“…Human lymphoblastoid CEM cells 41 were maintained in RPMI 1640 medium (Gibco-BRL, Life Technologies Italia, Milano, Italy), containing 10% fetal calf serum (FCS) plus 100 U/ml penicillin, 100 mg/ml streptomicin, at 371C in a humidified 5% CO 2 atmosphere. Apoptosis was induced by incubating cells at a concentration of 5 Â 10 5 per ml in serum-free medium supplemented with insulin (5 mg/l) and transferrin (5 mg/l), and by adding anti-Fas (CD95) IgM MoAb (clone CH11, Upstate Biotechnology, Lake Placid, NY USA) at 250 ng/ml for different incubation times (1, 10, 20, 40 min, 1 h, 2 h, 4 h).…”
Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltagedependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-b-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.
“…Human lymphoblastoid CEM cells 41 were maintained in RPMI 1640 medium (Gibco-BRL, Life Technologies Italia, Milano, Italy), containing 10% fetal calf serum (FCS) plus 100 U/ml penicillin, 100 mg/ml streptomicin, at 371C in a humidified 5% CO 2 atmosphere. Apoptosis was induced by incubating cells at a concentration of 5 Â 10 5 per ml in serum-free medium supplemented with insulin (5 mg/l) and transferrin (5 mg/l), and by adding anti-Fas (CD95) IgM MoAb (clone CH11, Upstate Biotechnology, Lake Placid, NY USA) at 250 ng/ml for different incubation times (1, 10, 20, 40 min, 1 h, 2 h, 4 h).…”
Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltagedependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-b-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.
“…CEM-C7-14 and CEM-C1-15 cells were kindly provided by Dr. E.B. Thompson (UTMB, Galveston, TX), and have been derived from the parental line CCRF-CEM, obtained from a patient with acute lymphoblastic leukemia [22]. Cells were cultured in RPMI 1640 medium supplemented with 5% FBS at 37 °C in a humidified 5% CO 2 incubator, and were maintained in log phase by passaging every 3 days.…”
Glucocorticoid (GC)-evoked apoptosis of T-lymphoid cells is preceded by increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which may contribute to apoptosis. This report demonstrates that GC-mediated upregulation of the bZIP transcriptional repressor gene, E4BP4, is dependent on [Ca 2+ ] i levels, and correlates with GC-evoked apoptosis of GC-sensitive CEM-C7-14 cells. Calcium chelators EGTA and BAPTA reduced [Ca 2+ ] i levels and protected CEM-C7-14 cells from Dex-evoked E4BP4 upregulation as well as apoptosis. In the GC-resistant sister clone, CEM-C1-15, Dex treatment did not induce [Ca 2+ ] i levels, E4BP4 expression or apoptosis, however, the calcium ionophore A23187 restored Dex-evoked E4BP4 upregulation and apoptosis. CEM-C7-14 cells were more sensitive to GC-independent increases in [Ca 2+ ] i levels by thapsigargin, and a corresponding increase in E4BP4 expression and cell death, compared to CEM-C1-15 cells, suggesting a direct correlation between [Ca 2+ ] i levels, E4BP4 expression, and apoptosis.
“…Cell lines and tissue culture The cell lines used were the human leukaemia T-cell line CCRF-CEM (CEM) (Foley et al, 1965) and its anthracycline-selected MDR subline CCRF-CEM/E1000 (E1000) which overexpresses mrp mRNA but does not express detectable levels of P-glycoprotein or mdrl mRNA (Davey et al, 1995). Cell cultures were maintained as previously described (Davey et al, 1995).…”
Summary Multidrug resistance (MDR) in cancer cells is a major contributor to the failure of chemotherapy treatment. This paper describes a novel protein named the anthracycline resistance associated (ARA) protein.The ara gene is amplified in the MDR leukaemia line CCRF-CEM/E1000 and its mRNA is overexpressed. ARA belongs to the ATP binding cassette (ABC) family of proteins. Another ABC protein, the multidrug resistance-associated protein (MRP), has previously been reported to be overexpressed in the CEM/E1000 subline. The primary amino acid sequence of ARA indicates that it is 49.5 kDa without glycosylation, and that it has one potential glycosylation site. ARA has one ATP binding site and associated transmembrane regions. This is in contrast to MRP (190 kDa, 172 kDa deglycosylated) and most other higher eukaryote ABC proteins, which consist of two similar halves, each having one ATP binding site. In addition to ARA being coexpressed with MRP, comparison of amino acid sequences showed that, among known proteins, ARA is most similar to the C-terminal half of MRP.
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