Protection against apoptosis in chicken bursa cells by phorbol ester in vitro

Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study we have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hr with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed particularly with bursa and, to a much smaller extent, with thymus or spleen cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristate acetate (PMA) but not by its inactive analogue 4 alpha-PMA during culture. Ionomycin is not required for this effect and, alone, appears to be slightly toxic for bursa cells, although it does not inhibit the effect of PMA. PMA did not affect the degree of DNA fragmentation in spleen or thymus cells. The addition of the protein kinase C (PKC) inhibitor staurosporine abolished both the preventive effect of PMA on apoptosis and its protective effect on bursa cells, as assayed by [3H]thymidine incorporation 24-48 hr after the initiation of cultures. PKC inhibitors also prevented the proliferation-inducing effect of PMA + ionomycin on spleen cells. It is concluded that the activation of protein kinase C and perhaps other kinases protects against apoptosis in cultured bursa cells.