A novel mechanism of synergistic cytotoxicity with 5-fluorocytosine and ganciclovir in double suicide gene therapy.
The combination of cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cells and animal models. In this study, we show that concurrent addition of prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV) was less efficacious than sequential treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. If cells were incubated for 24 hours with 5-FC followed by a 24-hour GCV treatment, GCV triphosphate levels were 2-fold higher, incorporation of GCV monophosphate into DNA was 2.5-fold higher, and growth inhibition was increased 4-fold compared with simultaneous treatment. As expected, cellular dTTP levels were reduced during the 5-FC preincubation. However, dGTP pools also declined parallel to the dTTP decrease. Similar results were obtained when 5-fluorouracil or 5-fluoro-2'-deoxyuridine was used instead of CD/5-FC. These data allowed us to propose a novel hypothesis for the synergistic interaction between CD/5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC-mediated reduction of dTTP results in a concurrent decrease of dGTP due to allosteric regulation of ribonucleotide reductase. Because dGTP is the endogenous competitor of GCV triphosphate, depleted dGTP at the time of GCV addition results in increased GCV in DNA and cell kill. In fact, addition of deoxyguanosine during the 5-FC incubation reverses the dGTP depletion, reduces the amount of GCV monophosphate incorporated into DNA, and prevents the CD/5-FC-mediated enhancement of HSV-TK/GCV cytotoxicity. Understanding this mechanistic interaction may help recognize better strategies for creating more efficacious clinical protocols.
Boucher
Paul D
PD
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0504, USA. pboucher@umich.edu
Im
Michael M
MM
Freytag
Svend O
SO
Shewach
Donna S
DS
D000256:Adenoviridae / Q000235:genetics; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage* / Q000493:pharmacokinetics; D045744:Cell Line, Tumor; D043525:Cytosine Deaminase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003849:Deoxyguanosine / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000378:metabolism; D004334:Drug Administration Schedule; D004357:Drug Synergism; D005437:Flucytosine / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D005472:Fluorouracil / Q000493:pharmacokinetics / Q000494:pharmacology; D015774:Ganciclovir / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics / Q000628:therapy*; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D003854:Deoxyribonucleotides; D005437:Flucytosine; D013937:Thymidine Kinase; D043525:Cytosine Deaminase; D003849:Deoxyguanosine; D015774:Ganciclovir; D005472:Fluorouracil