APOPTOSIS OF TERMINALLY DIFFERENTIATED CHONDROCYTES IN CULTURE

During the process of endochondral ossification chondrocytes progress through stages of terminal differentiation culminating in apoptotic de ath. We have developed a serum-free suspension culture that allows ter minal differentiation and facilitates the investigation of factors aff ecting chondrocyte apoptosis. We have found that chondrocytes not comm itted to terminal differentiation, i.e., those from the caudal region of chick embryo sterna, a region that remains cartilaginous for some m onths after the chick hatches, maintained high viability in serum-free suspension culture. A strong dependence of viability on culture densi ty and sensitivity to induction of apoptosis with the protein kinase i nhibitor, staurosporine, was consistent with the proposal that these c hondrocytes, like nearly all cells, require intercellular communicatio n for survival. Chondrocytes that were committed to terminal different iation, i.e., those from the cephalic region of chick embryo sterna, a region that is replaced by bone before the chick hatches, expressed t he hypertrophic phenotype but; maintained their viability in culture f or only approximately 6 days. Subsequent cell death was very consisten t between cultures and shown to occur by all apoptotic process by anal ysis of DNA fragmentation and cell morphology., Short-term viability o f hypertrophic chondrocytes was independent of culture density and rel atively resistant to treatment with staurosporine. Induction of the hy pertrophic phenotype in immature chondrocytes committed them to cell d eath and prevention of expression of the hypertrophic phenotype preven ted cell death. We conclude that commitment of chondrocytes to termina l differentiation is associated with a commitment to apoptosis and apo ptosis of hypertrophic chondrocytes in growth cartilage does not requi re initiation by external signals. (C) 1997 Academic Press.