Morphological modifications of apoptosis in HL-60 cells: effects of homocysteine and cytochalasins on apoptosis initiated by 3-deazaadenosine

Abstract

Using electron microscopy, confocal laser scanning microscopy and measurements of intact DNA we have studied the morphology and DNA degradation of human leukaemia HL-60 cells undergoing drug initiated apoptosis. Apoptosis was initiated by 100 microM 3-deazaadenosine (c3Ado), 25 microM c3Ado plus 1 mM homocysteine thiolactone (Hcy) and 100 microM c3Ado plus 5 micrograms/ml cytochalasin B (CB). Two different phenotypes of apoptotic cells (APC), blebbed and non-blebbed, were present in the cultures. Blebbed APC dominated in cultures exposed to c3Ado, whereas most APC in cultures treated with c3Ado plus Hcy and all the APC in cultures treated with c3Ado plus CB displayed a non-blebbed phenotype. A more pronounced reduction of the chromatin/cytoplasm ratio, lower volume fractions of uncondensed chromatin and higher volume fractions of highly condensed chromatin (micronuclei) were found in cultures exposed to c3Ado and c3Ado plus Hcy when compared with cultures exposed to c3Ado plus CB. A partial inhibition of c3Ado apoptosis by CB was confirmed by measurements of intact DNA. The inhibitory effect of CB was not reproducible by CE, indicating that CB exerts its effect by an actin independent mechanism. Both blebbed and non-blebbed APC displayed nuclear fragmentation, segregation of organelles and cytoplasmic vesiculation, suggesting that the differences between the phenotypes were restricted to the cytoplasmic membrane. We were not able to demonstrate the presence of F-actin by fluorescein isothiocyanate-phalloidin staining in blebbed APC nor in non-blebbed APC in cultures treated with c3Ado plus Hcy. Non-blebbed APC in cultures treated with c3Ado plus CB displayed foci of F-actin at the internal part of the cytoplasmic membrane. This suggests that F-actin is preserved by the mechanism by which CB inhibits blebbing, and may indicate that blebbing of the cytoplasmic membrane during apoptosis is associated with F-actin deficiency rather than a result of actin-myosin interactions.

Precisely delivered nanomechanical forces induce blebbing in undifferentiated mouse embryonic stem cells

“…21 Blebbing can be observed during apoptosis, but apoptotic blebs do not have F-actin around the edge of the bleb. 22,23 F-actin was observed at the leading edge of the bleb, supporting our earlier results showing that blebs were not apoptotic.…”

A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F‐actin and DNA

“…The cells in region R2 also displayed lower levels of F-actin compared to levels of control and region R 1 cells. Loss of F-actincontaining cell surface structures as pseudopodiums and microvilli is a common feature of apoptosis (2,4,22,54), and previously we reported an F-actin deficiency of HL-60 cells undergoing apoptosis after 3-deazaadenosine treatment (22) microscopy). Taken together, these findings support the conclusion that region R2 is constituted by apoptotic cells and that the proportion of cells in this region reflects the actual number of apoptotic cells present in the sample analyzed.…”

Effect of Staurosporine in the Morphology and Viability of Cerebellar Astrocytes: Role of Reactive Oxygen Species and NADPH Oxidase

“…These morphological changes could be due to a differential reorganization of cytoskeletal proteins such as actin and tubulin induced by cell death conditions. In this regard, some studies have established that during cell death there is rearrangement and accumulation of cytoskeletal proteins including actin and tubulin [65, 66]. This could be accompanied by the ability of microtubules to have spontaneous changes in the polymerization and depolymerization activities during apoptosis [67].…”