1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Replication-competent herpes simplex oncolytic viruses are promising anticancer agents that partly target increased DNA synthesis in tumor cells. Investigators have proposed that these DNA viruses may be combined with liver resection to enhance killing of liver malignancies. Whether or not the cellular alterations associated with hepatic regeneration affect the efficacy and toxicity of these promising anticancer agents is unknown. This study examined the behavior of two oncolytic viruses, NV1020 and G207, during liver regeneration. When delivered during the peak of liver regeneration, replication and appearance of both G207 and NV1020 in hepatic tissue are enhanced as demonstrated by histochemical staining for the marker gene lac Z, immunohistochemical staining, and quantitative polymerase chain reaction. This increased appearance of virus in liver tissue correlates with increases in cellular ribonucleotide reductase activity and DNA synthesis and is also associated with increased viral binding. However, increased viral presence is transient, and viral detection declines to baseline within 7 days. When these viruses were delivered to animals even as early as 7 days after hepatectomy, there proved to be no measurable viral replication in any organ and no increased morbidity or mortality. In conclusion, the early stages of hepatic regeneration after resection provide an environment suitable for viral replication. Administration of replication-competent herpes simplex virus during the peak of hepatocyte regeneration (24–48 hours) permits viral productivity in tissue that otherwise does not support viral growth. The increase in hepatotoxicity after hepatectomy is short-lived and can be predicted by peak hepatocyte DNA synthesis. (HEPATOLOGY 2004;39:1525–1532.)

Hepatic tumors are the most common gastrointestinal malignancy.1 Hepatic resection remains the only possibility of cure for hepatocellular carcinoma,1, 2 biliary cancer,3 or metastatic colorectal carcinoma.4 Nearly 65% of patients recur, likely from microscopic residual disease.5, 6 Marked increases in hepatocellular mitotic and metabolic activity due to the local milieu of the regenerating liver is thought to enhance growth of residual disease.5 Research efforts have been directed at targeting this residual disease.

Oncolytic viral therapy in the treatment of liver tumors has demonstrated promise in experimental models. In particular, herpes simplex virus type 1 (HSV-1)-based replication-competent vectors have been effective in a number of recent studies.3, 7, 8 Many of these viruses have been genetically engineered to exploit naturally high synthetic activity in tumor cells to complement the genetic deletions in these viruses.9 G207 and NV1020 are two such replication-competent, multimutated, oncolytic HSVs that have been designed to selectively replicate in tumor cells and produce tumor cell lysis. These viruses have shown efficacy and safety in preclinical testing8, 10–15 and are currently being studied in human clinical trials.16

After hepatectomy, greater than 95% of hepatocytes enter into S-phase within 48 hours. The current study investigates whether or not conditions present during hepatic regeneration enhance viral toxicity for regenerating hepatocytes. The binding and replication of G207 or NV1020 to hepatocytes throughout the peak of hepatic regeneration was also analyzed to identify mechanisms underlying altered viral presence.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References


G207, a gift from Drs. Rabkin and Martuza (Washington, DC), was constructed as described previously, with deletions of both copies of the neurovirulence γ134.5 gene and insertion of the Ecoli lac Z gene into the UL39 locus.17 This latter insertional mutation disables the viral enzyme ribonucleotide reductase (RR), making viral proliferation more dependent on host cellular proliferation.18, 19

NV1020 is a nonselected clonal derivative of R7020 originally obtained from B. Roizman (Chicago, IL).20, 21 It has a 15-kb deletion over the joint region of the HSV-1 genome. This deletion encompasses the region of the genome coding for the ICP0 and ICP4 latency-associated transcripts and one copy of γ134.5. Because it was originally designed as a vaccine against HSV-1 and HSV-2 infection,21 a fragment of HSV-2 DNA from the HindIIIL region encoding for several glycoprotein genes was inserted into the deleted joint region. It also has a 700-bp deletion of the endogenous TK locus that prevents expression of the overlapping transcripts belonging to the UL24 gene. An exogenous copy of the HSV-1 TK gene was inserted under control of the α4 promoter.

