Differential Ganciclovir-mediated Cell Killing by Glutamine 125 Mutants of Herpes Simplex Virus Type 1 Thymidine Kinase*

The therapeutic combination of the herpesvirus simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and the prodrug, ganciclovir (GCV), has found great utility for the treatment of many types of cancer. After initial phosphorylation of GCV by HSV-1 TK, cellular kinases generate the toxic GCV-triphosphate metabolite that is incorporated into DNA and eventually leads to tumor cell death. The cellular and pharmacological mechanisms by which metabolites of GCV lead to cell death are still poorly defined. To begin to address these mechanisms, different mutated forms of HSV-1 TK at residue Gln-125 that have distinct substrate properties were expressed in mammalian cell lines. It was found that expression of the Asn-125 HSV-1 TK mutant in two cell lines, NIH3T3 and HCT-116, was equally effective as wild-type HSV-1 TK for metabolism and sensitivity to GCV, bystander effect killing and induction of apoptosis. The major difference between the two enzymes was the lack of deoxypyrimidine metabolism in the Asn-125 TK-expressing cells. In HCT-116 cells expressing the Glu-125 TK mutant, GCV metabolism was greatly attenuated, yet at higher GCV concentrations, cell sensitivity to the drug and bystander effect killing were diminished but still effective. Cell cycle analysis, 4′,6′-diamidine-2′-phenylindoledihydrochloride staining, and caspase 3 activation assays indicated different cell death responses in the Glu-125 TK-expressing cells as compared with the wild-type HSV-1 TK or Asn-125 TK-expressing cells. A mechanistic hypothesis to explain these results based on the differences in GCV-triphosphate metabolite levels is presented.

Delivery and expression of herpesvirus thymidine kinase (HSV-1 TK) 1 in combination with ganciclovir (GCV) has shown great clinical promise as a gene therapy of different cancers (1)(2)(3)(4). GCV is a prodrug that must be initially phosphorylated by HSV-1 TK and then cellular kinases to the toxic triphosphate form, GCVTP, that incorporates into cellular DNA and may act as an inhibitor of DNA polymerase ␦ (5-7). The basis for the original clinical trials was the ability of 10% or less HSV-1 TK-expressing cells to mediate a bystander effect whereby non-TK-expressing cells also became sensitive to GCV killing (1,8,9). In vitro, the primary mechanism of the bystander effect has been determined to be the gap junction mediated transfer of GCV metabolites to neighboring non-HSV-1 TK-expressing cells (10 -13). In response to GCV phosphorylation and/or metabolite transfer, most cell types have been reported to undergo apoptosis, which appears to be the cellular mechanism by which both the HSV-1 TK-expressing cells and bystander cells ultimately die (5,11,14). Recently, GCV has been reported to induce S-and G 2 /M phase cell cycle arrest in HSV-1 TK-expressing cells (5,(15)(16)(17), and these changes were associated with modulation of Cdc2/cyclin B activities (16) and increased levels of cyclin B1 (15). In vivo, it is clear that initial HSV-1 TK/GCV tumor cell killing results in a complex inflammatory stimulation of the immune system that affects all tumor cells (1, 18 -21). Regression of tumors distant from the primary HSV-1 TK-expressing site and establishment of anti-tumor immunity has also been demonstrated, and this has been termed the distant bystander effect (22)(23)(24).
