End labeling studies of fragmented DNA in the avian growth plate

Evidence of apoptosis in terminally differentiated chondrocytes

M. Hatori, K. J. Klatte, Cristina Teixeira, I. M. Shapiro

Research output: Contribution to journalArticle

Abstract

The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag(TM) and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin, evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate far apoptosis. Dual parameteric flow cytometric contour plots 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 hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate 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 phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.

Original languageEnglish (US)
Pages (from-to)1960-1968
Number of pages9
JournalJournal of Bone and Mineral Research
Volume10
Issue number12
StatePublished - 1995

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Growth Plate
Chondrocytes
Apoptosis
DNA
Cell Death
Epiphyses
DNA Fragmentation
Chromatin
Electrophoresis
Flow Cytometry
Agar Gel Electrophoresis
Pulsed Field Gel Electrophoresis
Osteoblasts
Sepharose
Cartilage
Cell Count
Fluorescence
Gels
Electrons
Phenotype

ASJC Scopus subject areas

  • Surgery

Cite this

End labeling studies of fragmented DNA in the avian growth plate : Evidence of apoptosis in terminally differentiated chondrocytes. / Hatori, M.; Klatte, K. J.; Teixeira, Cristina; Shapiro, I. M.

In: Journal of Bone and Mineral Research, Vol. 10, No. 12, 1995, p. 1960-1968.

Research output: Contribution to journalArticle

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abstract = "The chondro-osseous junction has been the subject of considerable scrutiny, especially in terms of the fate and role of the terminally differentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteoblasts, evidence from a number of other studies suggests that chondrocytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the commercial reagent ApopTag(TM) and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin, evaluating fragmentation through alkaline and pulsed field agarose gel electrophoresis of DNA, and measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proximal tibial growth plate of the chick with ApopTag. Most of the chondrocytes in the hypertrophic region were labeled by the reagent; in contrast, few proliferative chondrocytes were stained by the end-labeling procedure. Both agarose and pulsed field electrophoresis were used to confirm that there was fragmentation of chondrocyte DNA. Alkaline gel electrophoresis indicated that there was more fragmentation of DNA from hypertrophic cells than from proliferative chondrocytes. Further evidence in support of apoptosis was provided by electron microscopic observation of cells in the hypertrophic region of the growth plate. We noted that many of the cells in this region of the growth plate appeared to be undergoing programmed cell death since their nuclei contained condensed chromatin. Finally, we used flow cytometry to analyze chondrocytes isolated from the proliferating and hypertrophic regions of the growth plate far apoptosis. Dual parameteric flow cytometric contour plots 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 hypertrophic cartilage. In summary, the results of this investigation indicate that chondrocytes terminate their life history by apoptosis. While it is possible that the terminal labeling studies may overestimate 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 phase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate events that lead to programmed cell death.",
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