of oncolytic viral therapy against MPM.19 We have demonstrated the efficacy of NV1066 and another HSV: G207 and NV1020 in the treatment of MPM both in vitro and in vivo.15,20

Recent studies have suggested a synergistic anti-tumor effect of HSV when combined with RT.21,22 Different molecular mechanisms underlying this interaction were proposed.23,24 One such mechanism may be related to radiation-induced upregulation of certain gene products whose function or functions are similar to deleted viral gene products, thereby promoting virus replication. One such potential protein is GADD34 (growth arrest and DNA damage repair gene), which is upregulated under conditions of DNA damage and cellular injury (such as RT) and shares marked homology at its carboxy terminus with the HSV protein ICP34.5 encoded by the gene {gamma}134.5.25,26

Previously, we have published the potentiating effect of RT on oncolytic HSV therapy in the treatment of localized cancers such as lung,13 head and neck,17 and cholangiocarcinoma.27 Unlike those cancers, MPM is a nonlocalized diffuse malignancy, where the delivery of low doses of either radiation or oncolytic viral therapy may not be effective. In this study, we hypothesized that by upregulating GADD34 in tumor cells, RT may result in greater oncolytic activity of NV1066 deficient in the {gamma}134.5 gene in the treatment of MPM.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Human malignant mesothelioma cell lines of various histological subtypes were studied, including: sarcomatoid (VAMT, H-2052, H-2373), epithelioid (H-2452, H-Meso), biphasic (JMN, H-Meso1A, MSTO-211H), and other pathological type (Meso9, Meso10). MSTO-211H and Vero cells (from the African green monkey kidney) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). H-Meso and H-Meso1A cell lines were obtained from National Cancer Institute (NCI, Bethesda, MD). JMN, VAMT, Meso9, and Meso10 cell lines were donated by Dr. Frank M. Sirotnik from Memorial Sloan-Kettering Cancer Center, New York. H-2052, H-2452, and H-2373 cell lines were donated by Dr. Harvey Pass from Karmanos Cancer Institute, Wayne State University (Detroit, MI). All the cells were maintained in appropriate media as recommended and were incubated in a humidified incubator supplied with 5% carbon dioxide.

Viruses
NV1066 is a replication-competent attenuated HSV-1 oncolytic virus with deletion of a single copy of the ICP4, ICP0, and {gamma}134.5 genes described in detail elsewhere.18 The virus contains the enhanced green fluorescent protein (GFP) sequence under the control of a cytomegalovirus promoter. Viral stocks were propagated on Vero cells, collected by freeze-thaw lysis and sonication, and titered by standard plaque assay.

In Vitro Cytotoxicity Assay
JMN cells were plated in 24-well flat-bottom plates (Becton Dickinson, Franklin Lakes, NJ) in 1 mL of media. Cells were treated with media alone (control wells), radiation alone (137Cs source irradiator, 224 cGy/minute), NV1066 alone, or in combination with both RT and NV1066. NV1066 infection was carried out at multiplicities of infection (MOI; ratio of viral plaque-forming units [PFU] per tumor cell) of .05, .1, .15, .2, or .25 in a total volume of 100 µL of medium. Combination therapy was performed with serial dilutions of RT (1, 2, 3, 4, and 5 Gy) and NV1066 (MOI = .05, .1, .15, .2, and .25) in a 20:1 ratio. This ratio was determined by estimating the 50% lethal dose (LD50) for each therapy in initial experiments and by using these doses to determine the ratio of combination therapy. Typically cells were plated overnight, radiated in the morning, and infected with virus within an hour of radiation. Percentage survival for each group was determined on each day for 4 or 5 days after treatment by use of a standard lactate dehydrogenase release bioassay. Results were expressed as surviving fraction, based on the measured absorbance of treated cellular lysates, compared with that of untreated, control cellular lysates. All samples were tested in triplicate. Cytotoxicity assays were also performed with all the other mesothelioma cell lines with NV1066 virus with and without radiation (combination ratio between both therapies was kept at 1:10 or 1:20).

