Annals of Surgical Oncology 15(8):2329–2335
DOI: 10.1245/s10434-008-9924-z
Myxoma Virus Is Oncolytic for Human Pancreatic Adenocarcinoma Cells Yanghee Woo, MD,1 Kaitlyn J. Kelly, MD,1 Marianne M. Stanford, PhD,3 Charles Galanis, MD,1 Yun Shin Chun, MD,1 Yuman Fong, MD,1 and Grant McFadden, PhD2
1
Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA 2 Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA 3 Biotherapeutics Research Group, Robarts Research Institute, and Department of Microbiology and Immunology, University of Western Ontario, Oshawa, ON, Canada
Background: Viral oncolytic therapy, which seeks to exploit the use of live viruses to treat cancer, has shown promise in the treatment of cancers resistant to conventional anticancer therapies. Among the most difficult to treat cancers is advanced pancreatic adenocarcinoma. Our study investigates the ability of a novel oncolytic agent, myxoma virus, to infect, productively replicate in, and kill human pancreatic cancer cells in vitro. Methods: The myxoma virus vMyxgfp was tested against a panel of human pancreatic adenocarcinoma cell lines. Infectivity, viral proliferation, and tumor cell kill were assessed. Results: Infection of tumor cells was assessed by expression of the marker gene enhanced green fluorescent protein (e-GFP). vMyxgfp had the ability to infect all pancreatic cancer cell lines tested. Killing of tumor cells varied among the 6 cell lines tested, ranging from [90% cell kill at 7 days for the most sensitive Panc-1 cells, to 39% in the most resistant cell line Capan-2. Sensitivity correlated to replication of virus, and was found to maximally exhibit a four-log increase in foci-forming units for the most sensitive Panc-1 cells within 72 h. Conclusion: Our study demonstrates for the first time the ability of the myxoma virus to productively infect, replicate in, and lyse human pancreatic adenocarcinoma cells in vitro. These data encourage further investigation of this virus, which is pathogenic only in rabbits, for treatment of this nearly uniformly fatal cancer. Key Words: Oncolytic therapy—Myxoma virus—Viral vectors—Pancreatic cancer—In vitro.
unresectable or recurrent disease, the increased survival is usually measured in months, and resistance to this standard first-line chemotherapeutic agent is already developing. It has been shown that Akt, a potent inhibitor of apoptosis, is constitutively activated in the majority of pancreatic cancer cell lines,4 and gemcitabine resistance in pancreatic cancer is often mediated by increased levels of phosphorylated, or activated, Akt.5 Viral oncolytic therapy is emerging as a promising anticancer treatment strategy which exploits the naturally occurring or genetically engineered ability of a virus to specifically target and kill cancer cells.6–8
Pancreatic adenocarcinoma is the fourth leading cancer killer in the USA, responsible for over 32,000 deaths this year.1 Patients diagnosed with metastatic pancreatic cancer have a median survival of 6–9 months with or without surgical resection2 and a dismal overall survival of less than 4% at 5 years.3 While some increased clinical benefit has been associated with gemcitabine treatment in patients with Published online May 3, 2008. Address correspondence and reprint requests to: Yuman Fong, MD; E-mail:
[email protected] Published by Springer Science+Business Media, LLC 2008 The Society of Surgical Oncology, Inc.
