Human recombinant interferon a increases oestrogen receptor expression in ZR-75-1 human breast cancer cells and sensitizes them to the anti-proliferative effects of tamoxifen

In the past ten years the oestrogen antagonist tamoxifen has played an increasingly important role in the management of primary and metastatic breast cancer. Although the mechanism of action of tamoxifen is imprecisely understood, there is considerable evidence that it acts primarily by blocking oestrogen action at its receptor. Interferon, in contrast to tamoxifen, has been disappointing as a single agent in the treatment of breast cancer. Two recent reports have suggested that interferon might enhance oestrogen receptor (ER) expression (Pouillart et al., 1982; Dimitrov et al., 1984). We have therefore investigated the effect of human recombinant interferon a on oestrogen receptor expression by the human breast cancer cell line ZR-75-1 and in addition determined the ability of interferon to potentiate the anti-proliferative effects of tamoxifen. ER levels were determined in viable cells at 37°C using a whole cell binding assay. Cells (50000 or 200000) were plated into a number of microwells and treated with interferon (10-1000 i.u./ml) for 48 h. After removal of interferon, control and treated cells were exposed to [2, 4, 6, 7, 16, I7-’H]oestradiol (0.25-3.5 n ~ , specific activity 140 pCi/ mmol) for 1 h. Non-specific binding was determined by simultaneously exposing parallel groups of cells to the radioactive ligand in the presence of a 200-fold excess of diet hylstilbestrol. Maximum specific binding capacity (B,,, ) and dissociation constant (K,,) were calculated after linearization of data by Woolf or Scatchard analysis. At the higher cell plating density there was little variability in control cell expression of ER (215 & 24fmol/mg of protein, Kd 0.5 5 0 . 1 5 n ~ ) , and interferon (1&1000i.u./ml) had no significant effect on receptor number or affinity for oestradiol. At the lower cell density there was some variation in control receptor levels (B,,, 36151 fmol/mg of protein), and interferon treatment (10 i.u./ml) consistently resulted in an increase in specific binding (BmaX 178-262 fmol/mg of protein). There was also a small decrease in affinity of oestrogen for its receptor hut this effect did not reach significance. Concentrations of interferon of > 100 i.u./ml were ineffective. Fig. l(a) shows that interferon at 10 i.u./ml had no significant effect on the proliferation of ZR-75-1 cells during a continuous 6 day treatment. Interferon at 500i.u./ml markedly inhibited cell proliferation. These data suggest that the effects of interferon on ER expression and cell proliferation are dissociated. Fig. 1 ( a ) also demonstrates that simultaneous exposure of cells to interferon and tamoxifen resulted in a degree of inhibition of cell proliferation greater than that observed with either drug alone, although there was no evidence of synergism. In contrast, a 5 day pre-exposure of cells to interferon (10 i.u./ml) markedly sensitized cells to a subsequent 6 day treatment with tamoxifen (Fig. 16). Sica et al. (1986), in a study reported simultaneously with our own preliminary data (van den Berg et al., 1986), demonstrated enhanced ER expression in an oestrogen supersensitive variant of the MCF-7 human breast cancer cell line after treatment with the j subtype of interferon. A