RESUMO
Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, E(DeltaNLS)M, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.E(DeltaNLS)M and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six- and 13-fold reduced for Ad.E(DeltaNLS)M and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.E(DeltaNLS)M and Ad.MEM in vivo. Induction of Ad.E(DeltaNLS)M inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. E(DeltaNLS)M and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.
Assuntos
Adenocarcinoma/terapia , Proteínas E1A de Adenovirus/genética , Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Receptores de Estrogênio/genética , Tamoxifeno/uso terapêutico , Adenocarcinoma/virologia , Animais , Efeito Citopatogênico Viral , Feminino , Regulação da Expressão Gênica , Engenharia Genética , Células HeLa , Humanos , Neoplasias Pulmonares/virologia , Camundongos , Camundongos Nus , Proteínas Recombinantes/uso terapêutico , Tamoxifeno/análogos & derivados , Transcrição Gênica , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosRESUMO
We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXalpha which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.