Methods

MCF7 cells (ECACC) were maintained in a pre-confluent state in Eagle's MEM supplemented with 1% penicillin/streptomycin, 1% glutamine, non essential amino acid solution, and pH'd to 7.2-7.4. Cells were seeded at lxlO6 in T25 flasks and allowed to attach overnight. The cells were subsequently grown in phenol red free medium and the fetal calf serum used was stripped with dextran coated activated charcoal (8). Cis-5,8,11,14,17 eicosapentaenoic acid was prepared in an equimolar NaHC03 and equal mass bovine serum albumin in PBS and stored under N2. Cells were treated with up to 100 pM EPA (9), repeat doses were applied every three days unless otherwise stated.

The measurement of DH activity was carried out according to the methods of Gompel, et al. (10) and Adams, et al. (11). To measure reductive and oxidative enzyme activities, cells were incubated in 2 nM/1 (40Ci/mM) [3H]- Et or [3H]- E2 respectively, for four h at 37°C . Reaction blanks in which cells were omitted were run in parallel. After the incubation, 2 ml ofthe media was removed and added to [14C]- E2 or [14C]- E! (5000 cpm) recovery labels. Steroids were then extracted into 4ml ether and dried at 40°C under N. Thin layer chromatography using 4:1 v/v dichloromethane:ethyl acetate solvent, separated the steroids and both product and recovery activity determined by scintillation counting according to James and Newton (12). Results are expressed as % control fmol product formed/cell and are a function of recovery and cell number. An n of 3 was used for each variable and each experiment was repeated at least twice. In all experiments cells were counted by measuring nuclei number as described by Butler, et al. (13). Statistical significance was determined with one-way ANOVA and Tuckey's post test as appropriate.

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