All of the effects of GCV metabolites must somehow be linked to their incorporation into DNA and the disruption of the cell cycle, which in many cell systems results in induction of apoptosis, yet these links are still not clear. It is apparent, however, that phosphorylation of GCV by HSV-1 TK leads to a broad range of diverse pharmacological, cellular, and physiological effects in vitro and in vivo. Optimizing these effects and understanding the biochemical mechanism by which GCV acts could lead to improved therapeutic and clinical outcomes for genetic therapies of cancer utilizing HSV-1 TK. A separate study has described the characterization of the enzymatic properties and cell killing properties of three site-specific mutations of Gln-125 in HSV-1 TK to Asp, Asn, or Glu. 2 It was observed that when expressed in the human colon tumor cell line HCT-116 and treated with GCV, the Glu-125 mutant was equally effective at cell killing as the wild-type (Gln-125) or Asn-125 HSV-1 TKs. This was despite the diminished enzymatic properties of Glu-125 HSV-1 TK compared with wild-type Gln-125 TK, which included a 7-fold higher K m for GCV and a 83-fold decrease in k cat /K m . 2 In this current report, further characterization of cell lines expressing these mutant HSV-1 TKs and analysis of the differences in cellular responses to GCV are evaluated. Besides determining that the Asn-125 TK enzyme acts just as efficiently as wild-type enzyme in these cell lines, we report differences in the cell cycle progression, apoptosis induction, bystander killing, and GCV dose effects of the Glu-125 TK enzyme. These studies demonstrate how cellular expression of different HSV-1 TK mutants with distinct enzymatic properties can be used to evaluate the unique pharmacological properties of GCV.

Expression and Characterization of HSV-1 TKs in Cell Lines-A
Moloney murine leukemia virus derived plasmid for the expression of HSV-1 TK, termed pLENTK, has been previously constructed (12). A unique BspEI-MluI restriction fragment within the HSV-1 TK sequence of pLENTK contains the Gln-125 mutation site. This fragment was removed from wild-type plasmid and replaced with the analogous fragments encoding each mutant. The new pLEN-mutant TK constructs were sequenced to confirm the presence of the mutation. Along with wild-type HSV-1 TK plasmid, each mutant TK plasmid was transfected individually into the murine fibroblast cell line, NIH3T3, and the human colon tumor cell line, HCT-116, using Lipofectin reagent (Life Technologies, Inc.) (2 g of plasmid, 14 l of lipid/1 ϫ 10 6 cells). Cells were maintained in RPMI 1640 medium and selected with G418 (200 g/ml for 2 weeks) as described previously (12). At least eight individual G418-resistant cell clones were picked and grown up for further characterization. Each clone was screened initially for growth inhibition by 25 M GCV. Those clones that were sensitive were further analyzed for HSV-1 TK protein expression by Western blot analysis with a polyclonal, rabbit anti-HSV-1 TK antibody (a gift from Dr. Margaret Black, Washington State University). For each clone, cell numbers were normalized to 1 ϫ 10 6 , and equal protein loading was confirmed for each sample by gel staining. Blotted HSV-1 TK protein bands were visualized on film using ECL chromophore reagents (Amersham Pharmacia Biotech). Each HSV-1 TK cell line set used was characterized for comparatively equal protein expression levels of HSV-1 TK to one another as determined by the Western blot analyses.
Metabolic Labeling with [ 3 H]Nucleosides-For metabolic labeling, cells (1-2 ϫ 10 6 ) were labeled in triplicate with 2 Ci of [ 3 H]GCV (8 M) for 18 h, and then nucleotides were extracted from pelleted cells in 0.2 ml of 70% methanol at 4°C for 15 min as described previously (12,25). An aliquot of each methanol-soluble supernatant was analyzed for radioactivity by scintillation counting. The methanol insoluble pellets, representative of a crude DNA fraction, were resuspended in 0.15 ml of water and also counted for radioactivity. For deoxypyrimidines, cells were grown to confluency in 60-cm 2 plates, and either 2 Ci of [ 3 H]thymidine (2 M final) or 2 Ci of [ 3 H]dC (10 M final) were added for 1 or 2 h, respectively, prior to extraction in 70% methanol. Methanol-soluble extracts were concentrated by evaporation under nitrogen and separated on polyethyleneimine-cellulose thin layer chromatography plates developed in 0.8 M LiCl for GCV or 0.35 M LiCl for thymidine/dC as described previously (25).