Quantitative Analysis of Synergy Between RT and NV1066
The combination effects of two therapies in terms of synergy or antagonism were analyzed by the median-effect plot by using the multiple therapeutic effect analysis of Chou and Talalay.28 This method defines the expected additive effect of two (or more) agents and then quantifies synergism or antagonism by determining how much the combination effect differs from the expected additive effect. Such an analysis involves plotting dose-effect curves for each therapy and multiplying diluted combinations of the therapies using the "median effect" equation: Fa/Fu = (D/Dm)m, where D is the dose, Dm is the dose required for 5% to 95% effect (i.e., 5% to 95% inhibition of cell proliferation at given time point), Fa and Fu are the fractions affected and unaffected, respectively, by dose D, and m is a coefficient signifying the sigmoidicity of the dose-effect curve. The dose-effect curve was plotted by a logarithmic conversion of this equation that determines the values of m and Dm. The conformity and reproducibility of the data to the median-effect principle can be readily shown by the linear correlation coefficient, r. A combination index (CI) was then determined by the following equation: D1/(Dx)1 + D2/(Dx)2 + {alpha}D1D2/(Dx)1(Dx)2, where (Dx)1 is the dose of the therapy "1" required to produce x% effect alone and D1 is the dose required to produce x% effect in combination with D2. Similarly, (Dx)2 is the dose of the therapy "2" required to produce x% effect alone, and D2 is the dose required to produce x% effect in combination. When the therapies are mutually exclusive (i.e., with similar modes of action), {alpha} = 0, or if they are mutually nonexclusive (i.e., with independent modes of action), {alpha} = 1. Finally, the CI was plotted as a function of the fraction affected (Fa). When CI = 1, the interaction is considered additive. When CI < 1, synergy is indicated, and when CI > 1, antagonism is indicated.

Data were also analyzed by the isobologram technique, which is dose-oriented. The axes on an isobologram represent the doses of each drug. Two points on the x- and y-axes are chosen that correspond to the doses of each drug necessary to generate that given Fa value. The straight line (hypotenuse) drawn between these two points on the x- and y-axes corresponds to the possible combination of doses that would be required to generate the same Fa value, indicating that the interaction between the two therapeutic agents is strictly additive. If these therapeutic combination points lie on the straight line, then the effect is additive at that Fa value. If the point lies to the lower left of the hypotenuse, then the effect is synergistic, and if the point lies to the upper right of the hypotenuse, then the effect is antagonistic at that Fa value. Another calculation available that uses the CI method is the dose-reduction index (DRI). The DRI is a determination of the fold of dose reduction allowed for each drug when given in synergistic combination, as compared with the concentration of single agent that is needed to achieve the same effect level. DRI > 1 signifies a favorable reduction in toxicity while still maintaining therapeutic efficacy.

In Vitro Viral Growth Analysis
The ability of NV1066 to replicate within JMN cells in the presence or absence of radiation was evaluated by viral growth analysis. A total of 5 x 104 cells per well were plated into six-well plates. Cells were then infected with either NV1066 (MOI = .05 or .1) alone, or with NV1066 after RT (2 Gy). Cells and media were collected at 48, 72, 96, 120, and 144 hours after infection. After three cycles of freeze-thaw lysis, standard plaque assay was performed on Vero cells to evaluate viral titers. All samples were performed in triplicate.

Vector Spread Assay by GFP Expression
Vector propagation as analyzed by GFP expression was determined by flow cytometric analysis at a viral infective dose of MOI = .01 or .1 after 0 or 2.5 Gy of RT. Percentage of GFP-positive live cells at 24, 48, 72, 96, and 108 hours after radiation, compared with control cells without radiation, was plotted to derive the GFP-expression trend. Cells were collected with .25% trypsin in .02% ethylenediaminetetra-acetic acid, centrifuged, washed in phosphate-buffered saline (PBS), and brought up in 100 µL of PBS. Five microliters of 7-amino-actinomycin D (BD Pharmingen, San Diego, CA) was added as an exclusion dye for cell viability. Data for GFP expression was acquired on a FACSCalibur machine equipped with Cell Quest software (Becton Dickinson, San Jose, CA). Results are reported as the percentage of live cells expressing GFP. All samples were performed in triplicate.

