The K562 chronic myeloid leukemia cell line undergoes apoptosis in response to interferon-alpha

BACKGROUND AND OBJECTIVE: The K562 cell line, derived from a chronic myeloid leukemia (CML) patient and expressing B3A2 bcr-abl hybrid gene, is known to be particularly resistant to apoptotic death. IFN-alpha treatment of CML patients impairs malignant cell clone, apparently protecting from progression to terminal blast crisis. The mechanisms underlying this kind of cell deletion are analyzed here by multiple technical approaches. DESIGN AND METHODS: K562 cells, variably treated with IFN-alpha, were examined by agarose gel DNA electrophoresis, light and electron microscopy. The presence of bcr-abl rearrangement was revealed by RT-PCR. RESULTS: At 4 day treatment both DNA ladder and apoptotic nuclear changes were identified, consistently in the presence of bcr-abl expression. INTERPRETATION AND CONCLUSIONS: Even cells expressing bcr-abl, such as K562, can be triggered to apoptosis. Therefore, this genetic condition, commonly preventing PCD, does not prevent IFN-alpha-mediated apoptosis. PCD seems thus to be the mechanism underlying IFN-alpha-treated K562 cell deletion and it could be the basis of malignant clone reduction in IFN-alpha treated CML patients.     

Interaction between a murine myeloma cell line and bone marrow stromal cells

Intravenous transplantation of an in vitro maintained murine myeloma cell line, 5T33, results in progressive growth in the bone marrow of C57Bl/KaLwRiJ mice. Concurrent with the growth of the tumor in vivo, the bone marrow stromal cells are inhibited, as assayed by their ability to form stromal cell foci and long-term monolayers in vitro. Inhibition of normal mouse marrow stromal cell growth also occurs when 5T33 cells are added to the marrow cells in vitro, and contact between the marrow and 5T33 cells is not necessary to achieve inhibition, indicating secretion of one or more diffusible inhibitory factors.     

Functional expression of Fas (CD95) in acute myeloid leukemia cells in the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy

Clinical studies of bone marrow transplantation (BMT) suggest that the immune system contributes to the eradication of acute myeloid leukemia (AML). A recent study also showed that the Fas (CD95/APO1) mediates apoptotic signal from cytotoxic T lymphocytes. Sixty-four patients with AML were studied for the expression of Fas in the context of CD34 and CD38 coexpression. The clinical relevance of Fas expression and function on AML was also investigated. Fas was expressed on 2% to 98% of AML cells (2% to 20% in 11 patients, 20% to 50% in 20 patients, 50% to 80% in 24 patients, and 80% to 98% in nine patients). Only 44.4% of patients with AML M1 (French-American-British [FAB] classification) were Fas+ (>/=20% of leukemia cells expressed Fas), whereas 89.1% of patients with AML M2, M3, M4, M5 were Fas+ (P < .01). Among 43 CD34+ patients (>/=20% leukemia cells were CD34+), 34 were Fas+, and 19 of 21 CD34- patients were Fas+ (P = NS). Thirteen cases were studied for their expression of Fas in the context of CD34 and CD38 using three-color analysis. Fas is expressed at a high level in the gated CD34+CD38+/- and CD34+CD38+ population. In 10 AML samples, Fas was expressed at a higher level in CD34+/CD38+ population than in CD34+/CD38+/- or CD34- cell populations. Fas-induced apoptosis by anti-Fas monoclonal antibody (MoAb) was determined by morphologic features and colorimetric DNA fragmentation assay. Induction of apoptosis was found in 14 of 24 cases. However, no statistically significant correlation was observed between Fas expression and induction of apoptosis. Leukemia colony-forming unit assays suggested that in some cases, Fas-induced apoptosis occurred in the clonogenic cell populations. Parameters such as laboratory and clinical data at initial diagnosis were correlated with Fas expression and only response to initial induction chemotherapy showed significant correlation with Fas expression (P < .05). We conclude that the majority of AML cells exhibit variable expression of Fas, and apoptosis could be induced by anti-Fas MoAb in some cases. Our results suggest the Fas-mediated apoptosis may be clinically relevant, whereas the issue of clonogenic leukemia cells and Fas expression needs further studies.     