Virus stocks were produced in World Health Organization Vero cells seeded in roller bottles and infected at a multiplicity of infection of 0.01, as previously described.3 G207 and NV1020 were purified using size exclusion chromatography (S500, Amersham Pharmacia Biotech, Piscataway, NJ) and concentrated using ultrafiltration (0.05-μM pore size, polysulfone hollow-fiber membrane). Viral preparations were formulated in dextrose (D)–phosphate-buffered saline (PBS)/10% glycerin and stored at −80°C.

Murine Hepatectomy Model.

Animal work was performed under guidelines approved by the Memorial Sloan-Kettering Institutional Animal Care and Use Committee. Male C57Bl/6 mice were purchased from the National Cancer Institute (Bethesda, MD), submitted to a 12:12-hour light/dark cycle, housed five per cage, and allowed food and water ad libitum. Animals to undergo hepatectomy were anesthetized with an intraperitoneal injection of ketamine/xylazine (70/10 mg/kg). A 70% partial hepatectomy was performed through a midline incision as described by Higgins and Anderson.22 Thirty-six hours or 7 days after hepatectomy the animals were treated with an injection of virus into the portal vein accessed via needle puncture of the spleen. Virus for injection (1 × 107 plaque-forming units of G207 or NV1020) was diluted in 300 μL of serum-free media and injected via a 26-gauge needle. Specimens were frozen in Tissue-Tek embedding medium (Sakura Finetek, Torrance, CA) and sectioned by cryotome. Sections fixed with 1% glutaraldehyde were evaluated for β-galactosidase expression by staining with X-gal solution. Tissues were subsequently counterstained with Nuclear Fast Red (Sigma, St. Louis, MO). Slides were then read by two independent blinded reviewers.

In Vivo Assay of Mitosis.

Liver tissue was harvested from animals at 12, 24, 36, 48, 72, and 96 hours after hepatectomy (n = 5–10). Specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Eight-micrometer-thick tissue sections were cut using a microtome (Model TP1050, Leica Microsystems, Deerfield, IL) and deparaffinized. Using Zymed (San Francisco, CA) reagents, the tissues were stained according to standard protocol for Ki-67.

Assay for Ribonucleotide Reductase Activity.

Mice were sacrificed between 9 and 11 A.M. and the right lobe of the liver was removed and washed in ice-cold PBS. Tissue was homogenized in four volumes of Tris buffer (0.1 M Tris-HCl; pH 7.0; 20 mM dithiothreitol [DTT]) with a motor-driven tissue homogenizer. The crude homogenate was centrifuged at 16,000g for 10 minutes and then at 100,000g for 1 hour. The clear supernatant was dialyzed against Tris buffer (20 mM Tris-HCl; pH 7.0; 2 mM DTT) for 6 hours with one buffer change after 3 hours using cassettes with a molecular weight cutoff of 10,000 (Slide-A-Lyzer Dialysis Cassettes, Pierce, Rockford, IL). Dialyzed extracts were snap-frozen in liquid nitrogen and stored at −80°C until analysis. The entire extraction procedure was performed at 4°C. Protein concentrations of extracts were measured by the method of Bradford using bovine gamma immunoglobulin as standard.23

Activity of RR was determined by monitoring conversion of CDP to dCDP using [14C]CDP as substrate and rattlesnake venom to hydrolyze nucleotides to nucleosides.19 The reaction mixture contained the following ingredients in a final volume of 150 μL: 40 μM CDP, 10 μM [14C]CDP (0.08 μCi), 6 mM DTT, 4 mM magnesium acetate, 2 mM adenosine triphosphate, 8.7 mM KPO4 pH 7, and 100 μL extract (0.7–1.2 mg protein). The enzyme reaction was carried out for 30 minutes at 37°C and stopped by boiling for 4 minutes. Nucleotides were hydrolyzed by adding 50 μL of carrier dCMP (6 mM dCMP; 2 mM MgCl2; 6 mM Tris-HCl, pH 8.8) and 25 μL snake venom suspension from Crotalus adamanteus. After 3 hours at 37°C, the reaction mixture was heat-inactivated by boiling for 4 minutes. Heat-precipitated material was removed by centrifugation at 14,000g for 10 minutes. [14C]deoxycytosine was separated from [14C]cytosine by covalent chromatography using phenylboronic acid-columns (BondElut PBA, Varian, Harbor City, CA). Supernatant fractions were mixed with triethenolamine buffer (pH 10) to a concentration of 0.4 M. One milliliter of this mixture was added to the cartridge. Fractions were collected and measured for radioactivity by liquid scintillation spectrometry (LS 6000IC Liquid Scintillation System, Beckman Instruments, Inc., Fullerton, CA). One unit of enzyme activity was defined as conversion of 1 nmol CDP to the product dCDP in 1 hour at 37°C.

