PubMed · 7390686
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M H Boyer. 1980. Schistosomiasis.. https://doi.org/10.1111/j.1365-4362.1980.tb00289.x
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Tetracycline-regulated vectors were used to obtain inducible expression in stable transfected B cell lines of two Epstein-Barr virus (EBV) latent genes, LMP1 and EBNA2. The transfected genes were tightly repressed by low, nontoxic concentrations of tetracycline (< or = 1 microgram/ml) and, following removal of tetracycline, were induced to levels comparable to or up to 3x that of EBV-transformed normal lymphoblastoid cell lines. In transfected DG75 cells, induced expression of LMP1, but not of EBNA2, led to the expected upregulation of various cell surface markers, including: CD40, CD54, CD58, and HLA class I.A novel observation was that both LMP1 and EBNA2 independently caused the downregulation of surface IgM, an effect mirrored in EBV-positive Burkitt lymphoma lines undergoing phenotypic drift during the transition from latency I to latency III in which both LMP1 and EBNA2 are upregulated. Most remarkably, induced LMP1 expression almost completely inhibited cell growth for 4 to 5 days, after which the cells recovered a limited proliferative capacity. The cytostatic effect of LMP1 was observed in all three B cell lines studied: DG75, BJAB, and Akata. Further analysis showed that induction of LMP1 coincided with a reduction in the levels of c-myc, and that the cytostatic effect was due to an accumulation of cells at the G2/M phase of the cell cycle. These data suggest a novel function for the LMP1 oncogene in controlling the proliferation of EBV-infected cells by regulating progress through G2/M phase.
Collagenase and Dispase enzymes are often used to disaggregate tissue. In this study, we examined by flow cytometry the effects of these enzyme preparations on peripheral blood lymphocyte surface marker expression. Peripheral blood mononuclear cells (PBMC) were obtained from seven healthy volunteers. Cells were incubated with collagenase (128 U/ml, type 1 A) for 3 h and overnight and with Dispase (1.6 mg/ml, grade II) for 15 min, 30 min, 1 h, 3 h and overnight. The intensity of expression of CD3, CD4, CD8, alpha beta and gamma delta T cell receptors was decreased by 25-40% in all cases, while CD4+ and CD8+ lymphocyte populations were undetectable after treatment with Dispase. Moreover, reappearance of these surface molecules did not occur following the incubation of cells in culture medium for 3 h. The results of this study emphasise the importance of considering the influence of isolation procedures on cell characteristics.
All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with acute promyelocytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiation- and apoptosis-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) inhibits proliferation and induces differentiation of myeloid leukemic cells. The 9-cis-RA, unlike all-trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D(3) analogue, 20-epi-22-oxa-24a,26a,27a-tri-homo-1alpha,25(OH) 2D, (KH 1060), which belongs to the family of potent 20-epi-1,25(OH),D3 analogues, with 9-cis-RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro. Our data show that KH 1060 alone is a very potent inhibitor of clonal proliferation of HL-60, but this effect is reversible, and that 9-cis-RA alone is a weak inhibitor of clonal proliferation of HL-60 cells. In contrast, the combination of KH 1060 and 9-cis-RA synergistically and irreversibly inhibited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation. This combination also affected the expression of apoptosis-related genes. The bcl-2 protein became nearly undetectable, and expression of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 cells was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for alpha-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentiation markers with two-color immunofluorescence. Treatment with the combination of KH 1060 and 9-cis-RA was a potent inducer of differentiation of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, induced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl-2 ratio. This drug combination may have important therapeutic significance.