Real-Time Reverse Transcriptase–Polymerase Chain Reaction Analysis for GADD34 in Cells Treated With Radiation
A total of 1 x 105 JMN cells per well were plated in six-well plates and incubated for 12 hours. Cells were treated with a radiation dose of 1, 2.5, or 5 Gy. Each sample was prepared in triplicate. After 24, 48, 72, and 96 hours of incubation, the cells from each well of the plate were collected after washing with cold PBS and frozen for RNA collection. RNA from each sample was collected and isolated with an RNeasy Protect Kit (Qiagen Inc., Valencia, CA), following the manufacturer’s protocol. GADD34 in each sample was measured quantitatively by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) by using a SYBR green fluorophores with a Bio-Rad iCycler iQ detection system (Bio-Rad Laboratories, Hercules, CA) and normalized by corresponding 18S ribosomal RNA. For GADD-34, the following primers were applied: GADD-34 forward 5'-GGA GGA AGA GAA TCA AGC CA-3'; GADD-34 reverse 5'-TGG GGT CGG AGC CTG AAG AT-3'; For 18-S: 18-S forward 5'-GTA ACC CGT TGA ACC CCA TT-3'; 18-S reverse 5'-CCA TCC AAT CGG TAG TAG CG-3'. A comparison between each treatment sample and the control group, which did not receive any radiation, was made to determine GADD34 upregulation. The results were represented as fold upregulation in the treatment sample compared with the control group.

GADD34 Small Inhibitory RNA (siRNA) Transfection
Duplex siRNAs targeting human GADD34 outside the viral homology domain were designed and tested for the ability to decrease GADD34 expression. After preliminary experiments, the following sequence targeting from codon 635 was chosen for further experiments: 5'-GUCAAUUUGCAGAU-GGCCATTUGGCCAUCUGCAAAUUGACTT-3'. JMN cells were plated at a concentration of 5 x 104 per well in 24-well plates 12 hours before transfection in appropriate medium without antibiotics. Standard siRNA transfection protocol as described before was used.29 Cells that were transfected with lacZ siRNA under similar protocol were used as controls.

Western Blot Test for GADD34 Protein
JMN cells (lacZ-transfected and GADD34 siRNA transfected) were incubated overnight and irradiated in the morning with either 2.5 or 5 Gy. Cells that received no RT served as a control. Cells were lysed and collected with cell lysis buffer (Cell Signaling Technology, Inc., Beverly, MD). Equal amounts of proteins were resolved on 10% sodium dodecyl sulfate polyacrylamide gels (Bio-Rad) under reducing conditions and blotted on Polyvinylidene fluoride (PVDF) membrane (Schleicher & Schuell Bioscience, Keene, NH). Expression of proteins was determined by using primary rabbit polyclonal anti-human GADD34 (Santa Cruz Biotechnology, Santa Cruz, CA) and primary goat polyclonal anti-human Actin (Santa Cruz Biotechnology). A secondary antibody conjugated to horse radish peroxidase (Santa Cruz Biotechnology) was used to visualize the expression level of GADD34 and actin on chemiluminescence film (Hyperfilm; Amersham Biosciences, Bucking-hamshire, England) by application of an ECL Plus Western Blotting Detection System (Amersham Biosciences).

Establishment and Treatment of Flank Tumors
Athymic male mice were purchased from the National Cancer Institute (Bethesda, MD) and were provided with food and water ad libitum. All animals received humane care in accordance with the National Institutes of Health’s "Guide for the Care and Use of Laboratory Animals," and the animal protocols were approved by the animal care committee of Memorial Sloan-Kettering Cancer Center. Mice were anesthetized with a mixture of 70 mg/kg ketamine and 10 mg/kg xylazine administered intraperitoneally. JMN flank tumor establishment and tumor measurements were conducted by the antitumor division of Memorial Sloan-Kettering Cancer Center core facility who were blinded to the treatment arms. Flank tumors were established in nude athymic mice by injecting 5 x 106 JMN cells in 50 µL of PBS. Mice were examined daily until tumor nodules reached approximately 125 or 500 mm3, at which time they were randomized into four groups 9 days after tumor implantation (six per group): (1) untreated control, (2) 2.5 Gy RT alone, (3) single intratumoral injection of 1 x 107 PFU NV1066 alone, and (4) 2.5 Gy RT followed by a single intratumoral injection of 1 x 107 PFU NV1066 (24 hours later). Mice were shielded with lead when flank tumors were exposed to external beam radiation (137Cs source irradiator, 224 cGy/ minute). The length and width of the tumors were measured every 3 days for 21 days. Tumor volume was calculated by the formula for an ellipsoid volume, [(4/3) x ({pi}) x (length/2) x (width/2)2]. Animals were humanely killed if the greatest tumor dimension exceeded 2 cm or if there was skin ulceration.

