Identification of human lymphocyte-derived lymphotoxins with binding and cell-lytic activity on NK-sensitive cell lines in vitro

Supernatants obtained from lectin-restimulated, preactivated, human peripheral blood lymphocytes rapidly released (5–24 hr) high levels of lymphotoxin (LT) activity in vitro. Peripheral blood lymphocytes were preactivated by coculturing with either fetal calf serum or with allogeneic continuous B-cell lines (LCCL) which were treated with mitomycin C. These Supernatants contained a population of L-929 cell-lytic LT forms which also selectively bind to the NK-sensitive K-562 cell. However, lytic LT forms for L-929 cells from cPBL and LCCL cultures did not bind to the NK-sensitive MOLT-4 or NK-resistant Raji cells. Additional studies reveal these supernatants contain a second set of LT forms which have cell-binding and cell-lytic activity detectable on MOLT-4 and K-562 cells in a 12 to 18 hr ⁵¹Cr-release assay. Cell-lytic form(s) for the MOLT-4 and K-562 cells were not stable for more than a week at ?20°C. These findings indicate that materials with LT activity are heterogeneous with respect to their capacity to recognize common and discrete cell-surface components on different types of target cells in vitro.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

In vitro depression of cellular immunity by Friend virus leukemic spleen cells.

Four distinct sublines of mouse L 929 cells (termed alpha, beta, gamma, and delta) were derived and shown to differ markedly in their in vitro sensitivity to human lymphotoxin (LT). The alpha L cell is most sensitive and is rapidly destroyed by very low dilutions of LT. This cell is 100 times more sensitive to LT than the most resistant (delta) L cell. The highly lymphotoxin-sensitive alpha cell makes it possible to reproducibly detect LT activity in as little as 0.0005 ml of supernatant medium. Additional studies revealed a direct correlation between the sensitivities of the four L cell sublines to LT and to direct cytolysis mediated by mitogen-stimulated human lymphocytes. The alpha, beta, gamma, and delta L cells were shown to be equally sensitive to antibody-mediated complement-dependent lysis, indicating that the sequence of sensitivities of these L cell sublines to the direct lymphocyte and to LT does not merely reflect a general susceptibility to cell destruction. These results lend further support to the view that lymphotoxin is an important mediator of in vitro target cell destruction by human effector lymphocytes.     

Synthesis and in vitro evaluation of N-alkyl-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)indolin-2-one analogs as potential anticancer agents

A series of novel 3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-yl)indolin-2-one analogs (3) have been synthesized under microwave irradiation and conventional heating methods. These analogs were evaluated for in vitro cytotoxicity against a panel of 57 human tumor cell lines. Compound 3o had GI₅₀ values of 190 nM and 750 nM against A549/ATTC non-small cell lung cancer and LOX IMVI melanoma cell lines, respectively, and both 3n and 3o exhibited GI₅₀ values ranging from 2 to 5 μM against CCRF-CEM, HL-60(TB), K-562, MOLT-4, and RPMI-8226 leukemia cell lines. These results indicate that N-4-methoxybenzyl-3-hydroxy-(2-imino-3-methyl-5-oxo-4-yl)indolin-2-one analogs may be useful leads for anticancer drug development.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Cytoagglutination and cytotoxicity of Wheat Germ Agglutinin isolectins against normal lymphocytes and cultured leukemic cell lines--relationship between structure and biological activity

The relationships between degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of three Wheat Germ Agglutinin isolectins (WGA-1, WGA-2, WGA-3) against normal lymphocytes and cultured leukemic cell lines (Jurkat, MOLT-4, Raji, Daudi, K-562) were studied. All WGA-isolectins interacted in a similar degree with normal lymphocytes, while in the case of leukemic cells, the degree of isolectin-cell binding increased in the order: WGA-1< or =WGA-3相似文献   

In vitro cytotoxicity of melleolide antibiotics: structural and mechanistic aspects

Melleolide sesquiterpene aryl esters are secondary products of the mushroom genus Armillaria. We compared the cytotoxicity of eleven melleolides—five thereof are new natural products—against four human cancer cell lines. Armillaridin, 4-O-methylarmillaridin, and dehydroarmillylorsellinate were most active, at IC₅₀ = 3.0, 4.1 and 5.0 μM, respectively, against Jurkat T cells for the former two compounds, and K-562 cells for the latter. Dehydroarmillylorsellinate did not inhibit respiration and RNA-synthesis of K-562 cells at 5 μM. However, replication of DNA dropped to 35% after 120 min at this concentration, and translational activity also decreased.     

Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.     

Stabilization and functional studies of high-molecular-weight murine lymphotoxins

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2–4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (α_H 130,000–160,000 daltons). It was found that the Cx and α_H LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr ⁷⁵Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell.     