Establishment of a novel human myeloid leukaemia cell line (FKH-1) with t(6;9)(p23;q34) and the expression of dek-can chimaeric transcript

Calcein-labeled B16 melanoma (B16M) cells were injected intraportally, and in vivo video microscopy was used to study the distribution and damage of cancer cells arrested in the liver microvasculature over a period of 4 hours. The contribution of glutathione (GSH)-dependent antioxidant machinery to the possible oxidative stress-resistance mechanism of B16M cell was determined by in vitro incubation with the selective inhibitor of GSH synthesis L-buthionine (S,R)-sulphoximine (BSO) before B16M cell injection in untreated and 0.5-mg/kg lipopolysaccharide (LPS)-treated mice. In addition, untreated and LPS-treated isolated syngeneic hepatic sinusoidal endothelial cells (HSE) were used to determine in vitro their specific contribution to B16M cell damage. Trauma inherent to intrasinusoidal lodgement damaged 35% of B16M cells in both normal and LPS-treated mouse liver. The rest of the arrested B16M cells remained intact in normal liver for at least 4 hours, although their damaged cell percentage significantly (P < .05) increased since the second hour in normal mice injected with BSO-treated cells and since the first hour in LPS-treated mice given untreated cells. Recombinant human interleukin-1 receptor antagonist (rHuIL-1-Ra) given to mice 15 minutes before LPS significantly (P < .05) abrogated B16M cell damage. On the other hand, 40% of the B16M cells co-cultured with unstimulated HSE and 70% of the co-cultured with LPS-treated HSE became sensitive to endothelial cell-mediated damage after BSO treatment. These results demonstrate that a high intracellular level of GSH protects B16M cells from possible in vivo and in vitro sinusoidal cell-mediated oxidative stress, contributing to the mechanism of metastatic cell survival within the hepatic microvasculature.     

Evidence for a critical role of DNA topoisomerase IIalpha in drug sensitivity revealed by inducible antisense RNA in a human leukaemia cell line

To examine the role of human DNA topoisomerase IIalpha (topo IIalpha) in drug resistance, we selectively inhibited topo IIalpha gene expression in U937 human monocytic leukaemia cells stably transfected with a plasmid that allowed for Zn-mediated conditional expression of a human alpha-topo IIalpha antisense sequence. Expression of topo IIalpha mRNA was reduced to <30%, whereas no significant alteration of topo IIbeta mRNA expression was observed. Under these conditions, drug sensitivity to the topo-II-directed agents, etoposide and daunorubicin, was reduced to approximately 50%, whereas sensitivity to 4-hydroperoxy-cyclophosphamide (4-HC) was not altered. This suggests that a reduced amount of topo IIalpha mRNA may be sufficient for the resistance to topo II inhibitors in leukaemia cells.     

Lack of correlation between bronchoconstrictor response and bronchodilator response in a population-based study

