Interferon production in human T lymphoblastoid cells

Human T lymphoblastoid cell (RPMI 8402 cell) produced interferon (IFN) through the induction by Sendai virus. The priming effect on the interferon production in the RPMI 8402 cell could be found by the pretreatment of human leukocyte IFN (Hu IFN-alpha), but not by that of the IFN produced in the RPMI 8402 cell (T-IFN). The superinduction by the irradiation of ultraviolet rays or the treatment of antimetabolites (actinomycin D and cycloheximide) or 5-bromodeoxyuridine was not found. The T-IFN was completely neutralized by the anti-Hu IFN-beta serum, but not by the anti-Hu IFN-alpha serum at all. In conclusion, it was confirmed that the IFN produced in the RPMI 8402 cell through the induction by Sendai virus was antigenically identical to Hu IFN-beta.     

Lack of expression on cultured human T-lymphocytes of a T-cell antigen shared by the molt-4 cell line and normal human thymocytes

Summary Four hematopoietic cell lines (CCRF-CEM, HSB-2, MOLT-4, and RPMI-8402), derived from acute lymphoblastic leukemia and expressing T-cell surface markers (T-HCL), were studied with two specific anti-T-cell sera. The sera were raised in rabbits against human thymocytes (anti-HTY) and against T-cells cultured in the presence of conditioned medium derived from lymphocytes stimulated with PHA (anti-CTC). Both sera were absorbed to obtain a T-cell specific pattern of reaction and were further absorbed with normal peripheral blood lymphocytes or with each of the four T-HCL. The anti-HTY sera absorbed with CEM, 8402, and HSB-2 still reacted with MOLT-4. A similar pattern of reactivity was found only with the anti-CTC absorbed with 8402, whereas, after absorptions with the other cell lines, this antiserum was unreactive against MOLT-4. After absorption with normal peripheral blood lymphocytes, anti-HTY still reacted with thymocytes and MOLT-4 but was negative on CTC. In contrast, anti-CTC absorbed with peripheral blood lymphocytes (PBL) was negative on thymocytes and MOLT-4 but still reacted against CTC. Our data confirm the existence of a T-cell antigen (probably an early T-cell differentiation antigen) shared between thymus and MOLT-4. This antigen is not expressed on CTC, although these cells express an antigenic pattern more complex than PBL. Antisera to CTC represents a source of anti-T-cell sera free of contamination with antibodies to early thymus-related antigens but containing other T-cell-related specificities. Supported in part by Naval Medical Research and Development Command, Research Task No. ZF51.524.013.1025, and National Cancer Institute Contract No. Y01-CB-00319. The opinions and assertions contained herein are the private ones of the writers and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal Resources, National Research Council.     

Induction of human malignant T-lymphoblastic cell lines MOLT-3 and jurkat by 12-O-tetradecanoylphorbol-13-acetate: Biochemical,physical, and morphological characterization

The process of induction of human malignant T-lymphoblastic cell line MOLT-3 by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined. It was found that the induction process by TPA, which included increase in cells with receptors to sheep red blood cells (E-rosette positive-E⁺) and decrease in the levels of the marker enzyme terminal deoxynucleotidyl transferase (TdT) was not affected by the presence of DNA synthesis inhibitor arabinofuranosylcytosine (Ara-C). The exposure time to TPA required to elicit these changes was found to be short, in the order of 1 hour or less. The kinetics of the increase in E⁺ cells, decrease in the levels of TdT in these cells, or decrease in the ability to proliferate as measured by colony formation were similar with exposure to TPA for 1, 6, 24, or 96 hours. We have examined the effect of antitumor promoter compounds on their ability to block induction of MOLT-3 cells by TPA. Results indicated that none of these compounds, dexamethasone, antipain, retinoic acid, and L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), was effective in reducing the number of E⁺ cells induced by TPA. Examination of three other leukemic T-cell lines indicated that, in addition to MOLT-3, the leukemic T-cell line Jurkat also responded to TPA, whereas two other leukemic T-cells lines CCRF-CEM and CCRF-HSB-2 did not. Certain physical and morphological changes were also observed after stimulation of MOLT-3 cells and Jurkat cells by TPA. We found that, following the addition of TPA, the cell volumes of MOLT-3 cells decreased from an average of 1150 μm³ to about 500 μm³, whereas those of Jurkat were reduced to about 700 μm³ from 1100 μm³. Electron microscopic studies of these lymphoblasts also revealed that after treatment with TPA the induced cells were generally smaller in size with increase in the density of the nuclear materials and condensation of the chromatin structures.     

Growth of measles virus in various human lymphoid cell lines.

Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.     

