Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

Effects of partially thiolated polycytidylic acid and liposomes on in vitro colony-forming cells of leukemic mice.

Partially thiolated polycytidylic acid (MPC), an antileukemic agent, when administered to leukemic RF/UN mice inhibited the clonogenicity of bone marrow progenitor cells in a time- and dose-dependent manner. The effect of a single dose of MPC disappeared within 40 hr due to the rapid degradation of this compound in mice. When MPC was encapsulated in liposomes before injection, its activity at 19 hr after inoculation was similar to that of free MPC. The inhibitory effect of this liposome-MPC complex, however, persisted for at least 40 hr, indicating that the MPC was protected from hydrolysis by the nucleases present in blood. Drug-free liposomes increased the number of clonogenic progenitor cells, whereas a mixture of plain liposomes and MPC decreased the number of clonogenic cells to a greater extent than did MPC alone or MPC within liposomes. A possible explantation for these observations is that the liposomes per se altered the clearance function of the reticuloendothelial system and completed with MPC for uptake by the reticuloendothelial system cells, thereby resulting in increased plasma levels of MPC which in turn resulted in greater killing of the target cells.

Decreased retention of actinomycin D as the basis for cross-resistance in anthracycline-resistant sublines of P388 leukemia.

Sublines of P388 leukemia resistant to Adriamycin and daunorubicin were cross-resistant to actinomycin D in vivo and in vitro. The Adriamycin-resistant cell line was 1000-fold resistant to actinomycin D on 1-hr exposure in vitro and 370-fold resistant when exposed to the drug for 16 hr. The immediate binding of radioactive actinomycin D to sensitive and resistant cells was similar, and the uptake of the drug by the resistant cells was only about 27% less than the rate of uptake by sensitive cells. There was a dramatic difference in efflux of drug from sensitive and resistant sublines. Equivalent cytotoxicity of actinomycin D for the sensitive and resistant sublines was obtained at concentrations of the drug that resulted in approximately equivalent levels of net retention of actinomycin D (retained drug minus background levels of immediate binding of the drug to the cells). Incubation of cells in the presence of actinomycin D plus either Tween 80 or acridine orange incresed the rate of uptake and the percentage of actinomycin D retained by the resistant cells on short-term assays but did not reverse the resistance. It is concluded that these tumors must retain appreciable concentrations of actinomycin D for several hr in order to be killed. The anthracycline-resistant sublines are cross-resistant to actinomycin D by virtue of their inability to retain the drug.

Anitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes.

Leukemic guinea pig lymphocytes (L2C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L2C cells. In contrast, greater than 5-10(9) molecules/cell of a nitroxide-derivative of androstane, (17 beta-hydroxy-4',4'-dimethylspiro [5 alpha-androstan-3,2'-oxazolidin]-3'-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol roduction by both normal and leukemic cells (maximum within 2 H). At less than 5-10(9) molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.

Catalytic hydrogenation of fatty acyl chains in plasma membranes; effect on membrane lipid fluidity and expression of cell surface antigens.

Optimal reaction conditions were established for hydrogenation of plasma membranes of living murine GRSL leukemia cells, using the water-soluble catalyst Pd(QS)2 (QS, sulphonated alizarine; C14H6O7NaS). Under these conditions more than 80% of the cells remained viable. Analysis by gas chromatography revealed that hydrogenation occurred predominantly in the 18:2, 20:4 and 22:6 fatty acyl chains of the membrane phospholipids. Under the same conditions hydrogenation was also performed in purified plasma membranes from GRSL cells and from rat liver, and in liposomes prepared from the total lipid extracts of these membranes. Hydrogenation increased the lipid structural order parameter in the membranes, as measured by fluorescence polarization. This increase was more pronounced in the liposomes (46%) than in the plasma membranes (17-25%). Hydrogenation increased the expression of a 15 kDa antigen on the surface of viable GRSL cells, as measured in a Fluorescence Activated Cell Sorter, using monoclonal antibodies. The expression of four other antigens, among which H-2k, was not or much less affected by this treatment.

Liposome encapsulation enhancement of methotrexate sensitivity in a transport resistant human leukemic cell line.

