Differential Ganciclovir-mediated Cell Killing by Glutamine 125 Mutants of Herpes Simplex Virus Type 1 Thymidine Kinase
1999; Elsevier BV; Volume: 274; Issue: 52 Linguagem: Inglês
10.1074/jbc.274.52.37186
ISSN1083-351X
AutoresRichard R. Drake, Tasha N. Wilbert, Trenton A. Hinds, Kathleen M. Gilbert,
Tópico(s)Virus-based gene therapy research
ResumoThe 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. 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. herpes simplex virus type 1 thymidine kinase ganciclovir toxic triphosphate form of GCV 4′,6′-diamidine-2′-phenylindoledihydrochloride 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 (1Freeman S.M. Whartenby K.A. Freeman J.L. Abboud C.N. Marrogi A.J. Semin. Onocol. 1996; 23: 31-45PubMed Google Scholar, 2Klatzmann D. Valery C.A. Bensimon G. Marro B. Boyer O. Mokhtari K. Diquet B. Salzmann J.L. Philippon J. Hum. Gene Ther. 1998; 9: 2595-2604Crossref PubMed Google Scholar, 3Sterman D.H. Treat J. Litzky L.A. Amin K.M. Coonrod L. Molnar-Kimber K. Recio A. Knox L. Wilson J.M. Albelda S.M. Kaiser L.R. Hum. Gene Ther. 1998; 9: 1083-1092Crossref PubMed Scopus (317) Google Scholar, 4Bonini C. Ferrari G. Verzeletti S. Servida P. Zappone E. Ruggieri L. Ponzoni M. Rossini S. Mavilio F. Traversari C. Bordignon C. Science. 1997; 276: 1719-1724Crossref PubMed Scopus (1028) Google Scholar). 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 δ (5Rubsam L.Z. Davidson B.L. Shewach D.S. Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar, 6Smee D.F. Boehme R. Chenow M. Binko B.P. Matthews T.R. Biochem. Pharmacol. 1985; 34: 1049-1056Crossref PubMed Scopus (104) Google Scholar, 7Ilsley D.D. Lee S.H. Miller W.H. Kuchta R.D. Biochemistry. 1995; 34: 2504-2510Crossref PubMed Scopus (118) Google Scholar). 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 (1Freeman S.M. Whartenby K.A. Freeman J.L. Abboud C.N. Marrogi A.J. Semin. Onocol. 1996; 23: 31-45PubMed Google Scholar, 8Culver K.W. Ram Z. Wallbridge S. Ishii H. Oldfield E.H. Blaese R.M. Science. 1992; 256: 1550-1552Crossref PubMed Scopus (1449) Google Scholar, 9Freeman S.M. Abboud C.N. Whartenby K.A. Packman C.H. Koeplin D.S. Moolten F.L. Abraham G.N. Cancer Res. 1993; 53: 5274-5283PubMed Google Scholar).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 (10Bi W.L. Parysek L.M. Warnick R. Stambrook P.J. Hum. Gene Ther. 1993; 4: 725-731Crossref PubMed Scopus (453) Google Scholar, 11Denning C. Pitts J, D. Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 13Rubsam L.Z. Boucher P.D. Murphy P.J. KuKuruga M. Shewach D.S. Cancer Res. 1999; 59: 669-675PubMed Google Scholar). 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 (5Rubsam L.Z. Davidson B.L. Shewach D.S. Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar, 11Denning C. Pitts J, D. Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 14Hamel W. Magnelli L. Chiarugi V, P. Israel M.A. Cancer Res. 1996; 56: 2697-2702PubMed Google Scholar). Recently, GCV has been reported to induce S- and G2/M phase cell cycle arrest in HSV-1 TK-expressing cells (5Rubsam L.Z. Davidson B.L. Shewach D.S. Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar, 15Wei S. Chao Y. Hung Y. Lin W. Yang D. Shih Y. Ch'ang L. Whang-Peng J. Yang W.K. Exp. Cell Res. 1998; 241: 66-75Crossref PubMed Scopus (75) Google Scholar, 16Halloran P.J. Fenton R.G. Cancer Res. 1998; 58: 3855-3865PubMed Google Scholar, 17Kaneko Y. Tsukamoto A. Cancer Lett. 1995; 96: 105-110Crossref PubMed Scopus (47) Google Scholar), and these changes were associated with modulation of Cdc2/cyclin B activities (16Halloran P.J. Fenton R.G. Cancer Res. 1998; 58: 3855-3865PubMed Google Scholar) and increased levels of cyclin B1 (15Wei S. Chao Y. Hung Y. Lin W. Yang D. Shih Y. Ch'ang L. Whang-Peng J. Yang W.K. Exp. Cell Res. 1998; 241: 66-75Crossref PubMed Scopus (75) Google Scholar). 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 (1Freeman S.M. Whartenby K.A. Freeman J.L. Abboud C.N. Marrogi A.J. Semin. Onocol. 1996; 23: 31-45PubMed Google Scholar, 18Freeman S.M. Ramesh R. Marrogi A.J. Lancet. 1997; 349: 2-3Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 19Vile R.G. Castleden S. Marshall J. Camplejohn R. Upton C. Chong H. Int. J. Cancer. 1997; 71: 267-274Crossref PubMed Scopus (177) Google Scholar, 20Hall S.J. Sanford M.A. Atkinson G. Chen S.-H. Cancer Res. 1998; 58: 3221-3225PubMed Google Scholar, 21Mullen C.A. Anderson L. Woods K. Nishino M. Petropoulos D. Hum. Gene Ther. 1998; 9: 2019-2030Crossref PubMed Scopus (44) Google Scholar). 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 (22Bi W. Kim Y.