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

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 alpha, beta and the novel epsilon isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (o.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-epsilon was observed within 39 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and and altered phosphorylation state of PKC-epsilon was seen. In contrast, the conventional alpha and beta isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the alpha and beta isoforms showed a significant down-regulation. The preferential alterations in PKC-epsilon observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca(2+)-independent epsilon 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.

Establishment and characterization of an EBNA-negative human lymphoma cell line (BHL-89).

BACKGROUND: The aim of the work was to establish human malignant lymphomas in culture, in order to study the biological characteristics and drug sensitivity of lymphomas of human lymphoid origin. MATERIALS AND METHODS: Lymph nodes of patients were explanted and kept in cultures using conventional tissue culture methods. Cytogenetic methods were used for karyotype analysis. Clonogenic assay was applied to test drug sensitivity. The tumorigenic capacity of the cells was determined by inoculating them into immunosuppressed mice. Immunological and other markers were examined with conventional techniques. RESULTS: A cell line, BHL-89, was established in culture from a patient with B-cell type non-Hodgkin's malignant lymphoma. Cells started to grow after a few days without a feeder layer in stationary suspension. The population doubling time was 48 h. The cells were hyperploid, and non-random aberrations were +1, -15, +14q+. Cloning efficiency in soft agar was found to be as high as 50-60%. The cells expressed markers characteristic of early B cells. The BHL-89 cells were Epstein-Barr nuclear antigen (EBNA) negative. They produced tumors when 10(7) cells were injected into immunosuppressed mice. The cells were sensitive to dibromodulcitol (Elobromol), an alkylating antitumor drug, and resistant to the phorbol ester TPA. CONCLUSIONS: The established EBNA-negative BHL-89 cell line has a few unique characteristics, e.g. rapid establishment without feeder cells, origin from the lymph node of an adult patient, high clonogenicity in soft agar, and resistance to TPA. The cell line is suitable for studying the nature of B lymphomas and testing compounds against lymphoproliferative disorders.

Peripheral blood leukocyte subpopulations a long time after posttraumatic splenectomy.

Peripheral blood leukocyte subpopulations have been determined in 50 patients a long time (2 to 20 years) after posttraumatic splenectomy. These otherwise healthy individuals had significant lymphocytosis and monocytosis, while the absolute number of granulocytes did not differ statistically from that of the controls. The absolute number of CD2+, CD3+ as well as CD4+ and CD8+ peripheral blood mononuclear cells was found to be elevated, while the number of CD21+, CD20+ and HLA-DR+ PBMN cells was significantly decreased. The absolute number of sIgM+ as well as CD16+ MN cells did not differ statistically from that of the controls. Two further patients were found to have developed B-chronic lymphocytic leukaemia 5 and 31 years following posttraumatic splenectomy, respectively.

[Heterogeneity of surface antigens in chronic B-cell lymphoid leukemia]. / A sejtfelszíni antigének heterogenitása B-sejtes krónikus lymphoid leukaemiában.

Clinical and immunological studies of fifty patients with CLL have been performed. No correlation was found either between the clinical stage or clinical course of the disease and the distribution of cell surface makers characteristic of CLL (CD19, CD20, CD21, HLA-DR, sIg). Therapy did not influence the distribution of B lymphocyte subpopulations. On the other hand we recognized differences when examining the B-cell specific features. The CD21 antigen was present in significantly lower proportion when compared to all other B-cell markers. This suggests the presence of immature B-cell population. Correlation studies showed a strong correlation between the presence of the CD5 antigen and the antigens CD19, CD20, HLA-DR and sIg, while a similar correlation could not be proved between the CD5 antigen and CD21 marker. Thus the application of the CD5 antibody together with any of the B-cell markers seems to be sufficient for the diagnostics of CLL with the exception of the CD21 antibody that marks only a small proportion of the B-cell population in CLL, so it can be used for purposes of clinical diagnostics.

Characterization of T lymphocyte subsets in hairy cell leukaemia: influence of splenectomy and correlations with the clinical stage of the disease.

Peripheral blood mononuclear cell surface markers were studied in a series of 26 hairy cell leukaemia patients 19 of whom were splenectomized previously. Patients with non-symptomatic and stable disease were distinguished from those with symptomatic and/or progressive disease (also termed "active" clinical stages). In all HCL patients as a group, the absolute number of CD4+ MN cells did not differ statistically from that of the controls, while the number of CD8+ MN cells was significantly increased. The reduction of the CD4/CD8 ratio in the peripheral blood of HCL patients as compared to the controls was explained by the reduction of this ratio in patients with "active disease", while the CD4/CD8 ratio of patients with non-symptomatic and stable disease did not differ statistically from that of the controls. The CD4/CD8 ratio was found to be influenced mainly by the clinical stage of the disease, and not by the effect of splenectomy.

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-l (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, 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.

[Cytochemical, immunologic and gene rearrangement studies in adult acute leukemia]. / Cytochemiai, immunológiai és génátrendezödés vizsgálatok felnöttkori akut leukaemiákban.

Results of morphological, cytochemical and immunological studies performed in adult acute leukaemias have been compared. Thirty one cases proved to be acute myeloid leukaemia, while 25 cases were shown to be acute lymphoid leukaemia. Based on our results we conclude that immunophenotyping with monoclonal antibodies does not help in distinguishing the subtypes of AML. For purposes of clinical diagnosis cytochemical methods are valuable. On the other hand the monoclonal antibodies are essential in distinguishing the very immature myeloid and lymphoid leukaemias and this is of great importance from the clinical point of view, in determining therapy. Moreover, the diagnosis of acute lymphoid leukaemias is not possible without the specific monoclonal antibodies. Their application is first of all in haematological centers caring for leukaemia patients nowadays already obligatory. Gene rearrangement studies make the diagnosis more accurate and help in the diagnosis of leukaemias of unknown immunological origin.

