Mechanism of cell death: Early morphological changes and DNA degradation of apoptosis in rat thymocytes.
thesisposted on 2015-11-19, 08:51 authored by Xiao-Ming. Sun
Apoptosis is an active process of gene-directed cellular self-destruction. It serves a biological meaningful and homeostatic function in diverse circumstances, including embryonic development, physiological involution or pathological atrophy of adult organs, normal tissue turnover, immune cell ontogeny and immune killing. A new flow cytometric method using Hoechst 33342/ propidium iodide staining has been established in this study. The basis of this method is the change of apoptotic thymocytes both in cell size and membrane permeability. This method has been used to study glucocorticoids or DNA topoisomerase inhibitor-induced apoptosis in immature rat thymocytes both in vitro (dexamethasone, etoposide, m-AMSA and camptothecin) and in vivo (etoposide). During establishment of the flow cytometric method, it was found that induction of apoptosis by dexamethasone was not inhibited by zinc as assessed by flow cytometry. Further studies indicated that apoptotic thymocytes induced by dexamethasone in the presence of zinc showed distinct morphology and other cellular changes characteristic of apoptosis but no DNA laddering. Thus, internucleosomal cleavage of DNA was, for the first time, dissociated experimentally from early nuclear morphological changes, as well as other cytoplasmic changes. Identification of a transitional preapoptotic population of thymocytes further confirmed this observation. In this population of cells, early nuclear morphological changes and other cytoplasmic changes were observed in the absence of DNA laddering. By using field inversion gel electrophoresis, it was found that formation of kilobase pair fragments of DNA preceded internucleosomal cleavage of DNA. Formation of large fragments of DNA was particularly visualised when thymocytes were treated with dexamethasone in the presence of zinc, due to an inhibitory effect of zinc on the internucleosomal cleavage of DNA. These large fragments of DNA were also present in the transitional preapoptotic population of thymocytes in the absence of oligonucleosomal fragments. Further studies with isolated thymocyte nuclei showed that formation of these large fragments of DNA was a Mg2+-dependent process that can be facilitated by Ca2+. The nature of these large fragments, the mechanism involved in their formation and their relevance to apoptosis reqire further investigation.
Date of award1994-01-01
Author affiliationCollege of Medicine, Biological Sciences and Psychology
Awarding institutionUniversity of Leicester