Immunohistochemistry Detection of G207 and NV1020 Viral Antigens.

Liver specimens harvested from animals 48 hours, 96 hours, or 7 days after NV1020 or G207 injection were fixed in 4% paraformaldehyde and embedded in paraffin and sectioned. Harvested tissue was then tested immunohistochemically to assess the presence of HSV-1. Specifically, a rabbit-raised polyclonal anti-HSV antibody directed against envelope proteins (Biogenex, San Ramon, CA, #PU084-UP) was used in conjunction with a Zymed (Histomouse-SP, San Francisco, CA) secondary antibody and detection kit used according to the specifications of the supplier.

Quantitative Polymerase Chain Reaction Analysis.

To demonstrate the amount of viral DNA within liver tissue, quantitative polymerase chain reaction (PCR) analysis was performed on genomic DNA extracted from livers of infected mice. Virus was administered at either 36 hours or 7 days after 70% hepatectomy. Specimens were obtained at 48 hours, 7 days, or 21 days after viral administration. Liver tissue from both hepatectomized and nonhepatectomized animals not treated with virus were used as controls. Standard curves were established by inoculating uninfected liver with known quantities of G207 virus prior to DNA extraction. Real-time quantitative PCR was performed using an ABI Prism 7700 Sequence Detector (PE Biosystems, Foster City, CA), as described previously. Sense (5′-ATGTTTCCCGTCTGGTCCAC-3′) and antisense (5′-CCCTGTCGCCTTACGTGAA-3′) primers and a dual-labeled fluorescent TaqMan probe (5′-6FAM-CCCCGTCTCCATGTCCAGGATGGTAMRA-3′) were designed to span the 111-bp fragment of the HSV ICP0 (immediate early gene). Additional sense (5′-CGCCTACCACATCCAAGGAA-3′) and antisense (5′-GCTGGAATTACCGCGGCT-3′) primers and TaqMan probes (5′-JOE-TGCTGGCACCAGCTTGCCCTC-TAMRA-3′) for the 187-bp 18S ribosomal RNA coding sequence were used in the same reaction to normalize the amount of total DNA. Samples were subjected to 40 cycles of PCR (stage 1, 50°C for 2 minutes; stage 2, 95°C for 10 minutes; stage 3, 95°C for 15 seconds; 60°C for 1 minute; stage 4, 25°C), cleaved with AmpliTaq gold nuclease releasing a fluorescent marker (FAM or JOE) from the nonextendable probe, therefore liberating it form the proximity of an associated quencher (TAMRA).

Viral Binding Assay.