Observations on NK cells, K cells and on their function a long time after posttraumatic splenectomy.

Natural killer (NK) cell activity and antibody-dependent cellular cytotoxicity of 48 posttraumatic splenectomy patients was found to be normal despite the increase in the proportion and the absolute number of large granular lymphocytes (LGLs). The NK cell activity of 2 further posttraumatic splenectomy cases was severely decreased, despite elevated number of LGLs and normal number of CD16+ peripheral blood mononuclear cells. Besides increased susceptibility to upper respiratory tract infections, 1 of these patients developed herpes genitalis recidivans. Two posttraumatic splenectomy patients were found to have developed chronic lymphocytic leukemia 5 and 31 years following splenectomy, respectively.

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells. The mixed lymphocyte culture of allo-sensitized individuals and the auto-tumor stimulation show similar kinetics of activation.

The kinetics of interleukin-2 receptor (IL-2R) expression and the [3H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R+ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R+ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.

Clinical value of cytomorphologic, immunologic and cytogenetic investigations of acute leukaemias.

The valuability of immunophenotyping of acute myeloid and lymphoid leukaemias in comparison to morphological and cytochemical classification were approached in 56 cases. In the case of acute myeloid leukaemias the immunophenotyping by monoclonal antibodies CD14, CD13, CD33 was less informative concerning the subtypes of the disease. The clinical diagnosis can be achieved on the basis of cytochemical investigation alone. In contrast, the diagnosis of lymphoid leukaemias requires all information obtained by immunophenotyping by a series of monoclonal antibodies CD3, CD2, CD4, CD8, CD1, CD19, CD20, CD21 and CD10. On the other hand, the monoclonal antibodies are essential in differentiation of the very immature myeloid and lymphoid leukaemias. This is of great importance from the clinical point of view for determining the therapy. Molecular genetic studies based on the characterisation of the state of gene rearrangement of immunoglobulin and T-cell receptor beta chains have basic importance in the confirmation of the result of immunophenotyping and in the determination of leukaemias of unknown origin.

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 Hodgkin's disease and T-cell lymphoma, are not resting, but activated B-cells or the neoplastic abberrants of activated cells.

Immunological and molecular biological identification of a true case of T-hairy cell leukaemia.

A hairy cell leukaemia (HCL) patient is presented in whom the peripheral blood mononuclear cells (PBMCs) carried suppressor T-cell markers (CD3+, CD2+, CD8+/CD4-, CD38+). Analysis of genomic DNA of PBMNC showed the presence of a monoclonal population of T cells, the T-cell receptor (TCR) beta-chain gene being rearranged on both alleles (DR/DR), while the immunoglobulin (Ig) heavy chain-genes were in germline configuration. The neoplastic cells were found to react with the monoclonal antibody RAB-1 - originally described as belonging to the B lineage-restricted monoclonal antibodies - and to carry RAB-1/CD-8 in a double marker assay. Natural killer activity of PBMNCs against K562 target cells was severely reduced, while the cells were found to exert strong antibody-dependent cellular cytotoxicity.

Hairy cell leukaemia: observations on natural killer activity in different clinical stages of the disease.

Follow-up studies of natural killer (NK) cells, NK activity and antibody dependent cellular cytotoxicity (ADCC) in the course of hairy cell leukaemia (HCL) were carried out in a series of patients affected by the disease. NK activity against K562 targets was found to be high in all the patients with non-symptomatic stable disease. On the other hand, absent or extremely low NK activity was found only in patients with symptomatic progressive disease. NK activity determined in the transitional stages showed values between these two extremes. Antibody dependent cellular cytotoxicity (ADCC) was less closely correlated with the clinical stage than NK activity. Our findings suggest that investigation of in vitro NK cell cytotoxicity might serve as a useful adjunct in determining clinical stage in HCL.

Cercopithecus aethiops monkey as a reliable model for in vitro study of T cell depletion of bone marrow with Campath-1 plus complement.

T-lymphocyte markers of peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) of C. aethiops monkeys were studied by using anti-human monoclonal antibodies. The results show that C. aethiops T lymphocytes express surface markers which react specifically with anti-human MoAbs including CD3, CD4, CD8, CD2. However, very few CD3-positive cells were found, in contrast to the abundance in CD8+ cells. There is a high conservation of receptors forming E rosettes with AET-treated SRBCs, and antigens reacting with the anti-human T and B cell monoclonal antibody (Campath-1). The present findings indicate that C. aethiops can be used as a new experimental model for studies on T-cell depletion from bone marrow with Campath-1 MoAb + rabbit C.

Human fetal liver as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation.

The CFU-GM and T cell contents of human fetal livers were studied at various times between 6-14 weeks of gestation. The number of CFU-GM increased parallel to gestational age, especially after week 10. Cells bearing mature T cell markers, however, were found only in one case out of 35 fetal liver samples. Cryopreservation of fetal liver cells hardly affected the viability and proliferative capacity of CFU-GM in the sample. According to these findings fetal liver is, at least up to the 14th gestational week, practically free of mature T cells but it does contain a considerable amount of CFU-GM (an accepted indicator of pluripotent stem cell content), consequently fetal liver can be considered as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation for children.