Cancer Research Cancer Epigenetics Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mezhir, J. J.
Right arrow Articles by Weichselbaum, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mezhir, J. J.
Right arrow Articles by Weichselbaum, R. R.
[Cancer Research 65, 9479-9484, October 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Ionizing Radiation Activates Late Herpes Simplex Virus 1 Promoters via the p38 Pathway in Tumors Treated with Oncolytic Viruses

James J. Mezhir1, Sunil J. Advani3, Kerrington D. Smith1, Thomas E. Darga2, Alice P.W. Poon3, Hank Schmidt1, Mitchell C. Posner1, Bernard Roizman3 and Ralph R. Weichselbaum2

Departments of 1 Surgery and 2 Radiation and Cellular Oncology and 3 The Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, Chicago, Illinois

Requests for reprints: Ralph R. Weichselbaum, Department of Radiation and Cellular Oncology, Center for Advanced Medicine, The University of Chicago Hospitals, Room 1329, Mail Code 9006, 5758 South Maryland Avenue, Chicago, IL 60637. Phone: 773-702-0817; Fax: 773-834-7233; E-mail: rrw{at}rover.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ionizing radiation potentiates the oncolytic activity of attenuated herpes simplex viruses in tumors exposed to irradiation at specific time intervals by inducing higher virus yields. Cell culture studies have shown that an attenuated virus lacking the viral {gamma}134.5 genes underproduces late proteins whose synthesis depends on sustained synthesis of viral DNA. Here we report that ionizing radiation enhances gene expression from late viral promoters in transduced cells in the absence of other viral gene products. Consistent with this result, we show that in tumors infected with the attenuated virus, ionizing radiation increases 13.6-fold above baseline the gene expression from a late viral promoter as early as 2 hours after virus infection, an interval too short to account for viral DNA synthesis. The radiation-dependent up-regulation of late viral genes is mediated by the p38 pathway, inasmuch as the enhancement is abolished by p38 inhibitors or a p38 dominant-negative construct. The p38 pathway is not essential for wild-type virus gene expression. The results suggest that ionizing radiation up-regulates late promoters active in the course of viral DNA synthesis and provide a rationale for use of radiation to up-regulate cytotoxic genes introduced into tumor cells by viral vectors for cytoreductive therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The genesis of the studies described in this report stems from preclinical and clinical studies using mutants of herpes simplex virus 1 (HSV-1) to target destruction of cancer cells (1–5). The mutant most effective in these studies lacks both copies of the {gamma}134.5 gene ({Delta}{gamma}134.5; ref. 6). The basis of the attenuation of this mutant is as follows. HSV-1 activates protein kinase R (PKR) concurrent with the onset of viral DNA synthesis. Thus, eukaryotic translation initiation factor 2-{alpha} (eIF-2{alpha}) becomes phosphorylated. The product of the {gamma}134.5 gene, infected cell protein 34.5 (ICP34.5), binds and redirects protein phosphatase I-{alpha} to dephosphorylate eIF-2{alpha} and protein synthesis continues. In cells infected with {Delta}{gamma}134.5 mutant virus, protein synthesis is shut off after the onset of viral DNA synthesis resulting in very little accumulation of late proteins, whose synthesis requires viral DNA synthesis (7–10). In experimental mouse models of malignant glioma, {Delta}{gamma}134.5 mutants replicate to a limited extent and destroy cancer cells but have no effect on normal tissues of the mouse (11). In a series of experiments, it was shown that ionizing radiation administered between 6 and 9 hours after intratumoral HSV-1 infection enhanced both the replication and spread of virus in the irradiated tumor (12). A central question is the mechanism by which ionizing radiation enhances the replication of {gamma}134.5-deficient viruses. In this report, we show that the combination of virus infection and ionizing radiation results in significant up-regulation of the p38 pathway and that the activated pathway plays a key role in stimulating the expression of late viral genes, even in the absence of other viral gene products. In effect, ionizing radiation complements the defect in {Delta}{gamma}134.5 viruses by enabling the synthesis of late viral proteins in sufficient amounts to augment viral replication. This report thus provides a molecular basis for combined use of genetically engineered and safe attenuated viruses and ionizing radiation to target cancer cells in a temporal and spatial fashion.

The mitogen activated protein kinase (MAPK) p38 is a serine-threonine kinase that upon activation results in a cascade of cellular events including phosphorylation of cellular kinases and transcription factors (13, 14). This protein is activated by numerous cellular stresses (13, 15, 16). It has been reported that activation of the p38 pathway is required for the transcription of HSV-1 genes and viral replication (17, 18). However, a recent study has convincingly shown that activation of p38 plays no role in the transcription of viral genes during the course of wild-type virus infection (19). Our studies are in accord with the latter conclusion but show that activation of the p38 protein is essential for activation of late promoters by ionizing radiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and viruses. MiaPaCa-2 (human pancreatic cancer), HeLa (human cervical cancer), rabbit skin, and Vero (green monkey kidney) cells were originally obtained from American Type Tissue Culture (Manassas, VA) and grown in DMEM (Life Technologies/Invitrogen Corp., Grand Island, NY) supplemented with 10% FCS (Intergen, Purchase, NY) and 1% penicillin-streptomycin at 37°C and 7% CO2. HSV-1(F) is the prototype wild-type HSV-1 strain used in our laboratory (20). The derivation and properties of the recombinant virus R3616 that lacks both copies of the {gamma}134.5 gene were reported elsewhere (6).