GCV Sensitivity and Bystander Effect Clonal Dilution Assays-For determination of GCV sensitivity, parental HCT-116 and each HSV-1 TK-expressing cells were seeded in 24-well plates (2 ϫ 10 5 /well) in 1 ml of medium. The next day, 0, 0.1, 1, or 10 M GCV was added to each cell line in triplicate. After 24 h, for each well the medium was removed, cells were rinsed twice in fresh medium and trypsinized, and then medium was added to 1 ml/well. Each well of cells was then sequentially diluted from 1:10 to 1:10,000 in 1 ml of fresh medium on a separate 24-well plate. After 7 days, surviving cell colonies were fixed in 100% methanol, stained with 0.1% methylene blue, and counted. For bystander effect assays, each of the three HSV-1 TK-expressing cell lines were plated with parental HCT-116 cells (total 2 ϫ 10 5 /well) in the following proportions: (parental:HSV-1 TK cells) 100%:0%, 95%:5%, 90%:10%, 75%:25%, 50%:50%, and 0%:100%. After 2 days, 25 M GCV was added in 1 ml of fresh medium. After 24 h, the medium was removed, and cells from each well were diluted from 1:10 to 1:10,000 as described above. After 7 days, surviving cell colonies were fixed and stained for counting.
DAPI Staining of Apoptotic Cells-Parental HCT-116 cells and each HSV-1 TK-expressing cell line were plated (5 ϫ 10 4 cells/well) in 8-well plastic chamber slides (Lab-Tek) and left untreated or treated with 25 M GCV for 36 or 84 h. At either time point, cells were washed with phosphate-buffered saline followed by staining in 1 g/ml DAPI in 100% methanol at 37°C for 10 min (26). After rinsing, the stained cells were visualized with a DAPI-specific filter on a Zeiss fluorescent microscope at 40ϫ magnification.
Caspase 3 Assay-Caspase 3-like activity was determined in parental HCT-116, and each HSV-1 TK-expressing cell line was treated for 50 h plus or minus 25 M GCV using an Apo-Alert CPP32/Caspase 3 Colorimetric Assay kit with the peptide substrate, DEVD-pNA, as per manufacturers instructions (CLONTECH). GCV-treated and untreated cells were grown in 25-cm 2 flasks, and cell numbers were determined using a hemocytometer prior to analysis. Assays were done in triplicate with protein extracts derived from 2 ϫ 10 6 cells. The amount of Caspase 3-like activities were quantitated using a Shimadzu UV/VIS spectophotometer set at 405 nm.
Cell Cycle Analysis-Parental HCT-116 cells and each HSV-1 TKexpressing cell line were grown to 60% confluency in 25-cm 2 flasks and treated for 24 h plus or minus 25 M GCV. Following drug incubation, cells were removed by trypsin, and total cell numbers were determined. The cells were then washed twice in phosphate-buffered saline and fixed in 1 ml of 70% ethanol at 4°C for at least 1 day. Just prior to cell cycle analysis, the ethanol was removed and cell pellets were resuspended in 0.1% bovine serum albumin plus RNase (0.1%) and propidium iodide (50 g/ml) for 30 min at room temperature to stain DNA. DNA content was measured using a FACScalibur flow cytometer (Becton Dickinson), and data were analyzed using MODFIT LT (Verity Software House) computer software.

