Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter

Abstract

The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the ␤-galactosidase (␤-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence Ϫ82 to Ϫ42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and ␤-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong ␤-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SR␣) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and ␤-gal genes, respectively (pTES.␤). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SR␣-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.

In vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice

“…Compared with conventional plasmid vectors, the EBV-based plasmid vectors enabled higher expression of the luciferase, as well as cytokine genes (Figures 2a and 3). This is consistent with our previous reports showing that the EBV-based plasmid vectors are quite useful in improving transfection efficiency by a variety of nonviral delivery systems, including cationic liposomes, 28,29 cationic polymers, 25,26,29,30 and particle bombardment. 31 The high level expression obtained by the EBV-based plasmid vectors may be ascribed to multiple functions of EBNA1, such as nuclear transfer of the plasmid, 32,33 binding of plasmid to nuclear matrix, 34 and transcriptional up-regulation.…”

Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo

“…16,17 Among the synthetic macromolecules, cationic polymers and cationic lipids conjugated with plasmid DNA (polyplex and lipoplex, respectively) are quite promising tools for cancer gene therapy. 18,19 Compared with conventional polyplex and lipoplex systems, EBV-based plasmid vectors conjugated with cationic polymers and cationic lipids (EBV/polyplex and EBV/lipoplex, respectively) achieve quite successful genetic delivery, resulting in strong and persistent transgene expression in various tumor cells both in vitro [6][7][8][9][10] and in vivo. 8,10,11 Fas ligand (FasL) is a type 2 transmembrane glycoprotein belonging to the tumor necrosis factor superfamily.…”

Evaluation of tumor-specific promoter activities in melanoma

“…12,35 Various promoters have been evaluated for transcriptional targeting. [36][37][38] However, the weakness of currently available TSPs, despite their name, is their lack of sufficient tumor specificity to avoid expression in normal tissues. Thus, a continued search for a true TSP, by which the transgene expression is induced at higher levels in tumor cells and at lower levels in normal host cells, including melanocytes, is warranted.…”