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• W1969203174 abstract "A novel gap junction-independent mechanism for ganciclovir-mediated bystander effect killing by a herpes simplex virus thymidine kinase (HSV-TK)-expressing SW620 human colon tumor cell line has been characterized. The mechanism of the HSV-TK/GCV bystander effect for many tumor cell lines has been demonstrated to be due to connexin gap junction transfer of phosphorylated ganciclovir (GCV) metabolites; however, there may be as yet uncharacterized connexin-independent mechanisms for the effect. To address this, the bystander effect was further evaluated in a panel of cell lines mixed with homologous HSV-TK-expressing cell lines, a SW620.TK cell line, or a high connexin43-expressing PA-317.TK cell line. Of the 10 cell lines tested, 4 were found to be resistant to bystander effect killing by their homologous HSV-TK-expressing cell lines and the PA-317.TK cells, but all of the cell lines were sensitive to GCV killing when mixed with the SW620.TK cells. The SW620.TK cells were then further evaluated for any indication of extracellular GCV metabolite efflux. Culture medium from SW620.TK cells labeled with [3H]GCV was evaluated for the presence of GCV nucleotides by ion-exchange column separation and HPLC analysis. The presence of GCV mono-, di-, and triphosphate metabolites in the medium was detected. Inclusion in the medium of inhibitors of extracellular phosphatases and ecto-ATPases increased the proportion of GCV metabolites recovered. These results indicate that phosphorylated GCV metabolites can be effluxed from SW620.TK cells and that some type of cellular uptake mechanism independent of gap junctions exists for nucleotide entry into neighboring cells. A novel gap junction-independent mechanism for ganciclovir-mediated bystander effect killing by a herpes simplex virus thymidine kinase (HSV-TK)-expressing SW620 human colon tumor cell line has been characterized. The mechanism of the HSV-TK/GCV bystander effect for many tumor cell lines has been demonstrated to be due to connexin gap junction transfer of phosphorylated ganciclovir (GCV) metabolites; however, there may be as yet uncharacterized connexin-independent mechanisms for the effect. To address this, the bystander effect was further evaluated in a panel of cell lines mixed with homologous HSV-TK-expressing cell lines, a SW620.TK cell line, or a high connexin43-expressing PA-317.TK cell line. Of the 10 cell lines tested, 4 were found to be resistant to bystander effect killing by their homologous HSV-TK-expressing cell lines and the PA-317.TK cells, but all of the cell lines were sensitive to GCV killing when mixed with the SW620.TK cells. The SW620.TK cells were then further evaluated for any indication of extracellular GCV metabolite efflux. Culture medium from SW620.TK cells labeled with [3H]GCV was evaluated for the presence of GCV nucleotides by ion-exchange column separation and HPLC analysis. The presence of GCV mono-, di-, and triphosphate metabolites in the medium was detected. Inclusion in the medium of inhibitors of extracellular phosphatases and ecto-ATPases increased the proportion of GCV metabolites recovered. These results indicate that phosphorylated GCV metabolites can be effluxed from SW620.TK cells and that some type of cellular uptake mechanism independent of gap junctions exists for nucleotide entry into neighboring cells. The herpes simplex virus thymidine kinase (HSV-TK) bystander effect describes the cell-killing phenomena by which tumor cells expressing HSV-TK are able to transfer ganciclovir (GCV) metabolites to adjacent tumor cells that do not express HSV-TK (1Culver K.W. Ram Z. Walbridge S. Ishii H. Oldfield E.H. Blaese R.M. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors.Science. 1992; 256: 1550-1552Crossref PubMed Scopus (1461) Google Scholar, 2Freeman S.M. et al.The “bystander effect”: Tumor regression when a fraction of the tumor mass is genetically modified.Cancer Res. 1993; 53: 5274-5283PubMed Google Scholar). To maximize the efficacy of gene delivery vectors currently used in clinical trials, the bystander effect is considered to be a major prerequisite for employing HSV-TK/GCV therapies (3Freeman S.M. Whartenby K.A. Freeman J.L. Abboud C.N. Marrogi A.J. In situ use of suicide genes for cancer therapy.Semin. Oncol. 