Increased proliferation, lytic activity, and purity of human natural killer cells cocultured with mitogen-activated feeder cells.

The addition of mitogen-prestimulated periferal blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) cultures to enriched populations of natural killer (NK) cells obtained from PBL of normal donors in the presence of rIL-2 resulted in highly significant increases in proliferation, purity, and cytolytic activity of cultured NK cells. Two sources of enriched NK cell preparations were used: (i) Adherent-lymphokine activated killer (A-LAK) cells obtained by adherence to plastic during 24 hr activation with 10(3) Cetus U/ml rIL-2; and (ii) NK cells negatively selected from PBL by removal of high-affinity rosette-forming cells and CD3+ lymphocytes. Coculture of A-LAK cells for 14 days with autologous or allogeneic Con A-activated PBL (10(6) cells/ml) or selected EBV-transformed LCL (2 x 10(5) cells/ml) as feeder cells increased fold expansion by a mean +/- SEM of 629 fold +/- 275 (P less than 0.019) and 267 fold +/- 54 (P less than 0.0001), respectively, compared to 55 +/- 20 in A-LAK cultures without feeder cells. The addition of either activated PBL or EBV lines to A-LAK cultures also led to a significant increase in the percentage of NK cells (CD3- CD56+) (84 +/- 2.4 and 84 +/- 2.6%, respectively, P less than 0.0001 for both), compared to 53 +/- 7.2% in cultures without feeders. The presence of feeder cells in cultures of A-LAK cells also led to significantly higher anti-tumor cytolytic activity compared to control cultures, as measured against NK-sensitive (K562) and NK-resistant (Daudi) target cells. Mitogen-stimulated CD4+ PBL purified by positive selection on antibody-coated flasks were better feeders than CD8+ or unseparated PBL. In the presence of feeder cells, it was possible to generate up to 6 x 10(9) activated NK cells from 2 x 10(8) fresh PBL by Day 13 of culture. Enhanced NK cell proliferation in the presence of feeder cells was not attributable to a detectable soluble factor. The improved method for generating A-LAK or activated-NK cells should facilitate cellular adoptive immunotherapy by providing sufficient numbers of highly enriched CD3- CD56+ effector cells with high anti-tumor activity.     

Induction of allogeneic cytolytic T lymphocytes by partially purified membrane glycoproteins

It has been previously shown that P815 (H-2^d) purified plasma membranes can induce cytolytic activity from primed C57BL/6 (H-2^b) spleen cells. The secondary cytolytic T lymphocyte (CTL) inducing activity is retained when these P815 plasma membranes are solubilized in deoxycholate. Evidence is now presented that the cell surface antigens responsible for CTL induction can be partially purified in active form and these antigens can be incorporated into reconstituted membranes and phospholipid vesicles. The active antigens have the properties expected for H-2 molecules on lentil lectin chromatography and gel filtration.     

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1×10⁻⁷ M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1×10⁻⁷ M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear³H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of Il-2 through its ability to enhance the expression of intermediate affinity Il-2R on these cells.     

Multiple cytolytic mechanisms displayed by activated human peripheral blood T cell subsets.