To assess the ability of HSV-1 mutants to bind to hepatocytes in different stages of regeneration and to further determine if adhesion played a role in the differences observed in viral presence, an in vivo viral binding assay was performed. Vero cells (1 × 107 cells per 10-cm dish) (ATCC, Rockville, MD) were rinsed two times with PBS followed by one rinse with adsorption medium (Dulbecco's Modified Eagle's Medium [DMEM], 2 mM L-glutamine, 20 mM Hepes, pH 7.4, 1% bovine serum albumin). Cells were infected for 1 hour at 37°C using a multiplicity of infection of 0.1 in adsorption medium (0.5 mL per 10-cm dish). Following adsorption, inoculum was removed, and cells were rinsed 3 times with PBS. Labeling medium (DMEM, 10% DMEM with methionine, 2 mM L-glutamine, 5% dialyzed FCS, 35S-L-methionine) (NEN Life Science Products, Inc., Boston, MA) was added (500 μCi in 8 mL per 10-cm dish) and cells were incubated at 37°C in a humidified incubator with 5% CO2 in air. When cytopathic effects were observable (≈2 d), cells and medium were harvested by scraping and centrifuged. Cell pellets then underwent three freeze–thaw cycles. Supernatant and pellet fractions were combined and centrifuged at 274,000g over a 30% sucrose cushion for 1.5 hours at 4°C. Virus pellets were resuspended in 10% glycerol in PBS with subsequent storage at −80°C. Virus was then titered by a standard plaque assay and titer was correlated with counts per minute as measured by scintillation counting. On the day of the assay, animals that had undergone 70% hepatectomy 36 hours or 96 hours before, or control animals that had not undergone hepatectomy, received 1 × 107 plaque-forming units of radiolabeled virus via intrasplenic injection. Thirty minutes after injection, the liver was flushed with PBS until blood was cleared from the vasculature. Specimens were then manually homogenized in a glass homogenizer. Volume of suspension was measured, as well as protein content via the Bradford technique.23 Five hundred microliters of homogenate was then placed in 4 cc of scintillation fluid (Ecolite, ICN Pharmaceuticals, Costa Mesa, CA) and then counted in a gamma counter for 5 minutes. Scintillant alone was used as a negative control. Results were converted to number of radioactive viruses normalized by cellular protein contents for a relative comparison.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling.

Liver tissue specimens harvested from animals at various time points after hepatectomy were fixed in 4% paraformaldehyde and embedded in paraffin. After slicing, drying overnight, and dewaxing, the 8-μm sections were then treated with 20 μg/mL of proteinase K for 2 minutes, and after PBS washes were fixed again in 4% paraformaldehyde for 10 minutes. After several PBS washes, endogenase peroxidase was quenched with 0.1% H2O2 for 15 minutes. After a dH2O rinse, slides were immersed in terminal transferase buffer (3 mM Tris, pH 7.2; 14 mM sodium cacodylate; 1 mM Cobalt Chloride) for 10 minutes. Subsequently, the liver sections were exposed to 30 units of terminal transferase (Roche) and 5 μM biotin-dUTP per slide at 37°C in a moist chamber for 1 hour. The reaction was stopped with 2× SSC and cells were blocked with 2% bovine serum albumin in PBS to eliminate nonspecific reactivity. Slides were then exposed to the secondary stain for 1 hour, washed in PBS, and treated with 0.5% Triton-X100 for 2.5 minutes. Slides were stained with a filtered diaminobenzidine/.0012% H2O2 solution, counterstained with Harris hematoxylin, washed in dH2O and mounted with Permount (ProSciTech, Kelso, Australia). Several fields were inspected under 40× magnification in bright-field mode using a Zeiss Axiophot-2 for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positivity.


Liver tissue specimens harvested from animals at various time points after viral administration were fixed in 4% paraformaldehyde and embedded in paraffin. After slicing, drying overnight, and dewaxing, the 8-μm sections were stained in Gill's hematoxylin-eosin. Slides were then dehydrated and mounted with Permount (ProSciTech) for histopathological analysis.

Determination of Liver Function Tests.

During the experiment, at 24 or 48 hours after G207 administration, blood was harvested via the retroorbital venous plexus. Prior to sacrifice at the end of the experiment, at 48 hours, or 7 days after G207 administration, animals were bled via cardiac puncture. The blood was spun at 1,500 rpm for 15 minutes; the serum was harvested and stored at −20°C. Liver function testing was performed by Idexx Veterinary Services (Totowa, NJ).


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Ki-67 Staining.

To identify the peak of hepatocyte DNA synthesis, Ki-67 staining was performed on liver tissue obtained from mice at 12, 24, 48, 72, and 96 hours after hepatectomy (Fig. 1). The peak of mitotic activity occurred between 24 and 48 hours after 70% hepatectomy and returned to baseline by 96 hours.

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Figure 1. Paraffin sections of liver tissue stained for the nuclear protein Ki-67. (A) No hepatectomy; (B) 48 hours after 70% hepatectomy; (C) 96 hours after 70% hepatectomy. Dark nuclear staining (>90%) in panel B is positive. (Original magnification ×10.)

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lac Z Staining.