Bronchodilator and bronchoconstrictor responsiveness have been considered physiological opposites in patients with obstructive airways disease. Provocation challenges have been replaced by bronchodilator tests in the assessment of cases of severe airways obstruction. The aim of this study was to examine the relationship between bronchoconstrictor and bronchodilator responsiveness, and their supposed interchangeability, in a general population. From the Vlagtwedde-Vlaardingen follow-up study, 101 adults were recruited (mean (SD) age 55 (11) yrs, 67 males and 34 females, and 31 were smokers). All completed a questionnaire on airways symptoms. Bronchoconstrictor and bronchodilator responsiveness were assessed with cumulative dose-response curves, using histamine and terbutaline, respectively. Thus, it was possible to relate histamine sensitivity of the airways (the concentration of histamine, at which forced expiratory volume in one second (FEV1) falls by 10% (PC10)) to the maximal bronchodilator response (delta FEV1) and the sensitivity to the bronchodilator (cumulative dose of inhaled terbutaline at which FEV1 increases by 10% (RD10)). Subjects with a bronchoconstrictor response (PC10 < or = 16 mg x mL(-1); n=38) had more respiratory symptoms than those without (n=63) (40 versus 21%) and also lower baseline FEV1 values (90 versus 96% predicted), but had comparable bronchodilator responsiveness. Subjects with a bronchodilator response (delta FEV1 > or = 9% of the predicted value; n=13) did not differ from those without (n=88) for all parameters, including symptoms, allergy and pulmonary function. In those with a bronchoconstrictor response, there was a weak but significant correlation between the PC10 and RD10 (rho=-0.32), but not between PC10 and delta FEV1. This study suggests that bronchoconstrictor and bronchodilator responsiveness are not highly correlated, even in subjects with airways obstruction. Symptoms were associated with the presence of a bronchoconstrictor, but not a bronchodilator, response. We conclude that bronchoconstrictor and bronchodilator responsiveness are two different phenotypic markers that are not interchangeable in epidemiological studies.     

Stimulation of G-CSF gene expression in the macrophage cell line by contact with extracellular matrix proteins and a pre-B leukaemia cell line

Freshly prepared dentine specimens of human teeth were perfused with either horse serum or physiologic saline. After application of AllBond2, ART Bond, Syntac or an experimental dentine bonding agent called P-Bond, a composite cylinder was added and cured at the same time. After 1500 thermal cycles with constant imitation of intrapulpal pressure, the shear bond strengths were measured. Resulting shear bond strength values were analysed with Students t-test or Mann-Whitney Test. The values for AllBond2 were not significantly different. The values for ART Bond (P < 0.05) and for Syntac (P < 0.05) were significantly higher if the dentine was perfused with horse serum. For P-Bond (P < 0.001) the values were significantly higher if the dentine was perfused with physiologic saline. According to these results it does not seem to be appropriate to take a clear decision as to which of the two perfusing media investigated might be more suitable to imitate in vivo conditions.     

Characterization of newly established human myeloid leukemia cell line (KF-19) and its drug resistant sublines

A new human myeloid leukemia cell line, designated KF-19, and its drug resistant sublines have been established. The KF-19 cell line was established from the pericardial effusion of a patient with acute myeloid leukemia clinically resistant to chemotherapy and KF-19 cells were characterized by expression of myeloid markers and differentiation into neutrophil- and macrophage-like cells upon optimal stimulations. KF-19AraC, KF-19ADR and KF-19VCR were established as sublines resistant to cytosine arabinoside (AraC), adriamycin (ADR) and vincristine (VCR), respectively. Efflux of the corresponding drugs was documented in each cell line. Expression of the MDR1 gene and the P-glycoprotein was found only in KF-19ADR, which showed a cross resistance to anthracyclines and vinca alkaloids; this resistance was reversed by verapamil or cyclosporin A. KF-19VCR lacking MDR1 gene and P-glycoprotein expression showed only resistance to vinca alkaloids, which was partially reversed by verapamil and cyclosporin A. Unexpectedly, KF-19ADR and KF-19VCR displayed cross resistance to AraC, despite lack of alterations of deoxycytidine kinase (dCK) and deaminase (dA) activities. KF-19AraC showed an efflux of AraC as well as a decreased level of dCK, but not of dA. In addition, KF-19AraC showed cross resistance to VCR in the efflux assay. The cell lines reported herein will provide new aspects on the mechanisms of drug resistance in leukemic cells.     

