In order to further characterize its role in pathogenesis and to

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested thus shedding 20-Hydroxyecdysone light on the function of SpvB in pathogenesis. In the first place the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of effectors during eukaryotic cells infection. In the second place the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents and eventually to construct novel potential tools for cancer therapy given that preferentially proliferates in tumors. Introduction serovar Typhimurium (is capable of preferentially colonizing Rabbit polyclonal to AGMAT. and proliferating in solid tumors to levels nearly 1000-fold higher than normal tissue a situation that usually results in tumor growth inhibition [1]. Additionally is not only able to colonize large solid tumors but also to accumulate in metastases when systemically administered [2 3 The genetic manipulation of is well developed and a variety of attenuated strains with mutations that render the bacteria safe for the host have been characterized [4 5 The administration of attenuated strains expressing different anti-tumor agents has been used in recent years with promising results in tumor regression [6-9]. After ingestion into the digestive tract 20-Hydroxyecdysone induces macropinocitosis by epithelial cells through the injection of bacterial effector molecules that manipulate the host cytoskeleton [10]. This injection is mediated by the Type Three Secretion System (TTSS) encoded in the pathogenicity island-1 locus (SPI-1). Inside the 20-Hydroxyecdysone eukaryotic cell bacteria remain enclosed in a membrane-bound vacuole termed Salmonella-containing vacuole (SCV). Effectors translocated by this TTSS and by a second TTSS (TTSS-2) encoded by the SPI-2 locus contribute to the intracellular 20-Hydroxyecdysone survival and replication of the bacteria (reviewed in 11). Once established inside epithelial cells is able to replicate and induce apoptosis after 18-24h [12 13 Many serovars such as [14] encoded by the virulence plasmid (or chromosomally in some strains) that enhances virulence in animals and humans [14-18]. This locus encodes among others the SpvB protein whose C-terminal domain confers ADP-ribosyl transferase activity [19 20 This activity covalently modifies G-actin monomers thus preventing their polymerization into F-actin filaments which causes the loss of the eukaryotic actin cytoskeleton [18 21 These results have been shown using different approaches such as adding purified SpvB protein to cell lysates transfecting epithelial cells and macrophages to transiently express the protein or infecting macrophages and epithelial cells with different SpvB mutants to analyze their efficiency in depolymerizing actin. It is thought that SpvB is delivered into the eukaryotic cytosol via the SPI-2 TTSS [18 23 and that both the SPI-2 TTSS and SpvB are required for the late apoptosis produced by in macrophages and epithelial cells [13 16 However the mechanism connecting SpvB to apoptosis induction remains unknown. In recent years the use of compounds that inhibit or prevent actin polymerization to reduce the growth of several tumor cell lines has been investigated [26 27 Cytotoxic agents that interfere with cytoskeleton dynamics have a recognized potential utility in the cancer treatment. For instance natural toxins such as pectenotoxin 2 isolated from dinoflagellates have been shown to have a potent apoptosis inducing effect on human cancer cells lines [28] together with G2/M arrest and endoreduplication [28-31]. Since purified SpvB is unable to enter eukaryotic cells 20-Hydroxyecdysone [22] here we have used to express SpvB in different cell lines to explore the possibility of its use in anti-tumor therapy. The ability to flexibly control expression levels and timing should help us better understand the role of SpvB in pathogenesis and apoptosis induction. To this end we have used a set of vectors and GFP-tagged strains recently developed in our laboratory that drive the expression of heterologous proteins inside the eukaryotic cytoplasm when salicylate or acetyl salicylate is added to the cell culture [32-35]..