Synergistic enhancement of herpes simplex virus thymidine kinase/ganciclovir-mediated cytoxicity by hydroxyurea.
We have previously demonstrated (L. Z. Rubsam et al., Cancer Res., 59: 669-675, 1999) that low ganciclovir (GCV) triphosphate (TP) levels similar to cellular deoxynucleotide concentrations can induce multilog killing in cells stably expressing herpes simplex virus thymidine kinase (HSV-TK). In this study, we evaluated whether reducing the endogenous competitor of GCV-TP, dGTP, enhanced GCV-mediated cytotoxicity. In SW620 human colon carcinoma cells stably expressing HSV-TK, the addition of the ribonucleotide reductase inhibitor, hydroxyurea (HU), decreased cellular dGTP pools and simultaneously increased the accumulation of GCV-TP levels. The amount of GCV nucleotide transfer from HSV-TK-expressing to nonexpressing (bystander) cells was quantitated in physically separated pHook-expressing bystander cells. Elevation of the GCV-TP:dGTP ratio by HU resulted in increased levels of GCV nucleotides transferred from HSV-TK-expressing to bystander cells during a 24 h drug incubation and enhanced GCV monophosphate incorporation into DNA after drug removal. Isobologram analysis demonstrated that the combination of GCV and HU was additive in 100% HSV-TK cultures and synergistic in HSV-TK/bystander mixtures. IC50 values for GCV in 1:1 cocultures of HSV-TK-expressing and nonexpressing SW620 cells were reduced from 1.5 microM to 0.07 microM with 2 mM HU. A similar reduction was also observed with HT-29 cells and U251 cells. With 2 mM HU, IC50 values for GCV in 10:90, 5:95, and 1:99 SW620 HSV-TK-expressing and nonexpressing cocultures were reduced from 55 microM to 0.3 microM, 71 microM to 0.8 microM, and 118 microM to 7 microM, respectively. These results demonstrate the ability to pharmacologically enhance HSV-TK/GCV-mediated bystander killing and may have an important therapeutic impact.
Boucher
P D
PD
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor 48109-0504, USA.
Ostruszka
L J
LJ
Shewach
D S
DS
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D015774:Ganciclovir / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000970:Antineoplastic Agents; D003854:Deoxyribonucleotides; D011993:Recombinant Fusion Proteins; C092309:ganciclovir triphosphate; D004247:DNA; D013937:Thymidine Kinase; D015774:Ganciclovir; D006918:Hydroxyurea