Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei

Nuclei of active cells (HeLa, mouse fibroblasts) partnered with chick erythrocyte nuclei in heterokaryons are suppressed, as judged by a decreased rate of ³H-uridine incorporation and diminished nuclear binding of ³H-actinomycin D. The extent to which active partner nuclei are suppressed, the extent to which erythrocyte nuclei are reactivated, and the degree of sensitivity of heterokaryons towards certain inhibitors of proteolytic enzymes, all correlate strongly with the ratios of erythrocyte nuclei to active nuclei. Thus, reactivation of individual erythrocyte nuclei is reduced progressively and active nuclei are suppressed progressively as the ratio of erythrocyte nuclei per active nucleus in heterokaryons increases. This erythrocyte nuclear-dose dependent suppression is markedly amplified when heterokaryons are grown in the presence of protease inhibitors. The protease inhibitors found to affect heterokaryons are low molecular weight (<400) inhibitors of trypsin-like enzymes: -1-tosylamide-2-leucyl chloromethyl ketone (TLCK), N-α-tosyl--arginine methyl ester (TAME) and N-benzoyl--arginine amide (BAA). They affect heterokaryons at concentrations comparable to the minimal concentrations at which they inhibit trypsin. Nonfused HeLa cells, mouse fibroblasts, or their homokaryons are refractory to protease inhibitors at these concentrations.Reactivation of chick erythrocyte nuclei in a heterokaryon may involve release of suppressors ordinarily confined to the erythrocyte nucleus, with subsequent redistribution of suppressor among all the nuclei of the heterokaryon. Under these circumstances the state of nuclear activity will depend on the quantity of suppressor per individual nucleus; within the erythrocyte nucleus the suppressors will decrease its rate of reactivation, when they migrate into an active nucleus they will suppress it. These suppressors, either in transit between the nuclei, or within the nuclei, may be hydrolysed by intracellular proteases.