1996; 23: 31-45PubMed Google Scholar, 4Denning C. Pitts J.D. Bystander effects of different enzyme–prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes.Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar). When expressed in cells, HSV-TK specifically phosphorylates GCV to its monophosphorylated form, GCVMP (5Moolten F.L. Wells J.M. Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors.J. Natl. Cancer Inst. 1990; 82: 297-300Crossref PubMed Scopus (500) Google Scholar). Cellular kinases further convert GCVMP to the toxic triphosphate metabolite, GCVTP, which becomes incorporated into DNA. This leads to cell cycle arrest and other cellular effects that eventually result in cell death (6Drake R.R. Wilbert T.N. Hinds T.A. Gilbert K. Differential ganciclovir mediated cell killing by glutamine-125 mutants of herpes simplex virus type 1 thymidine kinase.J. Biol. Chem. 1999; 274: 37186-37192Crossref Scopus (23) Google Scholar, 7Wei S. et al.S- and G2-phase cycle arrests and apoptosis induced by ganciclovir in murine melanoma cells transduced with herpes simplex virus thymidine kinase.Exp. Cell Res. 1998; 241: 66-75Crossref PubMed Scopus (75) Google Scholar, 8Rubsam L.Z. Davidson B.L. Shewach D.S. Superior cytotoxicity with ganciclovir compared with acyclovir and 1-β-D-arabinofuranosylthymine in herpes simplex virus–thymidine kinase-expressing cells: A novel paradigm for cell killing.Cancer Res. 1998; 58: 3873-3882PubMed Google Scholar, 9Halloran P.J. Fenton R.G. Irreversible G2-M arrest in cytoskeletal reorganization induced by cytotoxic nucleoside analogues.Cancer Res. 1998; 58: 3855-3865PubMed Google Scholar). In vitro evidence supports connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC) as a major mechanism for the transfer of GCV metabolites to neighboring cells (4Denning C. Pitts J.D. Bystander effects of different enzyme–prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes.Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 11Bi W.L. Parysek L.M. Warnick R. Stambrook P.J. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy.Hum. Gene Ther. 1993; 4: 725-731Crossref PubMed Scopus (455) Google Scholar, 12Elshami A.A. et al.Gap junctions play a role in the ‘bystander effect’ of the herpes simplex virus thymidine kinase/ganciclovir system in vitro.Gene Ther. 1996; 3: 85-92PubMed Google Scholar, 13Mesnil M. Piccoli C. Tiraby G. Willecke K. Yamasaki H. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.Proc. Natl. Acad. Sci. USA. 1996; 93: 1831-1835Crossref PubMed Scopus (402) Google Scholar, 14Yang L. et al.Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells.Hum. Gene Ther. 1998; 9: 719-728Crossref PubMed Scopus (85) Google Scholar). This transfer of GCV metabolites occurs within 2–4 h of GCV addition (4Denning C. Pitts J.D. Bystander effects of different enzyme–prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes.Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 15Boucher P.D. Ruch R.J. Shewach D.S. Differential ganciclovir mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.Hum. Gene Ther. 1998; 9: 801-814Crossref PubMed Scopus (62) Google Scholar), a process that precedes the onset of apoptotic cell death that generally occurs 24–96 h later (4Denning C. Pitts J.D. Bystander effects of different enzyme–prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes.Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 16McMasters R. et al.Evaluation of two-drug combinations that increase apoptosis and modulate Bak and BcI-XL expression in human colon tumor cell lines transduced with HSV-thymidine kinase.Cancer Gene Ther. 2000; 7: 563-573Crossref PubMed Scopus (16) Google Scholar). The ability of different malignant tumor systems and cell types to mediate gap junction transfer of GCV metabolites has proven to be quite variable (4Denning C. Pitts J.D. Bystander effects of different enzyme–prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes.Hum. Gene Ther. 1997; 8: 1825-1835Crossref PubMed Scopus (92) Google Scholar, 17Beck C. Cayeux S. Lupton S.D. Dorken B. Blankenstein T. The thymidine kinase/ganciclovir-mediated “suicide” effect is variable in different tumor cells.Hum. Gene Ther. 1995; 6: 1525-1530Crossref PubMed Scopus (86) Google Scholar, 18Ishii-Morita H. et al.Mechanism of ‘bystander