Short nucleotide sequence analysis of seven restriction fragments of murine herpesvirus 68 (MHV-68) DNA has been undertaken and used to determine the overall genome organization and relatedness of this virus to other well characterized representatives of the alpha-, beta-and gammaherpesvirus subgroups. Nine genes have been identified which encode amino acid sequences with greater similarity to proteins of the gammaherpesvirus Epstein-Barr virus (EBV) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simples virus type 1 or the betaherpesvirus human cytomegalovirus. In addition, the genome organization of MHV-68 is shown to have an overall collinearity with that of the gammaherpesviruses EBV and herpesvirus saimiri. In common with these viruses, dinucleotide frequency analysis of MHV-68 coding sequences reveals a marked reduction in CpG dinucleotide frequency thus implicating a dividing cell population as the site of latency in vivo.

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New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/ B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells. Cancer Res; 71(5); 1805-15. Ó2011 AACR.

SUMMARYPurified DNAs from Marek's disease virus (MDV) and the herpesvirus of turkeys (HVT) were randomly sheared and cloned into the M 13 bacteriophage. Two-hundred and ten MDV and 130 HVT clones were sequenced to give representative samples of the genome sequences. The predicted amino acid sequences from these gammaherpesviruses were compared to known sequences from other herpesviruses using computer analysis. Thirty-five MDV and 24 HVT genes were identified by comparison with varicella-zoster virus (VZV), an alphaherpesvirus. However, only 14 MDV and seven HVT genes, giving generally lower homology scores, were found by comparison with Epstein-Barr virus (EBV), a gammaherpesvirus, indicating that MDV and HVT sequences bear greater similarity to VZV than to EBV sequences. A number of sequences were mapped by hybridizing labelled M13 clones to Southern blots of restriction fragments of MDV or HVT DNA. The results were consistent with the MDV and HVT genomes being collinear with VZV.

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SUMMARYThe Marek's disease virus (MDV) homologue of the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) has been identified within BamHI fragments 13 and K 3 of the 'highly oncogenic' strain RB 1B of MDV. The entire nucleotide sequence of the gene has been determined and its predicted amino acid sequence shown to share gross overall structural features with the gB genes of HSV, varicella-zoster virus (VZV) and other mammalian herpesviruses. In particular, all 10 cysteine residues were conserved in MDV gB and there was extensive homology throughout the gene with VZV, HSV and pseudorabies virus except for the N and C termini. The overall percentage amino acid identity between MDV gB and gB of the alphaherpesviruses had a mean of 50% which was almost twice that between cytomegalovirus and Epstein-Barr virus. Northern blot analysis showed that the main RNA transcribed from this gene is approx. 2.7 kb in size. Antibodies raised against synthetic peptides (residues 250 to 271 and 304 to 330) allowed the identification of a family of serologically related glycoproteins of Mr 110K, 64K and 48K in extracts of MDVinfected cells using immunoblots. Furthermore, the antisera were able to differentiate between the antigens of MDV and herpesvirus of turkeys in immunoblots. Immunofluorescence tests indicated that MDV gB is associated with granules in the cytoplasm and is present at the surface of MDV-infected cells.

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Tumor hypoxia has long been recognized as a critical issue in oncology. Resistance of hypoxic areas has been shown to affect treatment outcome after radiation, chemotherapy, and surgery in a number of tumor sites. Two main strategies to overcome tumor hypoxia are to increase the delivery of oxygen (or oxygen-mimetic drugs), or to exploit this unique environmental condition of solid tumors for targeted therapy. The first strategy includes hyperbaric oxygen breathing, the administration of carbogen and nicotinamide, and the delivery of chemical radiosensitizers. In contrast, bioreductive drugs and hypoxia-targeted suicide gene therapy aim at activating cytotoxic agents at the tumor site, while sparing normal tissue from damage. The cellular machinery responds to hypoxia by activating the expression of genes involved in angiogenesis, anaerobic metabolism, vascular permeability, and inflammation. In most cases, transcription is initiated by the binding of the transcription factor hypoxia-inducible factor (HIF) to hypoxia responsive elements (HREs). Hypoxia-targeting for gene therapy has been achieved by utilizing promoters containing HREs, to induce selective and efficient transgene activation at the tumor site. Hypoxia-targeted delivery and prodrug activation may add additional levels of selectivity to the treatment. In this article, the latest developments of cancer gene therapy of the hypoxic environment are discussed, with particular attention to combined protocols with ionizing radiation. Ultimately, it is proposed that by adopting specific transgene activation and molecular amplification systems, resistant hypoxic tumor tissues may be effectively targeted with gene therapy.