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Some viruses, such as the herpes simplex virus (HSV), have been specifically designed to kill cancer.9–11 Other naturally occurring viruses, which have low pathogenicity for man, are also being tested as treatments for cancer. One such virus is the myxoma virus, a large double-stranded DNA (dsDNA) virus of the poxvirus family.12 In the European rabbit Oryctolagus cuniculus, the myxoma virus causes a rapidly lethal disease known as myxomatosis. While the myxoma virus is deadly to rabbits and has been used as an agent for pest control, it has never been documented to cause any disease in any host outside the rabbit, including man.13 Recent studies exploring the species specificity of the myxoma virus have discovered its ability to replicate in immortalized baby green monkey kidney (BGMK) cells and different varieties of human transformed cells,13 including malignant glioma and medulloblastoma.14,15 At present there are several host cell signaling pathway alterations implicated in increasing permissiveness to myxoma virus infection. As with other oncolytic viruses, a disrupted interferon pathway is required for successful infection. Normal murine fibroblasts are resistant to productive infection by myxoma virus. However, murine fibroblasts in which the interferon pathway has been artificially blocked are fully permissive.16 In addition, it has been shown that productive infection of transformed human cells requires constitutive activation of Akt,15 and high intracellular levels of phosphorylated Akt predict permissiveness to myxoma virus infection.17 The M-T5 gene product of myxoma virus is itself an activator of Akt.18 Finally, it has been shown that activation of p21–activated kinase 1 (PAK1) is required for viral replication.19 Our current study examines the potential for vMyxgfp to infect and kill human pancreatic adenocarcinoma cells in vitro. We hypothesized that vMyxgfp would be an efficacious oncolytic agent against pancreatic cancer given that the majority of pancreatic cancer cell lines have constitutively activated Akt. Further, we hypothesized that sensitivity to virus would be greatest in gemcitabine-resistant cell lines due to increased levels of phosphorylated Akt.
MATERIALS AND METHODS Cell Lines Human pancreatic ductal adenocarcinoma cell lines, Panc-1, Hs766T, AsPC-1, MiaPaCa-2, BxPC-3, Ann. Surg. Oncol. Vol. 15, No. 8, 2008
and Capan-2 were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in the recommended media. The selected cell lines all have constitutive Akt expression, but differ in baseline levels of phosphorylated Akt.4 Panc1 was previously shown to have high levels of phosphorylated Akt. Capan-2, MiaPaCa-2, and AsPC-1 cells express moderate levels, and Hs766T and BxPC3 cells do not express basal phosphorylated Akt.4 Cells were grown to 70–80% confluence in T225 flasks in a humidified incubator (37C, 5% CO2). BGMK cells were maintained in DME HG 10% FCS with penicillin and streptomycin for viral titering. Myxoma Virus, vMyxgfp The recombinant myxoma virus, vMyxgfp, is the oncolytic virus investigated in this study. The virus has previously been described.19 vMyxgfp is a replication-competent virus genetically engineered from strain Lausanne with an insertion of enhanced green fluorescent protein (eGFP) between open reading frames (ORF) M135R and M136R of its genome. Upon infection of a host cell, the expression of eGFP is regulated by a synthetic vaccinia virus early/late promoter. vMyxgfp was titered on BGMK cells by counting foci-forming units (FFU) under a Carl Zeiss fluorescent filter microscope, Axiovert 200 M (Carl Zeiss Inc., Thornwood, NY). Cytotoxicity Assay Panc-1, Hs766T, AsPC-1, MiaPaCa-2, BxPC-3, and Capan-2 were plated 2 · 104 cells per well in 12-well flat-bottom plates with 1 ml of media. After attachment of the cells, they were subsequently infected with vMyxgfp at multiplicities of infection (MOIs, or numbers of virus particles per cell) of 5.0, 10, 50, or 100 in triplicate. On day 1, 1 ml of media was added to each well. On days 1, 3, 5, and 7 after infection, cells were washed with phosphate buffered solution (PBS) and lysed with 1.5% Triton-X to release intracellular lactate dehydrogenase (LDH). The amount of LDH released in the lysate of the infected and uninfected tumor cells was compared to determine the cytotoxic effect of viral infection on tumor cells. The amount of LDH per well was quantified using a Cytotox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI) that measures the absorbance of the color change that occurs in the conversion of a tetrazolium salt into a formazan product. Absorbance was measured at 450 nm using a microplate reader (EL 312e: Bio-Tek Instruments, Winooski, VT). Results are
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expressed as the fraction of surviving cells in the infected wells versus the control untreated cells based on the LDH assay readings. Single-Step Viral Growth Curve Panc-1 cells were plated with 1 · 105 cells in 6-well flat bottom plates with 2 ml of media. After attachment and 80–95% confluence of cells, the media was removed and cells were infected with vMyxgfp at an MOI of 3 in a volume of 0.5 ml. The virus was then removed, and growth media supplied. Cells were maintained in a humidified incubator (37C, 5% CO2). At time points of 0, 4, 24, 48, and 72 h after viral infection, cells and supernatants were collected. The cells were subjected to freeze–thaw lysis for the release of viral particles. To assess viral titer from the Panc-1 cells, BGMK cells were plated to 90% confluence in 24-well plates. The respective lysates at the indicated time points were inoculated into the BGMK cells, and incubated at room temperature for 1 h and replaced by media. They were placed in the incubator, and foci-forming units were counted by fluorescence microscopy.