A stable mutant of human leukemia CCRF/CEM cells has recently been isolated which is transport resistant for methotrexate (MTX). Encapsulation of MTX in cationic unilamellar liposomes increased the association of the drug 5-fold with the sensitive, and 50-fold with the resistant, cells as compared to the uptake of free drug. The liposome-mediated associations of MTX with sensitive and transport deficient cell lines were similar. Cytostatic studies demonstrated that liposome encapsulation increased MTX activity 4-fold towards the transport resistant cell line. The addition of cholesterol to the vesicles decreased their effectiveness. A 4-fold increase in drug sensitivity due to encapsulation may allow such transport resistant tumor cells to become responsive to chemotherapeutic doses of MTX which are currently feasible in human clinical protocols.

Enhancement of the antitumor activity of adriamycin by Tween 80.

This paper describes the effect of Tween 80 on the antitumor activity and on the distribution of adriamycin in mice. The dilution of adriamycin in a 10% water solution of Tween 80 produced a significant increase of the antitumor activity in mice against ascites tumors (L 1210 leukemia), disseminated leukemias (transplanted leukemias originally induced by Gross leukemai virus and Moloney leukemia virus), and solid tumors (Sarcoma 180, MS-2 sarcoma). In all these experiments the drug was administered i.v., according to different schedules. Higher antitumor activity at the optimal dose and an increase of activity at lower doses were observed in different experimental systems. Toxicity was also slightly enhanced. Tissue distribution was studied in normal mice and in tumor-bearing mice (Gross leukemia and MS-2 sarcoma). In animals give i.v. adriamycin diluted in 10% Tween 80 there was a higher drug concentration in spleen, lung and kidney than there was in mice given the drug in a water solution. In all the other organs examined (heart, liver, small intestine) and in the MS-2 tumor tissue, no significant increase was observed. In L1210 leukemia-bearing mice, i.p. treatment with adriamycin diluted in 10% Tween 80 resulted in a significantly higher toxicity than that which resulted from treatment with adriamycin in a wa ter solution; no increase of antitumor activity was observed.

[Changes of cellular contractile proteins associated with differentiation of leukemic cells].

A myeloid leukemia cell line, M1, can be induced to differentiate to macrophages and gains motile and phagocytic functions which are not present before differentiation. The appearance of these differentiated functions were always accompanied with a loss of mitotic activity. It is well known that the intracellular contractile proteins play an important role in both cell motility and cytokinesis. Our question is, why the actomyosin system in the M1 cell line is utilized only for cell division and not for cell motility before differentiation, and why the opposite situation takes place after differentiation. Changes in contractile proteins hitherto revealed during the differentiation of this cell line are as follows: Actin; contents, synthetic rate, ability to polymerize, ratio of gamma-actin, activity to activate the myosin Mg2+ ATPase, and binding to plasma membrane all showed an increase after differentiation. Changes in the chemical structure of actin molecule were suggested by peptide mapping. In the undifferentiated M1 cytoplasms, an inhibitor protein for actin polymerization was identified. Myosin; contents, ratio of light chain L2, and binding to plasma membrane showed an increase after differentiation. Which one plays the most essential role in the transition from cell division to cell motility, and what interrelation is there among those changes are remained to be disclosed.

[Specificity of liposome uptake from target cell lipids]. / Spetsifichnost' zakhvata liposom iz lipidov kletok-myshenei.

A more considerable uptake of liposomes (1.5-2 times greater) from lipids of target cells was revealed in the cells of ascites lympholeukemia NKLy/LL and Ehrlich's ascites carcinoma as compared with liposomes from other tumor lipids. NKLy/LL cells uptake liposomes from their own lipids 8 times as effective, whereas Ehrlich's carcinoma cells 3 times as effective as widely applied vesicles from egg lecithin. Evidence is provided that the differences discovered are consequent on the different mechanisms of liposome interaction with the cells. As far as egg lecithin is concerned, the principal mechanism of interaction is endocytosis which is unmarked in these cells. Meanwhile uptake of target cell lipids is effected by the fusion mechanism.

Clearance of immune complexes formed in normal and leukaemic rats.

Immune complexes of 125I-HSA-rat anti-HSA formed in vivo under conditions of antibody excess were rapidly cleared from the circulation of both normal and leukaemic Hooded rats. In HSA-immune rats most of the 125I-HSA present in the blood was found to be cell-bound, but a proportion was present as circulating immune complexes that could be precipitated from plasma by 2.5% polyethylene glycol. There was no evidence that clearance of a soluble antigen was impaired in leukaemic animals.