-G. Feliciano E.S. Pavelic L. Wilson K.M. Pavelic Z.P. Stambrook P.J. Cancer Gene Ther. 1997; 4: 246-252PubMed Google Scholar, 23Kianmanesh A.R. Perrin H. Panis Y. Fabr M. Nagy H.J. Houssin D. Klatzmann D. Hum. Gene Ther. 1997; 8: 1807-1814Crossref PubMed Scopus (131) Google Scholar, 24Wei M.X. Bougnoux P. Sacre-Salem B. Peyrat M. Lhuillery C. Salzmann J. Klatzmann D. Cancer Res. 1998; 58: 3221-3225PubMed Google Scholar). 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. 2T. A. Hinds, C. Compadre, B. K. Hurlburt, and R. R. Drake, submitted for publication. 2T. A. Hinds, C. Compadre, B. K. Hurlburt, and R. R. Drake, submitted for publication. 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 higherK m for GCV and a 83-fold decrease ink 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. A Moloney murine leukemia virus derived plasmid for the expression of HSV-1 TK, termed pLENTK, has been previously constructed (12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). 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 μg of plasmid, 14 μl of lipid/1 × 106 cells). Cells were maintained in RPMI 1640 medium and selected with G418 (200 μg/ml for 2 weeks) as described previously (12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). 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 μm 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 × 106, 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. For metabolic labeling, cells (1–2 × 106) were labeled in triplicate with 2 μCi of [3H]GCV (8 μm) 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 (12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 25Drake R.R. McMasters R. Krisa S. Hume S.D. Rechtin T.M. Saylors R.L. Chiang Y. Govindarajan R. Munshi N.C. Antiviral Res. 1997; 35: 177-185Crossref PubMed Scopus (10) Google Scholar). 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-cm2 plates, and either 2 μCi of [3H]thymidine (2 μm final) or 2 μCi of [3H]dC (10 μm 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 (25Drake R.R. McMasters R. Krisa S. Hume S.D. Rechtin T.M. Saylors R.L. Chiang Y. Govindarajan R. Munshi N.C. Antiviral Res. 1997; 35: 177-185Crossref PubMed Scopus (10) Google Scholar). For determination of GCV sensitivity, parental HCT-116 and each HSV-1 TK-expressing cells were seeded in 24-well plates (2 × 105/well) in 1 ml of medium. The next day, 0, 0.1, 1, or 10 μm 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 × 105/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 μm 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. Parental HCT-116 cells and each HSV-1 TK-expressing cell line were plated (5 × 104 cells/well) in 8-well plastic chamber slides (Lab-Tek) and left untreated or treated with 25 μm GCV for 36 or 84 h. At either time point, cells were washed with phosphate-buffered saline followed by staining in 1 μg/ml DAPI in 100% methanol at 37 °C for 10 min (26Adam L. Crepin M. Savin C. Israel L. Cancer Res. 1995; 55: 5156-5160PubMed Google Scholar). After rinsing, the stained cells were visualized with a DAPI-specific filter on a Zeiss fluorescent microscope at 40× magnification. 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 μm 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-cm2 flasks, and cell numbers were determined using a hemocytometer prior to analysis. Assays were done in triplicate with protein extracts derived from 2 × 106 cells. The amount of Caspase 3-like activities were quantitated using a Shimadzu UV/VIS spectophotometer set at 405 nm. Parental HCT-116 cells and each HSV-1 TK-expressing cell line were grown to 60% confluency in 25-cm2 flasks and treated for 24 h plus or minus 25 μm 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 μg/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. 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. TheK m values for GCV varied from wild-type HSV-1 TK (69 μm), with the Asn-125 mutant decreasing (50 μm), the Glu-125 mutant increasing (473 μm), 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 (12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar) 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 [3H]GCV, [3H]thymidine, or [3H]dC. Cells were labeled with [3H]GCV for 18 h, and then nucleotides were extracted in ice-cold 70% methanol as described previously (12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar,25Drake R.R. McMasters R. Krisa S. Hume S.D. Rechtin T.M. Saylors R.L. Chiang Y. Govindarajan R. Munshi N.C. Antiviral Res. 1997; 35: 177-185Crossref PubMed Scopus (10) Google Scholar). The data in Table I summarize the amount of total nucleotide metabolites isolated in the methanol-soluble extracts (pmol/106 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 [3H]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 [3H]GCV metabolism was detected in the Asp-125 TK cells, and these cells were not further evaluated.Table ITotal methanol soluble and insoluble 3 H-labeled metabolitesCell LineTotal extracted metabolitesNIH3T3HCT-116[3H]GCV[3H]GvDNA[3H]GCV[3H]GvDNA[3H]T[3H]dCpmol/10 6 cellsParent2.82.81.11.22.41.1Gln-125 TK49848121614152.04.5Asn-125 TK5854210851338.01.2Glu-125 TK3.62.633273.30.9Asp-125 TKNDND1.01.6NDND Open table in a new tab The methanol-soluble metabolites were further separated into their constituent phosphorylated GCV metabolites by thin layer chromatography (25Drake R.R. McMasters R. Krisa S. Hume S.D. Rechtin T.M. Saylors R.L. Chiang Y. Govindarajan R. Munshi N.C. Antiviral Res. 1997; 35: 177-185Crossref PubMed Scopus (10) Google Scholar). 