Conditionally replicating adenovirus SG500-expressed mutant Dm-dNK gene             for breast cancer therapy.
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 10 amino 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.
Department of Breast Surgery, Fourth Affiliated Hospital, China Medical University, Shenyang, PR China.
Wenzhi
W
Qu
Zhi
Z
Zhu
Lei
L
Zhao
Anning
A
He
Xinyu
X
Zheng
D000256:Adenoviridae / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics* / Q000628:therapy*; D045744:Cell Line, Tumor; D003588:Cytopathogenic Effect, Viral; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D005260:Female; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics* / Q000633:toxicity; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D050505:Mutant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases