Effects of Insulin-like Growth Factor Binding Protein 7 on Apoptosis in Human Teratocarcinoma Cells In Vitro

Materials and Methods

Cell culture. Tera 2 cells were maintained and flasks and wells for experimental purpose were essentially prepared as previously described (12). All tissue culture material was obtained from NUNC (Roskilde, Denmark). The serum-free medium consisted of a 1/1 (v/v) mixture of α−modified Minimal Essential Medium (MEM) lacking nucleosides and deoxynucleosides and Hams F12 medium supplemented with 10 μg/ml of human transferrrin preloaded with iron, in accordance with the manufacturers instructions. This medium is subsequently referred to as α:Ham.

Two days before a growth experiment, the medium over the stock cultures was replaced by α:Ham supplemented with 10% heat inactivated foetal calf serum (FCS). This procedure allowed the cells to adapt to the basal medium that was used in the serum-free culture experiments. The cells were removed from the stock culture flasks by rinsing with phosphate-buffered saline (PBS) and then briefly exposing the cells to a mixture of trypsin, versene and chick plasma (TVP). The cells were taken up in a:Ham with 10% FCS and counted in a Coulter counter (Coulter Electronics, Bedford, UK). In the subsequent cell culture experiments, the media were pre-equilibrated for at least six hours in an atmosphere of 5% (v/v) CO₂ in 95% humidified air at 37°C. The day before the experiment, the cells were plated out in 10 ml of α:Ham with 10% FCS at a density that ranged between 1.4×10⁵ and 3.0×10⁵ cells per 60-mm diameter Primaria dish (Becton Dickinson, San Jose, Ca, USA). The number of cells plated out varied between experiments, but numbers were always comparable within each experimental series. At the start of each experiment, cells were rinsed twice in PBS and 10 ml of α:Ham were added to each dish. After one hour, the dishes were briefly removed from the incubator and the IGFs and binding protein added to the dishes. Two dishes were counted immediately after the PBS rinse to obtain a starting cell count, and on subsequent days, the effect of the additives was measured by counting duplicate dishes in each treatment. For these counts, the cells were exposed to trypsin (0.125%, w/v) and EDTA in PBS for up to 30 minutes to ensure complete detachment from the cell surface. When all cells had detached, any remaining trypsin was neutralised by adding soybean trypsin inhibitor. The cells were then counted in triplicate in a Coulter counter. The range of the cells of the duplicate rarely exceeded 8% of the mean cell count.

DNA synthesis and assessment of apoptosis. DNA synthesis was assayed by incorporating tritiated thymidine (Amersham, Sweden) of 25 Ci/mmol into cells growing on glass coverslips for one hour prior to fixation. Unincorporated thymidine was removed after fixation for 24 hours in 50/50 methanol/acetone (v/v) by washing with ice-cold trichloroacetic acid. The slides were then processed for autoradiography as decribed elsewhere (8). The assessment of intact vs apoptotic cells was performed by acridine orange staining and fluorescence microscopy as described elsewhere (9). Nuclear fragmentation corresponded to nucleosome laddering and FACS analysis as previously described in detail (9).

Growth factor and binding proteins. Recombinant IGF-II was purchased from British Biotechnology (Oxford, UK). Lyophilised samples were made up to stocks of 2 mg/ml by adding 0.5 ml of 0.1 M acetic acid to 10 mg, shaking every 5 minutes in a 37°C water-bath and then adding 4 ml of PBS with 1% (wt/vol) crystalline bovine serum albumin (BSA; Sigma, Stockholm, Sweden) adjusting the pH to 7.0 with 0.1 M NaOH, and finally bringing the volume up to 5 ml with PBS/BSA. These stocks were aliquoted into Eppendorf vials and stored at −70°C until further use. In the growth experiments, the IGF-containing vials were thawed on ice, and made up to intermediate stock solutions by diluting with α:Ham to a final concentration of 200 μg IGF-II/ml medium. 0.5 ml of this intermediate stock solution was added to each 60 mm dish containing 10 ml serum free α:Ham. Dishes used for experimental purpose were harvested after 24 hours. In each case, serum-free α:Ham and α:Ham supplemented with 10% serum were used as controls.

IGFBP-7 was purchased from R & D systems (MN, USA) and stock solutions made up to the relevant concentration (50 ng/ml) according to the manufacturer's instructions.