Acquisition of deoxyguanosine resistance by TPA-induced T lymphoid lines

The human leukemic T cell line 8402, which contains terminal deoxynucleotidyl transferase (TdT) and phenotypically resembles precursor thymocytes, when exposed to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) undergoes in vitro T maturation. TdT disappears from virtually all the cells and a fraction of TdT- -cells express specific T surface markers, such as the T3 determinant. Like T lymphocytes from the thymus, 8402 cells are extremely sensitive to the cytotoxic effect of deoxyguanosine (dGuo). As a consequence of TPA treatment, resistance to dGuo is observed in 8402, as well as in two other TdT+ lymphoid T cell lines, Molt-4 and CEM-10. These results suggest the occurrence of changes in deoxynucleoside metabolism in TPA-treated cells related to the in vitro maturation process. Maturation of 8402 cells, once started, progresses in the presence of additional physiologic stimuli provided by conditioned medium from lymphocyte culture, because a portion of cells display the T8+/T4- phenotype characteristic of cytotoxic/suppressor T cells. This in vitro lymphoid system may thus be used to study the relationships of molecular differentiation between precursor thymocytes and cytotoxic/suppressor T cells.     

Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Localization of the human angiogenin gene to chromosome band 14q11, proximal to the T cell receptor alpha/delta locus.

The gene encoding angiogenin, a potent inducer of blood vessel formation, has been localized within the human genome. It is present as a single copy per haploid genome and is located on chromosome 14, on the basis of discordancy analysis of human-rodent hybrid cell lines. This localization was refined to 14q11 by in situ hybridization of an angiogenin probe to metaphase chromosomes prepared from both normal human lymphocytes and RPMI 8402 cells. The results from the RPMI 8402 cells also establish that the angiogenin gene resides proximal to a translocation breakpoint within the T cell receptor alpha/delta locus and therefore upstream from that locus. An AvaII RFLP, present at a frequency of 29% in an unselected collection of human placental DNAs, was identified in the coding region of the gene and results from a single silent transversion.     

Implications of a 5′-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5′-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56°C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37°C for 1 h and it was destroyed completely by heating at 100°C for 30 min. When the heated (56°C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5′-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35 000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggests that the previously reported undetectability of 5′-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5′-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5′-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Cytocidal effect of tumor necrosis factor on cells chronically infected with human immunodeficiency virus (HIV): enhancement of HIV replication.

Tumor necrosis factor (TNF), a monokine initially described as a tumoricidal agent, facilitated the replication of human immunodeficiency virus (HIV) in vitro. The viability of human T-cell line MOLT-4/HIV, chronically infected with HIV, was affected by the addition of a low dose (10 ng/ml) of recombinant TNF-alpha (rTNF-alpha), while uninfected MOLT-4 cells were resistant to treatment with rTNF-alpha at concentrations up to 1,000 ng/ml. A marked increase in the level of HIV-specific RNA was detected in MOLT-4/HIV cells as early as 1 h after exposure to rTNF-alpha and reached almost maximum level within 6 h. Production of HIV particles from MOLT-4/HIV was also increased at 6 h after treatment with rTNF-alpha. Nearly identical phenomena were observed in CCRF-CEM/HIV, Jurkat/HIV, and H9/HIV cells, although the sensitivity of these cell lines to rTNF-alpha varied. A human T-lymphotropic virus type 1-infected cell line, MT-4, was insensitive to treatment with rTNF-alpha.     

Differential distribution of calpain in human lymphoid cells

Calpain, a calcium-activated neutral proteinase, is ubiquitously present in human tissues. To determine if lymphoid cells implicated in pathogenesis of demyelination may harbor calpain in a functionally active form, we determined both Calpain and mCalpain activities in human lymphoid cell lines. DEAE-cellulose and phenylsepharose column chromatography were used to isolate the enzyme from the natural inhibitor, calpastatin. Lymphocytic lines (CCRF-CEM, MOLT-3, MOLT-4, M.R.) showed predominance of Calpain (55–80%) whereas the monocytic line (U-937) showed prodominance of mCalpain (77%). Proportion and subcellular distribution of both isoforms varied among cell lines. Calpains isolated from U-937 cells degraded myelin basic protein. These results indicate that human lymphoid cells harbor functionally active calpain that can degrade myelin components in vitro. The study suggests a degradative role for calpain in demyelinating diseases.     

