This phenomenon was reproduced in in vitro studies where we incubated 
various tumor cell lines (murine 205, rat 9L, human U251) with HS-tk-vector producer 
cells for 24 - 72 hours. Following exposure to GCV, the supernates were removed and 
transferred to tissue cultures of the wild-type tumors (not transduced with the HS-tk 
gene). Thymidine incorporation assays revealed that the supernates significantly 
inhibited thymidine incorporation in all tumor types (Figure 7). The mechanism of this 
“bystander tumor kill” is not yet completely understood. This may involve the production 
of toxic triphosphates by the interaction of thymidine kinase and GCV leading to 
inhibition of DNA synthesis and death of replicating cells. It does not seem to involve 
generalized non-specific cellular toxicity since the overlying skin and other tissues 
surrounding these HS-tk treated tumors was not grossly injured while the tumors 
expressing the genes and the admixed wild-type tumor cells were completely 
destroyed. 
Both the 9L rat gliosarcoma and the human glioblastoma cell line U251 were also 
sensitive to this “Bystander” effect in an in vitro mixing experiment (see Appendix B; 
method by Hiroyuki Ishii). GITkSvNa transduced and non-transduced tumor cells 
were mixed at different ratios in 96 well microtiter dishes. GCV was added to the wells 
and 24 hours later, the cultures were pulsed with tritiated thymidine. These bar graphs 
depict a greater decrease in proliferation than would be expected at a GCV level of 5.1 
pg/ml in the medium (the numbers over the bars represent the percent of transduced 
tumor in the culture. This concentration is easily within the therapeutic range 
established in humans. Since it is unlikely that 100% of the tumor cells in the brains of 
our patients will be successfully gene-modified, this “bystander” effect is very important 
for the successful outcome of this treatment approach. 
Recombinant DNA Research, Volume 15 
[801] 
