Antitumor suicide gene therapy is one of the emerging strategies against cancer. The authors investigated the therapeutic potential of an adenovirally-delivered thymidine kinase/ganciclovir prodrug system expressed following vector delivery into a free flap. It is especially applicable for localized cancers, such as malignant glioma because of its restricted anatomical location and absence of metastasis. Background Ganciclovir (gan sye’ kloe vir) is an acyclic guanosine nucleoside analogue structurally related to acyclovir which has antiviral activity against many herpes viruses, including herpes simplex 1 and 2, Epstein-Barr virus and varicella-zoster, but is used largely in the therapy of cytomegalovirus (CMV) infections. Wilson, Steven M. Cancer is one of the most common worldwide diseases, with a high fatality rate. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting.
In a 50:50 coculture of U251tk and U251βgal cells, the survival of bystander cells was decreased by >99.5% with 3 μm GCV, whereas 30 μm GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251βgal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. The thymidine kinase of herpes simplex virus type-1 (HSV-1 TK) is a multifunctional enzyme that phosphorylates a broad range of pyrimidine nucleosides/nucleotides and purine nucleosides, including thymidine, thymidylate, deoxycytidine, acyclovir and ganciclovir (GCV) (Chen et al., 1979; Machida, 1986). With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. First, positive selection kills all cells that were not transformed, leaving a population of random and targeted recombinants. The proportion of GCVTP in bystander cells was consistently 50–80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. CT26 cells, an N-nitroso-N-methylurethane (NNMU)-induced undifferentiated colon carcinoma line from BALB/c mice, were obtained from the Korean Cell Line Bank (Seoul, Korea).
All patients provided written informed consent for all clinical and research aspects of the study according to institutional guidelines before treatment. However, the proportion of araT-5′-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population. (a) Scheme of the approach to engineering an HSVtk transgene specifically expressed in undifferentiated iPSCs by incorporation of MREs complementary to the let7 family of miRNAs in its 3′ end. Transfer of the HSV-TK 3 gene to tumor cells followed by GCV treatment has been widely used in vitro, where it has been shown to be active in many different tumor types (2, 3, 4, 5, 6, 7) . This strategy has resulted in impressive tumor regressions in several animal models, prompting clinical trials of HSV-TK/GCV therapy in patients with end-stage cancer including brain tumors (8) . This impressive antitumor activity appears to be mediated through the activation of GCV to its triphosphate derivative. HSV-TK phosphorylates GCV to its monophosphate form (GCVMP), which is further phosphorylated by cellular kinases to GCV diphosphate and the presumed cytotoxic metabolite GCVTP (9, 10, 11, 12, 13) .
Because GCV is a better substrate for HSV-TK than mammalian nucleoside kinases (14) , GCV cytotoxicity is selective for cells that express HSV-TK (2 , 15) . GCVTP mimics the endogenous DNA precursor dGTP and competes for mammalian DNA polymerases, resulting in inhibition of DNA synthesis and the incorporation of GCVMP into the nascent strand (9 , 13 , 16, 17, 18) . Previous work in this laboratory showed that GCVTP elicits a unique, multi-log cell killing distinct from the 1–2 log cytotoxicity induced by most clinically effective nucleoside analogues (19) . This multi-log cell killing occurs through a delayed mechanism that allows GCV-treated cells to complete one cell division and may be a consequence of GCVMP incorporation into the DNA template. An important component of enzyme-prodrug antitumor therapy is the ability to kill cells that do not express the transgene, a process termed the bystander effect (20) . This is critical for the clinical success of this approach, because currently available modes of gene transfer typically result in fewer than 10% of the cells expressing the gene of interest (8) . For HSV-TK/GCV, this bystander effect has been effective both in vitro and in vivo.