Statistical Analysis
All the data we present here is a result of at least three independent experiments unless stated otherwise. All data are expressed as mean ± standard error of the mean. Comparisons between groups were made by the two-tailed Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Cytotoxicity of RT and NV1066
Both RT and NV1066 demonstrated dose-dependent cytotoxicity against JMN malignant mesothelioma cancer cells. Combination therapy killed more tumor cells (96% ± 4%) than either single agent alone (1 Gy radiation killed 1% ± 3% and NV1066 at an of MOI = .05 killed 50% ± 6% cells). Combination therapy showed greater efficacy than the expected additive effect by day 5 (P < .001). Cytotoxicity derived by lactate dehydrogenase release assay on each day up to day 5 is represented in Fig. 1Go. Synergistic cytotoxicity (P <.01) is confirmed between RT and NV1066 for across a wide range of therapeutic doses (Fig. 2Go). At higher doses, either therapy alone killed more cells at an earlier time point, providing fewer cells for the virus replication cycle to continue, and therefore, the synergistic effect observed is not high (5 Gy killed 15% ± 4%, NV1066 at MOI = .25 killed 78% ± 8%, and combination therapy killed 92% ± 5%).


Figure 1
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  		FIG. 1. Cytotoxic effect of radiotherapy (RT), NV1066, or both on JMN cells in vitro. JMN cells were treated with RT (1 Gy), NV1066 (multiplicity of infection [MOI] = .05), or a combination. Results for the treated groups are expressed as cell survival compared with untreated control cells grown under identical conditions. Combination therapy enhanced cytotoxicity compared with either therapy alone.

 

Figure 2
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  		FIG. 2. Combination therapy with radiation and NV1066 on JMN cells as compared with single-agent therapy alone across a wide range of doses. JMN cells were treated with different doses of radiotherapy (RT) (Gy) or NV1066 (multiplicity of infection, MOI). Combination therapy was performed to keep the ratio of RT:NV1066 constant at 20:1. Standard lactate dehydrogenase colorimetric assay (on day 4) was used to assess cytotoxicity for each treatment group, with results represented as percentage survival as compared with untreated control cells. Combination therapy has demonstrated synergistic cytotoxicity as compared with single-agent therapy across a wide range of doses. (A) 1 Gy + MOI .05. (B) 2 Gy + MOI .1. (C) 3 Gy + MOI .15. (D) 4 Gy + MOI .2.

 
Pharmacological Analysis of Synergy Between RT and NV1066
Chou-Talalay analysis demonstrated that the CI values remained <1 over the entire range of Fa values for JMN cells (Table 1Go). The DRI was calculated for each Fa value. DRI > 1 indicated that a reduction in toxicity can be achieved without loss of efficacy. The radiation dose could be lowered 2- to 5470-fold and the NV1066 dose could be lowered 1- to 530-fold when provided as combination therapy. The synergism was present across an entire range of fractional cell kill from LD10 to LD90 (Table 1Go). Isobolograms were constructed for the doses of RT and NV1066 necessary to kill 75% of cells (LD75), 50% of cells (LD50), and 25% of cells (LD25) (Fig. 3Go). Experimental combination data points at drug and viral concentrations were well below the expected additive effect line for each of these Fa values (.25, .5, and .75).


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  		TABLE 1. Radiation and viral doses needed to kill various fractions (Fa) of JMN cells and fold dose reduction possible when delivered in combination
 

Figure 3
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  		FIG. 3. Isobolograms demonstrate synergism between radiotherapy (RT) and NV1066. The doses of radiation and NV1066 necessary to achieve 75% cell kill (open circles), 50% cell kill (open squares) and 25% cell kill (open triangles) are plotted on the axes, and the connecting solid lines represent the expected additive effects for combination therapy. Experimental combination therapy doses necessary to generate actual LD values of 75% (solid circle), 50% (solid square), and 25% (solid triangle) all lie to the lower left of the corresponding lines, indicating synergism.

 
In Vitro Viral Growth Analysis
NV1066 replicated well in JMN cells. After radiation, a 14-fold increase in viral titers compared with NV1066 infection alone (MOI = .05) was observed over 5 days after infection (P <.01, Fig. 4Go). At higher concentrations of virus, there was a 6-fold (MOI = .1) increase in virus yields in the presence of radiation (P = .02). Lower fold increases in virus yield measured with combination therapy at higher concentrations (MOI) of virus or RT may be the result of loss of cellular substrate at earlier time points, especially given the synergistic cytotoxicity of combination therapy.


