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A K Sullivan

Publications and source records attributed to A K Sullivan.

At least 55 records · Page 3Linked to original sources
PubMed1988

Changes in glycoprotein fucosylation in a concanavalin A-resistant variant of a human leukemia cell line (K562).

Previous studies from this and other laboratories have shown that variants of tumor cell lines can be selected for resistance to the lytic action of natural killer (NK) cells. One of these (K562-Clone I), when made resistant to the toxic effects of Concanavalin A (Con A-R1), regained its sensitivity to NK. Comparing the plasma membranes of Clone I and Con A-R1, we observed 1) a very similar electrophoretic pattern of membrane glycoproteins identified by binding to the lectins Con A, WGA, PNA, and SBA; 2) an increase in binding of Ulex europaeus lectin to a group of glycoproteins from Con A-R1 that were sensitive to treatment with fucosidase and N-glycanase and that had a diffuse mobility ranging in apparent molecular weight from 30 to 200 kDa; and 3) a marked decrease in binding of an antibody reactive with the lactoneofucopentaose III antigen (Lewis x). This constellation of changes is an unusual pattern to follow Con A resistance and may point to a pathway of glycosylation that a leukemic cell might use to modify its recognition by the NK mechanism.

Cell Membrane↗
PubMed1987

Augmentation of human natural cytotoxic cell activity by leukotriene B4 mediated by enhanced effector-target cell binding and increased lytic efficiency.

The addition of leukotriene B4 (LTB4) to cytotoxicity assays measuring natural killer (NK) or natural cytotoxic (NC) cell activities resulted in significantly augmented killing of K562 or herpes simplex virus (HSV)-infected target cells, respectively. Since the mechanism of cytotoxicity implies several steps, including the binding of effectors to targets which is Mg2+-dependent and the programming of lysis of the target which is Ca2+-dependent, we undertook to define the step(s) at which LTB4 acted in augmenting cytotoxicity. Our results showed that LTB4 significantly increased the percentage of effector-target conjugates when K562- or HSV-infected targets were incubated with lymphocytes. Maximal binding occurred at a concentration of LTB4 of 1 X 10(-10) M. Preincubation of lymphocytes and not target cells with LTB4 was sufficient to observe the increased binding. PBML binding to and killing of the NK-resistant target clone I, derived from K562, was not enhanced by LTB4. In the absence of Ca2+, cytotoxicity was impaired and LTB4 could not restore it. Use of a single cell lytic assay demonstrated augmented efficiency of lysis of both K562 and HSV-infected targets in the presence of LTB4. These findings suggest that LTB4 may augment natural cytotoxicity by enhancing target cell recognition by cytotoxic effector cells and subsequently by augmenting their lytic efficiency.

Antibodies, Monoclonal↗
PubMed1987

Different molecular forms of a glycoprotein antigen found on azurophilic granule membranes of cultured human HL60 promyelocytes and on the plasma membrane of a myeloblastoid variant line.

In previous immunohistological studies an antigen designated D46 was identified on the surface of an agranular, myeloblastoid cell line (HL60-D). It was not detected on the surface of either parental HL60 cells or variants with aberrant primary granules (HL60-A7). In these promyelocytes it was found to be intracytoplasmic, in a granular pattern. In the work presented here, this antigen and other granule-related glycoproteins were studied by radiolabeling, subcellular fractionation and gel electrophoresis. The major findings include the following: the anti-D46 antibody precipitated one major and one minor glycosylated component, each of which migrated with a different electrophoretic mobility, depending on whether it was derived from cells with or without granules. Incorporation of radioactive amino acids, but not monosaccharides, revealed an additional component of lower mobility. The D46 antigen(s) was recovered in the detergent-rich phase of a Triton X-114 extract of granules, suggesting that it is an integral membrane protein. As assessed by the relative activities of marker enzymes, the granules of both the HL60 and HL60-A7 cell lines exhibited a heterogeneous pattern of sedimentation in density gradients. The electrophoretic patterns of the major [2-3H]mannose-labeled glycoproteins of the granule-enriched fractions were similar, except for one diffuse band of 110-170 kD which was not detected in HL60-A7. These studies provide direct evidence for heterogeneity of enzymatic and membrane constituents of primary myeloid granules, and give the first indication that there may be glycoprotein changes associated with certain ultra-structural defects that occur in abnormal promyelocytes. They suggest also, in the example of the D46 antigen; that some glycoproteins may exist in different forms when localized to either the granules or the plasma membrane.