When only 1% of the cells in culture expressed HSV-TK, significant killing of bystander cells has been demonstrated (21, 22, 23) . Expression of HSV-TK in as few as 10% of the tumor cells in animal models has resulted in complete tumor regression after GCV treatment (5 , 24, 25, 26) . In vivo, the immune system may play a role in this bystander cell killing (27, 28, 29, 30) . However, induction of an immune response cannot account for bystander cytotoxicity in vitro, and another mechanism must account for efficient killing of bystander cells in culture and may also contribute to the bystander effect in vivo. Whereas it is generally recognized that the killing of bystander cells is crucial to the success of enzyme-prodrug therapy, the actual mechanism by which this occurs has not been clearly identified. Several laboratories have demonstrated that the proximity of bystander cells to HSV-TK-expressing cells is important (31, 32, 33, 34) . In addition, studies indicate that bystander cell killing appears to correlate with GJIC, a process that allows small molecules to diffuse between adjacent cells (22 , 33 , 35, 36, 37, 38, 39) .
These studies suggest that bystander cytotoxicity is mediated by GCV nucleotide transfer; however, there is little direct evidence documenting that such transfer occurs. Bi et al. (31) reported that tritium originally derived from GCV appeared in bystander cells cocultured with HSV-TK-expressing cells (31) , although the chemical nature of the radioactivity in bystander cells was not identified. Ishii-Morita et al. (40) recently demonstrated the transfer of GCV nucleotides from human HSV-TK-expressing cells to murine bystander cells, although only a single incubation condition was evaluated, with no information on the kinetics or mechanism of this transfer. To optimize HSV-TK/GCV therapy, more detailed studies of the mechanism of bystander cell killing are needed using bystander and HSV-TK cells developed from the same parental cell line. We recently documented the accumulation of GCVTP in bystander cells cocultured with HSV-TK-expressing cells, in which both cell lines were derived from the same human colon carcinoma cell line (34) .
We have now expanded these studies to evaluate GCV cytotoxicity and nucleotide transfer in HSV-TK-expressing and bystander cells generated from a human glioblastoma cell line. In the results presented here, we have performed a detailed characterization of GCV nucleotide transfer with respect to the length of incubation, the concentration of GCV, and the number of HSV-TK-expressing cells. Furthermore, we have compared the bystander cytotoxicity and nucleotide transfer of GCV with those of another HSV-TK substrate, araT. These studies were facilitated by the development of a new technique reported here for separating bystander cells from HSV-TK-expressing cells based on the presence of GFP in the bystander population. The results provide important insight into the reason for the excellent cytotoxic activity of GCV in cultures composed of HSV-TK-expressing cells and bystander cells. [8-3H]GCV (12.4 Ci/mmol) and [methyl-3H(N)]thymine-1-β-d-arabinofuranoside (2.9 Ci/mmol) were obtained from Moravek Biochemicals, Inc. (Brea, CA).
GCV (Cytovene) was a generous gift from Syntex (Palo Alto, CA). Propidium iodide, araT, and other nucleoside and nucleotide standards were purchased from Sigma Chemical Co. (St. Louis, MO). HPLC grade ammonium acetate and ammonium phosphate were purchased from J. (C) Anti-APO-1-induced apoptosis is inhibited by FADD-DN depending on the extent of expression of the mutated protein. Baker, Inc.
(Phillipsburg, NJ). Ammonium phosphate was also obtained from Fisher Scientific (Pittsburgh, PA). All other chemicals were of the highest purity available. U251 human glioblastoma cells were maintained in exponential growth in RPMI 1640 supplemented with 10% calf serum and 2 mm glutamine (Life Technologies, Grand Island, NY). To generate U251 clonal cell sublines that stably expressed HSV-TK (U251tk) or β-galactosidase (U251βgal) cDNA, cells were transduced with retrovirus vectors containing the corresponding cDNA. These vectors use the retrovirus long terminal repeat sequence as a promoter and also contain the cDNA for neomycin resistance (19 , 34) . Cells expressing GFP were generated by transfection with the pEGFP-N1 plasmid (Clontech Laboratories, Palo Alto, CA) and LipofectAMINE (Life Technologies).
Cells expressing each transgene were selected with 400 μg/ml G418, and individual colonies were expanded and subsequently maintained in medium containing 200 μg/ml G418. Expression of HSV-TK was confirmed by assaying cell lysates for GCV phosphorylation (7) , and β-galactosidase expression was verified by staining with the substrate X-gal. GFP fluorescence was visualized using a fluorescence microscope with excitation at 515 nm and emission at 525 nm. Cytotoxicity was assessed in the U251βgal and U251tk cell lines by a standard colony formation assay as described previously (7) . Exponentially growing cells were treated with the drug for up to 24 h, followed by plating 10–50 viable cells/well. Cells were allowed to grow for 7–11 days, at which time they were stained with crystal violet, and colonies of at least 50 cells were enumerated. In coculture experiments, U251βgal colonies were visualized by staining with X-gal and enumerated.
