Development and Characterization of a Human Colon Adenocarcinoma Xenograft Deficient in Thymidine Salvage

In order to determine the contribution of thymidine (dThd) salvage to intrinsic resistance to antimetabolites (5-fluoropyrimidines, antifolates) in the human colon adenocarcinoma xenograft, H×GCj, a subline deficient in thymidine kinase has been developed. A cell line (GC₃/M) was established in continuous culture that demonstrated a karyotype identical to that of the stem line of HxGC₃(46, X, -Y+12). After inoculation of GC₃/M cells into immune-deprived CBA/CaJ mice, the HxGC₃/M xenografts retained histological, histochemical, and growth characteristics of the H×GC₃xenograft. To develop a line deficient in dThd salvage, GC₃/M cells were selected with BrdUrd (100 μg/ml). Three clones characterized were unable to proliferate in HAT medium, and were deficient (<10% control) in the cytosolic form of thymidine kinase. Activities of dThd phosphorylase and dTMP synthase were unchanged from parental GC3/M cells. Of the three clones inoculated into mice, GC3/M TK” 100 C₃was tumorigenic, the xenografts demonstrating histological and growth characteristics similar to H×GC₃. The in vivo activity of the cytosolic form of dThd kinase was 3.5% of that in H×GC₃xenografts. Incorporation in vivo of [methyl-³H]dThd into acid insoluble material was 14% of that in HxGC₃tumors. Autoradiographs prepared from these tumors demonstrated that incorporation of radiolabel into nuclei occurred only in stromal cells derived from the host. It is anticipated that H×GC₃/M TK^-100 C₃will be a line valuable for determining the role of dThd salvage in intrinsic resistance to 5-fluoropyrimidines or antifolates in human colon adenocarcinomas growing as xenografts and also the relevance of dTMP synthase as a target for antimetabolites in this histiotype.