Miklós Benczúr

Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs.

Activation of lymphocytes after platelet allotransfusion possessing only class I MHC product

After platelet allotransfusion, we found a characteristic increase in the expression of interleukin‐2 receptor, dipeptydilpeptidase IV (CD26), activation‐inducer molecule (AIM, CD69) and transferrin receptors (CD71) on day 3 indicating that important functional molecules expressed on the activation of lymphocytes by allogeneic platelets. At the same time, no consistent increase of other activation molecules such as Ki‐1 (CD30), intercellular adhesion molecule (ICAM‐1, CD54) and Ki‐24 (CDw70) antigen expression was detected, probably as a result of the selective activation of some lymphocyte subsets. In order to obtain further evidence for the in vivo activation triggered by allogeneic platelets, a subsequent step of T cell activation towards differentiation was investigated with monoclonal antibodies to leucocyte common antigens. A sharp expression of the UCHL1, coupled with a decrease of the CD45R molecule was detected on day 7 or 14, suggesting a T cell priming.

Possible involvement of protein kinase C-ϵ in phorbol ester-induced growth inhibition of human lymphoblastic cells

Abstract Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional α, β and the novel ϵ isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (0.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5–1.0 ng/ml PMA. The translocation of PKC-ϵ was observed within 30 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and an altered phosphorylation state of PKC-ϵ was seen. In contrast, the conventional α and β isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the α and β isoforms showed a significant down-regulation. The preferential alterations in PKC-ϵ observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca 2+ -independent ϵ isoform in mediating the initial events of the phorbol ester stimulated cellular responses.

Modulation of drug-induced apoptosis in a human B-lymphoma cell line (HT58)

The cytotoxic effect of etoposide (ETO), a topoisomerase II inhibitor, and staurosporine (STA), a non-selective protein kinase inhibitor, were studied on a human lymphoma cell line of B-cell origin (HT58). Apoptosis, induced dose dependently by both drugs, was accompanied with nucleosomal DNA fragmentation detected by flow cytometry. On the other hand, induction of cell death failed using phorbol ester (PMA), anti-IgM antibody (a-IgM) or dexamethasone (DEX), although, all of these agents arrested the cells in G1. Furthermore, PMA pretreatment retarded ETO-induced apoptosis, but enhanced STA cytotoxicity. DEX increased the sensitivity of cells to STA, but did not to ETO. Activity of STA or DEX was only slightly modified by a-IgM pretreatment. The results support the possibility that different apoptotic pathways exist in HT58 cells. The differences in pathways could be manifested either in the signaling routes, or in the molecular effectors of apoptosis.

Detection of Activation Antigens on Chronic Lymphocytic Leukaemia Cells.

The peripheral blood mononuclear cells of patients with chronic lymphocytic leukaemia were characterized by the presence of a variety of cell surface differentiation antigens. The cells of 20 patients were found to be of B-cell phenotype when studied with antibodies directed against CD19, CD20, HLA-DR and sIg. Furthermore, a significant percentage of the cells gave a positive reaction with the monoclonal antibody to CD5. On the other hand, the CLL-cells did not express the CD21 antigen (C3d receptor, EBV receptor). We studied in parallel the presence of various activation antigens using 19 monoclonal antibodies grouped into 7 clusters (CD25, CD30, CD40, CD69, CD70, CD39, CD71). A significantly higher percentage of the CLL cells expressed activation antigens than lymphocytes from healthy controls. The percentage of CD3/HLA + DR + cells, compared to the healthy control lymphocytes was not increased in the CLL patients, and the activated cells in CLL were found to have characteristics of B-cells. Based on these results, we suggest that the CLL cells, like the cells in Hodgkins disease and T-cell lymphoma, are not resting, but activated B-cells or the neoplastic abberrants of activated cells.