Apoptosis Induction in Human Lymphoma and Leukemia Cell Lines by Transfection via Dendrosomes Carrying Wild-Type p53 cDNA

The efficiency of dendrosomes (novel dendritic spheroidal nanoparticle gene porters) were assessed in transferring wild-type p53 cDNA into two human acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) derived from T cells and erythroleukemic cell line K562. Flow cytometric studies showed a 65% and 45% enhancement in apoptosis and necrosis of K562 and CCRF-CEM cells transfected with complex of dendrosomes and wild-type p53 cDNA in comparison to controls. The cytotoxicity studies on T lymphoma cells revealed that dendrosomes have a low cytotoxicity in comparison to lipofectin.     

5′-nucleotidase activity in permanent human lymphoid cell lines Implication for cell proliferation and aging in vitro

5′-Nucleotidase of 11 human lymphoid cell lines was measured. These cell ines were homogeneous B, T and Null cells, had an unlimited lifespan in vitro, and were subcultivated from leukemic cells of patients with Burkitt lymphoma, the blastic phase of chronic myelocytic leukemia, or acute lymphoblastic leukemia. 5′-Nucleotidase activities in normal human lymphocytes and in human fibroblasts (VA-13 and IMR-90) could be determined at a cellular protein concentration as low as 0.025 and 0.007 mg/ml, respectively. In all the eleven lymphoid cell lines, including 8 B-cell lines (RPMI 8422, B46M, RPMI 1788, DAUDI, HRIK, B411-4, B85 and DND-3-9A), 2 T-cell lines (MOLT 3 and RPMI 8402) and 1 Null cell line (NALM-1) 5′-nucleotidase was undetectable with the protein concentration range from 0.033 to 8.543 mg/ml. Previously 5′-nucleotidase activity was found to increase 10-, 6- and 20-fold in normal human embryonic lung (WI-38 and IMR-90 cells) and chick embryo fibroblasts, respectively, from a young rapidly proliferative to a senescent non-proliferative stage (Sun, A.S., Aggarwal, B.B. and Packer, L. (1975) Arch. Biochem. Biophys. 170, 1 and Sun, A.S., Alvarez, L.J. Reinach, P.S. and Rubin, E. (1979) Lab. Invest. 41, 1). These data demonstrate that the large increase in 5′-nucleotidase activity occurs concomitantly with the in vitro senescence of these normal cell lines. The present study suggests that this large increase in 5′-nucleotidase activity during cell aging is absent in these permanently lymphoid cell lines. The undetectable 5′-nucleotidase activity may be a biochemical characteristic of these homogeneous B, T and Null cells originating from the aforesaid leukemias. The implications of these results for cell proliferation and aging are discussed.     

Leukotriene A4, conversion to leukotriene B4 in human T-cell lines

Human T-cell lines (HSB, MOLT-4 and CCRF-CEM) produced leukotriene B4 when incubated with leukotriene A4. The product was characterized by chromatographic properties, UV-spectroscopy and gas chromatography mass spectrometry. About 10 pmol of leukotriene B4 was obtained per 10(6) cells. When incubated with arachidonic acid plus the calcium ionophore A23187 however, no leukotriene B4 was found, indicating that the T-cell lines lack 5-lipoxygenase yet contain LTA4 hydrolase.     

Antibodies to human immunodeficiency virus in human sera induce cell-mediated lysis of human immunodeficiency virus-infected cells

The capacity of human immunodeficiency virus (HIV) antibody-positive sera from homosexually active men without acquired immune deficiency syndrome to lyse the HIV-infected T cell lines MOLT-4f and CCRF-CEM (CEM) in cooperation with lymphocytes from normal donors was investigated. Twenty-seven HIV antibody-positive sera, most of which enhanced the killing of HIV-infected MOLT-4f and CEM target cells by normal mononuclear cells were studied in detail. HIV antibody-positive sera resulted in lysis at dilutions as high as 1/10,000. HIV antibody-negative sera did not augment lysis of infected target cells. In addition, lysis of uninfected targets was not enhanced in the presence of HIV antibody-positive sera. Because fractionation of the HIV antibody-positive sera on a protein A affinity column resulted in recovery of the activity from the IgG fraction, the extra cytotoxic activity mediated by nonimmune cells in the presence of immune sera appears to be antibody-dependent. Furthermore, the cytotoxic effector cells were in the nonrosetting fraction of lymphocytes and expressed Leu-11 (cluster designation (CD)15) antigens, which is characteristic of cells participating in antibody-dependent cellular cytotoxicity reactions. The antibody specificity of the sera, determined by radioimmunoprecipitation, provides evidence that antibody-dependent cellular cytotoxicity can occur even when there are no detectable antibodies directed against gag proteins. Sera which lacked detectable antibodies to the envelope protein gp120 by radioimmunoprecipitation did not mediate antibody-dependent cellular cytotoxicity.     

Human cell membrane components bound to beta2-microglobulin in T cell-type cell lines.