It has been proposed that CTL-mediated cytotoxicity may involve multiple lytic mechanisms. We have examined both the antibody-redirected cytolytic potential and the direct cytotoxicity of purified human peripheral blood high buoyant density CD4+ and CD8+ T cells activated with IL-2 and anti-CD3 mAb. TNF-sensitive and TNF-resistant targets and various metabolic inhibitors were used to compare the antibody-redirected cytotoxicity of T cell subsets and discern the role of potential lytic mediators. In a 4-h assay against several different nitrophenyl-modified targets, the heteroconjugated antibody (anti-CD3-anti-nitrophenyl) redirected cytolytic potential of 72-h activated CD4+ T cells was inhibited by the continuous presence of actinomycin D, cycloheximide, and EGTA, but not mitomycin C, cyclosporin A, or cholera toxin (CT). Conversely, only CT and EGTA inhibited the antibody-redirected cytolytic potential of activated CD8+ T cells. Despite both CD4+ and CD8+ T cell subsets expressing granzymes, pore-forming protein, TNF-beta, and TNF-alpha, these T cell subsets displayed distinct pathways of antibody-redirected lysis against TNF-sensitive and TNF-resistant targets, even in the presence of anti-TNF antibodies. In addition, these same effector T cell subsets were also directly cytotoxic (in the absence of heteroconjugated antibody) against TNF-sensitive targets in an 18-h assay. Indeed, this direct cytotoxicity was completely abrogated by anti-TNF-alpha antibody and was sensitive to the metabolic inhibitors (cyclosporin A, CT, cycloheximide, and actinomycin D), all of which blocked CD4+/CD8+ T cell TNF-alpha production. Therefore, both CD4+ and CD8+ T cells were demonstrated to utilize antibody and lymphokine-mediated lytic mechanisms. CD4+ and CD8+ effector subsets were demonstrated to lyse the same TNF-sensitive target by these two different mechanisms. Although it cannot be excluded that the redirected lytic mechanisms of both CD4+ and CD8+ effectors share common elements, it is likely that other important events in this cytolytic process are fundamentally distinct between these subsets of T cells.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Purification and characterization of a doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) reductase from rat liver plasma membranes

Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.     

Cell cycle dependent resistance to staphylococcal delta toxin-induced lysis of cultured cells

Staphylococcal delta toxin is a protein capable of rapidly disrupting cell membranes. Synchronized populations of 3T3 mouse fibroblasts in mitosis and early G₁ phases of the cell cycle exhibit resistance to delta toxin at concentrations cytolytic to interphase cells. Similar results were obtained with HeLa cells grown attached or in suspension culture. Increased resistance appears to result from structural or biochemical features other than cell rounding or detachment. Delta toxin stimulated significantly less cellular phospholipase A₂ (a potentially lytic enzyme activity) in mitotic 3T3 cells than in interphase cells.     

Generation and characterization of purified adherent lymphokine-activated killer cells in mice

During the incubation of murine spleen, lymph node, or bone marrow cells with IL-2 (1000 U/ml) a small percentage of cells became adherent to the surface of plastic tissue culture flasks. After removal of the non-adherent lymphoid cells, plastic adherent lymphokine-activated killer (LAK) cells could be efficiently expanded in the presence of IL-2. Plastic adherent-derived A-LAK cells were characterized by high rates of proliferation and their cytotoxic activity was more than 10 fold higher than LAK cells generated in the bulk (unfractionated) spleen cell cultures. A-LAK cells could be continuously generated from the non-adherent cell population. Using multiple transfers (every 1 to 2 days) of non-adherent LAK cells into new flasks, new rounds of plastic adherent cells were generated with high expansion capability and high levels of cytotoxic activity. Morphologically, A-LAK cells were large granular lymphocyte and phenotypically expressed markers characteristic of NK cells (asialo GM1+, NK1.1+, Qa5+, Ly-6.2+, Thy-1.2+, but negative for Lyt-2.2 and L3T4). A-LAK cells generated from mice of different strains expressing low and high levels of NK cell activity were equally highly cytotoxic. However, A-LAK cells obtained from nude or beige mice had relatively lower levels of cytotoxicity. Stimulation of NK cell activity by poly I:C or inhibition by in vivo or in vitro treatment with anti-asialo GM1 serum did not affect the generation of A-LAK cells. A-LAK cells derived from spleen or bone marrow of C57BL/6 or nude mice treated with anti-asialo GM1 serum were found to be asialo GM1+ suggesting that A-LAK cell could be generated from the asialo GM1- precursor cells. Expansion of plastic adherent A-LAK cells in the presence of IL-2 could provide large numbers of highly purified cytotoxic A-LAK cells suitable for cancer immunotherapy.     

Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins

A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.     

Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

Autologous control of a highly malignant syngeneic CRNK-16 leukemia in the rat: a role for NK cells

It is unclear whether autologous immunity could be recruited to restrict the progression of leukemia. Patients harboring leukemia commonly display suppressed cell mediated immunity, which may contribute to their inability to control the disease. Immune response against leukemia is evident in allogeneic HLA-mismatched bone marrow transplantation, implicating the involvement of NK cells. This graft-versus-leukemia (GVL) activity suggests that, if not suppressed, an autologous NK cell response could potentially control acute leukemia that had down-regulated HLA expression. In the current study we assessed the role of non-suppressed autologous NK cells in controlling a syngeneic highly malignant leukemia, the CRNK-16 line, that constitute a major cause of natural death in aged F344 rats. A minuscule dose of 60 CRNK-16 leukemia cells per rat was sufficient to induce 50% mortality rates, and animals that survived this challenge did not show improved survival upon a second challenge. The CRNK-16 line was found to exhibit low levels of MHC-I, and selective in vivo depletion of NK cells nullified in vitro NK activity against the CRNK-16 line and reduced survival rates from this leukemia. In vivo activation of NK cells, employing low doses of poly I-C or IL-12, increased in vitro NK activity against the leukemia and dramatically improved survival rates when treatment was initiated before, but not after leukemia inoculation. These results indicate the ability of competent autologous NK cells to restrict highly malignant non-immunogenic leukemia. Thereby, this model presents an opportunity to study specific in vivo NK-leukemia interactions.     

Unimpaired ability to generate adherent lymphokine-activated killer (A-LAK) cells in patients with primary or metastatic liver tumors

Summary Adherent lymphokine-activated killer cells (A-LAK cells) obtained from human peripheral blood mononuclear cells represent a population of potent antitumor effectors enriched in interleukin-2(IL-2)-activated natural killer cells. This study shows that A-LAK cells can be successfully generated from the blood of patients with liver cancer not treated with adjuvant chemotherapy or irradiation. Mononuclear cells were isolated from the blood of 33 patients with liver tumors (6 benign, 10 primary malignant, 17 metastatic) at the time of liver resection. A-LAK cells were separated by adherence to plastic following activation of peripheral blood mononuclear cells in 1000 U/ml recombinant IL-2. A-LAK cells (enriched up to 92% in CD3^–CD56⁺ cells) showed better subsequent expansion and two to six times higher antitumor cytotoxicity per cell than unseparated LAK cells cultured under the same conditions. The ability to generate A-LAK cells with superior in vitro cytotoxicity from the blood of most patients with liver cancer indicates that adoptive cellular immunotherapy may be a feasible and new way of treatment for primary and secondary hepatic neoplasms in man.     

Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cells

Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts.     

Three-dimensional structure of the detergent-solubilized Vibrio cholerae cytolysin (VCC) heptamer by electron cryomicroscopy

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin that inserts a lytic water-filled channel into susceptible host membranes. Assembly of the toxin on cell surfaces may be enhanced by two tandem lectin domains, in addition to direct interactions with lipids and cholesterol within the membrane itself. We used single-particle electron cryomicroscopy (cryoEM) to generate a low-resolution molecular structure of the detergent-solubilized VCC oligomer to 20 Å resolution. After confirming a heptameric arrangement of individual protomers, sevenfold averaging around the central pore was utilized to improve the structure. Docking of the previously determined VCC protoxin crystal structure was possible with rigid-body rearrangements between the cytolytic and lectin domains. A second cryoEM reconstruction of a truncated VCC mutant supported the topology of our model in which the carboxyl-terminal lectin domain forms “spikes” around the toxin core with the putative carbohydrate receptor-binding site accessible on the surface of the oligomer. This finding points to an assembly mechanism in which lectin domains may remain bound to receptors on the cell surface throughout assembly of the cytolytic toxin core and explains the hemagglutinating activity of purified toxin. Our model provides an insight into the structural rearrangements that accompany VCC-mediated cytolysis and may aid in the engineering of novel pore-forming toxins to attack specific cells towards therapeutic ends.     