Antigens, Surface↗
PubMed1987

Increased fucosylation of glycolipids in a human leukaemia cell line (K562-Clone I) with decreased sensitivity to NK-mediated lysis.

A subpopulation of human lymphoid cells called natural killers is able to lyse certain normal and neoplastic targets in an in vitro cytotoxicity assay. The molecules which enable them to recognize sensitive cells, or permit tumour cells to escape remain unknown. In the studies described here we have compared some of the plasma membrane characteristics of a NK-sensitive human leukaemia cell line (K562) with those of a partially resistant subclone derived from it (K562-Clone I). Gel electrophoresis of cell-surface proteins radiolabelled by lactoperoxidase-catalysed iodination, periodate-borohydride tritiation, or biosynthetically by incubation with [3H]fucose did not reveal any reproducible differences between the sensitive and resistant lines. However, analysis of glycolipids showed that Clone I incorporated significantly more fucose than did the parental line, and that it synthesized a minor population of complex structures not found in the original K562. A subclone of Clone I (Clone I-Con Ar1), made resistant to the toxic effects of concanavalin A, became sensitive once again to NK, and showed the parental glycolipid profile. These results suggest that the Clone I line, selected for resistance to NK, may have altered one or more of its intermediate oligosaccharides or pathways of fucose incorporation into glycolipid, and points to one process by which a tumor cell might modulate its surface to escape recognition by natural killers.

Antigens, Neoplasm↗
PubMed1986

Target cell specificity of human natural killer cells. II. Apparent change with activation.

The activity of natural killer (NK) cells can be augmented by incubation with interferons, or with other compounds, such as staphylococcal protein A, which stimulate interferon production. In the experiments described here we compared the patterns of lytic activity of human lymphoid effector cells, before (NK-B) and after (NK-A) short-term activation. Target cells used were K562, Clone I (a partially NK-resistant K562 variant), and those that had been preincubated with neuraminidase or trypsin. The results obtained include the following: proteases, but not neuraminidase, decreased lysis by NK-B, but not by NK-A, of both K562 and Clone I in the standard Cr-release assay. In the single cell assay, trypsin minimally decreased conjugate formation and the fraction of bound cells that were killed by NK-B, but did not reverse the increased lytic efficiency of NK-A. In the cold-target competition assay, Clone I, which does not compete as well with K562 for NK-B, did so equally well for NK-A. Trypsinized targets, regardless of their equal sensitivity to lysis by NK-A, were not as active competitors for NK-A. We conclude that the most reasonable interpretation is that K562 cells bear surface structures which can induce release of lytic mediators from NK-A under conditions that are not sufficient to stimulate NK-B. Although it appears that NK-A may respond to a smaller number of the same target molecules recognized by NK-B, the process must be better defined at the molecular level to exclude the possibility that there are qualitative differences between the proposed recognition structures for these two states of NK activity.

Cell Line↗
PubMed1986

Deficiency of myeloperoxidase and abnormal chromosome 1 occurs in variant (HL60) promyelocytes.

Maturation of normal polymorphonuclear neutrophils is characterized by successive periods of granule synthesis, a process which frequently is abnormal in leukemia. Recently, the human leukemic cell line HL60, displaying a promyelocytic phenotype, has been used to study granulocyte maturation. We describe a variant line of HL60, called HL60-A7, resulting from growth in actinomycin D, which contains atypical large azurophilic granules deficient in myeloperoxidase. The products of in-vitro translation of A7 RNA contained less than 5% of the immunoreactive MPO found in the parent line. Electrophoresis of plasma membrane polypeptides radioiodinated by the lactoperoxidase technique revealed several differences. Karyotypic analysis identified a consistent chromosome 1q+ abnormality which was not found in any of the parental cells examined. This constellation of differences between HL60 and HL60-A7, i.e. MPO deficiency, abnormal granule morphology, cell surface changes, and further cytogenetic abnormalities, may point to a common site sensitive to altered regulation in some leukemic promyelocytes.