Construction of the recombinant virus R2636 carrying the luciferase gene driven by the glycoprotein C promoter (gC-Luc) in place of the {gamma}134.5 gene. R2636 was constructed in two steps. The first step involved the replacement of the thymidine kinase (TK) genes previously inserted in place of both copies of the {gamma}134.5 gene in the recombinant R3659 with the gC-Luc gene. The procedure involved was that described by Post and Roizman (21). The parent virus R3659 has the TK gene inserted at the BstEII site of {gamma}134.5. To construct the transfer plasmid, the plasmid gC-pGL3 was digested with restriction endonuclease KpnI and BamHI. The DNA fragment containing luciferase gene driven by the HSV-1 glycoprotein C promoter was purified by Qiagen gel extraction kit, blunt-ended by Klenow treatment, and subcloned into the BstEII site of plasmid pRB3616. pRB3616 contains the HSV-1 BamHI S fragment with a deletion extending from BstEII to StuI site in the {gamma}134.5 gene (22). The recombinant R2636 was isolated as follows. Rabbit skin cells were infected with R3659 viral DNA together with the gC-luciferase transfer plasmid pRB5968 using LipofectAMINE reagent (Life Technologies). Cells were harvested when they showed 100% cytopathic effect. The progeny was plated on 143 TK– cells in presence of bromodeoxyuridine to select for TK– viruses. Viruses (e.g., R2633) replicating under this condition were plaque purified through four successive cycles of single plaque purification, verified to express luciferase, and then amplified and tested for expression of the luciferase gene. In the second step, the TK gene was restored at its initial locus. In this step, rabbit skin cells were cotransfected with R2633 viral DNA and plasmid pRB103 carrying the viral TK gene in the BamHI Q fragment (21). The progeny of transfection was plated on 143 TK– cells in presence of hypoxanthine/aminopterin/thymidine media to select for TK+ viruses. Viruses growing under this condition were plaque purified through four cycles of single plaque purification and then amplified. Viral stocks were prepared and titered in Vero cells as described previously (22).

Preparation of cell lysates, electrophoretic separation of proteins, and immunoblotting. MiaPaCa-2 or HeLa cells were exposed at time 0 to 10 plaque forming units (pfu) of R3616 or HSV-1(F) per cell and irradiated with 5 Gy using a Pantak PCM 1000 X-ray generator (Pantak, East Haven, CT) at times after infection as indicated in Results. In some experiments, cells were maintained in medium containing metabolic inhibitors [20 µmol/L of SB203580 or SB202190 (EMD Biosciences, San Diego, CA) or 300 µg/mL of phosphonoacetic acid (Sigma Chemical Co., St. Louis, MO)] added at the time of infection or 1 hour before irradiation for promoter studies. The cells were harvested with lysis buffer [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerolphosphate, 100 µmol/L sodium orthovanadate, 1 µg leupeptin/mL, and 1 mmol/L phenylmethylsulfonyl fluoride]; proteins were quantified by the Bradford method of protein quantification (Bio-Rad Laboratories, Hercules, CA). Thirty micrograms of protein were electrophoretically separated in 12% denaturing polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA), blocked, and reacted with primary antibody followed by appropriate secondary antibody.

Phosphoprotein analysis. HeLa cells were exposed at time 0 to 10 pfu of HSV-1(F) per cell and irradiated with 5 Gy at 5 hours after infection as described above. At 10 hours after infection, the cells were harvested and analyzed with the Kinetworks Phospho-site Screen Version 1.3 according to the manufacturer's instructions (Kinexus Bioinformatics Corp., Vancouver, British Columbia, Canada).

Promoter cloning. The cloning of HSV-1(F) DNA sequences as BamHI fragments has been previously described (21). Reporter plasmids containing the luciferase gene driven by viral late (glycoprotein C, US11) or early (ICP0) promoter were constructed as follows. For glycoprotein C promoter, the HSV-1(F) BamHI fragment I (clone pRB130) was digested with BsiHKAI/NheI and ligated to the SacI/NheI-digested pGL3 basic (New England BioLabs, Beverly, MA). For US11 promoter, the HSV-1(F) BamHI fragment Z (clone pRB122) was digested with AgeI/XhoI and ligated to the XmaI/XhoI-digested pGL3 basic. For ICP0 promoter, HSV-1(F) BamHI fragment S2 clone (pRB144) was digested with SacI/NcoI and ligated to the SacI/NcoI-digested pGL3. Positive colonies identified by hybridization to synthesized oligonucleotide probes were amplified and the plasmids verified by sequencing.