RESULTS Infection and Replication of the Myxoma Virus in Pancreatic Cancer Cell Lines Six human pancreatic adenocarcinoma cell lines with constitutive Akt activation but with varying levels of phosphorylated Akt were tested. The myxoma virus was found to productively infect all cell lines tested. eGFP expression was detected in Panc-1, Hs766T, AsPC-1, MiaPaCa-2, BxPC-3, and Capan-2 cells, demonstrating vMxygfp infection by 24 h at MOI of 1.0. The infection was dose dependent. At lower MOIs of 0.1–1.0, initial infection efficacy was less than 10%, as demonstrated by little to no eGFP expression. However, early gene expression was present in nearly all the cells after exposure to higher MOIs of 10–100. Expression of eGFP occurred within 8–12 h after infection with vMyxgfp at an MOI 1.0, with earliest marker gene appearance in Panc-1 and MiaPaCa-2 cells. By 24–48 h all cell lines expressed eGFP, demonstrating the virus infection had proceeded to the phase of early gene expression. At an MOI of 10, all cell lines demonstrated prominent eGFP expression by 48 h after expression (Fig. 1). This high degree of GFP expression across all of the cell lines regardless of Akt status demon-
strates the nonspecific mechanism of entry of myxoma virus into target cells and subsequent virusmediated early gene expression. The replication of the myxoma virus in the pancreatic cancer cell lines was confirmed by one-step viral proliferation assay, demonstrating a four-log increase in foci-forming units per 1 · 105 BGMK cells over 72 h in the Panc-1 cell line (Fig. 2). Effective Oncolysis of Pancreatic Cancer Cells by Myxoma Virus in a Dose-Dependent Manner Killing of pancreatic cancer cells was dependent on viral dose. Figure 3 demonstrates a clear correlation of cell death with initial MOI in Panc-1 cells. In addition, the cell lines Panc-1, Hs766T, MiaPaCa-2, and AsPC-1 had the highest efficiency of cell kill (Fig. 4). Those cell lines that were relatively resistant to oncolysis by myxoma virus (BxPC-3 and Capan-2) still showed some degree of kill and were not completely resistant to the virus. While a minimal oncolytic effect of the myxoma virus was seen with MOI = 5 or 10, at a higher MOI of 50, 38% cell kill was demonstrated by day 7 after vMyxgfp treatment in Panc-1 cells. At the maximum dosage tested (MOI = 100), significant and increasing cytotoxic effect was observed in all the human pancreatic cancer cells over time. The myxoma virus killed 90% (±4.00) of the Panc-1 cells, 81% (±4.00) of the Hs766T cells, 75% (±7.87) of AsPC-1, 76% (±2.91) of MiaPaCa-2, 50% (±6.97) of BxPC-3, and 39% (±6.69) of Capan-2 cells by day 7 (Fig. 4). Correlation of Gemcitabine Sensitivity, vMyxgfp Sensitivity, and Basal p-Akt Expression in Pancreatic Cancer Cell Lines Our data demonstrate that, among the relatively gemcitabine-resistant cell lines tested, sensitivity to vMyxgfp is inversely related to gemcitabine sensitivity and is directly related to basal intracellular level of phosphorylated Akt (Table 1).