Cells, Cultured↗
PubMed1986

Differences in the plasma membrane glycoproteins of cultured myeloblastoid and promyelocytic human leukemia (HL60) cells.

Since the mechanisms that control synthesis of surface and internal granule membranes are closely-related within the Golgi apparatus, we have compared the plasma membrane proteins and glycolipids of cells of the human promyelocytic line HL-60 with those of its agranular myeloblastoid variant (HL60-D), and of other human myeloid lines (KG-1a, KG-1 and ML-2). Proteolytic degradation by granule enzymes altered the protein profiles unless multiple inhibitors were included in the cell suspension before lysis and during subsequent handling of the extracts. Polyacrylamide gel electrophoretic profiles of the proteins accessible to lactoperoxidase-catalyzed 125I-labeling or to periodate [3H]-borohydride labeling, as well as those of the glycoproteins bound to and eluted from immobilized concanavalin A, showed distinct patterns. The apparent molecular weights of the two major sialylated glycoproteins were larger in cell lines with a greater content of azurophilic granules. Also, the blastic line incorporated less fucose into glycolipid and contained less complex gangliosides and neutral glycolipids than did the parent. These data demonstrate that, within the limits of this culture model, cells capable of cytoplasmic granule production express a different constellation of surface components.

Cell Line↗
PubMed1985

Deficiency of a major myeloid antigen in neutrophils of patients with refractory anemia and excess myeloblasts.

The expression of a major granulocyte-related antigen was assessed on the peripheral blood neutrophils of patients with the syndrome of refractory anemia with excess blasts. Of thirteen patients studied six showed a marked decrease in the number of cells with detectable staining. These results indicate that, although the neutrophils may appear normal by standard morphological criteria in this disease, they may be defective in a major component associated with myeloid maturation.

Anemia, Aplastic↗
PubMed1985

Antigens differentially expressed on surface and cytoplasmic structures of human myeloid cells.

Specialized internal granules are a major feature of myeloid differentiation and are deficient in most acute myeloid leukemia cells. Although they arise from the same synthetic apparatus as does the plasma membrane, their relationship to it is not well characterized in human tissues. Using murine monoclonal antibodies, we have identified myeloid-related structures that illustrate three possible modes of antigen expression in these organelles. Immunocytochemical studies with the light microscope have shown that the first (D51) was restricted to the surface of neutrophils, monocytes, megakaryocytes and platelets; a second (D46) was found on the surface of blastic cell lines but on only internal components of mature granulocytes; the third (H36/71) appeared on both the surface and internal particles of promyelocytes, myelocytes and polymorphs. These model antigens may be used to study the control of granule synthesis in normal and leukemic cells.

Animals↗
PubMed1985

An antigen related to the phenotype of multi-drug resistance can be induced in vivo and used as a target for immunotherapy of rat leukemia.

Several laboratories have reported that new plasma membrane peptides appear in rodent and human cells after induction of in-vitro resistance to vinca alkaloids, anthracyclines and other anti-neoplastic drugs. Recently, murine monoclonal antibodies have been produced that recognize surface components of such drug-resistant cells. The work presented here describes the development of an in-vivo animal model of this phenomenon using a rat myeloid leukemia. Brown Norway rats were made leukemic with promyelocytes of the BNML line and subsequently were treated with 7.7 mg kg-1 of daunorubicin. After eight cycles of passage-treatment-regrowth, the resulting cells reacted with this antibody in immunofluorescence and cytotoxicity assays. Animals injected with cells that had been pre-incubated with antibody in the absence of complement survived significantly longer than did the controls. Further prolongation of survival occurred when the cells were treated with a second antibody of a different specificity. These results demonstrate that some of the changes associated with in-vitro drug resistance occur also in vivo and potentially may be exploited as a focus for immunotherapy.

Animals↗
PubMed1985

Membrane markers, target cell specificity, and sensitivity to biological response modifiers distinguish human natural cytotoxic from human natural killer cells.