Figure 4
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  		FIG. 4. Virus proliferation. JMN cells were infected with NV1066 at multiplicity of infection (MOI) = .05 after exposure to 1 Gy radiation (solid diamond), or without exposure to radiation (open diamond). Viral plaque-forming units were measured by plaque assay on consecutive days and plotted. Similar assay was conducted in JMN cells transfected with GADD34 small inhibitory RNA (siRNA) (open triangle).

 
Vector Spread After NV1066 Infection and Radiation as Measured by GFP Expression
Radiation-induced increased virus yields facilitated earlier and better viral spread among JMN cells (Fig. 5Go). After radiation, when JMN cells were infected with NV1066, a higher percentage of JMN cells were GFP positive at earlier time points. When cells were infected with NV1066, radiation of 2.5 Gy increased GFP-positive live cells to 39% ±4% at MOI = .01 or 74% ±6% at MOI = .1 when compared with cells infected without radiation 23% ±2% (MOI = .01) or 53% ±4% (MOI = .1) (P <.01) after 72 hours.


Figure 5
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  		FIG. 5. Vector spread assay by green fluorescent protein (GFP) expression. Vector propagation was analyzed by GFP expression in JMN cells infected with NV1066 at multiplicity of infection (MOI) = .05 after exposure to 1 Gy radiation (solid diamond), or without exposure to radiation (open diamond). GFP expressing cells were measured by flow cytometry and expressed as percentage of control. Similar assay was conducted in JMN cells transfected with GADD34 small inhibitory RNA (siRNA) (open triangle).

 
RT Upregulated GADD 34
RNA extracted from cells that were not treated with radiation served as negative controls in real-time RT-PCR analysis for GADD34 standardized by corresponding 18S control. In JMN cells, 1-, 2.5-, and 5-Gy radiation increased GADD34 by 1.7-, 3.2-, and 4.3-fold at 48 hours compared with untreated control cells (Fig. 4Go). Western blot analysis confirmed GADD34 upregulation at the protein level (Fig. 6Go).


Figure 6
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  		FIG. 6. GADD34 upregulation after radiation. JMN cells were radiated (1, 2.5, or 5 Gy), and GADD34 expression measured by real-time reverse transcriptase–polymerase chain reaction at various time points standardized by 18S control. GADD34 upregulation was expressed as fold upregulation compared with nonirradiated control cells at 48 hours.

 
GADD34 Knock-down by siRNA Eliminates Synergistic Cytotoxicity
Experiments were performed to determine whether the knock-down of GADD34 upregulation can decrease the expression of GADD34 in response to RT and whether this will decrease virus proliferation and combination therapy cytotoxicity. GADD34 is successfully downregulated by transfection of siRNA for GADD34 (60% to 80% knock-down) compared with lacZ-transfected JMN cells as confirmed by real time RT-PCR (data not shown). Western blot analysis confirmed radiation-induced upregulation of GADD34 protein and its inhibition by GADD34 siRNA (Fig. 7Go). In siRNA transfected cells, virus replication was decreased when compared with lacZ-transfected cells that were radiated (data not shown). Downregulation of GADD34 eliminated synergistic cytotoxicity (Fig. 8Go). In GADD34 siRNA transfected cells, by combination therapy, only 58% ± 7% of cells were killed compared with 96% ± 4% of lacZ-transfected cells (P <.01).


Figure 7
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  		FIG. 7. Western blot analysis confirmed GADD34 upregulation after radiation and its inhibition by GADD34 small inhibitory RNA (siRNA). lacZ-transfected and GADD34 siRNA–transfected JMN cells were irradiated with 2.5 or 5 Gy. Cells with no radiation served as control. GADD34 protein expression was quantified by Western blot analysis. Radiation upregulated GADD34 protein expression, and GADD34 siRNA knocked down the upregulation in siRNA-transfected cells.

 

Figure 8
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  		FIG. 8. Effect of GADD34 small inhibitory RNA (siRNA) transfection on combination therapy cytotoxicity. JMN cells were treated with radiotherapy (RT) (1 Gy), NV1066 (multiplicity of infection [MOI] = .05), or a combination, with GADD34 or lacZ siRNA transfection. Knock-down of GADD34 upregulation by siRNA eliminated the synergistic cytotoxicity in transfected cells.