Cell membrane components bound to beta2-microglobulin were isolated from Renex 30 (a nonionic detergent)-solubilized membrane materials of two human T cell-type cell lines, MOLT-4 and CCRF-CEM, by gel filtration and lectin affinity chromatography. The isolation was carried out by following the beta2-microglobulin activity by radioimmune inhibition assay. The T cell membrane components bound to beta2-microblogulin had a uniform molecular size of about 200,000 daltons and most of them showed an affinity to lentil lectin. The isolated membrane components were radioiodinated and examined for identity to HLA antigens by sequential precipitation with rabbit anti-HLA antiserum (specific to HLA large components) and with rabbit anti-beta2-microblogulin antiserum. In addition to HLA antigens, the beta2-microglobulin-bound components obtained from the MOLT-4 cells were found to contain certain membrane components that are the same in molecular size as the HLA large components but that are different antigenically from the HLA large components. On the other hand, the beta2-microglobulin-bound membrane components obtained from the CCRF-CEM cells were all HLA antigens. No other membrane components were involved in the binding.     

Ring-closing metathesis for the synthesis of a highly G-quadruplex selective macrocyclic hexaoxazole having enhanced cytotoxic potency

The synthesis of a 24-membered macrocyclic hexaoxazole via ring-closing metathesis is described. The target compound selectively stabilizes G-quadruplex DNA with no detectable stabilization of duplex DNA. An MTT cytotoxicity assay indicated that this unsaturated macrocyclic hexaoxazole exhibits significant cytotoxicity toward P388, RPMI 8402, and KB3-1 cell lines with IC50 values of 45, 25, and 38 nM, respectively.     

Expression of multiple mRNA species for choline acetyltransferase in human T-lymphocytes

Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in cholinergic neurons. However, both ACh and mRNA for ChAT are expressed in mononuclear leukocytes and various human leukemic T-cell lines. Multiple ChAT mRNA species (R-, N0-, N1-, N2-, and M-types) having an identical coding region and different 5'-noncoding regions have been discovered in human brain and spinal cord. These mRNAs are transcribed by a combination of use of different promoter regions and alternative splicing. However, which types of ChAT mRNA species are expressed in T-lymphocytes remains to be elucidated. In the present study, we used two human leukemic T-cell lines, CCRF-CEM (CEM) and MOLT-3, which express the same ChAT mRNA as that in the nervous system. Major mRNA species in CEM were N2- and M-type, and to a lesser extent N1-type, while MOLT-3 expressed only N2-type. Neither CEM nor MOLT-3 expressed R-type mRNA. We previously found a lack of mRNA expression encoding vesicular acetylcholine transporter (VAChT) in CEM and MOLT-3, which mediates ACh transport to synaptic vesicles in cholinergic neurons. These findings suggest that the mechanisms regulating ChAT mRNA expression in T-lymphocytes differ from those in cholinergic neurons.     

Production of macrophage activating factor by human leukemic T cell lines

Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies.     

Selection of carbohydrate-binding cell phenotypes using oligosaccharide-coated magnetic particles

Neoglycoconjugate coated magnetic beads were assessed for theirability to selectively isolate human cells with known anti-carbohydratereactivity. Four lung cancer cell lines, NCI-H146, NCI-N417D,SKMES-1, EKVX; two acute lymphoblastic leukemia lines, MOLT-4and CCRF-CEM; and the anti- Le^c (isolactosamine) hybridoma,LU-BCRU-G7, were tested. The neoglycoconjugates (biotinylatedpseudopolysaccharides) bound uniformly to streptavidin coatedmagnetic beads as demonstrated by FTTC labeled lectin. Streptavidinbeads alone did not bind to any of the cell types. The anti-Le^c hybridoma cell line, LU BCRU-G7, demonstrated binding onlyto Le^c pseudopolysaccharide coated magnetic beads. Subsequentincubation in the presence of unlabeled pseudopolysaccharideresulted in the release of the beads from the cell surface.Although there was some heterogeneity within the individuallung and leukemic cell lines, positive cells showed strong rosetteformation with the coated beads. The A_dl disaccharide coatedbeads showed binding in all four lung cancer cell lines, withthe Le^c and the H (type1) pseudopolysaccharide-bead conjugatesonly reactive in the N417 and H146 SCLC lines. The range ofL-selectin ligand-coated beads were all successful in bindingto the acute lymphoblastic leukemia cell lines MOLT4 and CCRF-CEM.This approach provides a versatile model for the study of cell-surfacecarbohydrate interactions that should find application in manyareas of cell biology. carbohydrate-coated magnetic beads cell selec-tion lectins neoglycoconjugates pseudopolysaccharides