The activation of interferon-regulatory-factor-1 (IRF-1) has been applied to regulate the cell growth of BHK cells. The constitutively expressed IRF-1-estrogen receptor fusion protein (IRF-1-hER) activated by the addition to the culture medium of an estrogen analogue (estradiol), enabled IRF-1 to gain its transcriptional activator function. By using a dicistronic stabilised self-selecting construct it was possible to control cell proliferation. With the addition of 100 nM of estradiol at the beginning of the exponential phase, the IRF-1 activation led to a rapid cell growth inhibition. Two days after estradiol addition cell concentration was still maintained but a decrease in cell viability was observed. This cell response is independent on clone (producer and non-producer) and culture system (static and stirred cultures). Specific recombinant-protein productivity of the producer clone was not significantly altered. Control experiments confirmed that IRF-1 activation effect was not due to the addition of estradiol per se, estradiol solvent or serum concentration. The extent of cell growth inhibition is dependent on estradiol concentration and estradiol addition time, although a decrease in cell viability was always observed. Reducing the time span of estradiol exposure allowed the decrease in the cell viability to be controlled and the stationary inhibited phase to be extended: when the time of contact between the cells and estradiol is reduced cell viability increases, archieving values similar to those obtained if no estradiol is added. During this recovery phase the cells passed two different phases: first a stationary phase extension where cell growth was still inhibited, followed by an increase of cell concentration. The IRF-1 system is reversible. This pattern can be repeated for an extended period when estradiol addition and removal are repeated, showing a cyclic response. Thus, it is possible to modulate the IRF-1 effect by manipulating cycles of addition/removal of estradiol and in this way the stationary phase can be maintained.
- Cell growth inhibition