An autocrine growth factor constitutively produced by a human lymphoblastoid B-cell line is serologically related to lymphotoxin (TNF-beta).

Conditioned medium of a human lymphoblastoid B-cell line RPMI-6410t contains a factor sufficient for maintainance and growth of these cells. At the same time RPMI-6410t cells secrete a soluble factor cytotoxic towards mouse L929 cells. Production of these activities by RPMI-6410t cell line and its subclones is significantly enhanced after activation with phorbol mirystate acetate (PMA). Both activities can be neutralized by antiserum raised against recombinant lymphotoxin (rTNF-beta) but not by antibodies against tumor necrosis factor (rTNF-alpha). Northern analysis showed the presence of lymphotoxin mRNA which is further induced after PMA treatment. These data suggest that both autocrine growth factor and cytotoxic activities correspond to the same molecule(s) probably identical to 25 kD lymphotoxin (TNF-beta).

Selective sensitivity to tiazofurin of human leukemic cells.

This study reports the selective sensitivity to tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC-286193) of human leukemic leukocytes as compared to normal ones in bone marrow and peripheral blood samples by comparing the production of the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), from labeled tiazofurin and the depression of GTP concentration. When labeled tiazofurin was incubated with leukocytes obtained from healthy volunteers or from leukemic patients (acute non-lymphocytic leukemia or acute lymphoblastic leukemia), the TAD production was 27.0 +/- 8.3, 551.3 +/- 71.8 and 755.9 +/- 94.1 pmoles/10(9) cells per hr, respectively. Thus, the leukemic cells produced over 20-fold higher concentrations of TAD than the normal leukocytes. Incubation with tiazofurin in leukemic leukocytes decreased the GTP pools (to 48-79%), whereas there was no change in the normal leukocytes. These results indicate a selectivity of response to tiazofurin in human normal and leukemic leukocytes. The procedure reported in this work may be suitable as a rapid predictive test for the sensitivity of leukemic leukocytes to tiazofurin. Such a diagnostic test should be helpful in identifying neoplastic cells sensitive to tiazofurin in the Phase II trials now being developed.

Identification of lymphocyte integral membrane proteins as substrates for protein kinase C. Phosphorylation of the interleukin-2 receptor, class I HLA antigens, and T200 glycoprotein.

The interleukin-2 (IL-2) receptor, the leukocyte-specific membrane glycoprotein, T200, and the class I major histocompatibility antigens (HLA) have been identified as substrates for protein kinase C in vitro. IL-2 receptors on normal human T lymphocytes and the leukemic cell line, HUT102B2, are rapidly phosphorylated in vivo in response to the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Tryptic peptide analysis showed that the in vitro and in vivo 32P-labeled IL-2 receptors were phosphorylated on the same sites. A synthetic peptide corresponding to the carboxyl-terminal cytoplasmic tail of the IL-2 receptor was shown to be phosphorylated in vitro by protein kinase C. Tryptic digestion of the peptide generated the same 32P-labeled species as those found for the IL-2 receptor. From these studies, it was concluded that Ser-247 is the major site of phosphorylation in the IL-2 receptor and that Thr-250 is a minor site. These results also provide direct evidence that the in vivo phosphorylation of the IL-2 receptor stimulated by TPA is catalyzed by protein kinase C. The sites phosphorylated in the HLA antigens in vitro by protein kinase C or in vivo after TPA stimulation were also localized to the carboxyl-terminal cytoplasmic domain of the heavy chain by limited proteolysis.

"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and partial characterization of human T-cell growth factor.

Human T-cell growth factor (TCGF) has been isolated from conditioned media of the Jurkat T-leukemia cell line. Using a high-efficiency isolation procedure involving hollow fiber concentration, gel filtration and 3 steps of reverse-phase HPLC we obtained 100 to 600 pmol TCGF per liter of conditioned medium. Jurkat cell-derived TCGF (jTCGF) has a molecular weight of 15,750. The amino acid composition of jTCGF agrees well with that derived from the cDNA sequence coding for this protein (Taniguchi et al, Nature 302, 305, 1983). jTCGF is highly active in vitro in stimulating the proliferation of T-cells as measured by 3H-thymidine incorporation into DNA (half-maximal stimulation with 3 fmol/100 microliters well).

1,25-dihydroxyvitamin D3 receptors in human leukocytes.

A 1,25-dihydroxyvitamin D3 receptor macromolecule was detected in peripheral mononuclear leukocytes from normal humans. This macromolecule was found to be present in monocytes but absent from normal resting peripheral B and T lymphocytes. However, it was present in established lines of malignant B, T, and non-B, non-T human lymphocytes, as well as in T and B lymphocytes obtained from normal humans and activated in vitro.

Localization of eosinophil granule major basic protein in human basophils.

In experiments using an immunofluorescent method to localize human eosinophil granule major basic protein (MBP) in cells and tissues, a small number of cells stained for MBP that subsequently could not be identified as eosinophils. Because the Charcot-Leyden crystal protein, another eosinophil protein, was recently identified in basophils, we tested whether MBP might also be a constituent of blood basophils. Highly purified, eosinophil-free basophil suspensions were prepared using the fluorescence-activated cell sorter (FACS) to sort basophil-containing mononuclear cell preparations stained with fluorescein-conjugated sheep IgG anti-human IgE antibody. Using these FACS-purified basophils, we found that: (a) enrichment for surface IgE-positive cells (greater than 95% basophils) by FACS also enriched for cells staining for MBP by immunofluorescence; (b) MBP appeared to be localized in the histamine-, heparin-containing granules; (c) significant quantities of MBP were measurable by radioimmunoassay (RIA) in freeze-thaw detergent extracts of purified basophils; and (d) RIA dose-response curves for extracts of purified eosinophils and basophils had identical slopes. The MBP content of basophils from normal individuals averaged 140 ng/10(6) cells, whereas purified eosinophils from normal donors and patients with the hyper-eosinophilic syndrome averaged 4,979 and 824 ng/10(6) cells, respectively. MBP was also detected by immunofluorescence and RIA in cells obtained from a patient with basophil leukemia, although the MBP content for basophil leukemia cells was lower than that for normal basophils. We conclude that basophils contain a protein that is immunochemically indistinguishable from eosinophil granule MBP.

Differential ability of mitogen-stimulated human leukocyte-conditioned media to induce Fc receptors in human leukemia cells.

The ability of mitogen-stimulated human leukocyte-conditioned media (M-CM) to induce the in vitro differentiation of various human leukemic cell lines was evaluated by measuring the appearance of Fc receptors (FcR) through their ability to form EA rosettes. Only cells of myeloid lineage were induced by M-CM to express FcR; T-, and B-, and non-T/non-B cells failed to respond. As determined with ML-1, a line of human myeloblastic leukemia cells, pokeweed mitogen-conditioned medium, at concentrations of 1-10%, stimulated the expression of FcR in 70-98% of the cells within 1 day after treatment. Phytohemagglutinin-, concanavalin A-, and lipopolysaccharide-conditioned media were less active. The FcR-inducing activity was partially separated from M-CM by chromatography on Sephadex G-75. It was stable between pH 4 and 10, and lost activity at temperatures above 40 degrees C.

Characterization of glycosphingolipids from cells of various types of human leukemia: occurrence of two glycosphingolipids, one reacting with anti-asialo GM1 antibody and one with anti-Forssman antibody.

Glycosphingolipids of neutrophils, lymphocytes and leukocytes from patients with various types of human leukemia [acute lymphoblastic (ALL), acute unclassified type (AUL), acute myeloblastic (AML), acute monocytic (AMoL), chronic myeloblastic (CML)] and the hypereosinophilic syndrome (HES) were analyzed chemically and immunochemically. No distinct difference was found in the molar ratio of lipid-bound sialic acid to lipid-bound phosphorus in these cells, but a low ratio of cholesterol to lipid-bound phosphorus was found in ALL (3 of 4 cases), AML, CML and AMoL (one of 2 cases). The predominant glycosphingolipid was ceramide dihexoside (CDH) in all cells analyzed, but the amount and the molar ratio of lipid-bound phosphorus to CDH were clearly different in different cell types, indicating that the molar ratio is a useful criterion in the classification of types of leukemia. In addition, molecular diversity of minor glycosphingolipid components was observed in various leukemic cells. Two of the neutral glycosphingolipids in AMoL were tentatively identified as asialo GM1 and Forssman glycolipids by comparing their mobilities on thin-layer chromatography with those of standard glycolipids and by observing the formation of precipitin lines on a double diffusion agar plate with anti-asialo GM1 and anti-Forssman antibodies.

Analysis of human leukemic cells by use of high-resolution two-dimensional electrophoresis. I: results of a pilot study.

We analyzed mononuclear leukocytes from patients with various human leukemias by high-resolution two-dimensional electrophoresis. Tumor cells of the granulocytic, monocytic, and lymphoid lineages [obtained from chronic granulocytic leukemia in blast transformation, acute monocytic leukemia, and chronic lymphocytic leukemia (CLL), respectively] can be easily recognized by using a series of cell-type marker proteins identified by comparison of fractionated normal cell populations. B and T cell types of CLL could be distinguished, the results correlating well with those obtained by use of monoclonal-antibody staining methods. In two cases representing almost pure B-cells (classical CLL; 0% T, 85% B) and T-cells (cutaneous T-cell leukemia; 77% T, 0% B), 27 of 29 marker proteins showed quantitative B/T differences comparable to those observed in comparisons of normal B-and T-lymphocytes prepared by cell sorting. These results indicate that cells from relatively well-differentiated leukemias show complex patterns of gene expression very similar to those of the corresponding normal cells and strongly support the use of large marker panels in cell-type determination. Less-well-differentiated acute leukemias [such as acute undifferentiated and acute granulocytic (FAB:M1)] appear to yield protein patterns corresponding less closely to recognizable mature cell types, and may show expression of novel proteins related to the state of differentiation.

