Conditionally replicating adenovirus SG500-expressed mutant Dm-dNK gene for breast cancer therapy

Abstract

The purpose of this analysis was to investigate the enzyme activity and specificity of using adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) mutants in combination with gemcitabine. Compared with herpes simplex type 1 thymidine kinase (HSV-TK) and other known dNKs, this Dm-dNK enzyme has a broader substrate specificity and a higher catalytic rate. We created the Dm-dNK mutants (dNKmu) by site-directed mutagenesis at the sites of 244E, 245S, 251S and 252R, with the last 10amino acids in the amino acid sequence randomly mutated. We evaluated the enzyme activity and substrate specificity. The engineered enzymes showed a relative increase in phosphorylation in the nucleoside analogs of BVDU ((E)-5‑(2-Bromovinyl)-2'-deoxyuridine) or gemcitabine (DFDC, 2',2'-difluoro-deoxycytidine) compared with the wild-type enzyme. The dNKmu enzymes were expressed in the breast cancer cell lines MDA-MB-231 (ER-) and MCF7 (ER+). In studying the sensitivity of the cell lines to DFDC, conditionally replicative adenovirus (CRAd) SG500-dNKmu showed higher expression and enzymatic activity than the replication-defective adenovirus SG500 in cancer cells, but with less cytotoxicity to cancer cells than that of SG500. Our data suggest that the triple phosphorylated DFDC catalyzed by dNKmu inhibited the replication of adenovirus with a simultaneous positive therapeutic effect to cancer cells. Therefore, concomitant use of the SG500‑dNKmu and DFDC could be a novel targeted strategy in suicide gene therapy with safe control of excessive virus replication.