Subsequently, the plates were stained with crystal violet to determine the total number of colonies formed, and the difference between the total number and the number of X-gal-stained colonies was used to calculate the survival of U251tk cells. After incubation with GCV or araT, cells were harvested, and nucleotides were extracted with perchloric acid and neutralized as described previously (41) . The phosphorylated derivatives of GCV and araT were separated from endogenous nucleotides and quantitated by strong anion exchange HPLC using a Waters (Milford, MA) gradient system composed of two Model 501 pumps, a U6K injector module, and a Model 996 photodiode array detector and controlled by Millenium 2010 software. Additionally, our data indicate that HSV1-derived sequences that regulate transgene expression from HCAd may differ from those previously shown to increase expression from intronless genes in plasmids (25, 36). Samples were loaded onto a 5-μm Partisphere 4.6 × 250-mm strong anion exchange column (Whatman, Hillsboro, OR), and nucleoside triphosphates were eluted with a linear gradient of ammonium phosphate buffer ranging from 0.15 m (pH 2.8 or pH 3.6) to 0.6 m (pH 3.7 or pH 2.8). For this purpose, we co-cultured either TK- or Tat11-TK–expressing cells (producers) with wild-type cells (targets) at different ratios. Schlichting , R.
In this series of experiments cells transfected with pTK1.0 exhibited only the basal TK activity found in cells transfected with a control plasmid. The amount of GCVTP in U251tk or U251gfp cells separated after coculture was corrected for the low level (99.8% decrease in cell survival with 1 μm GCV. Competent E. However, when cocultured with 90% bystander cells (a 10:90 ratio), 1 μm GCV decreased U251tk survival by only 54%, and 10 μm GCV was required to reduce survival by 96%. This suggests that the bystander cells rescued the U251tk cells from GCV toxicity in the 10:90 culture. Cytotoxicity of GCV or araT to HSV-TK-expressing and non-HSV-TK-expressing (bystander) cells in coculture. U251tk cells and U251βgal (bystander) cells were cocultured at ratios of 50:50 or 10:90 and incubated with drug at the indicated concentrations for 24 h.
After drug incubation, cells were harvested, and survival was measured using a colony formation assay. Bystander cell colonies were identified based on X-gal staining, and U251tk colonies were calculated by subtracting the number of X-gal-stained colonies from the total colony number (crystal violet staining). Cell survival is shown for U251tk (▪) and U251βgal (□) cells cocultured at a ratio of 50:50 and incubated with GCV, U251tk (•) and U251βgal (○) cells cocultured at a ratio of 10:90 and incubated with GCV, U251tk (▴) and U251βgal (▵) cells cocultured at a ratio of 50:50 and incubated with araT, and U251βgal cells incubated with GCV (×). U251βgal cells cultured alone were marginally sensitive to GCV at concentrations of up to 100 μm (survival ≥ 82%). However, in a 50:50 coculture with U251tk cells, 3 μm GCV decreased survival by >99.5% in the U251βgal cells. Whereas substantial bystander cell killing occurred in the 10:90 coculture, it was less efficient, requiring 30 μm GCV to effect a 99.5% decrease in U251βgal cell survival. Thus, increasing the proportion of bystander cells to 90% decreased cytotoxicity in both cell types, such that the sensitivity of U251βgal cells was similar to that of U251tk cells in the 10:90 coculture.
Studies with araT demonstrated that this drug did not induce bystander killing, even at high concentrations (Fig. 1) ⇓ . In 50:50 cocultures of U251tk and U251βgal cells incubated with up to 1000 μm araT, no significant bystander cell killing was observed. Furthermore, the presence of an equal number of bystander cells reduced the toxicity of araT to the U251tk cells. In the 50:50 coculture, >60% of the HSV-TK-expressing cells survived incubation with 1000 μm araT, whereas in 100% U251tk cultures, 97% pure and the U251gfp population was >99% pure (data not shown). A similar sorting and collection of cells was performed using cocultures of U251βgal (nonfluorescing) and U251gfp cells. Reanalysis of the U251βgal cells again indicated a >98% purity.