[Clinical and kinetic characteristics of a temporarily nonproliferating subpopulation of bone marrow blast cells from acute leukemia patients (based on data on DNA autoradiography and cytophotometry]. / Klinicheskaia i kineticheskaia kharakteristika vremenno neproliferiruiushchei subpopuliatsii blastov kostnogo mozga bol'nykh ostrym leikozom (po dannym avtoradiografii i tsitofotometrii DNK).

The morphology of Paramecium bursaria cells, both originally possessing 2 micronuclei (MI), or one MI, has been studied after a local UV irradiation of the germ nuclei. Elimination of one of the MI in bimicronuclear cells usually did not lead to general cytological damage in the UV progeny. Irradiation of MI in the unimicronuclear cells resulted in the following: producing of amicronuclear (MI-) UV subclones, 86% of which appeared to be non-viable. The MI- cells are characterized by the appearance of somatic deciliation and the loss of mobility, disorganization structures of the oral apparatus, the feeding process and the cell division. It is remarkable that the macronucleus (MA) of cells from those cultures displays fragmentation and aberrant division. The structure of MA was changed. Dimensions of the MI- ciliates decreased (by 1.7--1.8 times in average); sometimes these cells had 3--5 contractile vacuoles with one pore. These data suggest that the MI has important role during the vegetative phase of the cell life. Possible asexual cellular functions of the MI and the role of the break of nuclear interactions in the appearance of the morphological damage are discussed.

Decrease in human lymphocyte surface glycoconjugates in leukemia as demonstrated by lectin binding.

Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells.

[Urinary and fecal excretion of porphyrins and their precursors in leukemia patients]. / Prouchvane na izluchenite s urinata i izprazhneniiata porfirini i niakoi tekhni predshestveitsi pri levkozno bolni.

Delta-aminolevulinic acid (DALA), porphobilinogen (PBG) and porphyrins in urine and feces were determined in 40 healthy controls (20 males and 20 females) and in 60 patients with acute leukosis (27 males and 33 females), with chronic myeloleukosis - 23 (10 males and 13 females), and 25 patients with chronic lympholeukosis (II males and 14 females). Another 15 patients (7 males and 8 females) were studied at the initial stage, recurrence, recidivation of acute leukosis (AL). DALA excreted with the urine, manifested no significant discrepancy as compared with the controls of the three groups examined patients and in all the three stage of AL, whereas PBG was moderately reduced in the AL patients - initial stage and recurrence and was within the normal limits in the patients with chronic myeloleukosis (CML) and chronic lympholeukosis (CLL). The urine excreted coproporhyrin was increased, to various degrees, in the patients with CML and CLL and at the initial stage, recurrence and recidivation of AL, whereas uroporhyrin was within the norm. Coproporphyrin, excreted in the feces, was increased in all three groups of patients with leukosis and in the three stages of AL, whereas the other fractions showed no significant difference as compared with the controls. It could be concluded, from the results obtained, that porphyrins metabolism is disturbed in the patients with leukosis.

Unique cell surface glycoprotein expression in hairy cell leukemia: effect of phorbol ester tumor promoters on surface glycoproteins, cell morphology, and adherence.

Hairy cell leukemia cells from eight different patients exhibited a characteristic cell surface glycoprotein pattern when labeled by the neuraminidase-galactose oxidase-NaB3H4 procedure. The diffuse high-molecular-weight glycoprotein band (Mr 230,000 to 300,000) was not seen in other leukemic cell types and may represent a specific hairy cell leukemia antigen. Hairy cell leukemia cells can be maintained as cell suspension cultures, but treatment with a variety of tumor-promoting phorbol esters caused the cells to adhere to plates, assume a fibroblastic elongated shape, and extend long processes. This dramatic morphological change was not associated with any change in surface glycoproteins.

Murine cell surface transferrin receptor: studies with an anti-receptor monoclonal antibody.

A rat monoclonal antibody against the murine transferrin receptor has been identified. The receptor is a 95,000 molecular weight species that exists in the cell membrane as a disulphide-bonded dimer. Whereas 29 of 29 murine hematopoietic tumor cell lines express detectable numbers of transferrin receptors, less than 1% of adult thymocytes or spleen cells and only 5% of bone marrow cells are positive. However, fetal liver and neonatal spleen contain substantial numbers of transferrin receptor-positive cells. Induction of Friend cells in vitro with dimethyl-sulphoxide leads to an overall increase in the expression of transferrin receptors on the cell surface. The anti-transferrin receptor antibody we have obtained partially blocks iron uptake from 59Fe-transferrin by a variety of murine cell lines and inhibits the growth of a murine myeloma cell line in vitro.