effect’ killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment.Gene Ther. 1997; 4: 244-251Crossref PubMed Scopus (139) Google Scholar). In colon tumor systems, for example, the magnitude of the HSV-TK/GCV bystander effect in vivo and in vitro has been generally moderate (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 18Ishii-Morita H. et al.Mechanism of ‘bystander effect’ killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment.Gene Ther. 1997; 4: 244-251Crossref PubMed Scopus (139) Google Scholar, 19Trinh Q.T. Austin E.A. Murray D.M. Knick V.C. Huber B.E. Enzyme/prodrug gene therapy: Comparison of cytosine deaminase; 5-fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma line.Cancer Res. 1995; 55: 4808-4812PubMed Google Scholar). Of the many connexin isotypes that comprise gap junction channels, Cx43 appears to be the primary isotype responsible for the HSV-TK bystander effect in colon cells (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 14Yang L. et al.Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells.Hum. Gene Ther. 1998; 9: 719-728Crossref PubMed Scopus (85) Google Scholar, 18Ishii-Morita H. et al.Mechanism of ‘bystander effect’ killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment.Gene Ther. 1997; 4: 244-251Crossref PubMed Scopus (139) Google Scholar). A previous report evaluated the potential therapeutic application of coexpression of Cx43 with HSV-TK in three human colon tumor cell lines, of which, one line was sensitive and two were resistant to bystander effect killing. In the bystander sensitive cells, transfection of Cx43 increased the level of HSV-TK bystander effect killing, while in the two bystander-resistant cell lines, Cx43 transfection did not alter bystander resistance. This resistance was attributed to lack of surface localization of gap junctional plaques, even after stable transfection and expression of Cx43 (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). While it is clear that connexin gap junctions are major functional participants in the cell–cell transfer of GCV metabolites, it does not appear to be the only mechanism. One of the first reports of a different mechanism was reported in an HSV-TK-expressing human colon tumor cell line, SW620 (15Boucher P.D. Ruch R.J. Shewach D.S. Differential ganciclovir mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.Hum. Gene Ther. 1998; 9: 801-814Crossref PubMed Scopus (62) Google Scholar). This cell line was highly sensitive to HSV-TK/GCV bystander effect killing, even though it was demonstrated that these cells had both minimal gap junction dye transfer capability and diffuse Cx-32 expression. The expression and functionality of Cx43 in SW620.TK cells has not been previously reported. Additionally, this effect in SW620 cells was not due to apoptotic vesicle transfer of GCV metabolites, as has been proposed as a mechanism for bystander effect killing in other cell lines (2Freeman S.M. et al.The “bystander effect”: Tumor regression when a fraction of the tumor mass is genetically modified.Cancer Res. 1993; 53: 5274-5283PubMed Google Scholar). It was therefore hypothesized that a new mechanism distinct from gap junction transfer was responsible for bystander effect killing in the SW620 cells (15Boucher P.D. Ruch R.J. Shewach D.S. Differential ganciclovir mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.Hum. Gene Ther. 1998; 9: 801-814Crossref PubMed Scopus (62) Google Scholar). Other studies have examined the role of chemical inhibitors and enhancers of gap junction communication and their effects on HSV-TK/GCV bystander effect killing in human lung carcinoma cell lines (20Imaizumi K. et al.Bystander tumoricidal effect and gap junctional communication in lung cancer cell lines.Am. J. Respir. Cell Mol. Biol. 1998; 18: 205-212Crossref Scopus (33) Google Scholar), as well as a rat colon tumor line (21Princen F. et al.A cell type-specific and gap junction independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.Clin. Cancer Res. 1999; 5: 3639-3644PubMed Google Scholar). Similar to the SW620 cells, apparent gap junction-independent bystander killing was reported in these cell lines (20Imaizumi K. et al.Bystander tumoricidal effect and gap junctional communication in lung cancer cell lines.Am. J. Respir. Cell Mol. Biol. 1998; 