In the present report, we provide evidence for the distinct existence of a human natural cytotoxic (HNC) cell. This HNC cell can be identified by the monoclonal antibody HNC-1A3 and by the absence of the T10 antigen, other antigenic markers being shared, at least in part, with natural killer (NK) cells, T cells, or monocytes. In addition, the HNC cell preferentially kills the MA-160 target, the herpes simplex virus-1-infected MA-160 cell line, and the two human tumor cell lines HEp-2 and HF-2. It has weak lytic activity against the NK-sensitive K562 cell line or its relatively NK-resistant clone I subline. The cytotoxic activity of the HNC cell is not augmented by interferon but is markedly enhanced by interleukin 2 and by a measles-virus-induced factor (MVF). Furthermore, it is not inhibited by cyclosporin A (CsA), in contrast to NK cell cytotoxicity against the K562 target cell line which is augmented by interferon, inhibited by CsA, and not affected by MVF. These data suggest that spontaneously cytotoxic cells may belong to more than one subset of human lymphocytes, and that HNC cells may be defined in man using membrane markers, target cell specificity, and sensitivity to biological response modifiers.

Antibodies, Monoclonal↗
PubMed1984

Blastic variants of rat promyelocytic leukemia produce neurologic disease.

The characteristics of tumor cells that enable invasion into normal tissues are not well understood. We have derived variants of the promyelocytic leukemia of the Brown Norway rat (BNML) that exhibit a high incidence of growth in the epidural space and produce paralysis of the hind limbs. This unique animal model mimics a major cause of morbidity in human cancer and provides a reproducible system in which to study the determinants of metastasis localization.

Animals↗
PubMed1983

Target cell specificity of human natural killer (NK) cells. I. Development of an NK-resistant subline of K562.

By depletion of effector-target conjugates and cloning, a variant of the human leukemic line K562 that is partially resistant to lysis by natural killer (NK) cells when tested under conditions of culture and assay identical to that of the parent has been derived. Its karyotype shows markers similar to the original K562. The resistant phenotype remains stable after over 1 year of continuous passage and persists in multiple replicate subclones. However, it can be killed to a degree equal to the parent by antibody-activated complement, antibody-dependent cellular mechanisms, and by effector cells activated by staphylococcal protein A. These observations and experiments on cold target competition suggest that on its surface there is a significantly decreased, absent, or blocked effective target structure for a major population of unstimulated peripheral blood NK cells.

Animals↗
PubMed1983

In vitro lines of the BN rat promyelocytic leukemia that differ from the parent.

Cells from the spleens of Brown Norway rats made leukemic with the in vivo-passaged promyelocytic line BNML have been adapted to in vitro culture in RPMI-1640 medium supplemented with 4% rat serum and 6% fetal bovine serum. The presence of rat serum, which also was associated with suppression of fibroblasts, appeared to be required for initial growth. In contrast to the parent this new cell line (BNML-RS) was predominantly myeloblastic under standard conditions of culture with only 4-10% of cells showing granules or stainable peroxidase. However, when passaged through an animal, 60-70% contained both granules and peroxidase. Neither parent nor established line evolved to the polymorph stage when assessed for terminal maturation after exposure to dimethyl sulfoxide or retinoic acid. These cells now extend the limited number of myeloid lines available and potentially are a useful model in which to study the controlling events in early myeloblast maturation.

Animals↗
PubMed1983

Loss of promyelocytic maturation in HL60 sublines: a potential model for leukaemia progression.

The majority of cells of the human leukaemic line HL60 appear to be promyelocytes which can be stimulated to mature into functioning neutrophils. We report here the derivation of stable subclones from the parent line which differ. Two forms of variant were obtained: (1) those with a defined lesion, selected by resistance to kill by 6-thioguanine, which matured in the presence of DMSO and retinoic acid but not in hypoxanthine. They also continued to express peroxidase and a major granulocyte antigen, and (2) those obtained by clonal growth in DMSO which did not mature in the presence of any compound tested including hypoxanthine, DMSO, retinoic acid and actinomycin D and lacked granules, peroxidase and the granulocyte antigen. The concurrent loss of the characteristic of maturability and the phenotype of myeloid commitment suggests that the control of the two phenomena may be related. These variant cell lines provide a useful model in which to study how human leukaemic cells may become arrested in less differentiated stages of development.

Animals↗
PubMed1979

Isolation of a human B lymphocyte membrane protein with Ia-like properties.

A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with beta 2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radiodinated components from similarily treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.

Antigens, Surface↗