Transfection assays. MiaPaCa-2 cells grown in 12-well plates were transfected with luciferase reporter plasmids regulated by the glycoprotein C, US11, or ICP0 promoters in replicates of four using LipofectAMINE Plus reagent according to the manufacturer's instructions (Promega Corp., Madison, WI). Briefly, cells were transfected with 1.5 µg of plasmid DNA and maintained in unsupplemented DMEM for the first 6 hours after which cells were incubated in complete medium. Twenty-four hours after transfection, cells were exposed to 10 pfu of R3616 virus per cell. At 5 hours after infection, cells were lysed and the luciferase activity measured with the aid of the Luciferase Assay System (Promega) according to the manufacturer's recommendation. To study promoter activation in response to ionizing radiation, transfected cells were either left untreated or exposed to 5 Gy. Cells were harvested 5 hours after irradiation and luciferase assays done. To evaluate inhibition by p38, cells were transfected with 1.0 µg of either control plasmid DNA (pcDNA 3.1; Invitrogen, Carlsbad, CA) or p38 dominant-negative plasmid DNA (a kind gift of Roger J. Davis, University of Massachusetts, Worcester, MA; ref. 23) before transfection with the HSV-1 promoter plasmids. For chemical inhibition of p38, cells were exposed to either 20 µmol/L SB203580 or SB202190 or DMSO for 1 hour before irradiation.

Bioluminescence imaging. MiaPaCa-2 tumor xenografts were established in the hind limbs of athymic nude mice by injection of 1 x 107 cells in 100 µL of warm PBS. All animal studies were done in accordance with the University of Chicago Animal Care and Use Committee standards. Once tumors grew to an average volume of 250 mm3, 4.5 x 108 pfu of virus R2636 were injected intratumorally using a 30-guage needle. Animals were either left untreated or exposed to 10 Gy at a dose rate of 50 cGy/s using a Gamma Cell irradiator (Atomic Energy Corp. of Canada, Ltd., Ontario, Canada) in replicates of four animals per group. At time points posttreatment as indicated in Results, imaging of firefly luciferase in mice was done on a charge-coupled device camera (Roper Scientific Photometrics, Tucson, AZ). Animals were injected i.p. with 15 mg/kg body weight with D-luciferin (Biotium, Hayward, CA). After 5 minutes, animals were anesthetized with i.p. injection of ketamine (75 mg/kg) and xylazine (5 mg/kg) for imaging, which was done 10 minutes after the injection of D-luciferin. The relative intensity of transmitted light from tumors infected with virus R2636 are represented as a pseudocolor images with intensity ranging from low (blue) to high (red). Gray scale images were superimposed on the pseudocolor images using MetaMorph image analysis software (Fryer Co., Inc., Huntley, IL). Data for total photon flux was calculated using area under the curve analysis (MetaMorph).

Antibodies. Polyclonal antibodies to the phosphorylated and total forms of MAPK kinases 3 and 6 (MKK3/6, Ser189/207), p38 (Thr180/Tyr182), MAPK-activated protein kinase-2 (MAPKAPK-2, Thr334), and heat shock protein-27 (HSP27, Ser82) were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibody to infected cell protein No.27 (ICP27) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to US11 and UL42 were described elsewhere (24, 25). Secondary antibodies (Cell Signaling Technology) were conjugated to horseradish peroxidase. Protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL) and densitometric analysis was done using ImageJ analysis (NIH, Bethesda, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The accumulation of late ({gamma}2) proteins of HSV-1 is up-regulated in cells infected with R3616 ({Delta}{gamma}134.5) mutant and exposed to ionizing radiation. To test the hypothesis that ionizing radiation results in the accumulation of late viral proteins, MiaPaCa-2 cells infected with 10 PFU of R3616 mutant virus per cell were either untreated or irradiated with 5Gy 6h after infection. Cells were harvested 10h after infection and processed as described in Materials and Methods. Proteins were immunoblotted with antibodies representative of {alpha} (ICP27), ß (UL42), or {gamma}2 (US11 and glycoprotein C) viral proteins. As shown in Fig. 1A, there was a reproducible increase in accumulation of late proteins exemplified by US11 and glycoprotein C proteins as determined by densitometric analysis (35% and 30%, respectively) in two separate experiments. In contrast, there was no increase in the accumulation of {alpha} (ICP27) or ß (UL42) protein in infected and irradiated cells compared with unirradiated controls.



View larger version (35K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Ionizing radiation increases the accumulation of late viral proteins in cells infected with virus R3616. A, duplicate cultures of MiaPaCa2 cells were infected with 10 pfu of R3616 virus per cell and either irradiated (5 Gy) or left untreated at 6 hours after infection. Cells were harvested at 11 hours after infection and processed as described in Materials and Methods. The electrophoretically separated proteins were immunoblotted with antibodies for immediate-early ({alpha}, ICP27), early (ß, UL42), and late ({gamma}, US11 and glycoprotein C) viral proteins. B, replicate cultures of HeLa cells were infected with 10 pfu of R3616 virus per cell in presence or absence of 300 µg/mL of viral DNA replication inhibitor phosphonoacetic acid (PAA). Infected cells were either untreated or irradiated (5 Gy) at time points after infection as indicated. Cells were harvested at 12 hours after infection and processed as described in Materials and Methods. The electrophoretically separated proteins were immunoblotted with antibodies to immediate-early, early, and late viral proteins as described above.

