Evaluation of the deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) as a suicide gene for treatment of solid tumors

Abstract

Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors. The transfer of the gene encoding herpes simplex virus type-1 thymidine kinase (HSV-1 TK) into malignant cells and subsequent treatment with ganciclovir is one of the most commonly studied strategies of suicide gene therapy. The nucleoside kinase phosphorylates nucleoside analogs to its monophosphate form, and cellular enzymes further phosphorylate the compound to their triphosphate forms. The triphosphate interferes with DNA replication and induces cell death, probably by apoptosis. In addition to affecting the cells expressing HSV-1 TK, adjacent untransduced cancer cells are killed by so called "bystander effect".

One limiting factor for the efficiency of suicide gene therapy is the kinetic properties of the "suicide" nucleoside kinase. We have cloned a multisubstrate deoxyribonucleoside kinase of D. melanogaster (Dm-dNK) and shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity and its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present studies, we evaluated Dm-dNK for possible use in cancer gene therapy. We constructed a replication deficient retroviral vector that expresses the enzyme. A TK1-deficient human osteosarcoma cell line and a human pancreatic adenocarcinoma cell line were transduced with the retroviridae. We used autoradiography to in situ visualize incorporation of 3 H-dThd into DNA of the osteosarcoma cells, and Western blot analysis to detect the Dm-dNK protein expression. We determined the sensitivity of the untransduced cells to several cytotoxic nucleoside analogs. To determine the bystander effect, tumor cells expressing Dm-dNK were mixed at different ratios with their parental cell lines and treated with BVDU in different concentration.

We also created genetically engineered vectors that expressed Dm-dNK fused to GFP to facilitate detection of the expressed enzyme in the nucleus, cytosol and mitochondria of living cells and compared the cytotoxicity and bystander effects to some nucleoside analogs. Furthermore, we have investigated a novel strategy involving the direct delivery of recombinant Dm-dNK protein to cancer cells. We used lipid-based formulation, known as BioPorter, to form Dm-dNK protein-lipid complexes. We showed that Dm-dNK could be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its Cterminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs.

The bystander effect was observed in osteosarcoma cells, and interestingly, the cytotoxicity and bystander effect could be enhanced by the hydroxyurea. We also showed that the cells' sensitivity and the efficiency of bystander cell killing was not dependent on whether Dm-dNK was located in the nucleus or cytosol. Dm-dNK could also be expressed in mitochondria and retain its enzymatic activity, and increase the cells' sensitivity to some nucleoside analogs tested. In the study of liposome mediated protein delivery, we found that the protein was delivered in a functionally active form and that the sensitivity of the cell lines to nucleoside analogs increased. In summary, our findings suggest that multisubstrate deoxyribonucleoside kinase of D. melanogaster (Dm-dNK) may be used as a suicide gene in combined gene/chemotherapy of cancer.