DISCUSSION Pancreatic adenocarcinoma is a nearly uniformly fatal disease. Each year approximately 32,000 patients will present with this cancer in the USA with nearly an equal number of annual deaths. Our therapies, whether surgical,2 chemotherapeutic,20 or biologic,21,22 are merely palliative. Few patients survive beyond 18 months after diagnosis. The dismal outAnn. Surg. Oncol. Vol. 15, No. 8, 2008
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FIG. 1. Representative photograph of pancreatic cancer cells demonstrating eGFP expression after vMyxgfp infection with MOI 10. Panels (A, B, C), and (D) are images of eGFP expression from virus-infected cells under fluorescent filtered microscope overlaid on bright-field images of the same view. Panel (A) shows Panc-1 cells plated 2 · 105 in a 6-well plate 48 h after vMyxgfp infection at 50· magnification. Panel (B) shows Hs766T under the same conditions. Panels (C) and (D) show MiaPaCa-2 and Capan-2, respectively, 48 h after vMyxgfp infection, demonstrating eGFP expression. eGFP expression appearing to be in the extracellular space is the result of lysis of infected cells at this late time point.
FIG. 2. The myxoma virus proliferates in Panc-1 cells over time. Panc-1 cells harvested for viral proliferation assay at 4, 24, 48, and 72 h after infection with vMyxgfp at MOI 3 were titered on BGMK cells. The foci-forming units (FFU) of the myxoma virus per cells were counted on BGMK cells for the various time points. The graph shows a greater than four-log-fold replication of vMyxgfp in Panc-1 cells within 72 h.
come associated with this disease has led to active investigation for novel therapies. Oncolytic viruses are attractive agents for treatment of cancers refractory to conventional therapies because many of the cellular characteristics that predispose to chemotherapy resistance, such as resistance to apoptosis10 or high levels of alternate nucleotide synthetic path-
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ways,23 theoretically favor viral infection and replication. Cells that are resistant to apoptosis are able to withstand initial infection with oncolytic viruses and are able to support viral proliferation and spread to neighboring cells. If cells were to undergo apoptosis immediately after virus infection, viral proliferation would not occur and only those few apoptotic cells initially infected would die as a result of viral therapy. Similarly, cells with upregulated nucleotide pools and alternative nucleotide synthetic pathways benefit oncolytic viruses, many of which lack specific genes involved in nucleotide synthesis, for example, thymidine kinase. Viruses from the HSV family,24 and from the adenoviral family,25 have therefore been tested and found to be effective in killing pancreatic cancer cells. Our current data would indicate that the myxoma virus from the poxvirus family may also be an active agent for this challenging disease. The myxoma virus has been used extensively as an agent for pest control. In Australia, this virus was released into the wild to control the rabbit population after it was proven to be safe for other potential vertebrate hosts, including man.26 Myxoma virus demonstrated effective oncolytic ability against xenografts of human tumors of neural origin.14
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FIG. 3. vMyxgfp effectively kills Panc-1 human pancreatic adenocarcinoma cells at MOI 100. vMyxgfp kills Panc-1 cells in a dose-dependent manner. Panc-1 cells were placed 2 · 104 in 12-well plates and treated at different dosages reflected by the varying MOIs of 5, 10, 50, and 100. Comparing the percentage cell survival on D7, we can see that no oncolytic effect was seen at the lowest MOI of 5 with 100% cell survival. At a higher MOI 10, less than 10% cell kill was observed, in contrast to the 38% cell kill by the myxoma virus at MOI 50. Most impressively, at an MOI of 100 the myxoma virus almost completely killed all the Panc-1 cells with less than 10 ± 4% cells surviving. This demonstrates the dose-dependent effectiveness of the myxoma virus against pancreatic cancer cell lines.