The lac Z marker gene was used to detect cells infected by the G207 virus. Liver tissue was perfused with G207 either 36 hours or 7 days after 70% hepatectomy or sham laparotomy and harvested at 48 hours, 96 hours, or 7 days for histochemical staining. The reason for choosing 48-hour intervals for harvesting of specimens is that this represents the approximate time for one replication of HSV.24 In animals that received G207 36 hours after a 70% partial hepatectomy and subsequently had their livers harvested 48 hours later, there proved to be a marked increase in lac Z expression. This lac Z expression was transient, as livers harvested at 96 hours or 7 days after viral injection showed little staining. Livers that were injected with G207 7 days after hepatectomy as well as the injected sham laparotomy animals did not show any expression of lac Z. Mean expression (±SD) of blue plaques per low-power field in animals undergoing sham laparotomy was 0 (±0), while in the 70% hepatectomy animals that had their livers harvested 48 hours after viral inoculation, it was 67 (±22). Expression of blue plaques per low-power field again fell to zero in all other time points. The difference was statistically significant (P < .005). Representative samples are illustrated in Fig. 2A, B, and C.

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Figure 2. Histochemical and immunostaining for viral presence. (A–C) Livers stained for lac Z after G207 administration. All specimens were harvested 48 hours after viral injection. (A) Tissue without hepatectomy. (B) Liver inoculated with virus 36 hours after 70% hepatectomy. (C) Liver section from an animal that received virus 7 days after 70% hepatectomy. Cells stained blue indicate viral infection. (D–I) Liver sections that were immunostained to detect HSV antigens. Liver specimens were harvested 48 hours after inoculation with (D–F) G207 or (G–I) NV1020. Panels D and G show sections from animals without hepatectomy. Panels E and H show sections from animals that received virus 36 hours after 70% hepatectomy. Note the marked increase in detectable antigens (arrows). Panels F and I show sections from animals that received virus 7 days after 70% hepatectomy. Virus has returned to baseline levels.

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Immunohistochemistry (IHC) was used to detect the presence of herpes viral antigen in liver tissue after viral administration. Viral antigen was not detectable in specimens in nonhepatectomized animals that received either G207 or NV1020 (Fig. 2D and G). Animals that received virus 36 hours after 70% hepatectomy showed a marked presence of HSV viral antigens 48 hours after administration of G207 or NV1020 (Fig. 2E and H). Viral presence was nearly undetectable by 96 hours after administration (Fig. 2F and I) and was absent in liver samples harvested beyond 96 hours after viral administration. Animals that were injected with either virus a week after 70% hepatectomy did not have any detectable viral antigen by IHC at any harvest time point after viral delivery. This increase in viral antigen presence correlates to the lac Z staining results (Fig. 2B and C). Representative samples are illustrated in Fig. 2D–I).

Quantitative PCR.

PCR was used to confirm viral presence in the hepatic parenchyma. Quantitative PCR using the TaqMan system was performed on liver tissue samples 2, 7, and 21 days after G207 virus inoculation in animals that received virus 36 hours or 7 days after 70% hepatectomy and expressed as viral copies/10 ng genomic DNA. Animals that received virus 36 hours after 70% hepatectomy demonstrated a 24-fold increase in HSV DNA over that measured in the livers from animals that were perfused with virus after sham laparotomy. This viral appearance was transient. By 21 days, no viral particles were detectable at the threshold of the PCR (1 viral particle/10 ng genomic DNA) in livers of any animal injected at 36 hours (Fig. 3). This data from PCR correlated with staining for β-galactosidase at the 48-hour time point. Animals that received virus 96 hours after 70% hepatectomy demonstrated minimal appearance of virus, and no difference in the amount of HSV DNA than those animals receiving virus 96 hours after sham laparotomy. Liver samples taken 7 days after viral inoculation produced no detectable viral DNA by PCR analysis in animals that underwent sham laparotomy. Animals that received virus 7 days after hepatectomy showed minimal measurable viral copies (see Fig. 3).

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Figure 3. Results of quantitative PCR analysis. Specimens were taken from animals at 2, 7, and 21 days after viral inoculation. Open bars, nonhepatectomized animals; solid bars, liver tissue from animals that received virus 36 hours after 70% hepatectomy; striped bars, liver tissue from animals that received virus 7 days after 70% hepatectomy.

