Monitoring of sodium influx in cells expressing the mutant ASIC2^G430F channel. Gli36 cells were infected with either HET14B-ASIC2^wt or HET14B-ASIC2^G430F amplicon vectors. Twenty four hours postinfection, cells were incubated with dox and analyzed for sodium influx 6–8 hours later. (a,b) Representative whole-cell patch-clamping on cells expressing ASIC2^wt or ASIC2^G430F channels. (a) Current over time at two voltages as determined by voltage sweeps (black trace at ‐60 mV and red trace at +60 mV). The hatched bars above show presence of 250 µmol/l amiloride in the bath and white bar shows switching to regular extracellular solution to wash off the amiloride block. (b) Current voltage graph determined by single voltage sweeps in either 250 µmol/l amiloride (black line, taken at first vertical line in a) or after washout (red line, taken at second vertical line in a). (c,d) Gli36Gluc cells were infected to express ASIC2^G430F. (c) Images are shown from the same field. Expression of enhanced GFP indicated 10% transduction efficiency (top left). Cells were incubated with dox to induce the mutant sodium channel expression. Six hours later, cells were loaded with SBFI-AM (top right, red) and the intracellular sodium was imaged in both ASIC2^G430F-expressing cell (GFP positive; n = 63) and nontransduced control cells (GFP negative; n = 211) by monitoring the SBFI ratio of fluorescence intensities at 340/380 nm using an Olympus IX81 microscope. False colors representation of SBFI excitation ratio values recorded in Gli36Gluc cells infected with HET14B-ASIC2^G430F showing the Na elevation in ASIC2^G430F-expressing cells (yellow cells) and not the nonexpressing cells [(c) bottom; purple cells]. (d) Distribution of cells at various 340/380 ratios. A significant increase in the number of cells with higher intracellular sodium levels were seen in cells expressing the ASIC2^G430F (GFP) as compared to nonexpressing control cells. [*P = 1.6 × 10^‐11, χ²-test comparing ASIC2^G430F-expressing cells (green) and control (nongreen cells)]. GFP, green fluorescent protein.

In vivo tumor therapy with herpes simplex virus-1/Epstein–Barr virus amplicon vector encoding ASIC2^G430F mutant sodium channel. (a) Gli36Gluc cells were implanted subcutaneously in both flanks of nude mice. Ten days postimplantation (arrow) when tumors were ~80 mm³, the left side tumor received intratumoral injection of the vector carrying the expression cassette for the ASIC2^wt control channel and the right side received vector with the expression cassette for the ASIC2^G430F mutant channel (10⁵ tu in 10 µl/tumor). Dox (400 µg/ml with 5% sucrose) was included in the mice drinking water after the first injection. Two representative animals from this group are shown over time (top and middle panels). In another control group, both tumors were injected with PBS (bottom panel). Tumor growth and response to therapy was monitored over time by in vivo Gluc bioluminescence after intravenous injection of coelenterazine and acquisition of photon counts using a charge-coupled device camera. (b) Tumor volumes were quantitated by photon counting and averaging over tumors in the ASIC2^wt and ASIC2^G430F vector-treated animals (n = 5 per group). Mean photon counts ± SD are shown. Arrow indicates time of first vector injection. Asterisk indicates a level of significance of *P < 0.005 and **P < 0.001 (calculated by Student's t-test) between tumors treated with control and mutant vectors. (c) Caliper measurements of tumor diameter converted to volume immediately before vector injection (10 days postimplantation) and at the last time point of the experiment (34 days postimplantation) (**P < 0.001). (d) Amplicon vector (1 µl; 2 × 10⁴ tu) expressing ASIC2^G430F or PBS were stereotactically injected into the striatum of nude mice brain. Dox was included in the drinking water immediately after injection. Three days postinjection, mice were killed and brains were removed, postfixed in paraformaldehyde and formalin/sucrose, frozen, and sectioned into 20 µm sections. Sections were mounted on slides and evaluated by hematoxylin and eosin staining. Arrow shows the injection site. Bar = 100 µm. PBS, phosphate-buffered saline.