Overexpression of lung-resistance protein and increased P-glycoprotein function in acute myeloid leukaemia cells predict a poor response to chemotherapy and reduced patient survival

We investigated the role of the drug resistance-related proteins LRP, MRP and Pgp and the apoptotic suppressor, bcl-2, in relation to other clinical characteristics, with respect to response and survival in 91 patients with newly diagnosed AML, treated with standard chemotherapy. Multivariate analysis showed that poor response to chemotherapy was associated with increasing age (P=0.0004), LRP expression (P=0.0001) and Pgp function (P=0.015). The significant predictors of both leukaemia-free survival (LFS) and overall survival (OS) were LRP (LFS, P=0.01; OS, P=0.0001), Pgp function (LFS, P=0.0001; OS, P=0.0003) and cytogenetic abnormalities (LFS, P=0.0001; OS. P=0.0005). Patients with the lowest expression of LRP and Pgp function and favourable karyotype (group I) had an LFS of 30.2 months compared to 8 5 months in the group with the highest expression of LRP and Pgp and poor prognosis karyotype (group III, P=0.002). OS decreased from 75.4 months in group I to 7.9 months in group III patients (P <0.0001). Neither MRP nor bcl-2 were significantly associated with chemotherapy response and survival. Correlations were found between increasing expression of LRP and older age (P=0.05) and an unfavourable karyotype (P=0.005), but these variables were independent of each other in analysis of treatment response and patient survival. Our findings suggest that both LRP and Pgp are clinically relevant drug-resistance proteins and it may be necessary to modulate both LRP and Pgp functions in order to reverse the multidrug resistance phenotype in AML.     

A possible correlation between vertebral artery insufficiency and degenerative changes in the cervical spine

We studied 130 patients, aged 20 to 81 years, with symptoms of tinnitus, vertigo or dizziness. Radiological examinations revealed degenerative changes in the cervical spines of all patients such as discopathy or osteophytes. Head and neck and neurological examinations ruled out other symptoms apart from vertebrobasilar artery flow insufficiency. The vertebrobasilar arteries were examined by means of a color Doppler ultrasonograph using duplex scanning. The correlation coefficient (CC) defining the relationship between the number of patients with abnormal blood flow and the total number of patients with radiologically confirmed changes in the cervical spine was 41.5%. When patients were separated by age, the value of the CC coefficient increased proportionally according to age, changing from 0 to 79.1%. Use of the Doppler ultrasonograph was found to be a safe and non-invasive diagnostic method that enabled us to assess the influence of degenerative changes in the cervical spine on hemodynamic disturbances in the inner ear and brain stem. Our findings demonstrated a pathological decrease of vertebral artery flow velocity in relationship to degenerative changes in the cervical spine.     

Cyclosporin A and FK506 inhibit activation-induced cell death in the murine WEHI-231 B cell line

The patient presenting with a spinal meningioma usually has a slow indolent course of symptoms but at the time of diagnosis will have a range of neurological deficits. The diagnosis is most commonly secured with MR imaging. Safe resection of spinal meningiomas mandates a clear understanding of their growth characteristics as well as of regional anatomy. Surgical treatment of spinal meningiomas is gratifying, and the severity of preoperative neurological deficits should never deter one from withholding therapy.     

Effect on cell kill of addition of multidrug resistance modifiers cyclosporin A and PSC 833 to cytotoxic agents in acute myeloid leukaemia

