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This review summarizes our observations on the mechanism of induction of apoptosis in vitro in leukaemic cell lines and in vivo in patients with leukaemia undergoing chemotherapy, in relation to the cell cycle. Multiparameter flow cytometric methods allowed us to identify apoptotic cells and position them with respect to their cell cycle phase. Several antitumor agents of different classes have been...
We have addressed the possibility that Ca2+, Mg2+ and K+ ions play a central role in governing the morphological and biochemical changes attributed to apoptotic cell death. By removing Ca2+, Mg2+ or K+ ions from the cell culture medium we were able to assess the contribution of each ion to hybridoma cell growth and viability. The differences were explained in terms of a possible reduction in their...
The sequential occurrence of plasma and mitochondrial membrane alterations, intra-cellular pH shifts and changes in intracellular Ca2+ concentration after induction of cell death was monitored by flow cytometry in Jurkat and HSB2-cells. Cell death was induced by treatment with anti-Fas antibodies or by irradiation. Phosphatidylserine (PS) exposure and plasma membrane integrity were measured with FITC-Annexin...
Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the 51Cr release assay. However, measurement of target cell lysis by 51Cr release...
Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage...
Deficient activation of apoptosis signaling pathways may be responsible for treatment failure of malignant diseases. In primary leukemia samples the detection of deficient mitochondrial apoptosis signaling would enable identification of chemo-resistant cells. To investigate the key events of apoptosis at the mitochondrial level, we developed a flow cytometric method for simultaneous detection of mitochondrial...
Introduction: This paper presents a new method using natural cellular fluorescence (autofluorescence, AF) to study apoptosis. Measurement of AF reduces sample preparation time and avoids cellular toxicity due to the fact that no labelling is required. Methods: Human promyelocytic leukemic HL60 cells were incubated with camptothecin (CPT), tumour necrosis factor (TNF)-α in combination with cycloheximide...
The very early events of the intrinsic, damage-induced apoptotic pathway, i.e., upstream to Bax activation, probably consist of physico-chemical alterations (i.e., redox, pH or Ca2+ changes) rather then subtle molecular interactions, and in spite of many studies they remain unclear. One problem is that cells undergo apoptosis in an asynchronous way, leading to heterogeneity in the cell population...
There is increasing evidence that the active contribution of hepatocytes to liver disease is strongly dependent on local cytokine environment. It has been shown in vitro that TNFα can enhance hepatocyte FasLigand (FasL)-mediated cytotoxicity. Here, we demonstrate that TNFα-induced apoptosis was associated with Fas and FasL upregulation and that a FasL-neutralizing antibody prevented TNFα-induced apoptosis...
Polynuclear platinum(II) complexes represent a class of potential anticancer agents that have shown promising pharmacological properties in preclinical studies. The nature of cellular responses induced by these complexes, however, is poorly understood. In this research, the cellular responses of human ovarian cancer COC1 cells to dinuclear platinum(II) complexes {[cis-Pt(NH3)2Cl]2L1}(NO3)2 (1) and...
Apoptosis and necrosis play an important role in various aspects of preclinical pharmaceutical drug discovery and validation. The ability to quickly determine the cytotoxic effect of chemical compounds on cancer cells allows researchers to efficiently identify potential drug candidates for further development in the pharmaceutical discovery pipeline. Recently, a new imaging cytometry system has been...
Virulent Klebasiella pneumoniae (KP) inflicts severe liver abscesses in infected patients. This study investigated how the bacterial infection affected cell survival at the molecular level, in a cultured cell model. A strain of KP highly virulent in mice was isolated from a patient with liver abscess, and was used to infect HepG2 cells. The infected cells were examined for their viability, DNA fragmentation,...
Vaccines can have adverse side-effects, and these are predominantly associated with the inclusion of chemical additives such as aluminum hydroxide adjuvant. The objective of this study was to establish an in vitro model system amenable to mechanistic investigations of cytotoxicity induced by hepatitis B vaccine, and to investigate the mechanisms of vaccine-induced cell death. The mouse liver hepatoma...
Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection...
Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-d-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The...
Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3+ ve/RIP3− ve), necroptosis (RIP3Hi + ve/Caspase-3− ve) and...
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