18: 205-212Crossref Scopus (33) Google Scholar, 21Princen F. et al.A cell type-specific and gap junction independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.Clin. Cancer Res. 1999; 5: 3639-3644PubMed Google Scholar). It was also demonstrated that conditioned GCV-treated medium from the HSV-TK-expressing rat colon tumor cells could induce cell killing of the parent cell line that lacked HSV-TK expression (21Princen F. et al.A cell type-specific and gap junction independent mechanism for the herpes simplex virus-1 thymidine kinase gene/ganciclovir-mediated bystander effect.Clin. Cancer Res. 1999; 5: 3639-3644PubMed Google Scholar). Another study cocultured HSV-TK-expressing and parental rat glioma cells in separate chambers that shared the same medium. Besides killing the HSV-TK-expressing cells, the parental cells also displayed sensitivity to GCV treatment by some type of soluble factor, most likely a GCV metabolite or by-product of cellular apoptosis of the dying HSV-TK-expressing cells (22Bai S. Du L. Lui W. Whittle I.R. He L. Tentative novel mechanism of the bystander effect in glioma gene therapy with HSV-TK/GCV system.Biochem. Biophys. Res. Commun. 1999; 259: 455-459Crossref PubMed Scopus (26) Google Scholar). These preceding studies did not examine the mechanistic details of how the apparent gap junction-independent bystander effect was occurring. To begin to address this, we investigated whether HSV-TK-expressing SW620 (SW620.TK) cells were capable of exclusively transferring GCV metabolites to other SW620 cells, or could they transfer metabolites to other cell line types. This was evaluated in a panel of 10 cell lines that were also compared for their sensitivity to Cx43-mediated bystander effect killing. Of the 10 cell lines tested, 4 were shown to be bystander resistant and insensitive to Cx43-mediated gap junction metabolite transfer. When mixed with SW620.TK cells in different proportions, all 10 cell lines were found to be sensitive to HSV-TK/GCV bystander killing in a cell concentration-dependent manner. To determine whether efflux of phosphorylated GCV metabolites could be part of this mechanism in the SW620.TK cells, medium from GCV treated SW620.TK cells was collected and analyzed for GCV metabolites. Under different conditions alone or in the presence of phosphatase inhibitors, it was found that mono-, di-, and triphosphate metabolites of GCV were isolated extracellularly from these cells. This apparent efflux of GCV metabolites thus represents a new mechanism by which HSV-TK/GCV bystander killing occurs. The fact that coculturing of the SW620.TK cells with normally bystander-resistant cells resulted in GCV-mediated cell killing indicates a potential for future gene therapies with HSV-TK and whatever protein factor(s) responsible for GCV metabolite efflux and transfer. Cell lines. All human tumor cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Cellgro). The murine fibroblast retroviral vector packaging cell line PA317 and NIH-3T3 cell lines were maintained in DMEM with 10% heat inactivation. Two human colon cell lines derived from normal colon tissues (23Moyer M.P. Manzano L.A. Merriman R.L. Staufffer J.S. Tanzer L.R. NCM460, a normal colon mucosal epithelial cell line.In Vitro Cell. Dev. Biol. Animal. 1996; 32: 315-317Crossref PubMed Scopus (197) Google Scholar) were grown in M3:10 medium (INCELL Corp., San Antonio, TX). A bicistronic HSV-TK (+) Moloney murine leukemia virus (Mo-MuLV) vector, pLTKEN, was used to generate HSV-TK (+) cell lines by transduction with recombinant retrovirus (HCT-8, HT-29, CSC-1) or by plasmid transfections (HCT-116, NCM-460, SW-480, SW-620, PA317, MCF-7, Hela) as previously described (6Drake R.R. Wilbert T.N. Hinds T.A. Gilbert K. Differential ganciclovir mediated cell killing by glutamine-125 mutants of herpes simplex virus type 1 thymidine kinase.J. Biol. Chem. 1999; 274: 37186-37192Crossref Scopus (23) Google Scholar, 10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). Clonal lines for each HSV-TK-expressing cell line were selected by resistance to G418 (200–600 μg/ml) and characterized for protein expression, GCV metabolite profiles, and sensitivity to GCV killing as previously described (6Drake R.R. Wilbert T.N. Hinds T.A. Gilbert K. Differential ganciclovir mediated cell killing by