 
To verify that the radiation-mediated increase in late viral proteins was due to de novo protein synthesis rather than stabilization of proteins introduced into the cell by the infecting virus, replicated cell cultures were infected with R3616 as above and either left untreated or incubated in medium containing 300 µg of phosphonoacetic acid per mL. Infected cells were exposed to 5 Gy at 0 or 1.5 hours after infection and the cells were harvested at 11 hours after infection. At the concentration tested, phosphonoacetic acid blocks viral DNA synthesis and expression of {gamma}2 genes, decreases the accumulation of {gamma}1 proteins, and has no effect on the accumulation of {alpha} or ß proteins. In these experiments, there was a notable increase in the accumulation of US11 in irradiated cells compared with the unirradiated controls (Fig. 1B). As expected, phosphonoacetic acid blocked the accumulation of US11 protein in nonirradiated cells. The observation that phosphonoacetic acid blocked the accumulation of US11 in irradiated, infected cells indicates that the increased accumulation of the US11 protein in irradiated cells reflected de novo synthesis of the protein that was dependent on viral DNA synthesis.

Ionizing radiation does not affect the pattern of accumulation of late proteins in cells infected with wild-type virus. To determine whether ionizing radiation affects the accumulation of viral proteins in wild-type virus-infected cells, MiaPaCa-2 cells were exposed to 10 pfu per cell of HSV-1(F) and 5 Gy at 1.5, 3, or 6 hours after infection. Cells were harvested at multiple time points after infection and processed as described in Materials and Methods. This experiment showed no appreciable difference in the accumulation of US11 protein (see Fig. 2A). There was also no difference in the relative amounts of glycoprotein C, ICP 27, or UL42 proteins accumulating in irradiated, infected cells (data not shown). One hypothesis that may explain these results is that in cells infected with wild-type virus, the synthesis of late proteins is already at their highest levels and that this level cannot be altered by irradiation.



View larger version (46K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Ionizing radiation does not affect the accumulation of late viral proteins in cells infected with wild-type HSV-1(F) virus. A, replicate cultures of MiaPaCa-2 cells were infected with 10 pfu of HSV-1(F) virus per cell, either untreated or exposed to 5 Gy at 1.5, 3, and 6 hours after infection. Cells were harvested at 6, 8, or 10 hours after infection and processed as described in Materials and Methods. The electrophoretically separated proteins were immunoblotted with antibodies for the late protein US11. B, replicate cultures of MiaPaCa-2 cells were infected with 10 pfu of HSV-1(F) virus per cell in the presence or absence of 20 µmol/L SB203580. Cells were harvested at 3, 6, 9, or 12 hours after infection and processed as described in Materials and Methods. The electrophoretically separated proteins were immunoblotted with antibodies for immediate-early ({alpha}, ICP27) and late ({gamma}, US11) viral proteins.

 
Ionizing radiation up-regulates HSV-1 {gamma}2 promoters but not {alpha} promoters. To test the hypothesis that the increase in production of late viral proteins is mediated in part by activation of the promoters of late viral genes, we transfected MiaPaCa-2 cells with plasmids containing glycoprotein C or US11 ({gamma}2), or ICP0 ({alpha}) promoter fused to an open reading frame encoding luciferase. To insure that the chimeric genes transfected into the MiaPaCa-2 cells could be expressed, the cells were exposed to 10 pfu of R3616 mutant virus per cell and expression of the luciferase gene was measured. Based on the observation that the virus induced the expression of the transfected luciferase (data not shown), replicate uninfected cultures were exposed to ionizing radiation and luciferase was measured as described in Materials and Methods. In transfected cells that were irradiated, both the glycoprotein C and US11 promoter constructs consistently showed enhanced activation above that observed in unirradiated controls in serial experiments (Fig. 3A, compare lanes 1 and 2). In serial experiments, the expression of the ICP0 promoter driven luciferase was not affected by exposure to 5 Gy (data not shown).



View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 3. Late HSV-1 promoters are activated by ionizing radiation. A, replicate cultures of MiaPaCa-2 cells were cotransfected with luciferase reporter plasmid driven by the US11 promoter together with a p38 dominant-negative plasmid or a control plasmid, pcNDA. At 24 hours after transfection, one set of cultures was exposed to irradiation (5 Gy). Cells were harvested 3 hours later, lysed, and luciferase expression measured as described in Materials and Methods. Columns, mean relative luciferase activities; bars, ±SE. B, replicate cultures of MiaPaCa-2 cells were transfected with the luciferase reporter plasmid driven by glycoprotein C (gC) promoter. At 23 hours after transfection, cells were incubated in medium containing no or 20 µmol/L of inhibitor (SB203580 or SB202190) and exposed to 5 Gy 1 hour later. Cells were harvested 3 hours later, lysed, and luciferase expression measured as described in Materials and Methods. Columns, mean relative luciferase activities; bars, ±SE.