 
Antitumor Effect of NV1066 and Radiation on JMN Flanks Tumor Volume In Vivo
The improved tumor killing by NV1066 and radiation was demonstrated in a xenogenic flank tumor model established in athymic mice after treatment with 2.5 Gy of radiation and 1 x 107 PFU of NV1066 (Fig. 9Go). Combination therapy was statistically significantly better than single-agent therapy (P = .01 on day 21 by t-test). Twelve days after treatment, in mice with combination therapy, mean flank tumor volume changed to 1129 ± 210 mm3 from 140 mm3 compared with mice treated with NV1066 alone (3369 ± 1142 mm3), radiation alone (7481 ± 1711 mm3), or the control untreated mice (10,754 ± 2,900 mm3). The experiment was terminated after 13 days because of the overgrowth and ulceration of flank tumors.


Figure 9
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  		FIG. 9. Antitumor effect of radiation, NV1066, or a combination on JMN flank tumor volume in vivo. Flank tumors were grown in athymic mice and treated with (1) no treatment, (2) 2.5 Gy of external beam radiation, (3) single injection of 1 x 107 PFU NV1066 intratumorally, or (4) combination of therapies. Tumors treated with combination therapies had smaller average tumor volume than either treatment alone. Results represent average of sample group (n = 6 per group) and are shown with standard errors of mean. Shown in the inset are representative mice from each group.

 
Synergistic Cytotoxicity Is Confirmed in Multiple Malignant Mesothelioma Cells
To further confirm that the synergistic cytotoxicity is not isolated to one particular cell line or pathological type, cytotoxicity experiments were performed in multiple malignant mesothelioma cells. These cells have varied sensitivity to NV1066 (LD50 MOI = .16 to .70) and RT (LD50 = 2.7 to 21.8 Gy). Combination of therapies resulted in synergistic cytotoxicity in most of the cell lines and additive effect in others. Synergism and possible dose reductions were calculated by Chou-Talalay analysis and the isobologram method for 5% to 95% cell kill (LD5 to LD95) in all the cell lines. In Table 2Go, treatment doses for single agent therapy, combination therapy and dose-reduction fold achieved for 50% cell kill (LD50) with combination therapy are listed. With combination therapy, strong synergism was present in VAMT, Meso, Meso1A, and Meso10 cells. In MSTO-211H and H2373 cells, moderate synergism was demonstrated, and in other mesothelioma cells, additive effect was demonstrated.


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  		TABLE 2. Combination therapy with radiation and NV1066 to achieve IC50 (50% cell kill) in multiple malignant mesothelioma cells and fold dose reduction possible when delivered in combination
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
RT has been a well-established modality in the treatment of MPM.10,30–32 MPM pattern of growth and its large surface area, rather than localized bulk, makes it difficult to deliver sufficiently high doses of radiation and to avoid serious toxicity.10,33 Surgery has been the mainstay of treatment for MPM because chemotherapy and RT are relatively ineffective.34 However, only 10% to 20% of all patients presenting for evaluation are considered to be candidates for extrapleural pneumonectomy.35 Even in patients with extrapleural pneumonectomy, it is critical to anatomically reconstruct the diaphragm to allow optimal postoperative RT, thus allowing planned radiation dose to the entire hemithorax and minimizing the dose of radiation to adjacent organs such as liver and stomach. In patients with pleurectomy and decortication, local recurrence within the operated hemithorax is the most common form of recurrence.35 Delivery of tumoricidal dose of radiation to residual microscopic mesothelioma after pleurectomy and decortication is limited by the potential toxicity to the underlying lung and other vital structures, producing complications including radiation pneumonitis, myelitis, and hepatitis.33

Oncolytic viral gene therapy has been shown to be effective in many experimental cancers, including MPM.11,13–17,19–23,27,36–39 We and others have previously demonstrated that these oncolytic viruses can be delivered locally in the pleural cavity.11,40 Combining RT with viral gene therapy has many advantages.24,42 Because oncolytic HSV replication is specific to tumor cells, virus replication enhancement by ionizing radiation can be confined to the tumor from conformal RT, allowing higher intratumoral titers of the virus.41–43 Both RT and gene therapy are used in the treatment of local disease and may be used to kill tumor cells through independent mechanisms, thereby minimizing the evolution of treatment-resistant tumor cells at the initiation of treatment. Enhanced local tumor control may be achieved with lower doses of radiation, thereby reducing radiation damage to normal tissue parenchyma surrounding the tumor.