FIG. 4. vMyxgfp at an MOI 100 is an effective dose against pancreatic cancer. At an MOI of 100, the myxoma virus was effective in killing 90% (±4.00) of Panc-1, 81% of Hs766T (±4.00), 75% (±7.87) of AsPC-1, 76% (±2.91) of MiaPaCa-2, 50% (±6.97) of BxPC-3, and 39% (±6.69) of Capan-2 cells.
Productive infection of tumor cells by myxoma virus has been linked to aberrant activation of the Akt pathway.13 The level of phosphorylated Akt, whether endogenous or virus induced, is predictive of which cancer cell lines will support productive MV infection. This phenomenon can be enhanced by treatment of cancer cells with the drug rapamycin.26 Our current study extends the spectrum of potential cancer targets for this viral agent to include
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human pancreatic adenocarcinoma. In this study, we successfully demonstrated for the first time the oncolytic effect of the myxoma virus against human pancreatic adenocarcinoma cells in vitro. This virus killed a broad spectrum of pancreatic cancer cell lines. Tumor cell killing was dose dependent and progressive over time. Our current data demonstrate this virus to be a promising agent for treatment of pancreatic cancer and support further investigation toward this end. All of the cell lines tested displayed relatively equal infectivity and early gene expression by vMyxgfp as demonstrated by eGFP expression. It has been shown that entry of myxoma virus (and other poxviruses) into target cells is mediated primarily by nonspecific interactions of the virus with ubiquitously expressed cellular glycosaminoglycans.18 Once inside a target cell, myxoma virus begins transcribing its early genes using viral RNA polymerase and viral transcription factors. Up until and including this point, all events are mediated by components of the viral core. The next step, known as core uncoating, results in dissolution of the viral core structure and release of the dsDNA into the cytoplasm of the target cell. The following stages of intermediate and late transcription require interaction with endogenous host cell transcription factors and proteins. While these later stages are not well characterized, it is thought that the difference between nonpermissive and permissive target cells lies here, when host cell cooperation is required.18 All of the cell lines tested express Akt constitutively and, as hypothesized, all demonstrated sensitivity to oncolysis by vMyxgfp. Panc-1, the cell line with the highest levels of phosphorylated Akt, was the most sensitive to oncolysis by vMyxgfp. MiaPaCa-2 and AsPC-1 cells, with moderate levels of phosphorylated Akt, were also quite sensitive to virus. Hs766T and BxPC-3, which do not express basal phosphorylated Akt, differed in sensitivity, with about 30% greater kill of Hs766T cells at an MOI of 100 by day 7. Capan-2 cells, reported to have moderate basal levels of phosphorylated Akt, were least sensitive to vMyxgfp oncolysis. Interestingly, previously published data demonstrate the inhibitory concentration of 5 pancreatic cancer cell lines to gemcitabine to be (in order of increasing sensitivity to gemcitabine as indicated by IC50): Panc-1 (50 nM) \ MiaPaCa-2 (40 nM) \ BxPC-3 (18 nM) \ AsPC-1 (15 nM) \ Capan-2 (12 nM).27,28 The ability of vMyxgfp to kill these same cell lines is the opposite of the effectiveness of gemcitabine, indicating that vMyxgfp may be effecAnn. Surg. Oncol. Vol. 15, No. 8, 2008
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TABLE 1. Relationship among gemcitabine sensitivity, vMyxgfp sensitivity, and basal p-Akt expression in pancreatic cancer cell lines Gemcitabine sensitivity Capan-2 BxPC-2 MiaPaCa-2 AsPC-1 Panc-1
vMyxgfp sensitivity
Basal p-Akt level
+++
+
++
++
+
+
++
++
++
++
++
++
+
+++
+++
Oncolytic efficacy of vMyxgfp varies with basal phosphorylated Akt (p-Akt) levels in relatively gemcitabine-resistant pancreatic cancer cell lines. In the first column for gemcitabine sensitivity, +++ is IC50 \15 nM, ++ is IC50 15–40 nM, and + is IC50 [40 nM. For vMyxgfp sensitivity, +++ is [75% cell kill by day 7, ++ is 51% to 75%, and + is \50%. For basal p-Akt expression, + is little to no expression, ++ is moderate expression, and +++ is high expression, as reported by Bondar et al.4