 
Exposure of wild-type virus-infected cells to ionizing radiation results in increased phosphorylation of p38. One potential mechanism of radiation enhancement of viral replication is through activation of key cellular proteins. To test this hypothesis HeLa cells infected with 10 pfu of HSV-1(F) per cell and exposed to 5 Gy 5 hours after infection were harvested 10 hours after infection and analyzed by a global cellular phosphoprotein screen. The results obtained with lysates of cells exposed to 5 Gy alone, HSV-1(F) infection alone, or the combination of 5 Gy and HSV-1(F) were compared with uninfected, unirradiated cells (Fig. 4A). Relative to controls, the combination of HSV-1 and ionizing radiation resulted in an over 4-fold increase in p38 phosphorylation (T180/Y182; Fig. 4A).



View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 4. Enhanced activation of the p38 pathway in irradiated, HSV-1-infected cells. A, replicate cultures of HeLa cells were either mock infected or infected with 10 pfu of HSV-1(F) virus per cell at time 0, left untreated or exposed to irradiation (5 Gy) 5 hours later. Cells were harvested 10 hours after infection and analyzed with a variety of phosphoepitope-specific antibodies as described in Materials and Methods. The level of phosphorylation of p38 is shown. B, replicate cultures of MiaPaCa2 cells were either mock infected or infected with 10 pfu of R3616 virus per cell at time 0, left untreated or exposed to irradiation (5 Gy) at 6 hours after infection. Cells were harvested at 11 hours after infection and processed as described in Materials and Methods. The electrophoretically separated proteins were immunoblotted with the indicated phosphospecific antibodies. Lane 5, virus-infected cells were treated with 20 µmol/L of p38 inhibitor SB203580 1 hour before irradiation.

 
p38 is phosphorylated in R3616 mutant virus-infected cells, and this response is enhanced by ionizing radiation. The enhanced phosphorylation of p38 in cells infected with wild-type virus and exposed to ionizing radiation raised the question whether the combination of ionizing radiation and R3616 mutant virus infection also altered the p38 pathway. To investigate this possibility, MiaPaCa-2 cells infected with R3616 mutant virus and irradiated with 5 Gy at 6 hours after infection were harvested at 11 hours after infection and processed as described in Materials and Methods. The electrophoretically separated lysates were immunoblotted with antibody to phosphoproteins, which are substrates for activation by p38. As shown in Fig. 4B (lane 4), p38 and both upstream activators (MKK3/6) and downstream mediators (MAPKAPK-2 and HSP27) of the p38 pathway exhibited enhanced phosphorylation in cells infected with R3616 mutant virus and treated with ionizing radiation. Cells infected with R3616 that were treated with 20 µmol/L SB203580 before irradiation did not show enhanced phosphorylation of known downstream substrates of p38 (i.e., MAPKAPK-2 and HSP27; Fig. 4B, lane 5).

Inhibition of p38 abrogates activation of viral gene expression mediated by ionizing radiation. To further test the hypothesis that activation of late viral promoters is mediated by p38, we cotransfected plasmids encoding a p38 dominant-negative (p38DN) construct or a negative control plasmid and the US11 promoter linked to luciferase. Twenty-four hours later, cells were irradiated with 5 Gy and harvested 5 hours later. As shown in Fig. 3A (lanes 3 and 4), in cells transfected with the p38DN construct, the baseline levels of expression of the US11 promoter were decreased. Furthermore, the p38DN construct prevented the ionizing radiation–mediated enhancement of luciferase expression consistent with the hypothesis that ionizing radiation–mediated activation of the US11 promoter is p38 dependent (Fig. 3A). We further verified our results with two chemical inhibitors of p38, SB203580 and SB202190. Each of the two inhibitors suppressed the ionizing radiation–mediated activation of the glycoprotein C promoter (Fig. 3B, compare lanes 2 and 3 with lane 1 and lanes 5 and 6 with lane 4).

Inhibition of p38 does not affect the accumulation of late viral proteins in the context of infection with wild-type virus HSV-1(F). In Fig. 2A, we have shown that ionizing radiation did not affect the accumulation of viral proteins in cells infected with wild-type virus. To determine whether wild-type virus was dependent on activation by p38, we investigated the effect of inhibition of p38 activation on the accumulation of viral proteins during infection with HSV-1(F). As shown in Fig. 2B, the presence of 20 µmol/L SB203580 did not have a significant effect on viral protein expression by 12 hours after infection.

In these experiments, we have therefore established that enhancement of late gene expression by ionizing radiation correlates with a requirement for activation of the p38 pathway. Conversely, p38 activation was not required for optimal expression of late proteins in cells infected with wild-type virus, and in that instance, ionizing radiation had no stimulatory effect (Fig. 2A and B).