NV1066 is an attenuated multimutant HSV that has demonstrated cytotoxic activity against a wide variety of tumor cell types, including MPM.12–15,18,20,27 Strategic deletions have been made within the genome of NV1066 to improve the safety of this vector. One such deleted gene, {gamma}134.5, is a neurovirulent gene that codes for the protein ICP34.5, which functions to prevent the shutoff of protein synthesis that occurs as part of the host defense response to cellular infection by viruses (Fig. 10Go). Expression of ICP34.5 produces a cellular milieu greatly favoring virus replication. There is great homology between the carboxy terminus of the mammalian GADD34 and ICP34.5.44 We have shown that radiation-induced GADD34 upregulation enhances replication and antitumor efficacy of a {gamma}134.5-deficient HSV oncolytic virus (Fig. 10Go). Moreover, similar synergistic tumoricidal activity with RT was demonstrated with several malignant mesothelioma cell lines. As noted in other studies,24,45 the fact that RT results in high expression of GADD34 was confirmed in our study and is central to our strategy of combining RT with oncolytic viral therapy. Similar mechanism of synergy between chemotherapy and oncolytic HSV therapy was reported both by us and others in recent publications,14,36,38,39 including this mechanism in MPM.15


Figure 10
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  		FIG. 10. Schematic flow diagram demonstrating the possible mechanism of synergy between radiotherapy (RT) and oncolytic viral therapy. This flow diagram demonstrates the function of herpes virus {gamma}134.5 in dephosphorylating eIF2-{alpha}P to preserve cellular protein synthesis and to prevent apoptosis. Radiation-induced upregulation of cellular GADD34 prevents such phosphorylation and functionally complements loss of {gamma}134.5, thereby promoting virus replication and cytotoxicity.

 
Deletion of the {gamma}134.5 gene coding for ICP34.5 protein markedly reduces cytotoxicity.46,47 Our approach of inducing GADD34 function to replace the ICP34.5 function may provide a better safety profile. The use of RT in this strategy provides a selective means of restoring the GADD34 phenotype in tumor cells without the potential risk of increasing neurovirulence or the risk of enhancing virus replication in nonmalignant cells. Furthermore, such combination therapy may prove efficacious for tumor cells that may be resistant to either viral therapy or RT alone.

Synergy was examined in this study by using the CI and isobologram methods of Chou and Talalay.28,48 This type of analysis is one of the few methods available that determine synergy on the basis of an extrapolated equation. The possibility of predicting false-positive synergistic interactions, a problem inherent in many other methods, is minimized because the analysis takes into account both the potency and shapes of the dose-effect curves in precisely analyzing two therapeutic combinations. Synergistic therapeutic efficacy was demonstrated by using two different cell lines with varying sensitivities to virus therapy and RT. On the basis of these data, a marked DRI was also demonstrated. Ultimately, the DRI is the most important parameter in determining the clinical applicability of combination therapy because potential toxicity can be reduced without sacrificing any therapeutic effect.

Higher doses of radiation have been to shown to achieve better local control in advanced MPM at the cost of increased toxicity.49,50 Acute toxicity can lead to treatment interruptions and delays or alterations in the schedule of radiation delivery, resulting in reduction in total dose, thereby potentially compromising the effectiveness of the definitive treatment modality. Dose reduction achieved as a result of synergistic cytotoxicity may reduce treatment-related toxicities. Studies of malignant glioma in mice have demonstrated that radiation increases virus persistence, furthering the idea that the two therapies can be considered interactive.37,47 Our studies confirmed on a very different tumor type, namely mesothelioma, that a far greater reduction in volume of tumors can be achieved with such combined RT and viral therapy.

