Regulation of herpes simplex virus gamma(1)34.5 expression and oncolysis of diffuse liver metastases by Myb34.5

Abstract

Myb34.5 is a herpes simplex virus 1 (HSV-1) mutant deleted in the gene for ribonucleotide reductase (ICP6). It also carries a version of gamma(1)34.5 (a viral gene product that promotes the dephosphorylation of eIF-2alpha) that is under control of the E2F-responsive cellular B-myb promoter, rather than of its endogenous promoter. Myb34.5 replication in tumor cells results in their destruction (oncolysis). gamma(1)34.5 expression by HSV-1 subverts an important cell defense mechanism against viral replication by preventing shutoff of protein synthesis after viral infection. Infection of colon carcinoma cells with Myb34.5 results in greater eIF-2alpha dephosphorylation and viral replication compared with infection with HSV-1 mutants completely defective in gamma(1)34.5 expression. In contrast, infection of normal hepatocytes with Myb34.5 results in low levels of eIF-2alpha dephosphorylation and viral replication that are similar to those observed with HSV-1 mutants completely defective in gamma(1)34.5 and ICP6. When administered intravascularly into mice with diffuse liver metastases, Myb34.5 has greater antineoplastic activity than HSV-1 mutants with completely defective gamma(1)34.5 expression and more restricted biodistribution compared with HSV-1 mutants with wild-type gamma(1)34.5 expression. Myb34.5 displays reduced virulence and toxicity compared to HSV-1 mutants with wild-type gamma(1)34.5 expression. Portal venous administration of Myb34.5 significantly reduces liver tumor burden in and prolongs the life of mice with diffuse liver metastases. Preexisting Ab's to HSV-1 do not reduce the antitumor efficacy of Myb34.5 in vivo.

Figure 1

Diagram of the regulation of protein synthesis by γ₁34.5 in HSV-1–infected cells. Protein kinase R (PKR) recognizes viral double-strand RNA and is subsequently activated by autophosphorylation. Activated (phosphorylated) PKR phosphorylates eIF-2α, which inhibits initiation of protein translation within the cell and in turn inhibits viral replication. HSV-1 γ₁34.5 interacts with cellular protein phosphatase-1α (PP1α) to dephosphorylate eIF-2α, thus allowing continued protein synthesis.

Figure 2

In vitro eIF-2α dephosphorylation assay. (a) Autoradiograph of purified phosphorylated eIF-2α reacted with S10 fractions from mock-infected cells (lanes 1–3), F strain–infected cells (lanes 4–6), Myb34.5-infected cells (lanes 7–9), and MGH1-infected cells (lanes 10–12). S10 fractions were collected from HT29 colon carcinoma cells (upper panel) and primary cultures of normal human hepatocytes (lower panel). The reaction time measured in seconds is shown at the top of each panel. (b) The eIF-2α dephosphorylation rates of HT29 colon carcinoma cells (left) and human hepatocytes (right) was determined by densitometric quantification of the bands.

Figure 3

Replication of HSV-1 mutants in colon carcinoma cells and hepatocytes. (a) Single-step viral replication assays were performed using F strain, hrR3, Myb34.5, R3616, and MGH1 in HT29 colon carcinoma cells and primary cultures of human hepatocytes. Ribonucleotide reductase (RR) and β-actin expression of uninfected cells were measured by Western blot analysis. (b) Single-step viral replication assays were performed similarly in MC26 mouse colon carcinoma cells (MC26) and primary cultures of mouse hepatocytes.

Figure 4

Cytotoxicity assay in vitro. Five human colon carcinoma cell lines and one mouse colon carcinoma cell line (MC26) were infected with F strain, Myb34.5, MGH1, or hrR3 at several moi values, and surviving cells were quantitated after 6 days.

Figure 5

Treatment of diffuse liver metastases with Myb34.5. (a) Diffuse MC26 liver metastases were treated with 5 × 10⁷ pfu of either MGH1 (top row), heat-inactivated Myb34.5 (second row), Myb34.5 (third row), or hrR3 (bottom row). Mice were sacrificed, and livers and spleens were analyzed 11 days later. (b) Mice bearing diffuse liver metastases established as described were treated with either 5 × 10⁷ pfu Myb34.5, hrR3, MGH1, or heat-inactivated Myb34.5 (control) and followed for survival. Top graph: P = 0.24 by log-rank analysis for MGH1 versus control; Middle graph: P < 0.05 for hrR3 versus control; Bottom graph: P < 0.01 for Myb34.5 versus control. (c) Mice with subcutaneous spleens bearing diffuse liver metastases were treated with either 10⁷ pfu Myb34.5 or heat-inactivated Myb34.5 every 3 days for a total of four doses. P < 0.002 by log-rank analysis.

Figure 6

HSV-1 detection in mice by PCR assay. (a) Mice inoculated subcutaneously with F strain, Myb34.5, MGH1, or hrR3 were sacrificed 8 days later to harvest tissues for PCR amplification of a 220-bp portion of the HSV-1 DNA polymerase gene. A 1-kb marker is shown in lane M, with the location of the 220-bp bands indicated. A negative control reaction without template is shown in lane N, and a positive control reaction using HSV-1 genomic DNA as template is shown in lane P. (b) Mice inoculated intrasplenically with either 10⁵ pfu F strain or 5 × 10⁷ pfu Myb34.5, MGH1, or hrR3 were sacrificed 10 days later to harvest tissues for PCR analysis as above. (c) Mice bearing diffuse liver metastases were treated with an intrasplenic inoculation of either 10⁵ pfu F strain or 5 × 10⁷ pfu Myb34.5, MGH1, or hrR3, as above, and sacrificed 10 days later to harvest tissues for PCR analysis. In these animals, analysis of liver tissue included normal liver homogenized together with liver metastases. (d) Mice bearing a single liver metastases were treated with an intrasplenic inoculation of either 10⁵ pfu F strain (1 and 2) or 5 × 10⁷ pfu Myb34.5 (3 and 4), MGH1 (5 and 6), or hrR3 (7 and 8). Mice were sacrificed 12 days (rather than 8 days) later to harvest tissue for PCR analysis of either normal liver (N) or liver tumor (T). Positive (P) and negative (Ne) controls are shown. (e) Mice bearing a single liver metastases that had been treated with an intrasplenic injection of either 10⁵ pfu F strain (n = 3), 5 × 10⁷ pfu hrR3 (n = 3), or 5 × 10⁷ pfu Myb34.5 (n = 6). Normal liver and metastases were harvested separately and titered for the presence of HSV-1. The differences in viral titers in liver tumors are not statistically significant. The difference in viral titer in normal liver are statistically significant by t test as follows: F strain versus hrR3, P = 0.03; F strain versus Myb34.5, P = 0.03; hrR3 versus Myb34.5, P = 0.04.

Figure 7

Treatment of diffuse liver metastases with Myb34.5 in mice with neutralizing Ab’s to HSV-1. Mice were vaccinated with either Myb34.5 (top and middle row) or mock-infected media (lower row) 25 days before intrasplenic inoculation of MC26 cells. After confirmation of the presence of neutralizing Ab’s to HSV, mice were treated with 5 × 10⁷ pfu Myb34.5 3 days after tumor implantation and sacrificed 14 days after tumor implantation (middle and lower row). To serve as a control group, mice vaccinated with Myb34.5 were treated with heat-inactivated Myb34.5 (upper row).