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 (d CK), while Escherichia roll 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 deoxypy rimidine metabolic enzymes were mutated, and the cDNA for human dCK or TK1 under the control of the lac promoter was introduced. The TK leve l in extract from induced bacteria with cDNA for human TK1 was found t o 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) i nto bacterial DNA by the two recombinant strains was 20 and 40 times h igher, respectively, than that of the parental cells. A number of nucl eoside analogs, including cytosine arabinoside, 5-fluoro-dCyd, difluor o-dCyd, and several 5-halogenated deoxyuridine analogs, were tested wi th the bacterial system, as well as with human T-lymphoblast CEM cells . The results showed a close correlation between the inhibitory effect s of several important cytostatic and antiviral analogs on the recombi nant bacteria and the cellular system. Thus, E. coli expressing human salvage kinases is a rapid and convenient model system which may compl ement other screening methods in drug discovery projects.