In our study, the synergistic efficacy of RT with oncolytic viral therapy is demonstrated in several malignant mesothelioma cell lines irrespective of their radiation sensitivity. Previous studies have established that mesothelioma cells that are viable after 2 Gy radiation are relatively radiation resistant.51 In our study, more than half of the cell lines had LD50 > 5 Gy. The log-kill law states that the probability of an individual cell surviving a given dose of a particular radiation dose varies with the dose (i.e., the larger the dose, the lower the probability of the cell surviving). However, if the tumor cell population is fairly resistant so that the initial probability of survival is only .5 (IC50), trebling the dose of drug will yield only a (.5 x .5 x .5) = .125 probability of survival less than 1-log kill. This illustrates the futility of attempting to overcome resistant populations by pushing radiation dose to the limit. It also illustrates the point that high-dose strategies will be curative only if the residual tumor burden being treated is relatively low so that the higher dose of radiation can be delivered to a localized region without increasing toxicity. Our study demonstrated that synergistic cytotoxicity can be achieved even in RT-resistant MPM cells without increasing the dose. Furthermore, because of such synergism, both therapy doses can be markedly reduced in these cells. Also, sarcomatoid histology is known to be a poor prognostic factor in the treatment of MPM.35,52 In our study, synergistic effect is demonstrated in all MPM cells, irrespective of the pathological type. Therefore, the synergistic efficacy with RT and oncolytic HSV therapy is independent of the pathological type or radiation sensitivity.

One of the limitations of our study is that the experiments lack GADD34 transfection into MPM cells or the use of {gamma}134.5-restored NV1066. Although this would considerably strengthen the direct association of GADD34 with the observed synergy, we believe that this pathway is only one of the possible mechanisms of the synergistic effect observed. Several other possible mechanisms were elicited by several investigators who demonstrated RT-induced enhanced virus replication and cytotoxicity.23 Mezhir et al.23 suggested that ionizing radiation upregulates late promoters active in the course of viral DNA synthesis. Stanziale et al.12 showed that RT-mediated upregulation of ribonucleotide reductase augmented the tumoricidal activity of G207, an oncolytic HSV deficient in UL-39 (gene encoding the ICP6 protein that forms the large subunit of RR), as well as {gamma}134.5. The impact of RT on virus entry into tumor cells is also potentially important mechanistically and could also influence viral tumoricidal activity. In a similar study, while observing the synergistic effect between chemotherapy and HSV-1 mutants, Aghi et al.38 discussed similar limitations and the potential differences between stress-response–induced genes in vitro and in vivo. Our results in this study—that either specific DNA damage-repair protein or a DNA damage-response environment in general is beneficial for productive HSV-1 infection—can be supported by other studies with similar observations. Lilley et al.53 further demonstrated that HSV-1 replication is attenuated in the absence of DNA damage-repair response. Although we did not elicit GADD34 upregulation after RT in vivo in this particular tumor model, similar observations were elicited in head and neck tumor models in studies published from our laboratory.17 Further confirmation of the findings in this study will require additional corroboration in more stringent model systems such as primary tumor explants and tissue slices in advance of human trials.39

Nevertheless, these studies support for future clinical investigation of combined radiation and viral therapy for MPM that aim to increase efficacy while minimizing toxicity. Also, the synergistic effect observed has potentially important translational application during these combined therapies by administration of RT before viral therapy and by reduction of the dosage of both agents to elicit the maximal response.


   ACKNOWLEDGMENTS
 
Supported in part by AACR-Astra Zeneca Cancer Research and Prevention Foundation Fellowship (P.S.A), grants RO1 CA 75416 and RO1 CA/ DK80982 (Y.F.) from the National Institutes of Health, grant MBC-99366 (Y.F.) from the American Cancer Society, grant BC024118 from the U.S. Army (Y.F.), grant IMG0402501 from the Susan G. Komen Breast Cancer Foundation (Y.F. and P.S.A.), grant 032047 from the Flight Attendant Medical Research Institute (Y.F. and P.S.A.), and William H. Goodwin and Alice Goodwin and the Commonwealth Foundation for Cancer Research grant—The Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (Y.F).

The authors thank Medigene, Inc., for providing the NV1066 virus; Liza Marsh of the Department of Surgery at Memorial Sloan-Kettering Cancer Center for editorial assistance; and Yuhong She, MD, and Wong Wai, MS, of the Anti-Tumor Core Facility at Memorial Sloan-Kettering Cancer Center for their assistance with this project.


   FOOTNOTES
 
Presented at the 95th annual meeting of American Association of Cancer Research, March 2004, Orlando, FL.

Received for publication June 2, 2006. Accepted for publication June 26, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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