JM Cells Research Articles

Quantitative analysis of CD5 antigen modulation by 12- O-tetradecanoylphorbol-13-acetate in T-lymphoblastic leukemia cells: Individual response patterns and their relationships with both maturation and protein kinase C content

Modulation of CD5 expression by TPA was investigated on T-leukemic cell lines corresponding to different stages of ontogeny. These CD5 changes have been analyzed simultaneously with modifications of cell growth, cell cycle, cell surface phenotype, and PKC content. CD5 expression was found 6- to 17-fold increased by TPA in a dose-dependent manner on phenotypically mature T-cells (Jurkat, JM, and T-CLL) while T-cells from earlier stages of differentiation (CEM III, CEM 95, and CEM 44) were found unresponsive. CD5 upregulation on TPA-sensitive JM cells appears correlated with inhibition of cell growth, blockage in Gl phase, and phenotypic maturation (downregulation of CD7 and CD1 antigens) and seemed to be related to PKC activation since DiC 8 (a PKC activator) mimicked this TPA effect and H7 (a PKC inhibitor) partially reduced it. On the other hand, on CEM III cells TPA induced no modulation of CD5 antigen, a less dramatic effect on cell growth and cell cycle, but a CD7 downregulation. TPA appeared fully effective in binding and translocating PKC in both CEM III and JM cells, although the PKC activity level was three times higher in the latter. Finally, our study suggests that CD5 expression is at least partially under control of PKC in phenotypically mature neoplastic T-cells while PKC could not be directly involved in the regulation of CD5 antigen in leukemic cells arrested at earlier stages of differentiation.

Rational Design of Peptide-Based HIV Proteinase Inhibitors

A series of peptide derivatives based on the transition-state mimetic concept has been designed that inhibit the proteinase from the human immunodeficiency virus (HIV). The more active compounds inhibit both HIV-1 and HIV-2 proteinases in the nanomolar range with little effect at 10 micromolar against the structurally related human aspartic proteinases. Proteolytic cleavage of the HIV-1 gag polyprotein (p55) to the viral structural protein p24 was inhibited in chronically infected CEM cells. Antiviral activity was observed in the nanomolar range (with one compound active below 10 nanomolar) in three different cell systems, as assessed by p24 antigen and syncytium formation. Cytotoxicity was not detected at 10 and 5 micromolar in C8166 and JM cells, respectively, indicating a high therapeutic index for this new class of HIV proteinase inhibitors.

The down-regulation of the mitogenic fibrinogen receptor (MFR) in serum-containing medium does not occur in defined medium

Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 °C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

Thermal enhancement of tetraplatin and carboplatin in human leukaemic cells

The thermal enhancement of tetraplatin (racemic, d-trans and l-trans isomers) and carboplatin was studied as a function of temperature in vitro in JM, a human acute lymphoblastic leukaemia cell line. Exponentially growing JM cells were exposed to tetraplatin (0-10 micrograms/ml) or carboplatin (0-45 micrograms/ml) for 1 h at 37-43 degrees C in 1 degree C increments. Graphic analysis demonstrated a breakpoint for the onset of thermal enhancement for tetraplatin at 40 degrees C. Thermal enhancement was maximal at 42 degrees C with no significant increase at 43 degrees C. The tetraplatin thermal enhancement ratio (TER) was 2.7 at 42 degrees C. The TERs for d and l isomers at 41.8 degrees C were not significantly different. The relationship of TER to temperature for carboplatin closely paralleled that of tetraplatin. The implications of these results are discussed in the context of tetraplatin's unique properties.

Idiotype vaccines against human T cell leukemia. II. Generation and characterization of a monoclonal idiotype cascade (Ab1, Ab2, and Ab3).

Previously, we had generated anti-Id mAb (Ab2) binding to a hybridoma SN2 (Ab1), which recognizes a glycoprotein, gp37, expressed by human leukemic T cells. To characterize these anti-idiotopes further, they were used to immunize mice and rabbits. Several murine anti-anti-Idiotype mAb (Ab3), mostly of IgM-k isotype, were obtained. mAb3 and sera from rabbits immunized with Ab2 contained antibodies that bind to gp37 Ag and leukemic MOLT-4 and JM cells. Also, mAb3 and immune sera from rabbits competed with Ab1 for binding to MOLT-4 cells. They inhibited the binding of iodinated Ab1 to Ab2 indicating that Ab3 in mice and rabbits shares idiotopes with Ab1 (SN2). Furthermore, both the murine mAb3 and rabbit polyclonal Ab3 immunoprecipitated the same gp37 Ag as SN2 (Ab1). The production of Ag-specific Ab3 (Ab1') in mice and rabbits in absence of any exposure to gp37 indicates that these Ab2 may indeed carry the internal image of the gp37 Ag. Such anti-idiotopes (Ab2 beta) may be useful as Ag substitute for the induction of therapeutic immunity in T cell leukemia patients.

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line.

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.

Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes.

A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype vaccines against human T cell leukemia.

Fibrinogen mitogenic effect on hemopoietic cell lines: control via receptor modulation.

The possibility that the mitogenic effect of fibrinogen, a major plasma protein (3 mg/ml), is mediated by specific membrane receptors was studied. Specific binding analysis showed that fibrinogen receptors are present only on hemopoietic cell lines that respond to its mitogenic effect. The mitogenic fibrinogen receptor is not recognized by antibodies specific for the platelet fibrinogen receptor or is not competitively blocked by synthetic peptides containing the Arg-Gly-Asp sequence, which is common to fibronectin, fibrinogen, vitronectin, and other cell-attachment proteins. The lymphoma-derived pre-B-cells (Raji) have 149,000 receptors, whereas the lymphoma-derived T cells (JM), which are 3 times smaller, have 54,000 receptors. These receptors have a Kd of 2 X 10(-7) M. They are inducible by stimuli specific for the cell lineage: activators of the breakdown of phosphatidylinositol phosphates, such as platelet activating factor for Raji cells, and adenylate cyclase agonists and cAMP analogues for JM cells. The stimuli have no mitogenic effect in the absence of fibrinogen; they do not change the Kd. Each stimulus increases the number of fibrinogen receptors in a dose-dependent manner, which correlates strongly (r = -0.98, n = 5) with an increased growth rate of cells in the presence of fibrinogen. This correlation concludes that the mitogenic effect of fibrinogen is controlled via receptor modulation.

