Cytotoxic Effect of Drosophila Deoxynucleoside Kinase Gene on Replicating Plasmid in HeLa Cells

Abstract

Gene delivery with nonviral vectors is a promising approach, due to its excellent safety profile in comparison to that of viral vectors. [1][2][3][4][5] However, low and transient transgene expression is a major concern for nonviral vectors. One of the keys to overcome this problem would be the establishment of an efficient and targeted delivery system, in which intracellular trafficking is considered. In addition, the controlled intranuclear disposition of the delivered plasmid would be quite important for achieving practical gene therapy.5) Indeed, analyses of the intranuclear disposition of plasmid DNA have indicated that the reasons for the low and transient transgene expression are the decreases in the amount of the exogenous DNA and the expression efficiency from one copy of the transgene over time. [6][7][8] Thus, the maintenance of both the plasmid DNA and its expression efficiency is essential for the controlled intranuclear disposition.We focused on a replicating plasmid, to prevent the decrease in the amount of the exogenous DNA. The SV40 origin-SV40 large T antigen system was used as a model. The large T antigen binds the origin, acts as helicase that unwinds DNA, and recruits DNA polymerase a-primase, which initiates replication.9,10) The plasmid with the origin and the large T antigen gene is replicated in dividing cells, and the replication of the plasmid is conducted in the S phase of the cell cycle, along with the endogenous chromosomal DNA. The replicating plasmid could alleviate the decrease in transgene expression by maintaining the amount of the exogenous DNA. This type of plasmid DNA was previously used in extrachromosomal replication and transgene expression. 11,12) In addition, we focused on the deoxyribonucleoside kinase from the fruit fly Drosophila melanogaster (Dm-dNK). Previously, we reported that the presence of the Dm-dNK gene on a plasmid enhanced the cytotoxicity of nucleoside analogs. 13) We expected that this gene on a replicating plasmid would be more effective than that on a non-replicating plasmid for nucleoside analog-induced cell death.In this study, we transfected replicating plasmid DNAs containing the luciferase and Dm-dNK genes into human cells. Our results confirmed that the replicating plasmid is an excellent tool for efficient protein production. We serendipitously discovered that the Dm-dNK gene on the replicating plasmid was highly cytotoxic and caused cell death without nucleoside analogs. MATERIALS AND METHODS MaterialsOligodeoxyribonucleotides were purchased from Sigma Genosys Japan (Ishikari, Japan) and Invitrogen Japan (Tokyo, Japan) in purified forms.Construction of Plasmid DNAs pYK-CMV-luc (8.5 kb) and pCMV-dNK (7.5 kb), containing the luciferase and dNK genes, respectively, under the cytomegalovirus (CMV) promoter were used as the parental plasmids in this study. 6,13)The pCMV-luc-T plasmid (11.5 kb), containing the SV40 origin/promoter and the SV40 large T antigen gene as well as the CMV promoter-luciferase gene, was constructed by ligating the SV40 origi...

Lentivirus-mediated expression of Drosophila melanogaster deoxyribonucleoside kinase driven by the hTERT promoter combined with gemcitabine: A potential strategy for cancer therapy

“…These data reveal that the residues Tyr70 and Arg105 take into consideration the positioning of dFdC, suggesting its significant implications for therapeutic efforts due to the broad substrate acceptance of Dm-dNK. Characterized by its high catalytic property and broad substrate acceptance, Dm-dNK has therapeutic potential and is used in the treatment of several cancer types (3)(4)(5).…”

Cytotoxic effects of adenovirus- and lentivirus-mediated expression of Drosophila melanogaster deoxyribonucleoside kinase on Bcap37 breast cancer cells

“…Taken together, these data underline the importance of its potential role as a suicide gene. Therefore, Dm-dNK has been used successfully in suicide gene therapy against a wide variety of cancers (4)(5)(6)(7)(8).…”

Potent Anticancer Effects of Lentivirus Encoding a Drosophila Melanogaster Deoxyribonucleoside Kinase Mutant Combined with Brivudine

“…It has been reported that the catalytic rate of the enzyme for pyrimidines is 100-600 times higher than for purines (Munch-Petersen et al, 1998a;Munch-Petersen et al, 2000). All these data underline the importance of the potential role of Dm-dNK as a suicide gene, and therefore, this gene have been used (Ito et al, 2010;Ma et al, 2011;Zhu et al, 2011b;Ma et al, 2012).…”