tive against gemcitabine-resistant pancreatic cancer cells. In order of increasing sensitivity to vMyxgfp (as indicated by percentage cell kill by day 7): Capan-2 (39%) \ BxPC-3 (50%) \ AsPC-1 (74%) = MiaPaCa-2 (74%) \ Panc-1 (90%). In addition, our data show that, among these relatively gemcitabine-resistant cell lines, sensitivity to vMyxgfp is directly related to basal levels of p-Akt. This is not surprising as upregulation of p-Akt is a known pathway for development of gemcitabine resistance, yet simultaneously predicts permissiveness to myxoma virus infection.5,17 One cell line, Capan-2, is discrepant from our hypothesis, being the least sensitive to myxoma viral oncolysis but still with moderate baseline levels of phosphorylated Akt. Interestingly, Capan-2 cells are also discrepant in terms of gemcitabine sensitivity, being the most chemosensitive of all the lines tested in spite of moderate basal levels of activated Akt. These discrepancies indicate that Capan-2 cells may express some other, as yet uncharacterized, pro-apoptotic pathway that both decreases sensitivity to oncolytic virus and increases chemosensitivity. One possibility is that Capan-2 cells have low levels of c-Src, an upstream activator of Akt.27 The vMyxgfp sensitivity observed in Hs766T and BxPC-3 cells, neither of which express phosphorylated Akt at baseline, can theoretically be explained by baseline constitutive Akt expression and the Akt phosphorylating effect of the viral protein M-T5.18 Myxoma virus has been shown to achieve permissive infection of various human tumor types including colon, ovarian, lung, and renal cancers.29 No effective therapy is currently available for pancreatic cancer patients, especially those with chemoresistance to gemcitabine. Oncolytic therapy with myxoma virus Ann. Surg. Oncol. Vol. 15, No. 8, 2008
may represent a potential alternative. Among the most frequently documented mechanisms of development of gemcitabine resistance in pancreatic cancer is inhibition of apoptosis via dysregulation of the Akt/PI3 pathway.4 Inhibition of apoptosis and upregulation of phosphorylated Akt enhance productive infection by vMyxgfp. In conclusion, our study demonstrates for the first time that myxoma virus, a novel oncolytic agent, is able to enter, replicate in, and lyse human pancreatic adenocarcinoma cells in vitro. Furthermore, our findings warrant further investigation of myxoma virus as an oncolytic agent for gemcitabine-resistant disease. ACKNOWLEDGMENTS Supported in part by RO1 CA 75416 (Y.F.) from the National Institutes of Health, the Flight Attendant Medical Research Institute (FAMRI), and Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research grant—The Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (Y.F).
REFERENCES 1. Jemal A. Cancer statistics. CA Cancer J Clin 2007; 57:43–66. 2. Lillemoe KD. Current management of pancreatic carcinoma. Ann Surg 1995; 221:133–48. 3. Spanknebel K, Conlon KC. Advances in the surgical management of pancreatic cancer. Cancer J 2001; 7:312–23. 4. Bondar VM, Sweeney-Gotsch B, Andreeff M, et al. Inhibition of the phosphatidylinositol 3’-kinase-AKT pathway induces apoptosis in pancreatic carcinoma cells in vitro and in vivo. Mol Cancer Ther 2002; 1:989–97. 5. Pham NA, Tsao MS, Cao P, et al. Dissociation of gemcitabine sensitivity and protein kinase B signaling in pancreatic ductal adenocarcinoma models. Pancreas 2007; 35:e16–26. 6. Woo Y, Adusumilli PS, Fong Y. Advances in oncolytic viral therapy. Curr Opin Investig Drugs 2006; 7:549–59. 7. Bell JC, Lichty B, Stojdl D. Getting oncolytic virus therapies off the ground. Cancer Cell 2003; 4:7–11. 8. Lin E, Nemunaitis J. Oncolytic viral therapies. Cancer Gene Ther 2004; 11:643–64. 9. Stiles BM, et al. The replication-competent oncolytic herpes simplex mutant virus NV1066 is effective in the treatment of esophageal cancer. Surgery 2003; 134:357–64. 10. Stanziale SF, Fong Y. Novel approaches to cancer therapy using oncolytic viruses. Curr Mol Med 2003; 3:61–71. 11. Kooby DA, et al. Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207). FASEB J 1999; 13:1325–34. 12. Stanford MM, Werden SJ, McFadden G. Myxoma virus in the European rabbit: interactions between the virus and its susceptible host. Vet Res 2007; 38:299–318. 13. Johnston JB, Nazarian SH, Natale R, McFadden G. Myxoma virus infection of primary human fibroblasts varies with cellular age and is regulated by host interferon responses. Virology 2005; 332:235–48.