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After demonstrating that hepatic regeneration permits viral replication, the effect of increased hepatic viral presence on survival was investigated. Administration of the G207 virus during the peak of hepatic regeneration (24–48 hours) resulted in increased mortality compared with delivery of G207 after this peak. Four out of 10 animals receiving virus 36 hours after 70% hepatectomy died within 48 hours. No animals in the sham laparotomy group died, nor did any animals exhibit morbidity or mortality in the group that received virus 96 hours after 70% hepatectomy (P < .009) (Fig. 4).

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Figure 4. Kaplan-Meier survival curves for mice after viral treatment. Open circle, animals undergoing 70% hepatectomy and receiving a saline injection; solid triangle, animals receiving G207 7 days after 70% hepatectomy; solid squares, animals receiving G207 36 hours after 70% hepatectomy.

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Ribonucleotide Reductase Levels.

RR levels were assessed to determine what role cellular RR changes had in the observed enhanced viral replication. Livers from animals subjected to 70% hepatectomy 36 or 96 hours earlier as well as livers from animals that did not undergo a hepatectomy were assessed for RR activity. Additional livers were assessed from animals 7 and 14 days after 70% hepatectomy. The RR level was noted to markedly increase within 36 hours of resection (Fig. 5A), correlating with the results of the Ki-67 staining. RR levels remained elevated for a protracted period, lasting beyond 7 days (data not shown).

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Figure 5. (A) RR levels and (B) viral binding in regenerating liver over time. Liver tissue was harvested from nonhepatectomized animals or animals having undergone 70% hepatectomy 36 hours or 90 hours previously and then assayed for RR activity and viral binding. RR, ribonucleotide reductase.

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Viral Binding.

A viral binding assay was performed to determine whether or not enhanced viral binding was responsible for the increased viral presence. Virus was administered 36 hours after 70% hepatectomy; viral presence was measured 30 minutes thereafter. The results demonstrate that there was enhanced viral adhesion in livers that underwent 70% hepatectomy; however, the change was only twofold. This difference is inadequate to explain the marked increase in viral presence detected by lac Z staining and PCR (Fig. 5B).

Liver Chemistries.

Liver chemistries were performed to determine the amount of insult to the liver resulting from the hepatectomy and administration of the G207 virus at various time points. Animals that underwent hepatectomy with G207 administration within 36 hours had elevated laboratory values compared with animals that received partial hepatectomy only. In animals that received G207 via splenic injection concurrently with the surgery, the alanine aminotransferase (P = .02) and aspartate aminotransferase (P = .01) levels were significantly elevated when compared to control. Significant elevations were also seen in aspartate aminotransferase and alanine aminotransferase levels of mice that received G207 splenic or tail vein injections 36 hours after 70% hepatectomy. Laboratory values of mice receiving G207 administration 7 days after 70% hepatectomy revealed levels equivalent to or lower than those of control animals at 48 hours after viral administration. In fact, mice that underwent splenic injection of G207 1 week after 70% hepatectomy had significantly lower levels of alkaline phosphatase (P = .03) compared with control animals, and animals that had tail vein injections at the same time had significantly lower levels of alanine aminotransferase (P = .01). The degree of elevation in the first 36 hours was considerably higher in mice receiving portal administration compared with those receiving tail vein injections of virus (Fig. 6).

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Figure 6. Serum liver chemistries after partial hepatectomy with or without the administration of G207 virus. Animals underwent partial hepatectomy with G207 administration either at the time of resection (concurrent), 36 hours later, or 7 days later. Twenty-four hours after viral injection blood was harvested for measurement of liver function tests. *P ≤ .03 vs. control. ALT, alanine aminotransferase; AST, aspartate aminotransferase; AP, alkaline phosphatase.

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Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to detect the presence of apoptotic cells. This technique revealed large areas of apoptosis in specimens that had received G207 within the first 36 hours after hepatectomy. This was true in livers that had been harvested 48 hours after G207 administration as well as in those specimens that were harvested 7 days after G207 was given. Moreover, there was a large amount of apoptosis regardless of the route of G207 delivery (tail vein vs. splenic injection). When G207 was administered 1 week after hepatectomy, there was minimal evidence of apoptosis by 48 hours after viral administration (Fig. 7).

