Adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase mutants combined with gemcitabine harbor a safe cancer treatment profile

Abstract

Abstract. The purpose of this analysis was to investigate the enzyme activity and specificity of adenovirus-mediated Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) 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.

Gemcitabine resistance in triple‑negative breast cancer cells can be reverted by <em>Drosophila melanogaster</em> deoxyribonucleoside kinase in the nucleus or cytosol

“…Our research team has previously investigated Dm-dNK for its potential application as a suicide gene; the results have revealed that wild-type Dm-dNK retains its activity when it is expressed in human cells, and it is localized to the nucleus, resulting in high cell sensitivity to several cytotoxic nucleoside analogs, including araT, araC, BVDU and dFdC ( 25 , 27 , 37 , 40 ). In the present study, either the wild-type nuclear Dm-dNK (dNK-GFP) or the cytosolic arginine-247 Dm-dNK mutant (dNKmut-GFP) was expressed using lentiviral vectors and the mutant cytosolic Dm-dNK was also demonstrated to possess highly similar levels of enzymatic activity and cytotoxicity compared with the wild-type dNK ( 33 , 37 ), consistent with the findings of the present study. As one of the most effective substrates for Dm-dNK, dFdC exerts strong effects on Dm-dNK-expressing MDA-MB-231R cells, which exhibit a 50-fold decrease in IC 50 value for dFdC compared with that of untransfected cells ( 40 ), suggesting that the nucleoside analogs (prodrug)/Dm-dNK system may overcome drug resistance by lowering the IC 50 values for these chemotherapeutic agents.…”

New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

“…In parallel, N- and C-terminal truncations were done. C-terminal truncation had been shown for DmdNK to stabilize the enzyme [ 30 ] and DmdNK mutated at the very C-terminus is also used in suicide gene therapy testing [ 20 ]. In total 11 different N- and C-terminal deletions were made ( Table 1 , Figure 1 ) and tested in KY895 cells on screening plates with and without AZT.…”

“…The use of non-viral dNKs in suicide gene therapy has also been reviewed recently [ 2 ]. Among the three non-viral dNKs that have been tested in in vivo animal models so far, we find enzymes from the non-TK1-like group, the ultrafast multisubstrate dNK from Drosophila melanogaster (DmdNK) and the human dCK (HsdCK) [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. The third dNK is the above described ToTK1 and belongs to a different structural class, the TK1-like family of dNKs.…”

“…Both enzymes belonging to the non-TK1-like family: DmdNK and HsdCK have been targets for directed evolution and knowledge based protein engineering with the aim to improve performance and optimizing enzyme prodrug combinations. Mutants of DmdNK have been created by directed evolution [ 28 , 29 ], site directed mutagenesis [ 20 , 21 ] or C-terminal truncations have been made [ 20 , 30 ]. In several animal tumor models, wt HsdCK with gemcitabine and a mutated HsdCK with BVDU were tested and showed positive effects on treatment of the tumors [ 25 , 26 , 27 ].…”

Recombinant deoxyribonucleoside kinase from Drosophila melanogaster can improve gemcitabine based combined gene/chemotherapy for targeting cancer cells

“…DmdNK and its improved variants, produced by site-directed mutagenesis, have been tested for their optimum application in suicide gene therapy [38][39][40]. Both viral (adeno-and lentivirus-based vectors) and non-viral delivery systems have been used for transfecting different cancer cells with DmdNK and its mutants, to study the combined cytotoxic effect of gene/prodrug and to reverse drug resistance in various cancer cell lines [9,[41][42][43].…”