title: Adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase mutants combined with gemcitabine harbor a safe cancer treatment profile.
abstract: The purpose of this analysis was to investigate the enzyme activity and specificity of adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) mutants in combination with gemcitabine. Compared to herpes simplex type 1 thymidine kinases (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 (dNKmut) by site-directed mutagenesis at the sites of 244E, 245S, 251S, and 252R, with the last 10 amino acids in the amino acid sequence randomly alternated. We subsequently evaluated the enzyme activity and substrate specificity. The engineered enzymes showed a relative increase in phosphorylation in the nucleoside analogs of gemcitabine (dFdC, 2',2'-difluoro-deoxycytidine) compared to the wild-type enzyme. The dNKmut enzymes were expressed in breast (Bcap37) and gastric (SGC-7901) cancer cell lines. In studying the sensitivity of the cell lines to dFdC, conditionally replicative adenovirus (CRAd) ZD55-dNKmut showed higher expression and enzymatic activity than the replication-defective adenovirus Ad-dNKmut in cancer cells, but with less cytotoxicity to cancer cells than that of Ad-dNKmut. Our data suggest that the triple phosphorylated dFdC catalyzed by dNKmut inhibited the replication of adenovirus with a simultaneous positive therapeutic effect on cancer cells. Therefore, concomitant use of the ZD55-dNKmut and dFdC could be a novel targeted strategy in suicide gene therapy with safe control of excessive virus replication.
authors.affiliation: Department of Surgical Oncology, China Medical University, Shenyang, People's Republic of China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zhu
authors.forename: Shuai
authors.initials: S
authors.lastname: Ma
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Zhe
authors.initials: Z
authors.lastname: Sun
authors.forename: Anning
authors.initials: A
authors.lastname: He
authors.forename: Huimian
authors.initials: H
authors.lastname: Xu
authors.forename: Xinyu
authors.initials: X
authors.lastname: Zheng
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D003841:Deoxycytidine / Q000009:adverse effects / Q000031:analogs & derivatives* / Q000627:therapeutic use; D004331:Drosophila melanogaster / Q000235:genetics*; D005260:Female; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D050505:Mutant Proteins / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics; D009369:Neoplasms / Q000235:genetics / Q000628:therapy*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000008:administration & dosage* / Q000009:adverse effects / Q000235:genetics*; D000091402:Prohibitins; D023041:Xenograft Model Antitumor Assays; D000093542:Gemcitabine
chemical_list: D000970:Antineoplastic Agents; D050505:Mutant Proteins; C000717331:PHB2 protein, human; C000717333:Phb2 protein, mouse; D000091402:Prohibitins; D003841:Deoxycytidine; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D000093542:Gemcitabine