Cloning and expression of the mutant and wild-type sodium channel in HSV/EBV hybrid vector. (a) cDNAs for the mutant (G430F) and the wild-type ASIC2a sodium channels were cloned into the HSV/EBV hybrid amplicon, pHET14B under the control of a bidirectional tet responsive promoter. Components of the amplicon are described in Methods. (b,c) CHO cells were transduced with pHET14B-ASIC2^wt or pHET14B-ASIC2^G430F in the presence of dox with or without amiloride. (b) Viable cells expressing the wild-type channel were replated on coverslips, fixed, and immunostained for the ASIC2 channel. Green fluorescence shows that the infected cells are expressing eGFP (top left); red fluorescence signals that the transgene is turned on by doxy (top right); blue represents the ASIC2 immunostaining (bottom left). An overlay of all images is also shown (bottom right). Bar = 20 µm. (c) Cells were lysed 16 hours post-transfection and proteins (100 µg/sample) were analyzed by SDS-PAGE and western blotting using the ASIC2 antibodies. The ASIC2 monomer is 59 kd. CNV, cytomegalovirus; eGFP, green fluorescent protein; HSV/EBV, herpes simplex virus-1/Epstein–Barr virus; IE, immediate early; RFP, red fluorescent protein.

Tumor cell death caused by expression of the mutant ASIC2^G430F sodium channel. Gli36Gluc cells were infected with the amplicon vectors carrying the expression cassettes for either wild-type ASIC2^wt or the mutant ASIC2^G430F channel. (a) Sixteen hours postinfection and dox treatment, 10 µl aliquots of the conditioned medium were analyzed for Gluc activity as a measure of cell viability. (b) WST-1 viability assay was carried out in the same wells used for Gluc assessment, confirming cell death only in the ASIC2^G430F + dox wells. (c) To assess the bystander killing effect, cells infected with vector encoding either the wild type ASIC2^wt or mutant ASIC2^G430F channel were mixed in a 1:3 ratio with noninfected Gli36Fluc cells and plated together as a monolayer in the presence or absence of AGA, an inhibitor of gap junctions, and then gene expression was induced with dox. Sixteen hours later, cells were lysed and analyzed for Fluc activity. (d) To evaluate gap junctional communication between Gli36Gluc cells, these cells were labeled with both Calcein-AM (green) and DiI (red) (top panel) and plated on top of a monolayer of nonlabeled cells (lower panel). Four hours later, clear transfer of the Calcein-AM dye to non-DiI-labeled cells was evident, supporting transfer through functional gap junctions. (e) Cells were infected with HET14B-ASIC2^G430F or HET14B-ASIC2^wt vectors and 24 hours later were placed in a 37 °C chamber on a confocal microscope and images were captured every 5 minutes over 12 hours after adding dox to the medium. Cells expressing the mutant channel as well as neighboring noninfected cells (arrows) became round 3 hours after induction with dox, indicating sodium and water influx, and were completely lysed by 12 hours with no sign of dsRed expression. On the contrary, the cells expressing the wild-type channel did not show any signs of toxicity and were positive for both dsRed and green fluorescent protein (yellow) by 12 hours after dox treatment. (f) Different types of tumor cells expressing Gluc were infected with HET14B-ASIC2^G430F or HET14B-ASIC2^wt control vector and incubated with dox. Twenty four hours later, an aliquot of the conditioned medium was assayed for Gluc activity. (g) Gli36Gluc cells were infected with either HET14B-ASIC2^G430F or herpes simplex virus-TK amplicon vectors and treated with dox or ganciclovir, respectively. At different time points, aliquots of the conditioned medium were assayed for Gluc activity. Results are expressed as percentage, in which the control cells of each type were set at 100%. In a–c and f,g, values are given as the mean ± SD with **P < 0.001 and *P < 0.005 as calculated by two-tailed Student's t-test. AGA, α-glycyrrhetinic acid; TK, thymidine kinase.