An Escherichia coli system expressing human deoxyribonucleoside salvage enzymes for evaluation of potential antiproliferative nucleoside analogs.
Deoxyribonucleoside salvage in animal cells is mainly dependent on two cytosolic enzymes, thymidine kinase (TK1) and deoxycytidine kinase (dCK), while Escherichia coli expresses only one type of deoxynucleoside kinase, i.e., TK. A bacterial whole-cell system based on genetically modified E. coli was developed in which the relevant bacterial deoxypyrimidine metabolic enzymes were mutated, and the cDNA for human dCK or TK1 under the control of the lac promoter was introduced. The TK level in extract from induced bacteria with cDNA for human TK1 was found to be 20,000-fold higher than that in the parental strain, and for the strain with human dCK, the enzyme activity was 160-fold higher. The in vivo incorporation of deoxythymidine (Thd) and deoxycytidine (dCyd) into bacterial DNA by the two recombinant strains was 20 and 40 times higher, respectively, than that of the parental cells. A number of nucleoside analogs, including cytosine arabinoside, 5-fluoro-dCyd, difluoro-dCyd, and several 5-halogenated deoxyuridine analogs, were tested with the bacterial system, as well as with human T-lymphoblast CEM cells. The results showed a close correlation between the inhibitory effects of several important cytostatic and antiviral analogs on the recombinant bacteria and the cellular system. Thus, E. coli expressing human salvage kinases is a rapid and convenient model system which may complement other screening methods in drug discovery projects.
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Center, S-751 23 Uppsala, Sweden.
J
J
Wang
J
J
Neuhard
S
S
Eriksson
D000970:Antineoplastic Agents / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology*; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D009705:Nucleosides / Q000494:pharmacology*; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D009705:Nucleosides; D003841:Deoxycytidine; D013937:Thymidine Kinase; D003842:Deoxycytidine Kinase; D013936:Thymidine