Ionizing radiation activates premature expression of gC-Luc gene in human tumor xenografts in mice infected with R2636 mutant virus. To investigate the radioinducibility of the glycoprotein C promoter in vivo, MiaPaCa-2 tumor xenografts were treated with a single intratumoral injection of 4.5 x 108 pfu/mL of virus R2636 and then either left untreated or exposed to 10 Gy at the time of infection. Animals were then imaged at 2, 5, and 24 hours after infection and exposure to ionizing radiation. Figure 5A shows the average increase in photon flux in four mice imaged at 2 hours after viral infection and irradiation compared with infected but unirradiated controls. The 13.6-fold increase in photon flux is significant (P < 0.05, t test). Figure 5B shows the image of a pair of mice imaged at 2, 5, and 24 hours after infection and irradiation.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 5. Bioluminescence of mice infected with R2636 and exposed to 10 Gy. MiaPaCa-2 tumors were established in the hind limbs of athymic nude mice and infected by direct intratumoral injection of 4.5 x 108 pfu of virus R2636. Immediately following injection of virus, tumors were exposed to 10 Gy. A, bioluminescence was measured from four animals at 2 hours posttreatment and data is plotted as photons/mm2/s. Columns, means; bars, ±SE. B, a pair of unirradiated and irradiated animals imaged at 2, 5, and 24 hours posttreatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The activation of PKR in HSV-1-infected cells is concordant with the onset of viral DNA synthesis and the accumulation of complementary mRNA representing a large portion of HSV DNA (6, 8, 26–29). In wild-type virus-infected cells, ICP34.5, the product of {gamma}134.5, blocks the shutoff of protein synthesis induced by the phosphorylation of eIF-2{alpha} by activated PKR. In these cells, ICP34.5 recruits phosphatase 1{alpha} to dephosphorylate eIF-2{alpha} and enables continuous synthesis of proteins (30). In cells infected with the mutant R3616 lacking the {gamma}134.5 genes, activation of PKR results in near total shutoff of synthesis of late proteins, but particularly, the {gamma}2 proteins exemplified by US11 or glycoprotein C and whose synthesis depends on the onset and ongoing synthesis of viral DNA (8). Consequently, viral yields are grossly reduced in PKR competent cells. As noted in the Introduction, the genesis of this report stems from the observation that the {Delta}{gamma}134.5 mutant R3616 replicates to higher levels in tumors irradiated between 6 and 9 hours after infection (12, 31). Because the optimal time for irradiation coincides with the synthesis of late proteins, the objective of the studies described here was to analyze the target and mechanism by which ionizing radiation alters the course of replication of R3616 mutant. The salient features of our results are as follows:

(i) Irradiation enhanced the expression of {gamma}2 genes (glycoprotein C and US11) in transduced cells in the absence of other viral gene products or virus-directed DNA synthesis. Irradiation had no effect on transduced viral {alpha} genes. The activation of late genes in the absence of viral DNA synthesis may be inferred from the observation that irradiation enhanced 13.6-fold the expression of glycoprotein C promoter in tumors 2 hours after administration of a mutant virus derived by substitution of the {gamma}134.5 gene with a chimeric gene consisting of the luciferase open reading frame driven by the glycoprotein C promoter. The 2-hour interval from the time of inoculation to the appearance of luciferase is too short to reflect viral DNA synthesis.

(ii) Several lines of evidence support the conclusion that the p38 pathway plays a significant role in the activation of late promoters by ionizing radiation. Specifically, (a) the baseline activity of late promoters in transient transfection assays is attenuated by inhibition of the p38 pathway. The inhibitors used in our assays included chemical (imidazole) inhibitors of p38 as well as a dominant-negative p38 construct. (b) The combination of HSV-1 infection and ionizing radiation results in enhanced phosphorylation of p38 above that of either treatment alone. This was shown by direct measurement of labeled protein as well as by immunoblotting with antibody specific for phosphorylated p38. Inhibition of p38 resulted in attenuation of late promoter activation by ionizing radiation.

The significance of the results obtained in these studies stems from the observation that promoters of late {gamma}2 genes seem located downstream from the TATA box and do not seem to share common response elements (32, 33). In recent studies, it has been shown that the expression of glycoprotein C and US11 is enhanced by the ICP22-mediated interaction of the viral DNA processivity factor UL42 (a viral functional homologue of proliferating cell nuclear antigen), cyclin-dependent kinase cdc2, and topoisomerase II{alpha} (34–36). One hypothesis that could explain the mechanism of activation of late promoters and the role of ionizing radiation in inducing these promoters is that these are activated by modifications in the structure of the DNA, either as a result of nicks, gaps, or other perturbations in the DNA structure.

Two key questions remain unanswered. The first concerns the mechanism of activation of the p38 pathway by the combined effects of viral infection and irradiation. It seems likely that the requirement for both virus infection and irradiation reflects activation of different pathways that converge on activation of p38 rather than a synergistic effect on a single pathway. The second key unanswered question concerns the mechanism by which p38 acts to enhance the expression of late viral genes. The p38 pathway seems to play a significant role in the activation of late promoters in the absence of other viral genes or in cells infected with the {Delta}{gamma}134.5 mutant R3616 but not in wild-type virus-infected cells possibly by mimicking the structural modification that take place during viral replication. The implication of these results is that in wild-type virus-infected cells, other mechanisms insure optimal expression of late genes. The studies described here do not support the hypothesis that the p38 pathway alters the accumulation of activated PKR or of phosphorylated eIF-2{alpha}, and in any event, we can exclude the involvement of PKR in cells transduced with late viral genes.

Finally, two features of these and earlier studies should be stressed. Foremost, in the absence of viruses that specifically target cancer cells, current approaches to therapy of cancer with viruses hinges on the use of attenuated virus that replicate better in dividing tumor cells than in normal, resting cells, but their replication in both types of cells is abysmal. In the case of mutant viruses defective in the expression of late genes, ionizing radiation can stimulate the replication of these viruses in a specific portion of the tissues exposed to ionizing radiation.

