High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model

Copyright: Copyright 2014 Elsevier B.V., All rights reserved.Background: The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1).Methods: Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase.Results: Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression.Conclusions: Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.publishersversionPeer reviewe

Staphylococcus aureus sortase A cyclization and evaluation of enzymatic activity in vitro

Abstract Protein cyclization has been shown to increase the stability of molecule structure by reducing the number of possible conformation variations. Cyclization of an enzyme can significantly enlarge the spectrum of its application in specific conditions, including high temperature, presence of a chaotropic agent, and denaturing environment. In this study the cyclic form of Staphylococcus aureus sortase A enzyme (srtA) was obtained by introducing a peptide bond between amino acids at N-and C-termini. The catalytic activity of purified cyclic srtA was tested in vitro by protein-protein ligation reaction. In contrast to the linear srtA, the cyclic form of the enzyme was active in presence of chaotropic agent. This finding suggests the potential use of srtA enzyme in protein engineering techniques

Structural basis for 5'-end-specific recognition of single-stranded DNA by the R3H domain from human Sμbp-2

The R3H domain is a conserved sequence motif in nucleic acid binding proteins. Previously, we reported the solution structure of the R3H domain and identified a putative nucleic acid binding site composed of three conserved basic residues [Liepinsh, E., Leonchiks, A., Sharipo, A., Guignard, L. & Otting, G. (2003). Solution structure of the R3H domain from human Sμbp-2. J. Mol. Biol. 326, 217-223]. Here, we determine the binding affinities of mononucleotides and dinucleotides for the R3H domain from human Sμbp-2 (Sμbp2-R3H) and map their binding sites on the protein's surface. Although the binding affinities show up to 260-fold selectivity between different nucleotides, their binding sites and conformations seem very similar. Further, we report the NMR structure of the Sμbp2-R3H in complex with deoxyguanosine 5′-monophosphate (dGMP) mimicking the 5′-end of single-stranded DNA. Pseudocontact shifts from a paramagnetic lanthanide tag attached to residue 731 in the mutant A731C confirmed that binding of dGMP brings a loop of the protein into closer proximity. The structure provides the first structural insight into single-stranded nucleic acid recognition by the R3H domain and shows that the R3H domain specifically binds the phosphorylated 5′-end through electrostatic interactions with the two conserved arginines and stacking interactions with the highly conserved histidine

Structural basis for 5′-end-specific recognition of single-stranded DNA by the R3H domain from human Sμbp-2

The R3H domain is a conserved sequence motif in nucleic acid binding proteins. Previously, we reported the solution structure of the R3H domain and identified a putative nucleic acid binding site composed of three conserved basic residues [Liepinsh, E., Leonchiks, A., Sharipo, A., Guignard, L. & Otting, G. (2003). Solution structure of the R3H domain from human Sμbp-2. J. Mol. Biol. 326, 217-223]. Here, we determine the binding affinities of mononucleotides and dinucleotides for the R3H domain from human Sμbp-2 (Sμbp2-R3H) and map their binding sites on the protein's surface. Although the binding affinities show up to 260-fold selectivity between different nucleotides, their binding sites and conformations seem very similar. Further, we report the NMR structure of the Sμbp2-R3H in complex with deoxyguanosine 5′-monophosphate (dGMP) mimicking the 5′-end of single-stranded DNA. Pseudocontact shifts from a paramagnetic lanthanide tag attached to residue 731 in the mutant A731C confirmed that binding of dGMP brings a loop of the protein into closer proximity. The structure provides the first structural insight into single-stranded nucleic acid recognition by the R3H domain and shows that the R3H domain specifically binds the phosphorylated 5′-end through electrostatic interactions with the two conserved arginines and stacking interactions with the highly conserved histidine