Metabolic labeling with [ 3 H]GCV, [ 3 H]Thymidine, and [ 3 H]dC-
In a separate study, three site-specific mutations of Asp, Asn, and Gln were substituted for amino acid Gln-125 in HSV-1 TK. 2 In contrast to wild-type HSV-1 TK, these mutant enzymes all displayed increased K m values for thymidine and minimal phosphorylation activities for TMP, dC, and AZT. The K m values for GCV varied from wild-type HSV-1 TK (69 M), with the Asn-125 mutant decreasing (50 M), the Glu-125 mutant increasing (473 M), and the Asp-125 mutant having minimal activity. 2 For this study, the cDNAs for these three mutants and the wild-type HSV-1 TK were incorporated into a retroviral plasmid (12) and used to stably transfect NIH3T3 fibroblasts and the human colon tumor cell line HCT-116 as described under "Materials and Methods." From a panel of multiple HSV-1 TK-expressing clones, a subset of clones from each cell line expressing wild-type HSV-1 TK, the Asn-125 HSV-1 TK mutant (Asn-125 TK), the Glu-125 HSV-1 TK mutant (Glu-125 TK), and the Asp-125 HSV-1 TK mutant (Asp-125 TK) were selected for comparatively equivalent levels of HSV-1 TK protein expression based on Western blot determinations. Initially, these two sets of HSV-1 TK-expressing NIH3T3 and HCT-116 cell lines were evaluated for intracellular metabolism of [ 3 H]GCV, [ 3 H]thymidine, or [ 3 H]dC. Cells were labeled with [ 3 H]GCV for 18 h, and then nucleotides were extracted in ice-cold 70% methanol as described previously (12,25). The data in Table I summarize the amount of total nucleotide metabolites isolated in the methanol-soluble extracts (pmol/10 6 cells) as well as the total amount of methanol-insoluble metabolites representative of incorporation into DNA. The Asn-125 TK metabolizes GCV at (or near) equal levels to the wild-type HSV-1 TK in both cell lines. The methanol-insoluble data, although only a crude indicator of [ 3 H]GCV incorporation into DNA, reflects the numbers obtained with the soluble extracts. As compared with the non-HSV-1 TK-expressing HCT-116 cells and consistent with minimal enzymatic activity, 2 minimal [ 3 H]GCV metabolism was detected in the Asp-125 TK cells, and these cells were not further evaluated.
The methanol-soluble metabolites were further separated into their constituent phosphorylated GCV metabolites by thin layer chromatography (25). As presented in Table II, the predominant metabolite in each HSV-1 TK-expressing cell line was GCVTP. In both HCT-116 and NIH3T3 cell lines, the Asn-125 TK cells indicated slightly higher levels of GCVTP as compared with wild-type HSV-1 TK cell lines. The Glu125-TK in HCT-116 cells resulted in a 23-fold or greater decrease in GCVTP levels, whereas levels of GCVTP in the NIH3T3 cells was only weakly detected. This difference in the levels of GCVTP in the two Glu-125 TK-expressing cell lines could explain the lack of sensitivity to GCV killing observed for the NIH3T3 Glu-125 TK cell lines.
Because previous enzymatic data indicated that the Asn-125 and Glu-125 mutations had altered deoxypyrimidine substrate utilization, the metabolism of thymidine and dC in the HCT-116 cell set were examined. Cells were grown to confluency and labeled with either [ 3 H]thymidine or [ 3 H]dC for 1 or 2 h, respectively, prior to methanol extraction. If labeling was done in subconfluent, dividing cultures, we found that the metabolite numbers reflected cell growth rates and therefore cellular kinase activities rather than that of HSV-1 TK activity (data not shown). As presented in the last two columns of Table I, the levels of deoxypyrimidine metabolites extracted from the mutant HSV-1 TK cells were analogous to those isolated from parental HCT-116 cells rather than the wild-type HSV-1 TK cells. As shown in Table II, the levels of TMP and TTP separated from the methanol-soluble fractions of the [ 3 H]thymidine-labeled HCT-116 Asn-125 TK and Glu-125 TK cells were similar to parental HCT116 cells rather than the wild-type HSV-1 TK-expressing 116 cell line. These metabolite levels reflect the enzymatic properties and altered substrate specificities of the Glu-125 and Asn-125 HSV-1 TKs. Thus when expressed in cell lines, these mutant forms of HSV-1 TK appear to function more as GCV kinases rather than thymidine kinases.
