RESUMEN
BACKGROUND: The potential use of gene therapy for cancer treatment is being intensively studied. One approach utilises the expression of genes encoding cytotoxic proteins. Such proteins can affect cellular viability, for example by inhibiting the translation machinery or disturbing membrane integrity. The bacteriophage Lambda (lambda)-holin protein is known to form a lesion in the cytoplasmic membrane of E. coli, triggering bacterial cell lysis and thereby enabling the release of new bacteriophage particles. The aim of this study was to evaluate whether the lambda-holin protein has a cytotoxic impact on eukaryotic cells and whether it holds potential as a new therapeutic protein for cancer gene therapy. METHODS: To explore this possibility, stably transfected human cell lines were established that harbour a tetracycline (Tet)-inducible system for controlled expression of the lambda-holin gene. The effect of the lambda-holin protein on eukaryotic cells was studied in vitro by applying several viability assays. We also investigated the effect of lambda-holin gene expression in vivo using a human breast cancer cell tumour xenograft as well as a syngeneic mammary adenocarcinoma mouse model. RESULTS: The lambda-holin-encoding gene was inducibly expressed in eukaryotic cells in vitro. Expression led to a substantial reduction of cell viability of more than 98%. In mouse models, lambda-holin-expressing tumour cell xenografts revealed significantly reduced growth rates in comparison to xenografts not expressing the lambda-holin gene. CONCLUSIONS: The lambda-holin protein is cytotoxic for eukaryotic cells in vitro and inhibits tumour growth in vivo suggesting potential therapeutic use in cancer gene therapy.
Asunto(s)
Bacteriófago lambda/genética , Neoplasias de la Mama/terapia , Terapia Genética , Proteínas Virales/genética , Animales , Bacteriófago lambda/metabolismo , Supervivencia Celular , Células HeLa , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Trasplante Heterólogo , Proteínas Virales/metabolismoRESUMEN
With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate Lactobacillus casei phage phiFSW-TI in Escherichia coli. The phiFSW-TI promoter fragment was abutted to the plasmid-borne promoterless beta-glucuronidase (gusA) reporter gene and shown to direct its transcription in L. casei. In addition, the functionality of the promoter-repressor system was verified in the L. casei phiFSW-TI lysogen by showing that the gusA reporter gene, controlled by the isolated phiFSW-TI promoter, was repressed at 28 degrees C and expressed at 42 degrees C. Moreover, a homology search revealed that the C terminus of the isolated phiFSW repressor shows a high similarity to the small mutS-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.