The second development exemplified in Fig. 5 is the use of bioluminescence to track the distribution and accumulation of virus in tumors in situ. The ability to image HSV-1 with bioluminescence for superficial tumors or positron emission tomography for deep-lying tumors allows for adaptive image guidance for both viral oncolysis and radiotherapy. In this concept, subsequent administration of viral therapy or radiotherapy can be guided by the distribution of virus and ionizing radiation in the tumor. In summary, these data provide the basis for the construction of new oncolytic viruses that take advantage of the host cell response to stress and thereby can be targeted by ionizing radiation.


   Acknowledgments
 
Grant support: NIH grant 5-P01 CA 7193307-07.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. David Smith (Fryer) for help with animal imaging and analysis, Dr. Samuel Hellman for helpful discussion, and Edwardine Lebay and Marija Pejovic for technical help.


   Footnotes
 
Note: J.J. Mezhir and S.J. Advani are equal contributors to this article.

The authors declare no conflicts of interest.

Received 6/ 2/05. Revised 7/18/05. Accepted 8/ 2/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experimental therapy of human glioma by means of a genetically engineered virus mutant. Science 1991;252:854–6.[Abstract/Free Full Text]
  2. Andreansky SS, He B, Gillespie GY, et al. The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors. Proc Natl Acad Sci U S A 1996;93:11313–8.[Abstract/Free Full Text]
  3. Advani SJ, Chung SM, Yan SY, et al. Replication-competent, nonneuroinvasive genetically engineered herpes virus is highly effective in the treatment of therapy-resistant experimental human tumors. Cancer Res 1999;59:2055–8.[Abstract/Free Full Text]
  4. Markert JM, Medlock MD, Rabkin SD, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial. Gene Ther 2000;7:867–74.[CrossRef][Medline]
  5. Shah AC, Benos D, Gillespie GY, Markert JM. Oncolytic viruses: clinical applications as vectors for the treatment of malignant gliomas. J Neurooncol 2003;65:203–26.[CrossRef][Medline]
  6. Chou J, Kern ER, Whitley RJ, Roizman B. Mapping of herpes simplex virus-1 neurovirulence to {gamma} 134.5, a gene nonessential for growth in culture. Science 1990;250:1262–6.[Abstract/Free Full Text]
  7. Gale M, Jr., Katze MG. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol Ther 1998;78:29–46.[CrossRef][Medline]
  8. Chou J, Roizman B. The {gamma} 1(34.5) gene of herpes simplex virus 1 precludes neuroblastoma cells from triggering total shutoff of protein synthesis characteristic of programmed cell death in neuronal cells. Proc Natl Acad Sci U S A 1992;89:3266–70.[Abstract/Free Full Text]
  9. He B, Gross M, Roizman B. The {gamma}(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1{alpha} to dephosphorylate the {alpha} subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 1997;94:843–8.[Abstract/Free Full Text]
  10. He B, Gross M, Roizman B. The {gamma}134.5 protein of herpes simplex virus 1 has the structural and functional attributes of a protein phosphatase 1 regulatory subunit and is present in a high molecular weight complex with the enzyme in infected cells. J Biol Chem 1998;273:20737–43.[Abstract/Free Full Text]
  11. Chambers R, Gillespie GY, Soroceanu L, et al. Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma. Proc Natl Acad Sci U S A 1995;92:1411–5.[Abstract/Free Full Text]
  12. Advani SJ, Sibley GS, Song PY, et al. Enhancement of replication of genetically engineered herpes simplex viruses by ionizing radiation: a new paradigm for destruction of therapeutically intractable tumors. Gene Ther 1998;5:160–5.[CrossRef][Medline]
  13. Brancho D, Tanaka N, Jaeschke A, et al. Mechanism of p38 MAP kinase activation in vivo. Genes Dev 2003;17:1969–78.[Abstract/Free Full Text]
  14. Olson JM, Hallahan AR. p38 MAP kinase: a convergence point in cancer therapy. Trends Mol Med 2004;10:125–9.[CrossRef][Medline]
  15. Dent P, Yacoub A, Fisher PB, Hagan MP, Grant S. MAPK pathways in radiation responses. Oncogene 2003;22:5885–96.[CrossRef][Medline]
  16. Wang X, McGowan CH, Zhao M, et al. Involvement of the MKK6-p38{gamma} cascade in {gamma}-radiation-induced cell cycle arrest. Mol Cell Biol 2000;20:4543–52.[Abstract/Free Full Text]
  17. Zachos G, Clements B, Conner J. Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1. J Biol Chem 1999;274:5097–103.[Abstract/Free Full Text]
  18. Zachos G, Koffa M, Preston CM, Clements JB, Conner J. Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication. J Virol 2001;75:2710–28.[Abstract/Free Full Text]
  19. Karaca G, Hargett D, McLean TI, et al. Inhibition of the stress-activated kinase, p38, does not affect the virus transcriptional program of herpes simplex virus type 1. Virology 2004;329:142–56.[CrossRef][Medline]