Comparative GCV Sensitivities and Bystander Effect-During characterization of the different HSV-1 TK-expressing cell lines, it was observed using an 3-(4,5-dimethylthiazol-2-yl)-2,,5-diphenyltetrazolium bromide cell viability assay that HCT-116 cells expressing the poor GCV metabolizing Glu-125 TK were just as sensitive to GCV killing as the high GCV metabolizing wild-type or Asn-125 TK enzyme. 2 Therefore, the HCT-116 cell panel was further evaluated for sensitivity to GCV using a more sensitive clonal dilution assay. Because the Glu-125 TK expressed in NIH3T3 cells had little effect on their GCV sensitivities, only the HCT-116 cell panel was evaluated in the rest of the study. Cells previously plated in 24-well plates were treated with GCV (0 -10 M) in triplicate for 24 h. Following drug removal, cells were diluted and replated in fresh medium from dilutions of 1:10 to 1:10,000. Surviving cell colonies were counted 6 -7 days later. As shown in Fig. 1   HCT-116 parental cells. Cell populations were treated with 25 M GCV for 24 h and then diluted from 1:10 to 1:10,000. As shown in Fig. 2, only 5% wild-type or Asn-125 TK-expressing cells were required to cause a greater than 1-log decrease in cell colony numbers. In these same cell lines, a greater than 4-log reduction in cell colony numbers was detected with 25 and 50% proportions of HSV-1 TK-expressing cells. For the Glu-125 TK-expressing cells, a 1-log GCV-mediated bystander effect was observed at 10% proportions, and a near 3-log decrease was detected with the 50% proportions. Even though the bystander effect with the Glu-125 TK-expressing cells was clearly attenuated relative to the other two cell lines, the Glu-125 mutant was still able to generate significant bystander effect cell killing. Cell Cycle Analysis-GCV has also been previously reported to induce S and G 2 /M phase cell cycle arrest in HSV-1 TKexpressing glioma and melanoma cell lines (5,(15)(16)(17). Therefore, the effect of 24 h of GCV treatment on the cell cycling of parental and the three HSV-1 TK-expressing cell lines was examined by flow cytometry of propidium iodide-stained cells. As shown in Fig. 3 (A and B) and Table III, GCV treatment of HCT-116 parental, non-HSV-1 TK-expressing cells had little effect on the percentage of cells in each phase of the cell cycle as compared with untreated cells. In the wild-type and Asn-125 TK-expressing cell lines, GCV treatment (Fig. 3, D and F) led to an increase in the proportion of cells in the S phase and undetectable percentages in G 2 /M phase when compared with untreated cultures (Fig. 3, C and E). For the Glu-125 TK-expressing cells, over 60% of the GCV treated cells were in S phase, and 0% were in G 2 /M (Fig. 3H). The doubling rate of growth for each HCT-116 cell line was in the 15-18-h range (data not shown), thus these results are consistent with a previous study that found that HSV-1 TK-expressing glioma cells treated with GCV underwent one replication cycle prior to an S phase arrest (5).

DAPI Staining and Caspase 3 Apoptosis Assays-
The differential dose responses, morphological features, and cell cycle patterns associated with the Glu-125 HSV-1 TK-expressing cells treated with GCV suggested induction of a distinct cell death response different from that observed in wild-type HSV-1 TK-expressing cells. It has been previously established that GCV treatment of wild-type HSV-1 TK-expressing cell lines results in induction of apoptosis (5,11,14,15). Therefore, two late stage apoptosis assays, nuclear DAPI staining and caspase-3 activation, were done for GCV treatments of the three HSV-1 TK-expressing HCT-116 cell lines. As shown in Fig. 4, DAPI-stained nuclei of wild-type and Asn-125 TK-expressing cells treated with GCV for 36 or 84 h indicated progressive increases in condensed and fragmented nuclei characteristic of apoptosis. Also, the DAPI staining of these cell lines indicates a GCV-specific nuclear swelling of preapoptotic cells and enhanced staining of nucleoli. This nuclear swelling in response to GCV has been observed within 12 h of GCV administration in wild-type HSV-1 TK HCT-116 cells (data not shown) and has also been reported for other GCV treated HSV-1 TK cell lines (16,17). For the Glu-125 HSV-1 TKexpressing cells, 36 h of GCV treatment led to fewer swelled nuclei and little evidence of apoptotic nuclei, although distinct staining of condensed nucleoli was observed. Even after 84 h of GCV treatment of these cells, there were still comparatively fewer changes in nuclear morphologies of the Glu-125 TK cells compared with the wild-type or Asn-125 HSV-1 TK-expressing cells, although there was more apparent nuclear swelling. Under identical treatment conditions, GCV treatments of parental, non-HSV-1 TK-expressing HCT-116 cells indicated none of the nuclear swelling or apoptotic fragmentations seen in the three HSV-1 TK-expressing cell lines (data not shown).
