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T G Cotter

Publications and source records attributed to T G Cotter.

At least 91 records · Page 5Linked to original sources
PubMed1992

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.

Actin Cytoskeleton↗
PubMed1992

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.

Calcimycin↗
PubMed1992

Induction of apoptosis in cells of the immune system by cytotoxic stimuli.

Apoptosis is a physiological mode of death where the dying cell plays an active part in its own demise, which contrasts sharply with what is seen in necrosis. In the present paper I have shown that when cells of the immune system are exposed to a range of cytotoxic agents, such as the RNA synthesis inhibitor actinomycin D, the DNA polymerase inhibitor aphidicolin or the topoisomerase I inhibitor campthothecin, they rapidly undergo cell death via apoptosis. This is characterized by DNA fragmentation to yield the now hallmark ladder pattern of death by this mechanism. All of these agents are capable of inducing apoptosis irrespective of what phase of the cell cycle a cell is in. These studies also indicate that apoptosis can occur in immune cells without recourse to macromolecular synthesis.

Animals↗
PubMed1991

Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis.

UV radiation is known to be a potent agent for the induction of programmed cell death (apoptosis) in human skin. However, the mechanistic aspects of UV-induced apoptosis remain ill-defined. In this study the effects of varying periods of UV-irradiation on the human leukaemia HL-60 cell line and on five other human cell lines were investigated. HL-60 cells were found to rapidly undergo apoptosis en masse after short periods of UV-irradiation, whereas prolonged exposure of these cells to this form of radiation induced a more rapid form of cell death which was suggestive of necrosis, the pathological mode of cell death. Similar effects were observed on the U937 (myelomonocytic), Molt-4 (T-lymphoblastoid), and Molt-3 (T-lymphoblastoid) cell lines, whereas the K562 (pre-erythroid) and Daudi (B-lymphoblastoid) cell lines proved to be relatively resistant to the death-inducing properties of UV-irradiation by comparison. UV-induced apoptosis in cell lines was characterized by morphological changes as well as DNA fragmentation into unit multiples of approximately 200 bp, which was indicative of endogenous endonuclease activation. This DNA fragmentation pattern was not detected in cells immediately after UV-irradiation, and was therefore not the result of direct UV-induced DNA damage. UV-induced apoptosis of the HL-60 cell line was found to require extracellular calcium and to be inhibited in a dose-dependent way by zinc added to the culture medium.

Calcium↗
PubMed1991

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.

Calcium↗
PubMed1991

Programmed cell death (apoptosis) in lymphoid and myeloid cell lines during zinc deficiency.

Three human cell lines of lymphoid (Molt-3 and Raji) or myeloid (HL-60) origin were maintained in vitro under zinc-sufficient or zinc-deficient conditions. Under these conditions, cell proliferation, viability and mode of death (apoptotic or necrotic) were assessed. All three cell types decreased their proliferative capacity and viability under conditions of zinc deficiency. Cell death in the HL-60 and Raji cultures occurred primarily via apoptosis, while most cells in zinc-deficient Molt-3 cultures died via necrosis. Apoptosis in zinc-deficient cultures of HL-60 and Raji cells was characterized by a slow decline in culture viability as cells with condensed and fragmented nuclear DNA appeared. These morphological changes were accompanied by an increase in cell buoyant density, which allowed separation of viable apoptotic cells from their non-apoptotic counterparts by means of percoll stepdensity gradients. Necrosis in zinc-deficient Molt-3 cultures was characterized by rapid loss of cell culture viability as these cells underwent direct lysis. Intact necrotic cells were easily identified by the flocculated state of their chromatin as well as the decreased basophilia of their cytoplasm. Analysis of DNA from apoptotic HL-60 and Raji cells revealed that internucleosomal DNA degradation, indicative of endogenous endonuclease activation, had occurred, whereas the nuclear DNA of necrotic Molt-3 cells remained relatively unfragmented. The different modes of cell death evoked may reflect the relative sensitivities of cells of these lineages to zinc levels in vivo.

Cell Division↗
PubMed1990

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.

Calcimycin↗
PubMed1990

Biochemical characterisation studies on a leukocyte alloantigen expressed with high frequency in leukaemia patients.

In this report we describe, using a previously characterised monoclonal antibody (NC-2), the biochemical characteristics of a human leukaemia-associated alloantigen. Two proteins with molecular weights of 50 kDa and 15 kDa were immunoprecipitated from 125I surface labelled HL-60 cells. Both proteins appeared to be sensitive to digestion with trypsin, the 50 kDa protein in particular. Treatment with glycopeptidase F indicated the presence of N-linked oligosaccharides, whereas treatment with neuraminidase had no effect on the mobility of the antigens in SDS-PAGE indicating the absence of detectable sialic acid residues. Sensitivity to glycopeptidase F indicates that the reacting antigens are glycoproteins in nature. The antibody reacts with a range of normal tissues and appears to be associated with cytoplasmic granules in HL-60 cells.

Antibodies, Monoclonal↗
PubMed1990

Disruption of microtubules induces an endogenous suicide pathway in human leukaemia HL-60 cells.

Terminally differentiated HL-60 cells undergoing programmed cell death (apoptosis) in culture were found to have a disrupted microtubular network. Treatment of undifferentiated HL-60 cells with microtubule-disrupting agents alone was found to induce apoptosis en masse in these cells. In contrast, disruption of microfilaments did not induce apoptosis; instead these cells underwent necrosis, the pathological mode of cell death. Apoptosis in response to microtubule disruption in HL-60 cells was characterized by cell shape changes, nuclear condensation followed by fragmentation and the separation of the cell into numerous intact fragments, termed apoptotic bodies. Apoptosis of these cells was further confirmed by DNA analysis, which demonstrated the activation of an endogenous endonuclease which cleaved the DNA of these cells into oligonucleosomal fragments. Microtubule disrupting agents were found to exert these effects over a wide range of doses. Apoptosis was also inducible in HL-60 cells, in a dose-dependent manner, by the calcium ionophore A23187. Since microtubules are known to be highly sensitive to intracellular calcium fluctuations, this suggests that calcium influx could act at the microtubule level in effecting apoptosis.

Actin Cytoskeleton↗