An aliquot of each sorted cell population was plated and stained with X-gal, indicating that >97% of the low GFP-fluorescing population were β-galactosidase-expressing cells, whereas 97% purity. Analysis of U251tk and U251gfp cells by flow cytometry. Cells in exponential growth were harvested by trypsinization, resuspended in complete culture medium, and analyzed for fluorescence within 1 h of harvest. (A, U251gfp cells; B, U251tk cells; C, U251tk and U251gfp cells analyzed after coculture at a ratio of 10:90). Bars, the portion of each population that was reanalyzed for purity. Because only the highest fluorescing U251gfp cells were collected (representing less than half of the total U251gfp cells separated), it was important to ensure that this represented a heterogeneous population and did not select for cells in a specific phase of the cell cycle. U251gfp cells were treated with Hoechst 33342 to stain the DNA before cell cycle analysis by flow cytometry.
As illustrated in Fig. 3 ⇓ , representative samples of cells with low, mid-level, or high GFP fluorescence exhibited a similar cell cycle distribution. Thus, the collection of high GFP fluorescing cells did not bias the sample for a specific phase of the cell cycle. These studies demonstrated that U251gfp (bystander) cells were separated from U251tk cells with high purity, which allowed us to assess directly whether phosphorylated GCV could accumulate in U251gfp cells cocultured with U251tk cells. Analysis of the cell cycle distribution of cells based on GFP fluorescence intensity. U251gfp cells were harvested, and the DNA was stained with Hoechst 33342. Cells were then separated and collected based on the intensity of GFP fluorescence (A).
Cells representing low, mid-level, or high fluorescence were reanalyzed for DNA content (Hoechst fluorescence; B). To determine whether GCVTP accumulated in bystander cells, U251tk and U251gfp cells were cocultured in the presence of GCV. U251tk and U251gfp cells were combined at ratios of either 50:50 or 10:90 and allowed to grow for 2 days before drug addition. At that time, cells were examined under a fluorescence microscope, which indicated that the two cell types were still present at the desired ratio (data not shown). The cells in coculture appeared to be well mixed, and each culture was ≤ 50% confluent at the time of drug addition. After the drug incubation period, the cocultures were trypsinized, and the two cell types were separated and collected by cell sorting based on GFP fluorescence. Nucleotides in separated cell populations were then extracted and analyzed by HPLC.
As illustrated in Fig. 4 ⇓ , GCVTP was readily distinguished from the endogenous nucleoside triphosphates after the incubation of U251tk cells with GCV. “Regulation of thymidine kinase synthesis in human cells”. HPLC analysis of GCVTP in U251 glioblastoma cells. U251tk cells were incubated with no drug (top) or 100 μm GCV (bottom) for 1 h. All of the 143B TK− cells grown in HAT medium died. The chromatograms show the region in which the endogenous and analogue nucleoside triphosphates elute.
Although not visible in the chromatograms, dGTP would elute after GTP. No endogenous nucleotides coelute with GCVTP under these conditions. Fig. 6 ⇓ demonstrates that the accumulation of GCVTP in the U251gfp cells occurred readily, with detectable levels of phosphorylated GCV apparent within 4 h after drug addition. The amount of GCVTP in both the U251tk and U251gfp bystander cells increased continuously throughout the 24-h incubation period, without evidence of saturation. Furthermore, the proportion of GCVTP in the bystander cells compared to that in U251tk cells was relatively constant throughout the incubation (54–71% of the amount in U251tk cells). Accumulation of GCVTP with time in U251gfp bystander cells.
U251tk and U251gfp cells were cocultured at a ratio of 50:50 for 24 h with 10 μm GCV. At the time points indicated, U251tk and U251gfp cells were harvested, separated by cell sorting, and extracted, and the nucleotides were analyzed by HPLC. The graph represents the amount of GCVTP in U251tk (▪) and U251gfp (•) cells after coculture.