Bis(4-hydroxy-2H-chromen-2-one): Synthesis and effects on leukemic cell lines proliferation and NF-κB regulation

Synthesis of the bis-4-hydroxycoumarin-type compound, 3,3′-[3-(2-hydroxyphenyl)-3-oxopropane-1,1-diyl]bis(4-hydroxy-2H-chromen-2-one), was performed by two alternative pathways, either involving a basic organocatalyzed 1,4-conjugate addition tandem reaction of 4-hydroxycoumarin on chromone-3-carboxylic acid, or a double condensation of 4-hydroxycoumarin on ω-formyl-2′-hydroxyacetophenone. The anti-proliferative effects of the bis-4-hydroxycoumarin-type compound on human K-562 (chronic myeloid leukaemia) and JURKAT (acute T-cell leukaemia) cell lines using trypan blue staining, as well as its involvement in nuclear factor-kappa B (NF-κB) regulation analyzed by luciferase reporter gene assay, gene expression analysis and western blots were analysed. This compound inhibited TNFα-induced NF-κB activation in K-562 (IC₅₀ 17.5 μM) and JURKAT (IC₅₀ 19.0 μM) cell lines, after 8 h of incubation. Interestingly, it exerted mainly cytostatic effects at low doses on both cell lines tested, whereas it decreased JURKAT cell viability starting at 50 μM from 24 h of treatment. Importantly, it did not affect the viability of peripheral blood mononuclear cells (PBMCs) from healthy donors, even at concentrations above 100 μM.     

The abietane diterpenoid parvifloron D from Plectranthus ecklonii is a potent apoptotic inducer in human leukemia cells

BackgroundAbietane diterpenes have attracted much attention because they display a wide range of biological activities, including antitumor activities. These compounds are the most diverse of the diterpenoids isolated from species of Plectranthus. Naturally occurring diterpene parvifloron D is the main phytochemical constituent of Plectranthus ecklonii. To examine the therapeutic potential of the plant, we evaluated whether parvifloron D displays cytotoxicity against human tumor cells.MethodsThe cytotoxicity was analyzed by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was evaluated by fluorescent microscopy, transmission electron microscopy, flow cytometric analysis of annexin V-FITC and propidium iodide-stained cells and DNA fragmentation. Protein expression and processing and release of mitochondrial proteins were analyzed by Western blot. Caspase activity was determined using colorimetric substrates. The membrane potential and intracellular reactive oxygen species were detected by flow cytometry.ResultsParvifloron D displays strong cytotoxic properties against leukemia cells (HL-60, U-937, MOLT-3 and K-562) and in particular P-glycoprotein-overexpressing K-562/ADR cells, but has only weak cytotoxic effects on peripheral blood mononuclear cells (PBMCs). Overexpression of the protective mitochondrial proteins Bcl-2 and Bcl-x_L did not confer resistance to parvifloron D-induced cytotoxicity. Growth inhibition of HL-60 cells that was triggered by parvifloron D was found to be caused by a rapid induction of apoptotic cell death. This apoptosis was prevented by the non-specific caspase inhibitor z-VAD-fmk, and by the selective caspase-9 inhibitor z-LEHD-fmk. Cell death induced by parvifloron D was found to be (i) associated with the dissipation of the mitochondrial membrane potential and the release of cytochrome c, (ii) amplified by inhibition of extracellular signal-regulated kinases (ERKs) 1/2 signaling and (iii) caused by a mechanism dependent on intracellular reactive oxygen species generation.ConclusionParvifloron D is a potent cytotoxic compound against several human tumor cells and also a fast and potent apoptotic inducer in leukemia cells.     

Synthesis and biological properties of α-thymidine 5′-aryl phosphonates

Diethyl(N-arylaminocarbonyl)methyl phosphonates have been obtained by the reaction of diethylphosphonoacetic acid imidazolides with methyl-4-aminobenzoate or 3,5-bis(trifluoromethyl)phenylamine. Their treatment with Me₃SiBr in DMF led to a mixture of the corresponding (N-arylaminocarbonylmethyl)phosphonic acids and their monoethyl esters. After separation, they were condensed with 3′-O-acetyl-α-thymidine, which, after the removal of the acetyl protecting group, gave (α-D-thymidine-5′-yl)-[4-aminocarbonyl-, methoxycarbonyl-, or carboxy)phenylaminocarbonyl]methyl phosphonates and (α-D-thymidine-5′-yl)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. It was shown that the compounds are stable under different conditions, low toxic (in Vero and K-562 cell cultures), and capable of penetrating into K-562 cells. Only ethyl (α-D-thymidine-5′-yl)-[4-(methoxycarbonyl)phenylaminocarbonyl]methyl phosphonate at a high concentration (200 μg/mL) inhibited in vitro the growth of the laboratory strain M. tuberculosis H37Rv.     

The human LT system. II. Immunological relationships of LT molecules released by mitogen activated human lymphocytes in vitro.

Materials with LT activity present in supernatants from PHA stimulated human lymphocytes in vitro are very heterogeneous and can be separated into multiple molecular weight classes, termed complex, α, β, and γ. Several of these classes can be further resolved into subclasses by other physical and chemical methods. The immunologic relationships of these materials one to another were examined employing various rabbit anti-humn LT sera which will neutralize LT activity on L-929 cells in vitro. These studies reveal: (a) LT activities are due to a distinct group of substances which are immunologically related one to another and can exist in several molecular weight forms; (b) a high MW class of molecules, termed complex, appears to contain all currently known LT classes and subclasses; (c) LT classes and subclasses both have common (public) and discrete (private) antigenic specificities; (d) human LT classes and subclasses do not appear to share Ag determinants with materials with LT activity released by lectin stimulated lymphoid cells from rabbit, rat, hamster, guinea pig, or mouse; and (e) human LT molecules are not immunologically related to cell toxins released by glass adherent human peripheral blood monocytes or PMN cells. These data indicate human LT molecules form a “discrete system” of lymphocyte derived cell toxins, which can associate together into various related but different MW forms in the supernatant.     