A more direct analysis of apoptotic activity was done by assaying the activation of the executioner protease, caspase 3. Activation of the zymogen form of caspase 3 has been implicated as a component of the late execution phase of apoptosis, and the substrate proteins cleaved by activated caspase 3 and related enzymes are responsible for the end stage morphological and intracellular changes associated with apoptotic cell death (27,28). Caspase 3 activity was determined in different cell extracts derived from GCV-treated and control cells using a colorimetric assay with the peptide substrate DVED as described under "Materials and Methods." As shown in Fig. 5, the DVEDase activity of GCV-treated Glu-125 HSV-1 TK cells was three times lower than that observed for GCV-treated wildtype or Asn-125 HSV-1 TK-expressing cells. Co-incubation of GCV-treated wild-type HSV-1 TK-expressing cells with the competing peptide DVED resulted in caspase 3 activities near untreated control cell values (data not shown). Thus, the results of the DAPI staining and caspase 3 assays are consistent with an altered apoptotic response and cell death pathway in GCV-treated Glu-125 HSV-1 TK-expressing cells. The cumulative results of this study are consistent with two distinct cell death responses induced by GCV treatment in the same HCT-116 cell line background that is dependent on the distinct enzymatic properties of HSV-1 TK. DISCUSSION The killing of tumor cells with HSV-1 TK and GCV is a complex interactive sequence of biochemical and cellular events involving incorporation and accumulation of GCVMP into DNA, disruption and inhibition of the cell cycle, gap junction metabolite transfer, and apoptosis. Because of all these interactions, defining the exact sequential processes involved and the primary cellular targets of GCV metabolites has proven difficult. In our report, we describe a new approach to characterizing the action of GCV by using HSV-1 TKs with altered and distinct enzymatic properties. By using the HCT-116 cells that have been previously characterized for bystander effect sensitivity (12) and apoptosis events, 3 and by normalizing expression levels of the HSV-1 TKs in these cell lines, we sought to establish a cell system in which the major variable for GCV sensitivity would be the inherent activity of the expressed HSV-1 TK. Comparing the effects of the expressed Glu-125 TK Each of the three HSV-1 TK-expressing cell lines were plated with parental HCT-116 cells (total 2 ϫ 10 5 /well) in the following proportions: (parental:HSV-1 TK cells) 100%:0%, 95%:5%, 90%:10%, 75%:25%, 50%: 50%, and 0%:100%. Cells were treated with 25 M GCV for 24 h, and then each well of cells was sequentially diluted from 1:10 to 1:10,000 in 1 ml of fresh medium on a separate 24-well plate as described under "Materials and Methods." After 7 days, surviving cell colonies were fixed in 100% methanol, stained with 0.1% methylene blue, and counted. enzymes with wild-type HSV-1 TK in the HCT-116 cells led to the unexpected observations of differential intracellular responses and cell death mechanisms in response to GCV as indicated by the DAPI staining, cell cycle analysis, and caspase 3 assays.
