Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene.
Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.
Balzarini
J
J
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.
De Clercq
E
E
Verbruggen
A
A
Ayusawa
D
D
Shimizu
K
K
Seno
T
T
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects*; D002461:Cell Line, Transformed; D004247:DNA / Q000096:biosynthesis; D005814:Genes, Viral; D007065:Idoxuridine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D018139:Simplexvirus / Q000187:drug effects / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000998:Antiviral Agents; C020235:brivudine; C030360:5-(2-iodovinyl)-2'-deoxyuridine; D004247:DNA; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D007065:Idoxuridine