The ability of HSV-TK-expressing cells to cause cytotoxicity with GCV in neighboring non-transgene expressing (bystander) cells is crucial to the success of this enzyme-prodrug gene therapy strategy. Through protein channels known as gap junctions, HSV-TK-expressing cells can transfer phosphorylated GCV metabolites to neighboring bystander cells. Phosphorylated GCV then competes with dGTP for incorporation into DNA, an event correlated with cytotoxicity. Previous studies in our laboratory have shown that co-incubation of a ribonucleotide reductase inhibitor, such as hydroxyurea (HU), with GCV increases bystander cytotoxicity, synergistically, in a variety of cell types. Synergy has been attributed to the reduction of dGTP, the endogenous competitor for GCVTP incorporation into DNA, to ≥25% of the control value for at least 8 hours. However, in SW480 colon carcinoma cells that exhibited good bystander cytotoxicity in cultures with 50% HSV-TK-expressing cells, co-incubation with HU produced only an additive increase in bystander cytotoxicity. Analysis of GCVTP and dNTP pools in SW480 cells incubated with GCV and HU for 24 hours demonstrated that HU had a greatly reduced effect on dGTP levels, although effects on other dNTP pools was similar to that in other cell lines. While HU decreased dGTP levels to 40% of the control within 4 hours of drug incubation, dGTP levels increased to 50% of control by 8 hours, recovered to control levels by 12 hours, and remained at control levels for the remainder of the drug incubation period. Thus, compared to other cell lines, HU was able to effect only a small and transient decrease in dGTP. We also noted that SW480 HSV-TK-expressing cells had high GCVTP levels throughout the 24 hour drug incubation with a relatively slow degradation (t1/2 = 24.7 hours). However, degradation of GCVTP was considerably more rapid in bystander cells (t1/2 = 8.3 hours) co-cultured with HSV-TK-expressing cells. These data suggest that lack of synergistic bystander cytotoxicity can be attributed to the limited effect of HU on dGTP pools combined with the rapid degradation of GCVTP in bystander cells.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]