M. Hatori et al., END LABELING STUDIES OF FRAGMENTED DNA IN THE AVIAN GROWTH-PLATE - EVIDENCE OF APOPTOSIS IN TERMINALLY DIFFERENTIATED CHONDROCYTES, Journal of bone and mineral research, 10(12), 1995, pp. 1960-1968
The chondro-osseous junction has been the subject of considerable scru
tiny, especially in terms of the fate and role of the terminally diffe
rentiated chondrocyte. Although it has been proposed that these cells
change their phenotype and survive in the epiphysis, possibly as osteo
blasts, evidence from a number of other studies suggests that chondroc
ytes may undergo apoptosis or programmed cell death. A useful test for
programmed cell death is to end label DNA in cryosections using the c
ommercial reagent ApopTagTM and detect antibody binding to fragmented
DNA by epifluorescence; more direct assessments include examination of
the nucleus for condensation of chromatin, evaluating fragmentation t
hrough alkaline and pulsed field agarose gel electrophoresis of DNA, a
nd measuring apoptosis by flow cytometry. We found that we could label
cells in the proliferative and the hypertrophic region of the proxima
l tibial growth plate of the chick with ApopTag. Most of the chondrocy
tes in the hypertrophic region were labeled by the reagent; in contras
t, few proliferative chondrocytes were stained by the end-labeling pro
cedure. Both agarose and pulsed field electrophoresis were used to con
firm that there was fragmentation of chondrocyte DNA. Alkaline gel ele
ctrophoresis indicated that there was more fragmentation of DNA from h
ypertrophic cells than from proliferative chondrocytes. Further eviden
ce in support of apoptosis was provided by electron microscopic observ
ation of cells in the hypertrophic region of the growth plate. We note
d that many of the cells in this region of the growth plate appeared t
o be undergoing programmed cell death since their nuclei contained con
densed chromatin. Finally, we used flow cytometry to analyze chondrocy
tes isolated from the proliferating and hypertrophic regions of the gr
owth plate for apoptosis. Dual parameteric now cytometric contour plot
s of Hoechst and 7-amino-actinomycin D fluorescence showed that about
8% of cells in the plate were apoptotic. Most of these cells were in h
ypertrophic cartilage. In summary, the results of this investigation i
ndicate that chondrocytes terminate their life history by apoptosis. W
hile it is possible that the terminal labeling studies may overestimat
e the number of cells undergoing this event, the data lend credence to
the view that cells are removed from the epiphysis through apoptosis.
If this is the case, then chondrocytes probably enter the terminal ph
ase of their life as fully functioning cells and genomic, and/or local
environmental conditions provide termination signals that initiate ev
ents that lead to programmed cell death.