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14. Lun X, et al. Myxoma virus is a novel oncolytic virus with significant antitumor activity against experimental human gliomas. Cancer Res 2005; 65:9982–90. 15. Lun XQ, et al. Targeting human medulloblastoma: oncolytic virotherapy with myxoma virus is enhanced by rapamycin. Cancer Res 2007; 67:8818–27. 16. Wang F, et al. Disruption of Erk-dependent type I interferon induction breaks the myxoma virus species barrier. Nat Immunol 2004; 5:1266–74. 17. Wang G, et al. Infection of human cancer cells with myxoma virus requires Akt activation via interaction with a viral ankyrin-repeat host range factor. Proc Natl Acad Sci USA 2006; 103:4640–5. 18. McFadden G. Poxvirus tropism. Nat Rev Microbiol 2005; 3:201–13. 19. Johnston JB, et al. Role of the serine-threonine kinase PAK-1 in myxoma virus replication. J Virol 2003; 77:5877–88. 20. Schnall SF, Macdonald JS. Chemotherapy of adenocarcinoma of the pancreas. Semin Oncol 1996; 23:220–8. 21. Heinemann V. Present and future treatment of pancreatic cancer. Semin Oncol 2003; 29:23–31. 22. Evans JD, et al. A phase II trial of marimastat in advanced pancreatic cancer. Br J Cancer 2001; 85:1865–70. 23. Petrowsky H, et al. Functional interaction between fluorodeoxyuridine-induced cellular alterations and replication of a
24.
25.
26.
27. 28.
29.
2335
ribonucleotide reductase-negative herpes simplex virus. J Virol 2001; 75:7050–8. Eisenberg DP, et al. 5-fluorouracil and gemcitabine potentiate the efficacy of oncolytic herpes viral gene therapy in the treatment of pancreatic cancer. J Gastrointest Surg 2005; 9:1068–77; discussion 1077–9. Ogura Y, et al. Peritumoral injection of adenovirus vector expressing NK4 combined with gemcitabine treatment suppresses growth and metastasis of human pancreatic cancer cells implanted orthotopically in nude mice and prolongs survival. Cancer Gene Ther 2006; 13:520–9. Stanford MM, Barrett JW, Nazarian SH, et al. Oncolytic virotherapy synergism with signaling inhibitors: rapamycin increases myxoma virus tropism for human tumor cells. J Virol 2007; 81:1251–60. Duxbury MS, Ito H, Zinner MJ, et al. siRNA directed against c-Src enhances pancreatic adenocarcinoma cell gemcitabine chemosensitivity. J Am Coll Surg 2004; 198:953–9. Shi X, Liu S, Kleeff J, et al. Acquired resistance of pancreatic cancer cells towards 5-Fluorouracil and gemcitabine is associated with altered expression of apoptosis-regulating genes. Oncology 2002; 62:354–62. Sypula J, Wang F, Ma Y, et al. Myxoma virus tropism in human tumor cells. Gene Ther Mol Biol 2004; 8:103–14.
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