Cell lines
Human mesothelioma cell lines, CRL-5915, 5820, and 5830 were obtained from the American type culture collection (Rockville, MD, USA) with the permission of Prof. A Gazdar (MD Anderson Cancer Center, Texas, USA) [23]. Mouse mesothelioma cell lines, ABI (H-2d) from BALB/c mice, AE17 (H-2k) from CBA mice, and AC29 (H-2b) from C57BL/6 mice were obtained from Prof. B. Robinson, QEII Medical Centre, University of Western Australia, Nedlands, Australia [24]. Human ovarian tumor (teratocarcinoma) cell line, PA-1 and PA-STK were obtained from Prof. S Freeman, Tulane University Medical School, New Orleans, USA [17]. Three ovarian tumor cell lines already available in the laboratory were: OVC-432 (ovarian carcinoma, a kind gift from Dr C. Dohring, Basel Institute for Immunology, Basel, Switzerland [25], SKOV-3 (ovarian adenocarcinoma) [26], and OIB, an ovarian tumor cell line established by Prof. F Farzaneh in the Molecular Medicine Department [27]. All human cell lines were maintained in DMEM, supplemented with 10% Fetal Calf Serum (FCS) and 1% sodium pyruvate. In the case of mouse mesothelioma cells, the RPMI-medium was supplemented with 5% FCS, 20 mM HEPES, 50 mM 2-mercaptoethanol (2ME) and 2 mM glutamine.
Retroviral infection
Cells to be infected were seeded at a density of 5 × 105 cells per 10 cm plate, 24 h before infection. Virus producing cells were also grown to about 90% confluence, and the medium changed 10 h prior to harvest of the culture medium to ensure that fresh virus containing supernatant was used [28].
The medium from virus producing cells was removed and filtered through a 0.45 μm pore-size filter to remove cell debris but allowing passage of the viral vector through the filter. To enhance the retroviral infection, 8 μg/ml of polybrene (Sigma Aldrich, Poole, UK) was added to the culture medium. Polybrene is required for coating of target cells in order to neutralise their negative surface charge, therefore increasing the efficiency of infection [29, 30]. The medium from the cells to be infected was removed prior to infection, and the vector-polybrene mixture added. After 10 h of infection the medium was changed. 48 h after infection, the medium was removed and replaced with fresh medium containing G418 at 1 mg/ml. G418 resistant clones were expanded.
In vitro GCV-sensitivity studies on the tumor cell lines
The in vitro studies examining the effect of GCV on the HSV-TK expressing mesothelioma and ovarian carcinoma cells, as well as the untransduced cells, were performed in 96-well plates. Transduced and untransduced cells were plated in triplicate at two densities, 104 cells/well and 105 cells/well for comparison. After 2 days, the medium was replaced by fresh DMEM containing the indicated concentrations of GCV in the range 10,000–0.01 μM. Cells were then incubated at 37 °C in humidified 5% CO2 for 5 days. Sensitivity to GCV treatment was measured by using a colorimetric cell proliferation assay that measures viable cell dehydrogenase activity, the microculture tetrazolium cell proliferation assay [31]. 20 μl of 5 mg/ml MTT (Sigma Aldrich, Poole, UK) was added to each well for 3 h and the cells in each well were solubilised in 150 μl of MTT solubilisation solution. After overnight incubation, the optical density of each well was measured on a 96-well plate reader (Dynatech, Reading, UK) set at 570 nm wavelength. Known concentrations of cells were also plated, cultured in the presence of MTT, and similarly solubilized. The absorbance reading of these control cells represents the metabolic activity of a known number of cells and was used to generate a standard curve in Microsoft Excel. The absorbance reading for each sample (well) was directly compared with the standard curve, and the numbers of viable cells were determined accordingly.
Bystander killing effect studies
The bystander effect was determined by mixing HSV-TK expressing cells with untransfected cells at the indicated ratios. Cells were then plated in triplicate in 10 cm plates at two densities, 1 × 105 and 5 × 105 cell/plate to ensure cell–cell contact and to compare the in vitro effect of cell densities on the bystander effect. Two days later, the cells were treated with 50 μM GCV and incubated at 37 °C, 5% CO2 for 10–14 days. The plates were then stained with 2% methylene blue and stained cells were counted.
In order to calculate the effect of GCV on the mixed PA-STK and PA-1 populations, the number of colonies counted were expressed as a percentage of the total number of colonies in the co-cultured TK + ve and TK − ve tumor cells at the indicated ratios of the two cell populations in the presence of the indicated concentrations of GCV. A graph was obtained by plotting the percentage of surviving colonies.
γ-irradiation of HSV-TK modified tumor cells
2 expressed as 106 tumor cells resuspended in 5 ml of DMEM were γ-irradiated (100 Gy) using a Gamma cell-1000 (Atomic Energy of Canada Ltd. Source: 137Cs).
Cytospin histological analysis
A 100 μl aliquot of 104 cells/ml in PBS was used to prepare cytospin slides using a cytospin system (Hettich, Salford, UK), fixed in 10% formalin for 10 min, left overnight to air-dry and then stained by the Papanicolaou staining method [32, 33].
Measurement of apoptosis and necrosis by FACS analysis
Tumor cells were resuspended in 100 μl of working labelling solution and incubated for 15 min in the dark. Two control tubes were used: cells stained with Annexin V-FITC (Becton–Dickinson, Oxford, UK) alone and cells stained with PI alone [34, 35]. The cells were then analysed by flow cytometry using a FACScan analyser (Becton–Dickinson, Oxford, UK).
Cell cycle analysis
Cells were irradiated at 100 Gy and incubated overnight at 37 °C, in 5% CO2, resuspended in 1 ml of staining buffer (0.1% sodium citrate, 0.1% Triton-X100 and 10 μg/ml propidium iodide in dH2O), vortexed and left overnight at 4 °C in the dark. The cells were then analysed by flow cytometry using a FACScan analyser.
In vivo studies: inoculation, establishment and treatment of the tumors intraperitoneally (IP) in mice
AB1 mouse mesothelioma adherent tumor cells were harvested by washing with versene only and re-suspended in 0.2 ml of PBS. Tumors were established IP in female BALB/c mice by injecting the mesothelioma cells (AB1 tumor cell line), at different doses (1 × 105, 5 × 105 and 1 × 106/100 ml PBS) using a 26-gauge needle, to establish the TD50 for the tumor cell line, according to Home Office regulations. Mice (n = 45) were injected i.p. with AB1 tumor cells on day 0. Nine days later, animals were assigned to nine groups (n = 5 per group). GCV treatment was started at day 10. GCV (Cymevene® 500 mg; Roche, Switzerland) was diluted in sterile DMEM to a stock concentration of 50 mg/ml. The stock solution of GCV was diluted in DMEM to a concentration of 2 mg/ml, and 1 ml of the stock was injected IP once a day for 5 consecutive days. Mice were monitored every 2 days to palpate the tumor. At post-mortem, all tumor nodules were counted and measured using a calliper (for each nodule, 2 perpendicular diameters were recorded). Tumor volume was calculated for each nodule assuming spherical shape and the total tumor volume was calculated by adding all the calculated values for each mouse.
Statistical analysis
Statistical analysis was performed using the Microsoft Excel program. Differences between groups were analysed using Student’s paired t test. A P value of < 0.05 was considered as significant.