glutamine-125 mutants of herpes simplex virus type 1 thymidine kinase.J. Biol. Chem. 1999; 274: 37186-37192Crossref Scopus (23) Google Scholar, 10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). Based on these analyses, the 10 HSV-TK expressing cell lines can be grouped as follows regarding GCV metabolite production and HSV-TK expression: high, SW620.TK, HCT-116.TK, PA317.TK; medium, HT29.TK, MCF7.TK, SW480.TK, Hela.TK; low, HCT-8.TK, CSC.TK, NCM460.TK. A stably transfected SW620 cell line expressing connexin-43 was generated and characterized as previously described (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). Bystander effect clonal dilution assays. An HSV-1 TK expressing cell line was seeded in 24-well plates with one of the 10 parental cell lines (total 2 × 105/well) in the following proportions: (parental: HSV-1TK cells) 100%:0%; 90%:10%; 75%:25%; 50%:50% and 0%:100%. After 1 day, 10 μM GCV was added in 1 ml fresh medium. After 24 h, the medium was removed, and the cells were rinsed and then treated with trypsin. After addition of 1 ml fresh medium, each well of cells was then sequentially diluted from 1:10 to 1:10,000 in 1 ml of medium in a separate 24-well plate. After 7 days, surviving cell colonies were fixed in 100% methanol, stained with 0.1% methylene blue, and counted (6Drake R.R. Wilbert T.N. Hinds T.A. Gilbert K. Differential ganciclovir mediated cell killing by glutamine-125 mutants of herpes simplex virus type 1 thymidine kinase.J. Biol. Chem. 1999; 274: 37186-37192Crossref Scopus (23) Google Scholar). To evaluate cell plating density and the bystander effect, SW620 or HT29 cells were mixed in 1:1 ratios with the SW620.TK cells (2.5 × 105 total, n = 6) in plates having growth surface areas of either 1.8 cm2 (24-well plate), 4.9 cm2 (12-well plate), 9.6 cm2 (6-well plate) or 19.6 cm2 (60-cm dish). In triplicate for each condition, half of the cell samples were treated with 25 μM GCV for 24 h, while the other half received no drug. Following rinsing and trypsinization, the cells were then dilution plated as described above and surviving cell colonies were counted after 7 days. GCV metabolite efflux from SW-620.TK cells. SW620.TK cells were seeded in 6 well plates (0.5 × 106 cells/well). Two days later, 5 μCi [3H]GCV and 10 μM GCV were added in 1.5 ml fresh medium for 11 h. The labeling medium was removed and the cells rinsed twice with base medium (no added FBS). Each well received 1.25 ml of base medium plus the following: 10 μM GCV; 200 μM dATP + 10 μM GCV; 100 μM vanadate + 10 μM GCV; or no addition controls (three wells each per condition). Immediately after addition of the 1.25 ml medium and additives, a 150-μl aliquot from each well was removed for scintillation counting (time zero), and additional aliquots were removed at 2 and 5 h postaddition. At 5 h, the medium from each of the sets was pooled and diluted with water to 12 ml. Each pooled solution was loaded on an individual small Pasteur pipet DEAE-column. After loading, each column was rinsed with four column volumes of water. Bound nucleotides were eluted from the columns with 0.5 M ammonium bicarbonate. The unbound/washed and bound/eluted fractions were collected and aliquots counted for total radioactivity. For analysis of individual metabolites, the eluted samples (approximately 4 ml each) were coevaporated with methanol (3×) and triethylamine (to remove ammonia) under reduced pressure. The final samples were resuspended in 0.5–0.7 ml of methanol and stored at –20° C. Metabolites were further analyzed by thin layer chromatography separation or by HPLC analysis as previously described (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 15Boucher P.D. Ruch R.J. Shewach D.S. Differential ganciclovir mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.Hum. Gene Ther. 1998; 9: 801-814Crossref PubMed Scopus (62) Google Scholar). Radiolabeled nucleotides consistent with the migration of GCT/MP, GCVDP, and GCVTP were detected. To determine the amount of labeled GCV metabolite remaining within the cells on the plates (after 5 h efflux), the cells were removed with trypsin and cell numbers were determined with trypan blue counting. The cells were then pelleted and extracted in 70% methanol for 10 min as previously described (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). The