The expressed Asn-125 TK mutant appears to act identically to wild-type HSV-1 TK when expressed in HCT-116 and NIH3T3 cells, the only major difference being the lower metabolism of deoxypyrimidine substrates by the Asn-125 TK. The catalytic efficiency (k cat /K m ) of the purified Asn-125 TK and Glu-125 TK enzymes for GCV have been determined to be four times and 82 times lower, respectively, than wild-type HSV-1 TK, 2 yet both enzymes were still effective at mediating GCV killing of HCT-116 tumor cells. This lower efficiency did not seem to affect the amount of GCV metabolites generated by the expressed Asn-125 TK, because they were equivalent to wildtype HSV-1 TK (Tables I and II). The Glu-125 TK proved ineffective when expressed in NIH3T3 cells, which was also reflected by the GCV metabolite levels. These very low GCV metabolite levels and minimal DNA incorporation are likely the reasons for this inactivity in the NIH3T3 cells, but why this occurs is not clear. It is possible that the NIH3T3 cells have comparatively more active nucleotide phosphatase or efflux activities than the HCT-116 cell lines. These activities could be saturated in the wild-type or Asn-125 TK-expressing cells but not in the lower metabolizing Glu-125 TK-expressing cells. In contrast, the low amount of GCVTP generated in the Glu-125 TK-expressing HCT-116 cells was sufficient to cause over a 2-log reduction in cell colonies (Fig. 1) and generate effective bystander killing (Fig. 2).
In several studies that analyzed the effects of GCV on cell cycle inhibition and DNA synthesis in HSV-1 TK-expressing tumor cells, the general conclusion was that the primary mechanistic action of GCV was related to its accumulation and incorporation into DNA (5,15,16). The resulting DNA damage and instability activates cell cycle regulatory proteins that lead to cell cycle arrest in S and early G 2 phases (15)(16)(17). Eventually this cell cycle inhibition leads to cell death via apoptosis (5,15) or by a nonapoptotic process (16,17). Interestingly, two different cell death mechanisms were described for the same murine melanoma B16F10 cell lines in response to GCV. One study demonstrated induction of apoptosis in response to GCV (15), whereas the other concluded that cell cycle arrest caused cell death and did not require induction of apoptosis (16). This is intriguing because our data suggest that distinct cell death responses to GCV are occurring in the same HCT-116 cell line. It was also demonstrated that GCV metabolites had no direct effects on the synthesis of DNA (5), this despite in vitro studies showing that GCVTP is a selective inhibitor of DNA polymerase ␦ (K i ϭ 2 M) (7). Another critical pharmacological feature of GCV related to its incorporation into DNA is its ability to cause multi-log cell killing at short exposure times and very low drug doses compared with other nucleoside drugs like 1-␤-Darabinofuranosylthymine or acyclovir (5,25). As can also be seen in our results in Figs. 1 and 2, low doses of GCV can cause significant cell killing (5). It is likely this low dose aspect of GCV that is the primary reason for its effectiveness in mediating the bystander effect (13). Using the same GCV metabolite analysis procedure as described herein, we have detected a range of GCVTP metabolite levels that vary widely between different tumor cell lines (ranging from 0.5 to 533 pmol/10 6 cells) (12,29). Even the lowest range of GCVTP levels were effective for the particular cell line analyzed. In regards to our mutant Glu-125 TK, it is likely that expression of this gene in other cell lines will result in similar variable killing and dose effects (as seen with the NIH3T3 and HCT-116 cells) depending on the inherent properties of each cell line.