LDL-mediated targeting of liposomes to leukemic lymphocytes in vitro.

We describe a method for the covalent coupling of low-density lipoproteins (LDL) to the surface of small unilamellar vesicles, and the delivery of the liposome content to leukemic L2C lymphocytes in vitro. We demonstrate the stability of the linkage between LDL and liposomes, the preservation of vesicle integrity and the affinity of the LDL for their specific receptors after the coupling reaction. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted liposomes, and delivered into the cytoplasm of leukemic L2C lymphocytes by the LDL pathway, as demonstrated by the lethal effect on cells measured by 51chromium-release assay.

Effect of N-methylation of phosphatidylethanolamine on the fluidity of phospholipid bilayers.

The effect of N-methylphosphatidylethanolamine on phase transition and the fluidity of the liposomes made of dipalmitoylphosphatidylcholine or phosphatidylethanolamine was studied by the steady-state fluorescence polarization method and differential scanning calorimetry. N-methylation of phosphatidylethanolamine caused a decrease of fluidity of liposomes made of dipalmitoylphosphatidylcholine, but had little effect on dipalmitoylphosphatidylethanolamine. The liposomes prepared with both phosphatidylcholine and N-methylphosphatidylethanolamine and also phosphatidylethanolamine and N-methylphosphatidylethanolamine could be composed of solid solution and exhibited symmetric phase diagram.

Mobile haptens in liposomes stimulate serotonin release by rat basophil leukemia cells in the presence of specific immunoglobulin E.

A new method for studying the physicochemical determinants of IgE-mediated activation of basophils is described. Rat basophil leukemia cells having IgE receptors were preincubated with monoclonal anti-dinitrophenyl IgE. These cells were then exposed to liposomes containing dinitrophenyl conjugated to phosphatidylethanolamine. The release of serotonin was measured. Using this system it was observed that liposomes containing dinitrophenyl conjugated to phosphatidylethanolamine by aminoethylformamidomethoxy acetyl (but not by caproic acid) were able to trigger the release of serotonin. "Solid" liposomes composed of dipalmitoylphosphatidylcholine and 2 mol % hapten were more potent inducers of serotonin release than "fluid" liposomes composed of dimyristoylphosphatidylcholine and hapten, but the fluid liposomes definitely triggered the cells to release serotonin. The addition of cholesterol to both types of liposomes enhanced their potency as activators and diminished the difference between the two types of phospholipid. This occurred despite the fact that cholesterol renders the solid liposomes fluid. Since it is unlikely that these fluid membranes can provide lateral forces that produce IgE-Fc receptor molecular clustering, we conclude that either receptor clustering is not necessary for basophil triggering, or molecular clustering is driven by molecular forces derived from IgE and components of the basophil cell.

Heterogeneous pathways of oxidizing radical production in human neutrophils and the HL-60 cell line.

Oxygen-derived free radicals with hydroxyl radical (OH.)-like reactivity are products of the human neutrophil respiratory burst. Such radicals, although dependent on O2-generation arise from complex oxidation reactions that may be independent of an iron-or lactoferrin-catalyzed Haber-Weiss mechanism. Differentiated HL-60 promyelocytic leukemia cells completely deficient in lactoferrin generate oxidizing radicals at a rate greater than that of human neutrophils, indicating lactoferrin-independent pathways for OH. generation. The further heterogeneity of pathways generating OH. activity in neutrophils is indicated by the cell concentration dependence of the reaction, the variability of H2O2 as a precursor reactant, and the various proportions of oxidizing radical activity to O2-detected in human neutrophils stimulated to a variety of agonists. The ethylene assay for oxidizing radical activity may detect different classes of oxidizing species capable of reacting with the aldehyde substrate. The complexity of defining the oxygen-derived radicals of the ethylene assay suggests methodologic difficulties of either quantitating or precisely defining the radicals generated in the respiratory burst by this method.

A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate).

A photosensitive alpha-cyanocinnamylideneacetyl group was coupled to poly(allylamine) to obtain a photosensitive polymer. This photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) can cross-link upon light exposure. Microcapsules were fabricated from alginate in contact with Ca+2 ion, followed by coating with the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). The microcapsules, thus formed, can be strengthened significantly by the light-induced cross-linking of poly(allylamine alpha-cyanocinnamylideneacetate). Only 16 capsules (out of 50) prepared from the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) fractured after 48 h of agitation. For microcapsules prepared from the unmodified poly(allylamine), 32 capsules fractured. The photo cross-linked capsular membrane was permeable to cytochrome C, moderately permeable to myoglobin, and least permeable to serum albumin. IW32 (a mouse leukaemia cell line) cells were entrapped and cultured within these microcapsules. The cells proliferated to a density of about 9 x 10(6) cells/ml in the capsules after 7 days of cultivation.

