ABSTRACT
Oncolytic viruses (OVs) are promising therapeutic agents for cancer treatment, with recent studies emphasizing the combined use of chemotherapeutic compounds and prodrug suicide gene strategies to improve OV efficacy. In the present study, the synergistic activity of recombinant vesicular stomatitis virus (VSV)-MΔ51 virus expressing the cytosine deaminase/uracil phosphoribosyltransferase (CD::UPRT) suicide gene and 5-fluorocytosine (5FC) prodrug was investigated in triggering tumor cell oncolysis. In a panel of VSV-sensitive and -resistant cells-prostate PC3, breast MCF7 and TSA, B-lymphoma Karpas and melanoma B16-F10-the combination treatment increased killing of non-infected bystander cells in vitro via the release of 5FC toxic derivatives. In addition, we showed a synergistic effect on cancer cell killing with VSV-MΔ51 and the active form of the drug 5-fluorouracil. Furthermore, by monitoring VSV replication at the tumor site and maximizing 5FC bioavailability, we optimized the treatment regimen and improved survival of animals bearing TSA mammary adenocarcinoma. Altogether, this study emphasizes the potency of the VSV-CD::UPRT and 5FC combination, and demonstrates the necessity of optimizing each step of a multicomponent therapy to design efficient treatment.
Subject(s)
Cytosine Deaminase/genetics , Genes, Transgenic, Suicide , Genetic Therapy/methods , Oncolytic Viruses/genetics , Vesiculovirus/genetics , Combined Modality Therapy , Flucytosine/pharmacology , Fluorouracil/pharmacology , Humans , Oncolytic Viruses/metabolism , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Recombinant Fusion Proteins/genetics , Vesiculovirus/metabolismABSTRACT
The BALB/c inbred mouse is widely used in models of infectious disease, transplantation, and cancer. The differences in the immune responses of BALB/c compared to C57BL/6 mice are especially valuable for the identification of immune regulation genes. One striking immune variance between these mice is in the function of natural killer (NK) cells, and there is strong evidence implicating differential expression of Ly49 genes. In this study, the complete BALB/c Ly49 gene cluster has been sequenced and found to contain six functional genes and two pseudogenes. Compared to C57BL/6 mice, there is a 200 kb region absent in the BALB/c cluster including a complete lack of Ly49h-related genes, which explains the increased susceptibility of BALB/c to cytomegalovirus infection. In addition, there is no BALB/c Ly49d allele, explaining the inability of BALB/c NK cells to kill certain tumor cells. The Ly49 region has now been sequenced in three different inbred mouse strains, and comparisons indicate that the evolution of each haplotype is not straightforward and has involved large-scale deletions/insertions, gene recombination, and unequal crossing over between divergent haplotypes. This study confirms that relatively small murine class I MHC receptor haplotypes exist, analogous to observations made of human killer cell Ig-like receptor gene haplotypes.
Subject(s)
Antigens, Ly/genetics , Killer Cells, Natural/immunology , Multigene Family , Receptors, Immunologic/genetics , Animals , Base Sequence , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/immunology , Evolution, Molecular , Exons , Haplotypes , Histocompatibility Antigens Class I/metabolism , Lectins, C-Type , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily A , Promoter Regions, Genetic , Pseudogenes , Receptors, NK Cell Lectin-Like , Sequence Analysis, DNAABSTRACT
The murine Ly49 gene family encoding natural killer cell receptors for class I MHC is an example of a rapidly evolving cluster of immune response genes. Determining the genomic sequence of the 129S6/SvEvTac (129S6) Ly49 cluster and comparing it to the known sequence of the C57BL/6 (B6) region provided insight into the mechanisms of Ly49 gene evolution. 129S6 contains 20 Ly49, many of which are pseudogenes and 40% of the genes have no counterpart in the B6 genome. The difference in gene content between these two strains is primarily the result of distinct patterns of gene duplication. Phylogenetic analyses of individual exons showed that Ly49 genes form distinct sub-families and an ancestral haplotype can be surmised. Dotplot analysis supports limited allelism in the two haplotypes; however, large regions of variation punctuate these islands of co-linearity. These variable regions contain a high concentration of repetitive elements that are predicted to contribute to the dynamic evolution of this cluster. The extreme variation in Ly49 haplotype content between mouse strains provides a genetic explanation for the documented differences in natural killer cell phenotype, and also indicates that differences in natural killer cell function observed between B6 and 129-derived gene-targeted mice should be interpreted with caution.