RESUMEN
Tumor-specific promoters are predominantly active and ensure expression of the gene under control exclusively in cancer cells. However, a low activity of the promoters is an essential disadvantage for their therapy usage. To achieve a higher expression level of the therapeutic gene, herpes simplex virus thymidine kinase (HSV-tk), the Tat-TAR-system being utilized by HIV-1 for increasing own gene expression was developed. A potentiating activity of tat gene under control of two different cancer-specific gene (human survivin gene and human telomerase reverse transcriptase) promoters for increasing of the HSV-tk gene expression being regulated by TAR-element was evaluated, and activity of the cancer-specific promoters in the Tat-TAR-system was compared. Co-transfection of the cells with the both constructions led to the tat protein synthesis and its affect the HIV-1 TAR-element. An expression level of HSV-tk gene ensured by the both promoters in the binary system was close to that for strong non-specific cytomegalovirus (CMV) promoter. Enzymatic activity of HSV-tk protein in cells having both elements of Tat-TAR-system was two orders of magnitude higher than that in the cells transfected with HSV-tk gene under control of the cancer-specific promoter. Notably, the effect was independent of p53-status of transfected cells: HSV-tk expression level was almost the same in p53(+) and p53(-) cells. The obtained results show that system may be used for therapy of different cancer types both p53-defective and p53-positive ones inhibiting cancer-specific promoters activity.
Asunto(s)
Expresión Génica , VIH-1/genética , Herpesviridae/enzimología , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/genética , Telomerasa/genética , Timidina Quinasa/biosíntesis , Proteína p53 Supresora de Tumor/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Línea Celular Tumoral , Herpesviridae/genética , Humanos , Proteínas Inhibidoras de la Apoptosis , Neoplasias/genética , Neoplasias/metabolismo , Survivin , Timidina Quinasa/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
An important problem in the development of gene therapy approaches in oncology is the necessity of using promoters providing specific and high level of gene expression in tumor cells. To solve this problem, we used inducible system of gene expression regulation (Tat-TAR-system), which is utilized by human immunodeficiency virus (HIV). tat and tk-HSV genes, as well as a fragment of LTR HIV-1, were cloned in the retrovirus vector, tk-HSV gene was under control of the LTR HIV-1 fragment. Potential capacity of these constructions for transactivating tk-HSV gene transcription was studied. Basal expression level of this gene was defined in transient transfection of HEK293 cells. It was shown that specific transactivation of the tk-HSV gene was controlled by the LTR HIV-1 fragment in lung carcinoma cells Calu-1, permanently transfected by the tat gene construction. The effect of transactivation of tk-HSV transcription in Tat-TAR-system was demonstrated in Calu-1 cells in conditions of control of cancer-specific tat gene over BIRC5 promoter.