[Effect of gamma-irradiation on malonic dialdehyde formation in the interaction of liposomes with cells]. / Vliianie gamma-oblucheniia na obrazovanie malonovogo dial'degida pri vzaimodeistvii liposom s kletkami.

A study was made of the effect of relatively low gamma-radiation doses (up to 10 Gy) on the ability of Ehrlich ascites tumor cells to induce peroxidation in lipids of liposomes from egg lecithin. The mechanism of this peroxidation is probably associated with the release from cells of catalyzers which perform free-radical oxidation of higher unsaturated fatty acids. Two processes occur upon irradiation of cells: disintegration of the released catalyzers, and stimulation of their release from cells. Correspondingly, the formation of malonic dialdehyde was inhibited or stimulated depending on the radiation dose and time of the combined incubation of liposomes and cells. On the basis of the data obtained a conclusion is made that the modification of the effect of malonic dialdehyde formation upon oxidation of liposomes by the exposed cells is conditioned by the effect of radiation on cell membranes.

Cross-linking of immunoglobulin E-receptor complexes induces their interaction with the cytoskeleton of rat basophilic leukemia cells.

Bridging of immunoglobulin E (IgE)-receptor complexes on rat basophilic leukemia cells by polyclonal anti-IgE antibodies induces a detergent-resistant association of these complexes with the cellular cytoskeleton. In dose-response curves the extent of the cytoskeletal association appears to follow the extent of bridging, continuing to increase beyond where stimulated degranulation is maximal. This stable association is maintained after the aggregated IgE-receptor complexes have been internalized by the cell. Multivalent antigen and trimeric IgE cause less extensive receptor cross-linking than anti-IgE and stimulate degranulation; they also induce receptor association with the cytoskeleton that is revealed only after stabilization by addition of a chemical cross-linking reagent. The ability of a membrane impermeant chemical cross-linker to stabilize this association suggests that the receptor-cytoskeletal interaction may be mediated by a transmembrane protein that is exposed at the cell surface. Monomeric and dimeric IgE bound to receptors fail to induce a stable interaction with the cytoskeleton even in the presence of chemical cross-linkers and are poor (dimers) or insignificant (monomers) stimulators of cellular degranulation. These findings are consistent with a possible relationship between receptor attachment to the cytoskeleton, receptor immobilization as measured by fluorescence photobleaching recovery, and the triggering of cellular degranulation.

Regulation of cholesterol biosynthesis by normal and leukemic (L2C) guinea pig lymphocytes.

The cholesterol production of guinea pig leukemic (L2C) lymphocytes preceeds at greater than 30 times the rate found in normal cells. Fatty acid biosynthesis is also enhanced in L2C cells. Exposure of L2C cells to cholesterol/lecithin liposomes does not depress their sterol biosynthesis, in contrast to the behavior of normal lymphocytes [Philippot, J.R., Cooper, A.G. & Wallach, D. F. H. (1975) Biochim. Biophys. Acta 406, 161-166]. However, 25-hydroxycholesterol, an inhibitor of hydroxymethylglutaryl-CoA reductase (NADPH) [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34], the rate limiting enzyme in cholesterogenesis, and 25-hydroxycholecalciferol, a biologically potent form of vitamin D3, block sterol biosynthesis of both normal and L2C lymphocytes [Philippot, j.r., cooper, A.G. & Wallach, D.F.H. (1976) Biochem. Biophys. Res. Commun. 72, 1035-1041]. Moreover, both cell types exchange cholesterol equivalently with cholesterol/lecithin liposomes. The only difference in sterol biosynthesis observed between the two cell types is in the temperature response of the enzyme. Arrhenius plots of this enzyme activity exhibit a prominent discontinuity at about 24 degrees in the case of normal cells, but none in the case of L2C. The activation energies for L2C cells and normal cells, above the normal cell transition temperature, were not significantly different. All of the data suggest that the regulatory defect in L2C lymphocytes arises from a deficiency in these cells' internal membranes.