How do the results from the Asn-125 TK-and Glu-125 TKexpressing HCT-116 cells relate to the previously reported mechanistic studies of GCV? Clearly the metabolic labeling and cell killing data for the poor GCV metabolizing Glu-125 TK highlights the low dose effectiveness of the drug in killing tumor cells. Especially noteworthy is the ability of 10% Glu-125 TK-expressing cells to cause a 1-log reduction in cell colonies in the bystander effect assays (Fig. 2). Whatever the primary cellular target of GCVTP is in the HCT-116 cells, it is ex- tremely sensitive to low levels of this metabolite. Results of our cell cycle analysis of the three HSV-1 TK-expressing cell lines (Fig. 3), although not extensive, were still consistent with the previous reports of GCV causing accumulation of cells in S phase (5,(15)(16)(17). The profile differences between the Glu-125 TK cells and the wild-type and Asn-125 TK cells underscore the observed differences in GCV dose responses between the three cell lines (Fig. 3 and Table III). The nuclear swelling detected after DAPI staining of the GCV-treated wild-type and Asn-125 TK-expressing cells has been previously described for GCVtreated B16F10.TK cells in culture or from the same cells extracted from in vivo tumor studies (16). In this same study, it was noted that once the cells became swelled in response to GCV, this was an irreversible event that eventually led to cell death (16). For our Glu-125 TK cells treated with GCV, this nuclear swelling was delayed and not as extensive as compared with the wild-type or Asn-125 TK cells (Fig. 4). Also, the numbers of fragmented nuclei indicative of apoptosis were much less in the GCV-treated Glu-125 TK cells as compared with the other two cell lines (Fig. 4). Lastly, consistent with the DAPI staining results, there was approximately three times lower activation of caspase 3 in the Glu-125 TK cells treated with GCV as compared with the wild-type and Asn-125 TK cell lines. The cumulative data indicate a comprehensively different response of the Glu-125 TK-expressing HCT-116 cells to GCV than for HCT-116 cells expressing wild-type HSV-1 TK or Asn-125 TK. We hypothesize that the Glu-125 TK-expressing HCT-116 cells are undergoing necrosis in response to GCV rather than apoptosis; however, it remains to be proven. A necrotic response to GCV has been demonstrated in vivo with the B16 melanoma model, and it was determined that a necrotic cell death in response to GCV was more immunostimulatory than apoptotic cell death (30).
Based on the fact that the Glu-125 TK-expressing cells only require minimal levels of GCVTP to ultimately lead to some form of cell death, it is apparent that the levels of GCVTP generated in the wild-type and Asn-125 TK-expressing cells are in large excess of those required to kill the cell. We propose that in the wild-type and Asn-125 TK-expressing cells, the excess GCVTP and possibly other GCV metabolites saturate the primary cellular target and with time accumulate and interact with other secondary or tertiary cellular targets. As an acyclic analog of both dGTP and GTP, GCVTP could act as a mimic of GTP and inhibit GTPases or other GTP-binding proteins involved in the regulation of the cell cycle, signal transduction cascades, and/or cytoskeletal organization. This secondary or tertiary inhibition of cellular targets by GCVTP could be the cause of, or at least potentiate, the induction of apoptosis. This excess in GCVTP would not accumulate to the levels observed for the wild-type or Asn-125 TK cell lines in the Glu-125 TK cells, and thus the result is less of an apoptotic response. We have observed that GCV treatment of wild-type HSV-1 TKexpressing HCT-116 cells leads to modulation of protein levels of the Bcl-2 family members, Bak and Bcl-X L . 3 Also, co-incubation of GCV with the protein kinase inhibitor, UCN-01 (7hydroxystaurosporine), in these same cells can increase the amount and rate of the apoptotic response. 3 Although these latter observations are consistent with the proposed secondary and tertiary effects of GCVTP on apoptosis, our hypothesis remains to be tested. Utilizing the Glu-125 TK mutant for comparative analysis with wild-type expressing HSV-1 TK cells should allow us to test this hypothesis in the HCT-116 cells and other cell lines. More extensive cell cycle studies and analysis of changes in cell cycle regulatory proteins in response to GCV should prove most informative in this regard. Because of the in vivo stimulation of the immune system in response to Parental HCT-116, wild-type HSV-1 TK-, Asn-125 TK-, and Glu-125 TK-expressing HCT-116 cells were treated with 0 (black bars) or 25 M GCV (gray bars) for 4 days. Cell lysates were prepared and caspase 3 assays performed using a colorimetric ApoAlert CPP32/Caspase-3 kit according to the manufacturer's instructions (CLONTECH). Relative caspase 3 activity was determined by A 405 nm readings of the cleaved DEVD-pNA substrate and presented as DVEDase activity.  GCV treatment in many animal studies (18 -24, 30), it will also be interesting to determine whether the altered cell death response in tumors expressing the Glu-125 TK translates to any differences in the in vivo immune response in animal tumor models.