The effect of 3′, 5′-C-GMP and 3′, 5′-C-CMP on cell growth of the T-lymphoblastic JM cell line. An in vitro study on parasynchronized cells by flow cytometry

The effect of 3′, 5′-C-GMP and 3′, 5′-C-CMP on cell growth of the T-lymphoblastic JM cell line. An in vitro study on parasynchronized cells by flow cytometry

Human T-cell receptor β-chain genes

cDNA clones encoding the beta chain of the T-cell antigen receptor from the human leukaemic cell line JM (which extend through the constant region and into the variable region of the receptor) show hybridization only to RNA from JM cells, and not in five other T-cell leukaemias. Multiple hybridizing bands were detected in genomic DNA digests by a V beta probe, suggesting that inherited V beta gene diversity will be significant. Two beta-chain constant region genes (designated C beta 1 and C beta 2) occur in human genomic DNA, and both of these genes are capable of rearrangement in T-cell DNA. When the downstream C beta 2 gene is rearranged, deletion of the C beta 1 gene can occur. These characteristics of rearrangement and deletion of beta-chain genes show analogies to the immunoglobulin gene loci.

Antibodies to rheumatoid arthritis nuclear antigen (RANA) in Japanese patients with rheumatoid arthritis.

We studied antibodies to rheumatoid arthritis nuclear antigen (RANA) by the Ouchterlony method in 0.5% agarose plates, using soluble antigen extracted with 0.25 M sucrose solution from cultured Raji cells. Anti-RANA antibody was found in sera from 24 to 40 (60%) patients with rheumatoid arthritis (RA), from 4 of 20 (20%) patients with systemic lupus erythematosus (SLE), and from 2 of 30 (7%) healthy controls. When sucrose extracts from BJAB, Ramos, and JM cells were used as the cellular antigens, no anti-RANA precipitin lines were detected. Indirect immunofluorescence study, using Raji cells or human B lymphocytes transformed by EB virus as substrate tissues, demonstrated anti-RANA antibody as fine speckled nuclear staining. Although RA patients with positive anti-RANA antibody usually had high titers of anti-Epstein-Barr nuclear antigen (EBNA) and anti-viral capsid antigen (VCA) IgG antibodies, the Wilcoxon ranks sum test showed no close statistical correlation between the presence of anti-RANA antibodies and the titers of anti-EBNA or anti-VCA (IgG) antibodies. These data showed that the incidence of positivity of anti-RANA antibodies in Japanese RA patients is almost the same as that in American and European RA patients.

Polycationic polypeptides: a possible model for the penetration-enhancing factor in the invasion of host cells by Toxoplasma gondii.

The effect of polycationic polypeptides (polylysine, polyarginine and polyhistidine) on the invasion of mammalian cells and plant protoplasts by Toxoplasma gondii was studied. In JM cells, a human lymphoblastoid cell line with T cell characteristics, all polycationic polypeptides used increased the invasion rate in a concentration-dependent manner. This effect and the morphological changes revealed by electron microscopy resembled the action of the penetration-enhancing factor previously described by E. Lycke and co-workers. Plant protoplasts of Catharanthus roseus, which are resistant to T. gondii invasion, showed the same morphological changes in the presence of polycationic polypeptides as observed for JM cells, but were not invaded.

Glutamine requirements for purine metabolism in leukemic lymphoblasts

The two pathways of purine metabolism that include glutamine-dependent reactions, purine synthesis de novo and guanine nucleotide synthesis, were studied in cultured lymphoblasts derived from patients with T cell (JM), B cell (BALL) or null cell (NALL) acute lymphoblastic leukemia (ALL). When glutamine was omitted from the incubation medium, purine, synthesis de novo, measured by the incorporation of 14C-formate into purine compounds, was depressed to barely measurable rates in BALL and NALL cells, but proceeded at moderate through reduced rates in JM cells, when compared to synthesis in the presence of 2 mM glutamine. Similarly, the incorporation of 14C-hypoxanthine into guanine nucleotides was arrested at the glutanine-requiring XMP-aminase reaction in the BALL and NALL lines but not in the JM line, when exogenous glutamine was absent. The data suggest that glutamine deprivation, whether by omission from the culture medium in vitro or by glutaminase treatment in vivo, will have more profound biochemical consequences in B and null cell-derived ALL than in T ALL.

The induction of antichromatin antibodies with specificity for nuclear antigens of transformed human lymphocytes

An antibody was produced in rabbits using human dehistonized chromatin from T lymphocytes in continuous cell culture (Jm) as immunogen. The antibody was characterized by a micro-complement fixation (CF) method against chromatin from Jm cells as well as other sources. The antibody reacted only with chromatin of transformed lymphocytes, obtained either in vitro from tissue culture, or in vivo specifically from chronic lymphocytic leukemia (CLL). This antibody did not reset with chromatin of normal human or non-human lymphocytes, or with chromatin of two other related non-lymphoid human tumor types, namely chronic myelocytic leukemia (CML) and HeLa cells in continuous culture. The data suggest that there are common antigens specific to the nucleus of transformed human lymphocytes which may be related to the maintenance and/or development of this particular state of differentiation.