  20. Roizman B, Knipe DM. The replication of herpes simplex viruses. 4th ed. In: Knipe D, Howley P, Griffin DE, Lamb RA, Martin MA, Roizman B, Strauss SE editors. Fields' virology. New York: Lippincott-Williams and Wilkins; 2001. p. 2399–459.
  21. Post LE, Conley AJ, Mocarski ES, Roizman B. Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as BamHI fragments. Proc Natl Acad Sci U S A 1980;77:4201–5.[Abstract/Free Full Text]
  22. Chou J, Poon AP, Johnson J, Roizman B. Differential response of human cells to deletions and stop codons in the {gamma}(1)34.5 gene of herpes simplex virus. J Virol 1994;68:8304–11.[Abstract/Free Full Text]
  23. Lee TH, Huang Q, Oikemus S, et al. The death domain kinase RIP1 is essential for tumor necrosis factor {alpha} signaling to p38 mitogen-activated protein kinase. Mol Cell Biol 2003;23:8377–85.[Abstract/Free Full Text]
  24. Roller RJ, Roizman B. The herpes simplex virus Us11 open reading frame encodes a sequence-specific RNA-binding protein. J Virol 1990;64:3463–70.[Abstract/Free Full Text]
  25. Sheaffer AK, Hurlburt WW, Stevens JT, et al. Characterization of monoclonal antibodies recognizing amino- and carboxy-terminal epitopes of the herpes simplex virus UL42 protein. Virus Res 1995;38:305–14.[CrossRef][Medline]
  26. Kozak M, Roizman B. RNA synthesis in cells infected with herpes simplex virus. IX. Evidence for accumulation of abundant symmetric transcripts in nuclei. J Virol 1975;15:36–40.[Abstract/Free Full Text]
  27. Kozak M, Roizman B. Regulation of herpesvirus macromolecular synthesis: nuclear retention of nontranslated viral RNA sequences. Proc Natl Acad Sci U S A 1974;71:4322–6.[Abstract/Free Full Text]
  28. Jacquemont B, Roizman B. RNA synthesis in cells infected with herpes simplex virus. X. Properties of viral symmetric transcripts and of double-stranded RNA prepared from them. J Virol 1975;15:707–13.[Abstract/Free Full Text]
  29. Jacquemont B, Roizman B. Ribonucleic acid synthesis in cells infected with herpes simplex virus: characterization of viral high molecular weight nuclear RNA. J Gen Virol 1975;29:155–65.[Abstract/Free Full Text]
  30. Roizman B, Markovitz N. Herpes simplex virus virulence: the functions of the {gamma} (1)34.5 gene. J Neurovirol 1997;3 Suppl 1:S1–2.
  31. Chung SM, Advani SJ, Bradley JD, et al. The use of a genetically engineered herpes simplex virus (R7020) with ionizing radiation for experimental hepatoma. Gene Ther 2002;9:75–80.[CrossRef][Medline]
  32. Michael N, Roizman B. Binding of the herpes simplex virus major regulatory protein to viral DNA. Proc Natl Acad Sci U S A 1989;86:9808–12.[Abstract/Free Full Text]
  33. Guzowski JF, Wagner EK. Mutational analysis of the herpes simplex virus type 1 strict late UL38 promoter/leader reveals two regions critical in transcriptional regulation. J Virol 1993;67:5098–108.[Abstract/Free Full Text]
  34. Advani SJ, Weichselbaum RR, Roizman B. The role of cdc2 in the expression of herpes simplex virus genes. Proc Natl Acad Sci U S A 2000;97:10996–1001.[Abstract/Free Full Text]
  35. Advani SJ, Weichselbaum RR, Roizman B. cdc2 cyclin-dependent kinase binds and phosphorylates herpes simplex virus 1 U(L)42 DNA synthesis processivity factor. J Virol 2001;75:10326–33.[Abstract/Free Full Text]
  36. Advani SJ, Weichselbaum RR, Roizman B. Herpes simplex virus 1 activates cdc2 to recruit topoisomerase II {alpha} for post-DNA synthesis expression of late genes. Proc Natl Acad Sci U S A 2003;100:4825–30.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
C. Liu, J. N. Sarkaria, C. A. Petell, G. Paraskevakou, P. J. Zollman, M. Schroeder, B. Carlson, P. A. Decker, W. Wu, C. D. James, et al.
Combination of Measles Virus Virotherapy and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme
Clin. Cancer Res., December 1, 2007; 13(23): 7155 - 7165.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Veerapong, K. A. Bickenbach, M. Y. Shao, K. D. Smith, M. C. Posner, B. Roizman, and R. R. Weichselbaum
Systemic Delivery of {gamma}134.5-Deleted Herpes Simplex Virus-1 Selectively Targets and Treats Distant Human Xenograft Tumors That Express High MEK Activity
Cancer Res., September 1, 2007; 67(17): 8301 - 8306.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. Zhou and B. Roizman
Construction and properties of a herpes simplex virus 1 designed to enter cells solely via the IL-13{alpha}2 receptor
PNAS, April 4, 2006; 103(14): 5508 - 5513.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mezhir, J. J.
Right arrow Articles by Weichselbaum, R. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mezhir, J. J.
Right arrow Articles by Weichselbaum, R. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online