Microfilament-disrupting agents prevent the formation of apoptotic bodies in tumor cells undergoing apoptosis.

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Dose-dependent induction of apoptosis in human tumour cell lines by widely diverging stimuli.

Cell death may occur by either of two mechanisms: apoptosis or necrosis. Necrosis, the first type of cell death to be recognized, is an uncontrolled degenerative phenomenon invariably caused by noxious stimuli and is the result of irreversible failure of membrane function. Apoptosis, on the other hand, is a death process which involves a series of well-organized events which require active cell participation, and is primarily caused by physiological stimuli. In the present study we show that cell death induced by a range of varied agents may take the form of either apoptosis or necrosis. Apoptotic cell death was found to occur at low levels of these agents, while at higher levels necrosis occurred. Hence, cells which are not killed directly, but merely injured by these agents, have the capacity to activate an internally programmed suicide death mechanism, whereas cells receiving greater injuries apparently do not. In addition, the presence of extracellular calcium was found to be necessary for the induction of apoptosis with all agents tested.

Cell death via apoptosis and its relationship to growth, development and differentiation of both tumour and normal cells.

Cell death can occur by two possible mechanisms, necrosis or apoptosis. Necrosis is the classically recognised form of cell death and is characterised by high amplitude swelling of the mitochondria, nuclear flocculation and uncontrolled cell lysis. Tissue necrosis is normally seen following severe trauma to cells. The alternative form of cell death is via a programmed sequence of events and is termed apoptosis. Apoptosis occurs under a variety of physiological conditions and cells dying by this process undergo cytoplasmic and nuclear condensation, coupled with cleavage of the cell's DNA into nucleosome size fragments. DNA cleavage is due to the activation of a specific endogenous endonuclease. The cell finally fragments into apoptotic bodies which are engulfed by neighbouring cells and degraded. Apoptosis is an energy requiring process requiring intact energy generating systems, unlike that of necrosis. In relation to malignant disease, apoptosis is the mechanism by which cytotoxic T cells kill tumour target cells; it may also be the mechanism which accounts for the high loss of cells in growing tumour masses. Extensive apoptosis is seen in regressing tumours and also in those treated with chemotherapeutic agents. This form of death may require the activation of specific death genes, although in view of work carried out in this and other laboratories, demonstrating that inhibitors of both protein and RNA synthesis will readily induce apoptosis, this may not be universal. Finally, apoptosis has far reaching implications for the treatment of malignant disease, since only by understanding how cells die will be able to develop more effective means of killing them.

Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis.

Apoptosis is regarded as a suicidal cell response since the dying cell appears to be an active participant. Previous studies have shown that apoptosis of various murine cell types, induced by a variety of stimuli, required RNA and/or protein synthesis. However, when human promyelocytic leukemia HL-60 cells were induced to undergo apoptosis by treatment with the calcium ionophore A23187 or microtubule-disrupting agents, in the presence of inhibitors of macromolecular synthesis, apoptosis of these cells was neither abrogated nor delayed. Furthermore, the presence of either cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an RNA synthesis inhibitor, alone was found to induce large scale apoptosis of these cells. Apoptosis in these cells was characterized by cell and chromatin condensation followed by nuclear and DNA fragmentation. In common with many other studies, this DNA fragmentation was found to have an approximately 200-bp multiple pattern, which is consistent with the activation of an endogenous endonuclease which cleaves at internucleosomal sites. Calcium-dependent endonuclease activity of this type was also detected in the isolated nuclei of untreated HL-60 cells. The morphologic and biochemical changes characteristic of apoptosis were found to precede cell death, as measured by trypan blue uptake and were completely distinct from death caused by toxic stimuli such as azide, ethanol, or heat treatment. Similar experiments with six other human cell lines confirmed that this phenomenon was not peculiar to the HL-60 cell line. These results suggest that certain dividing cell populations do not require RNA or protein synthesis to undergo apoptosis and further, that continuous transcription and translation of some regulatory protein(s) may be required to maintain control over the apoptotic "machinery" of such cells.

Elevations in cytosolic free Ca2+ are not required to trigger apoptosis in human leukaemia cells.

Previous studies have indicated that Ca2+ is a trigger for apoptosis (programmed cell death) in thymocytes and related cell lines. Recently we have shown that levels of apoptosis in leukaemic cells are diminished in Ca(2+)-deficient conditions, indicating that Ca2+ may be important in the mechanism of apoptosis in these cells. In the present study we investigated the possibility that Ca2+ serves as a trigger for apoptosis in the human leukaemic cell line, HL-60. Using fura-2 to measure cytosolic free Ca2+ concentrations, [Ca2+]i, in cell suspensions, and by using ratio imaging of fura-2 in single cells, we did not observe an early significant increase in [Ca2+]i in HL-60 cells undergoing apoptosis. The latter stages of apoptosis were, however, accompanied by increasing [Ca2+]i; these increases were apparently a result of, rather than a cause of, apoptosis. Furthermore, apoptosis could be induced in HL-60 cells under conditions of vastly reduced [Ca2+]i achieved by loading these cells with fura-2 in the presence of EGTA. These results indicate that elevation of [Ca2+]i is not a prerequisite for apoptosis in HL-60 cells and that apoptosis can occur in these cells in the presence of low [Ca2+]i.