Human natural killer cell activity is reversibly inhibited by antagonists of lipoxygenation

We here demonstrate that NK cell activity by human peripheral blood mononuclear cells (PBMC) against K562 or MOLT-4 target cells is rapidly and reversibly inhibited by two agents that inhibit the lipoxygenation of fatty acids, BW755C and nordihydroguaiaretic acid (NDGA). Natural killing by nonadherent PBMC was similarly inhibited by both agents, indicating that monocytes were not required for the effect. The inhibition of natural killing was not seen with indomethacin at concentrations that inhibit prostaglandin synthesis but not the lipoxygenation of arachidonic acid. Moreover, indomethacin did not alter inhibition by either BW755C or NDGA. Thus, suppression of natural killing by these agents was not mediated by the effects on prostaglandin synthesis; neither agent inhibited target cell binding. These results suggest that products of lipoxygenation are required for target cell lysis by human NK cells.     

PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in colonization and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was constructed in a pneumococcal TIGR4 background. When the mutant and wild type strains were compared for their adherence to D562 and A549 cell lines, a reduction in adherence by the mutant was observed (p = 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host Escherichia coli resulting in augmented adherence to D562 (p = 0.002) and A549 (p = 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p = 0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p = 0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.     

Lymphocyte function-associated antigen 1 (LFA-1) and natural killer (NK) cell activity: LFA-1 is not necessary for all killer: target cell interactions

A panel of five monoclonal antibodies detecting human lymphocyte function-associated antigen 1 (LFA-1) was generated and shown by competitive binding studies to react with at least four distinct epitopes on this molecule. The antibodies were then tested for their ability to inhibit the lytic activity of a variety of different human natural killer (NK) populations on a panel of four NK-susceptible target cells (K562, MOLT-4, HSB-2, and Jurkat). When heterogeneous NK populations derived from fresh peripheral blood and mixed-lymphocyte culture (MLC)-generated lines were used, these anti-LFA-1 monoclonal antibodies (MAbs) inhibited lysis of all four NK targets; this finding supports the notion that LFA-1 molecules play an important role in NK-mediated lysis. When tested on a cloned line of NK cells (NK 3.3), lysis of K562 was inhibited by these MAbs, but lysis of the other three targets was not affected. This represents an instance where a MAb specific for LFA-1 inhibits the lytic activity of NK cells against some but not all targets; thus the LFA-1 molecule cannot be considered under all circumstances to be an absolute requirement in NK-mediated lysis.     

Leukotrienes Are Upregulated and Associated with Human T-Lymphotropic Virus Type 1 (HTLV-1)-Associated Neuroinflammatory Disease

Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB₄ and CysLTs were both increased in the plasma and in the supernatant of peripheral blood mononuclear cell cultures of HTLV-1-infected when compared to non-infected. Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB₄ and LTC₄ positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4⁺ and CD8⁺ T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.     

A sulfoximine-based inhibitor of human asparagine synthetase kills l-asparaginase-resistant leukemia cells

An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase (hASNS) with nanomolar potency, has been reported to suppress the proliferation of an l-asparagine amidohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when l-asparagine is depleted in the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of 1 that have been studied as part of systematic efforts to identify compounds with improved cell permeability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net charge at cellular pH, is a better hASNS inhibitor (K_I¹ = 8 nM) than 1 and suppresses proliferation of MOLT-4R cells at 10-fold lower concentration (IC₅₀ = 0.1 mM). More importantly, and in contrast to the lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25 mM causes the death of MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits different dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), supporting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment of leukemia, and perhaps other cancers, including those of the prostate.     

SAR studies of brasilicardin A for immunosuppressive and cytotoxic activities

Eleven derivatives (5-13, 15, and 16) of an immunosuppressive and cytotoxic tricyclic terpenoid, brasilicardin A (1), were prepared and assayed for inhibitory effects to the mouse mixed lymphocyte reaction (MLR) and seven human tumor cell lines. The 17N-methyl form (8) of 1 showed the most potent immunosuppressive activity in mouse MLR, while induction of more bulky group for N-17 resulted in significant decrease of the activity. Compound 8 also showed potent cytotoxic activity against DLD-1, Lu-65, A549, K562, and MOLT-4 cells, while the benzyl ester (13) of 1 exhibited potent cytotoxicity against K562, MOLT-4, and jarkat leukemia cell lines. The 17N-acetyl derivative (11) of 1 selectively inhibited the cell growth of DLD-1 cells. The methyl ester (5) of 1 showed potent cytotoxic activity against K562, MOLT-4, and Ball-1 cell lines, the last of which was resistant to 1, 8, and 13.