Hydroxyurea induces bystander cytotoxicity in cocultures of herpes simplex virus thymidine kinase-expressing and nonexpressing HeLa cells incubated with ganciclovir.
Suicide gene therapy with the herpes simplex virus thymidine kinase (HSV-TK) cDNA and ganciclovir can elicit cytotoxicity to transgene-expressing and nonexpressing bystander cells via transfer of ganciclovir phosphates through gap junctions. HeLa cells do not exhibit bystander cytotoxicity, although we showed recently that they transfer low levels of ganciclovir phosphates to bystander cells. Here, we attempted to induce bystander cytotoxicity using hydroxyurea, an inhibitor of ribonucleotide reductase, to decrease the endogenous dGTP pool, which should lessen competition with ganciclovir triphosphate for DNA incorporation. Addition of hydroxyurea to cocultures of HSV-TK-expressing and bystander cells synergistically increased ganciclovir-mediated cytotoxicity to both cell populations while producing primarily an additive effect in cultures of 100% HSV-TK-expressing cells. Whereas HSV-TK-expressing cells in coculture were approximately 50-fold less sensitive to ganciclovir compared with cultures of 100% HSV-TK-expressing cells, addition of hydroxyurea restored ganciclovir sensitivity. Quantification of deoxynucleoside triphosphate pools showed that hydroxyurea decreased dGTP pools without significantly affecting ganciclovir triphosphate levels. Although hydroxyurea significantly increased the ganciclovir triphosphate:dGTP value for 12 to 24 hours in HSV-TK-expressing and bystander cells from coculture (1.4- to 4.9-fold), this value was increased for <12 hours (2.5-fold) in 100% HSV-TK-expressing cells. These data suggest that the prolonged increase in the ganciclovir triphosphate:dGTP value in cells in coculture resulted in synergistic cytotoxicity. Compared with enhancement of bystander cytotoxicity through modulation of gap junction intercellular communication, this strategy is superior because it increased cytotoxicity to both HSV-TK-expressing and bystander cells in coculture. This approach may improve clinical efficacy.
Department of Pharmacology, University of Michigan Medical Center, 4713 Upjohn Center, 1310 East Catherine, Ann Arbor, MI 48109, USA.
Brian G
BG
Gentry
Paul D
PD
Boucher
Donna S
DS
Shewach
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000493:pharmacokinetics / Q000494:pharmacology*; D018920:Coculture Techniques; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D004357:Drug Synergism; D015774:Ganciclovir / Q000009:adverse effects / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000009:adverse effects / Q000494:pharmacology*; D009711:Nucleotides / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D003848:Deoxyguanine Nucleotides; D009711:Nucleotides; C092309:ganciclovir triphosphate; C029603:deoxyguanosine triphosphate; D013937:Thymidine Kinase; D015774:Ganciclovir; D006918:Hydroxyurea