resulting pellet (representing crude DNA) and supernatant (soluble nucleotide metabolites) were counted for total radioactivity. The role of Cx43 in the HSV-TK/GCV-mediated bystander effect killing has been firmly established for many tumor cell lines (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar, 11Bi W.L. Parysek L.M. Warnick R. Stambrook P.J. In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy.Hum. Gene Ther. 1993; 4: 725-731Crossref PubMed Scopus (455) Google Scholar, 12Elshami A.A. et al.Gap junctions play a role in the ‘bystander effect’ of the herpes simplex virus thymidine kinase/ganciclovir system in vitro.Gene Ther. 1996; 3: 85-92PubMed Google Scholar, 13Mesnil M. Piccoli C. Tiraby G. Willecke K. Yamasaki H. Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.Proc. Natl. Acad. Sci. USA. 1996; 93: 1831-1835Crossref PubMed Scopus (402) Google Scholar, 14Yang L. et al.Intercellular communication mediates the bystander effect during herpes simplex thymidine kinase/ganciclovir-based gene therapy of human gastrointestinal tumor cells.Hum. Gene Ther. 1998; 9: 719-728Crossref PubMed Scopus (85) Google Scholar). In a previous study, the function of Cx43 in HT-29, HCT-8, and HCT-116 human colon tumor cell lines was evaluated (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). One main conclusion from this study was that expression of Cx43 alone was not enough to confer bystander effect killing, it also had to be localized to the cell surface (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar). In a separate study, the colon tumor SW-620 cell line was reported to be highly sensitive to HSV-TK/GCV bystander effect killing, but only minimal gap junction communication between cells was detected by dye transfer analysis (15Boucher P.D. Ruch R.J. Shewach D.S. Differential ganciclovir mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.Hum. Gene Ther. 1998; 9: 801-814Crossref PubMed Scopus (62) Google Scholar). The role, if any, of Cx43 in the HSV-TK bystander effect in SW620 cells has not been previously reported. To further evaluate the HSV-TK/GCV bystander effect mechanism in the SW-620.TK cell lines, a series of cell-mixing experiments were performed with 10 cell lines (5 colon tumor, 2 normal colon, 1 breast tumor, 1 cervical tumor, 1 mouse fibroblast). Three experimental approaches involving mixing of the 10 parental cell lines with different proportions of an HSV-TK-expressing cell line (10, 25, 50%) were done. The HSV-TK-expressing cell line used was either an HSV-TK cell line homologous to the parental cell line, the SW-620.TK cell line, or the PA317.TK cell line. The PA-317.TK cells functionally express a high level of Cx43 (10McMasters R. Jones K.E. Saylors R.L. Hendrix E.M. Moyer M.P. Drake R.R. Lack of bystander killing in HSV-TK transduced colon cell lines due to deficient connexin 43 gap junction formation.Hum. Gene Ther. 1998; 9: 2253-2261Crossref PubMed Scopus (69) Google Scholar) and thus were used as indicators for Cx43-mediated bystander effect killing. Using a modified clonal dilution assay (6Drake R.R. Wilbert T.N. Hinds T.A. Gilbert K. Differential ganciclovir mediated cell killing by glutamine-125 mutants of herpes simplex virus type 1 thymidine kinase.J. Biol. Chem. 1999; 274: 37186-37192Crossref Scopus (23) Google Scholar), confluent cells were treated with 10 μM GCV for 24 h, followed by drug removal and cell dilutions (1:10 to 1:10,000). Resulting cell colonies were counted after 7 days to assess cell survival and the amount of bystander effect killing (6Drake R.R. Wilbert T.N. Hinds T.A. Gilbert K. Differential ganciclovir mediated cell killing by glutamine-125 mutants of herpes simplex virus type 1 thymidine kinase.J. Biol. Chem. 1999; 274: 37186-37192Crossref Scopus (23) Google Scholar). The bystander effect sensitivities of the 10 cel" @default.
• W1969203174 created "2016-06-24" @default.
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• W1969203174 date "2000-11-01" @default.
• W1969203174 modified "2023-09-27" @default.
• W1969203174 title "Connexin-Independent Ganciclovir-Mediated Killing Conferred on Bystander Effect-Resistant Cell Lines by a Herpes Simplex Virus–Thymidine Kinase-Expressing Colon Cell Line" @default.
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