Cytolytic activity of cytotoxin isolated from Indian cobra venom against experimental tumor cells

Cytolytic activity of cytotoxin isolated from the venom of the Indian cobra (Naja naja) on experimental tumor cells was far stronger than that on normal cells such as peritoneal exudate cells, spleen cells, and erythrocytes of the rat. The effect on Yoshida sarcoma cells was temperature-dependent, being stronger at 37 degrees C than at 0 degrees C. Intramolecular disulfide linkages and free amino groups in the cytotoxin molecule were shown to be essential for the lytic action on the cell membrane. Yoshida sarcoma cells treated with 0.1 mM N-ethylmaleimide reduced the cytolytic action of the toxin. Antitumor activity of the cytotoxin toward a Yoshida sarcoma inoculated intraperitoneally into a rat was not observed.     

Physical characterization of the pore forming cytolysine from Gardnerella vaginalis

Abstract The cytolytic toxin (CTox) produced by Gardnerella vaginalis is able to form voltage-dependent cationic channels when incorporated in lipid membranes (Moran et al, 1991) FEBS Lett. 283, 317–320). Osmotic protection experiments show that toxin incorporated in human erythrocytes forms pores between 18 Å and 28 Å in diameter. A hypothesis of pore formation as a primary event to produce cytolysis is proposed. The CTox activity increases when cells are depolarized by increasing the extracellular K⁺ concentration, probably reflecting the voltage dependent character of CTox formed channels. The cytolytic effect of the toxin was prevented by low temperatures and was a function of the extracellular Ca²⁺ concentration, suggesting a Ca²⁺ influx as part of the lytic mechanism. Binding of CTox to erythrocytes was dependent on external Ca²⁺ and was less temperature-dependent. Dose-response analysis suggests cooperativity of the toxin for the lytic activity, although no direct evidence of oligomerization has been found.     

EpsinR: an ENTH domain-containing protein that interacts with AP-1

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the gamma-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH(2)-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPgammaS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.     

Activation of insect cell adenylate cyclase by Bacillus thuringiensis delta-endotoxins and melittin. Toxicity is independent of cyclic AMP.

Insecticidal Bacillus thuringiensis (Bt) delta-endotoxins are cytolytic to a range of insect cell lines in vitro. Addition of Bt var. aizawai or var. israelensis toxins to Mamestra brassicae (cabbage moth) cells in vitro increased intracellular cyclic AMP, which was paralleled by activation of adenylate cyclase in isolated membranes. Var. kurstaki toxin, which does not lyse M. brassicae cells, had no effect on cyclic AMP concentrations in intact cells, but was able to stimulate adenylate cyclase in membrane preparations. In contrast, the bee-venom toxin melittin, which is also cytolytic, increased intracellular cyclic AMP in whole cells, but inhibited adenylate cyclase in isolated membranes. Octopamine and forskolin also increased cyclic AMP in cells, but were not cytolytic. When added to cells at concentrations exceeding their LC90 (concentration causing 90% cell death), melittin and var. israelensis toxins caused cell lysis without a concomitant increase in intracellular cyclic AMP. Taken together, these results suggest that activation of adenylate cyclase by cytolytic toxins is a secondary effect (related perhaps to interactions of these toxins with membrane lipids) and is neither necessary nor sufficient for cytolysis.     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Comparative analysis of cell surface proteins in chronic and acute leukemia cell lines

This study was designed to identify the cell surface protein markers that can differentiate between chronic myeloid leukemia (CML) and acute promyelocytic leukemia cells (APL). The differentially expressed plasma membrane proteins were analyzed between CML cell line (K562) and APL cell line (NB4) using the comparative proteomic approach. The cell membrane proteins were enriched by labeling with a membrane-impermeable biotinylation reagent, sulfo-NHS-SS-Biotin, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). By comparative proteomic analysis of K562 and NB4 cells, we identified 25 membrane and 14 membrane-associated proteins. The result of LC-MS/MS combined with chemical tagging method was validated by confirming the expression and localization of one of the differentially expressed plasma membrane proteins, CD43, by FACS and confocal microscopy. Our results indicate that CD43 could be a potential candidate for differentiating CML from APL.