5-(2-iodovinyl)-2--deoxyuridine and Cell-Transformation--Viral

To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively.

Topics: Acyclovir; Animals; Cell Line; Cell Transformation, Viral; Cloning, Molecular; Genes, Viral; Idoxuridine; Mammary Neoplasms, Experimental; Mice; Mutation; Simplexvirus; Thymidine Kinase

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.

Topics: Animals; Antiviral Agents; Bromodeoxyuridine; Cell Division; Cell Transformation, Viral; DNA; Idoxuridine; Iodine Radioisotopes; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C3H; Simplexvirus; Thymidine Kinase; Thymidylate Synthase; Tritium

E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) and E-5-(2-iodovinyl)-2'-deoxyuridine (IvdUrd) are among the most potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) replication. To elucidate the site of inhibition, we examined whether the halovinyl analogs are incorporated into DNA using two approaches. (i) In assays with purified DNA polymerases omitting dTTP from the reaction system, addition of either BrvdUTP or IvdUTP increased the polymerization reaction, indicating that these two analog triphosphates can be alternate substrates. (ii) When HSV-1-infected Vero cells were grown in the presence of either BrvdUrd or IvdUrd, there was an increase in the density of both the viral and cellular DNA. The viral DNA had 40% of its thymidine moiety substituted by IvdUrd when the concentration of [125I]IvdUrd was 24 microM (in the absence of added thymidine). At 30 microM BrvdUrd and 1 microM [2-14C]thymidine, the viral DNA had only 11% of its thymidine moiety substituted by BrvdUrd, presumably because of the presence of added thymidine. Following digestion of [125I]IvdUrd-substituted DNA with DNase 1, venom phosphodiesterase, and alkaline phosphatase, the radioactivity co-migrated with nonradioactive IvdUrd in thin layer chromatography. Under similar conditions, no detectable incorporation of either [125I]IvdUrd or BrvdUrd into mock-infected Vero cell DNA was observed. Thus, IvdUrd and BrvdUrd are incorporated into DNA of HSV-1 infected cells but not into DNA of uninfected cells.

Topics: Animals; Bromodeoxyuridine; Cell Line; Cell Transformation, Viral; Deoxyuridine; DNA Polymerase II; DNA Replication; DNA, Viral; Halogens; Idoxuridine; Kidney; Simplexvirus; Structure-Activity Relationship; Vinyl Compounds; Virus Replication