Manipulation of culture conditions for BHK cell growth inhibition by IRF-1activation

The activation of interferon-regulatory-factor-1 (IRF-1) has been applied t o 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 ga in its transcriptional activator function. By using a dicistronic stabilise d self-selecting construct it was possible to control cell proliferation. W ith 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 day s after estradiol addition cell concentration was still maintained but a de crease in cell viability was observed. This cell response is independent on clone (producer and non-producer) and culture system (static and stirred c ultures). Specific recombinant-protein productivity of the producer clone w as not significantly altered. Control experiments confirmed that IRF-1 acti vation effect was not due to the addition of estradiol per se, estradiol so lvent or serum concentration. The extent of cell growth inhibition is depen dent on estradiol concentration and estradiol addition time, although a dec rease in cell viability was always observed. Reducing the time span of estr adiol 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, archi eving values similar to those obtained if no estradiol is added. During thi s recovery phase the cells passed two different phases: first a stationary phase extension where cell growth was still inhibited, followed by an incre ase 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.