Multidrug resistance (MDR) mediated by the drug efflux pump P-glycoprotein (Pgp), may cause remission failure and relapse in patients with acute myeloid leukaemia (AML) by extruding cytotoxic agents such as anthracyclines from leukaemic cells thus allowing them to survive. Cell line data suggest that reversal of MDR is possible using modifying drugs such as cyclosporin A (CSA) and its analogue PSC 833. We have investigated the effects on cell kill of the addition of CSA and PSC 833 to daunorubicin, idarubicin, mitozantrone, etoposide and cytarabine in 52 fresh cell samples from AML patients using an MTT assay. Pgp status was determined by using monoclonal antibodies JSB-1 and MRK-16 and by assessment of rhodamine efflux. Although overall each cytotoxic-modifier combination produced significant improvements in cell kill compared to cytotoxic alone (P values ranged from P < 0.001 to P = 0.017), modifiers also produced significant cytotoxicity in their own right, and no consistent difference was seen between responses in Pgp-positive and negative groups. Up to one in three Pgp-positive samples failed to show any improvement in cell kill with the addition of CSA or PSC 833, possibly owing to co-expression of alternative resistance mechanisms not affected by the MDR modifiers. The best responses were seen when PSC 833 was added to idarubicin, with 7 out of 22 Pgp-positive cases (32%) showing five-fold improvements in cell kill or better compared to idarubicin alone. Comparison of equimolar concentrations of the two modifiers in the Pgp positive group failed to show a significant difference in cell kill, though PSC 833 was markedly superior to CSA in a minority of highly responsive samples which demonstrated clear evidence of MDR reversal. Our in vitro data suggest that MDR modifiers such as CSA and PSC 833 could play an important role in the therapy of AML and indicate the need for prospective randomised trials to assess their clinical efficacy.     

Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis

To investigate the role of chemokines during the initial local response to Mycobacterium tuberculosis in the human lung, we studied chemokine production by the human alveolar epithelial cell line A549 after infection with M. tuberculosis. M. tuberculosis-infected A549 cells produced mRNAs and protein for monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) but not mRNAs for macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES. Chemokine production in response to M. tuberculosis was not dependent on production of tumor necrosis factor alpha, IL-1beta, or IL-6. Two virulent clinical M. tuberculosis isolates, the virulent laboratory strain H37Rv, and the avirulent strain H37Ra elicited production of comparable concentrations of MCP-1 and IL-8, whereas killed M. tuberculosis and three Mycobacterium avium strains did not. The three virulent M. tuberculosis strains grew more rapidly than the avirulent M. tuberculosis strain in the alveolar epithelial cell line, and the three M. avium strains did not grow intracellularly. These findings suggest that intracellular growth is necessary for mycobacteria to elicit production of MCP-1 and IL-8 by alveolar epithelial cells but that virulence and the rate of intracellular growth do not correlate with chemokine production. Alveolar epithelial cells may contribute to the local inflammatory response in human tuberculosis by producing chemokines which attract monocytes, lymphocytes, and polymorphonuclear cells.     

Structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) in a murine macrophage-like cell line, J774.1

The structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) were examined in a murine macrophage-like cell line, J774.1. TNF-alpha production is one of the characteristic phenotypes of LPS-activated macrophages, and is observed upon incubation with LPS. On the contrary, macrophage cell death, which had been found in our laboratory (Amano F., Karahashi H., J. Endotoxin Res., 3, 415423 (1996)) and was examined as the release of lactate dehydrogenase (LDH) from cells into the culture supernatant, was observed 2.5 h after the addition of LPS in the presence of CHX. However, both TNF-alpha production and macrophage cell death were similarly dependent on the structures of LPS and lipid A. At more than 10 ng/ml, wild-type LPS from E.coli and S. minnesota exhibited the highest activity, and LPS as well as diphosphoryl lipid A from S. minnesota rough mutants also exhibited activity, but E. coli LPS detoxified by alkaline treatment or monophosphoryl lipid A from S. minnesota exhibited no activity even at 100 ng/ml. These results suggest that LPS-induced macrophage cell death in the presence of CHX shows similar requirements for LPS and/or lipid A structures as for the macrophage activation at higher doses than 10 ng/ml, although the former was not observed at 1 ng/ml LPS, while the latter was. However, TNF-alpha does not seem to be involved in the induction of macrophage cell death, because a neutralizing anti-TNF-alpha antibody failed to block the macrophage cell death and because recombinant TNF-alpha had little effect on the extent of LDH release in the presence or absence of LPS and/or CHX, and also because TNF-alpha production by LPS was abolished in the presence of CHX.     

Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1)

The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4, 5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.