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.Table IIPhosphorylated metabolites of [3 H]GCV and [3 H]thymidine from HCT-116 and NIH3T3 cell clonesPhosphorylated metabolitesNIH3T3HCT-116GCVMPGCVDPGCVTPGCVMPGCVDPGCVTPTMPTTPpmol/10 6 cellsParent0.2000.400.40.10.2Gln-125TK WT1222232 (48)96101415 (30)1.29.5Asn-125TK1121272 (71)95104533 (44)0.20.9Glu-125TK001.6 (0.4)3.02.518 (5)0.20.4 Open table in a new tab 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 [3H]thymidine or [3H]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 [3H]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. 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 μm) 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, 0.1 μm GCV treatment led to a 2-log decrease in the wild-type and Asn-125 TK-expressing cell colony numbers. In the same cell lines treated with 10 μm GCV, a 4-log decrease in colony numbers were determined. In contrast, the Glu-125 TK-expressing cells treated with 0.1 GCV μm caused no reduction in cell colony numbers, whereas 1 or 10 μm GCV led to 0.7-log and 2.5-log decreases in colony numbers, respectively. At higher concentrations of GCV (20–110 μm), the the expressed Glu-125 TK led to over 3-log reductions in colony numbers (data not shown) It has been previously established that HCT-116 cells expressing HSV-1 TK are sensitive to bystander effect cell death via a connexin-43 gap junction-mediated transfer of GCV metabolites (12McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). To evaluate this effect in the HSV-1 TK 116 cell panel, clonal dilution assays were performed with different proportions of HSV-1 TK-expressing cells (5–50%) mixed with HCT-116 parental cells. Cell populations were treated with 25 μm 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. GCV has also been previously reported to induce S and G2/M phase cell cycle arrest in HSV-1 TK-expressing glioma and melanoma cell lines (5Rubsam L.Z. Davidson B.L. Shewach D.S. Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar, 15Wei S. Chao Y. Hung Y. Lin W. Yang D. Shih Y. Ch'ang L. Whang-Peng J. Yang W.K. Exp. Cell Res. 1998; 241: 66-75Crossref PubMed Scopus (75) Google Scholar, 16Halloran P.J. Fenton R.G. Cancer Res. 1998; 58: 3855-3865PubMed Google Scholar, 17Kaneko Y. Tsukamoto A. Cancer Lett. 1995; 96: 105-110Crossref PubMed Scopus (47) Google Scholar). 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, Dand F) led to an increase in the proportion of cells in the S phase and undetectable percentages in G2/M phase when compared with untreated cultures (Fig. 3, C andE). For the Glu-125 TK-expressing cells, over 60% of the GCV treated cells were in S phase, and 0% were in G2/M (Fig. 3 H). 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 (5Rubsam L.Z. Davidson B.L. Shewach D.S. Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar).Table IIICell cycle profiles of GCV-treated cell linesCell lineCell cycle phaseG0/G1SG2/M%HCT-116 (−GCV)43.335.021.7HCT-116 (+GCV)39.044.017.1WTGln-125TK (−GCV)65.818.415.8WTGln-125TK (+GCV)60.139.90Asn-125TK (−GCV)63.418.018.6Asn-125TK (+GCV)70.030.00Glu-125TK (−GCV)62.48.529.1Glu-125TK (+GCV)39.061.00 Open table in a new tab 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 (5Rubsam L.Z. Davidson B.L. Shewach D.S. Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar, 11Denning C. Pitts J, D. Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 14Hamel W. Magnelli L. Chiarugi V, P. Israel M.A. Cancer Res. 1996; 56: 2697-2702PubMed Google Scholar,15Wei S. Chao Y. Hung Y. Lin W. Yang D. Shih Y. Ch'ang L. Whang-Peng J. Yang W.K. Exp. Cell Res. 1998; 241: 66-75Crossref PubMed Scopus (75) Google Scholar). 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 (16Halloran P.J. Fenton R.G. Cancer Res. 1998; 58: 3855-3865PubMed Google Scholar, 17Kaneko Y. Tsukamoto A. Cancer Lett. 1995; 96: 105-110Crossref PubMed Scopus (47) Google Scholar). For the Glu-125 HSV-1 TK-expressing cells, 36 h of GCV treatment led to fewer swelled nuclei and little evidence of apoptotic nuclei, although disti
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