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Figure 7. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to detect the presence of apoptosis in the harvested liver specimens. (A) This technique revealed large areas of apoptosis in liver specimens that had received the G207 virus 36 hours after hepatectomy. (B) When G207 was administered 1 week after partial hepatectomy, there was minimal evidence of apoptosis.

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  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References

Experimental evidence suggests that HSV-1–based oncolytic viruses selectively target neoplastic tissues.8, 9, 25–27 This specificity has been attributed to high levels of tumor cell enzymes for nucleotide synthesis and cell reproduction shown to complement the genetic deletions in the viruses.8, 9, 25–27 As a result of this specificity, such viral agents have demonstrated considerable promise as anti-neoplastic therapy while producing minimal toxicity.14, 28 In particular, G207, NV1020, and other similar oncolytic viruses have proven effective in safely treating both primary and metastatic experimental liver tumors.3, 7, 8

Surgical excision remains the only proven, potentially curative therapy for liver tumors.1, 29 However, resection is curative in only one third of patients. Adjuvant chemotherapeutic treatment only minimally improves outcome.30 The majority of anti-neoplastic agents currently used exploit cellular DNA synthesis for effect.31–33 5-Fluorouracil has been shown to adversely affect liver regeneration when given during the peak of hepatic regeneration.31–35 Oncolytic HSVs also depend on increased cellular proliferation for replication and thus may also induce toxicity during liver regeneration. Therefore, examination of the mechanics and adverse effects on the regenerating liver of adjuvant HSV oncolytic therapy is warranted as this type of therapy approaches clinical trials.

During regeneration, more than 95% of hepatocytes engage in DNA synthesis and replication.36 Some investigators have shown the peak of hepatic regeneration to occur by 48 hours after resection.22, 37–39 PCR, IHC, and staining for β-galactosidase demonstrated that there is an increased amount of virus present at 48 hours after injection in animals that received G207 or NV1020 during this period of enhanced DNA synthesis. Animals who are treated after the peak of regeneration (beyond 96 hours after 70% partial hepatectomy), as well as nonhepatectomized animals, had no detectable viral presence by IHC, PCR, or lac Z staining techniques. Therefore, our data demonstrate that a short-lived increased susceptibility to herpes oncolytic viral infection and replication occurs during peak regeneration (24–48 hours after 70% partial hepatectomy).

The current data as well as that in the literature demonstrate that hepatic regeneration is associated with an increase in hepatocyte RR activity peaking between 24 and 48 hours after hepatectomy.40–42 This enzyme regulates production of nucleotides that are important for DNA and viral synthesis. Both G207, which is deficient in viral RR, as well as NV1020, which retains a functional RR gene, infected hepatocytes with the same efficiency at the peak of hepatocyte RR expression. These data suggest that amplification of viral replication during regeneration is not solely dependent on RR activity.

The pattern of viral adhesion to hepatocytes in hepatectomized and nonhepatectomized livers could have led to differences in viral presence. To address this, we studied viral binding to hepatocytes. The data showed there to be a twofold increase in viral binding to hepatocytes at the peak of liver regeneration. Whether this is due to increased vascular permeability during regeneration changes in cell surface heparin sulfate or to increased receptors for HSV cannot be determined from the current studies. Of note, this increase in binding was not enough to explain the large differences in viral presence seen by PCR and IHC. This further implicates viral replication as the major factor accounting for the increased presence of virus after 70% hepatectomy.

Survival studies were conducted to determine if viral administration during peak hepatic regeneration led to increased morbidity or mortality. Mortality increased to 40% in animals that were injected with virus 36 hours after 70% hepatectomy. Animals that were given virus 1 week after 70% hepatectomy as well as nonhepatectomized animals demonstrated no mortality or morbidity. These data suggest that in the adjuvant setting immediately after partial hepatectomy, virus should be administered with caution but can be safely employed if given beyond the peak of hepatic regeneration. Noninvasive tests such as magnetic resonance spectroscopy have been shown to reliably predict completeness of liver regeneration.43 Such markers of hepatocellular proliferation may be useful in determining safe timing of viral administration.


  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
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