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WO1999036536A2 - Methodes et compositions permettant de modifier la sensibilite des tissus face aux lesions immunitaires, a la mort cellulaire programmee et a la clairance par le systeme reticulo-endothelial - Google Patents

Methodes et compositions permettant de modifier la sensibilite des tissus face aux lesions immunitaires, a la mort cellulaire programmee et a la clairance par le systeme reticulo-endothelial Download PDF

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Publication number
WO1999036536A2
WO1999036536A2 PCT/US1999/001087 US9901087W WO9936536A2 WO 1999036536 A2 WO1999036536 A2 WO 1999036536A2 US 9901087 W US9901087 W US 9901087W WO 9936536 A2 WO9936536 A2 WO 9936536A2
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scramblase
cell
protein
cells
human
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PCT/US1999/001087
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WO1999036536A9 (fr
WO1999036536A3 (fr
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Peter J. Sims
Therese Wiedmer
Ji Zhao
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Blood Center Research Foundation
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Priority to AU23262/99A priority Critical patent/AU2326299A/en
Priority to EP99903179A priority patent/EP1047779A2/fr
Publication of WO1999036536A2 publication Critical patent/WO1999036536A2/fr
Publication of WO1999036536A9 publication Critical patent/WO1999036536A9/fr
Publication of WO1999036536A3 publication Critical patent/WO1999036536A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • cell surface PS has a role in coagulation, programmed cell death and clearance by the reticuloendothelial system.
  • U.S. Serial No. 08/790,186 also describes regulation of the transmeubrane distribution of PS, the role of calcium in the collapse of phospholipid asymmetry, and the role PL translocation in Scott Syndrome.
  • Programmed cell death or apoptosis is an important process that is now recognized to be central to the selective elimination of mammalian cells during embryogenesis, tissue remodeling, the clonal selection of immune cells, metamorphosis and hormone-dependent atrophy, as well as cell death induced by chemicals or irradiation (reviewed by G.M. Cohen, Biochem. J. 326:1- 16, 1997; D.L. Vaux and A Strasser, Proc . Natl . Acad. Sci . USA 93:2239-2244; D. J. McConkey and S. Orrenius , Biochem Biophys . Res . Commun . 239:357-366, 1997, and references therein) . Inappropriate apoptosis (either unregulated excessive or inappropriately low rate) is now implicated in many human diseases, including Alzheimer's
  • the specific pathways leading to cell death may vary for different cell types and cell stimuli, it is convenient to divide the process into distinct phases.
  • the earliest phase is the stimulus that initiates the apoptotic response. This may be an external signal delivered through surface receptors such as CD95 (Fas) or tumor necrosis factor receptor, or may originate inside the cell in response to a drug, irradiation or toxin.
  • the signal is detected and transduced to the cell death effector machinery.
  • the third phase of the apoptotic process includes activation of proteases (including caspases) , as well as their positive and negative regulators (including Bcl-2, Bcl-x L , and their inhibitors Bax, Bad, and Bak) (J.C. Reed, Nature 387:773- 776, 1997; T. Patel, et . al., FASEB J. 10:587-597, 1996; G. M. Cohen, supra , 1997) .
  • Activation of the "execution phase” results in morphological changes including cell shrinkage, cell surface exposure of aminophospholipids resulting in the recognition and phagocytosis of apoptotic cells, and plasma membrane blebbing.
  • PS plasma membrane phosphatidylserine
  • PS exposure has been reported to be inhibited by the caspase inhibitor zVAD, implicating this family of "executioner” proteases in this process (S.J. Martin, et. al. , J. Biol . Chem. 271:28753-28756, 1996) .
  • inhibitors of calpain have also been reported to inhibit externalization of PS.
  • the molecular link between protease activation and PS exposure remains to be elucidated.
  • US Patent Application Serial No. 08/949,246 and reported in Zhou, et. al. (Q. Zhou, et al., J. Biol. Chem.
  • sphingomyelin cycle results in activation of sphingomyelinase which hydrolyses plasma membrane sphingomyelin to generate cellular ceramide.
  • Ceramide in turn, has emerged as a candidate intracellular mediator, affecting the activities of several enzymes, including a ceramide-activated protein phosphatase (CAPP) , ceramide-activated kinases (CAPK, JNK/SAPKs) , and protein kinase C (I. Herr, et al . . , EMBO J. 16:6200-6208, 1997) .
  • CAPP ceramide-activated protein phosphatase
  • CAPK ceramide-activated kinases
  • JNK/SAPKs protein kinase C
  • cytokine response modifier A (CrmA) , a potent inhibitor of caspases, inhibited ceramide formation and tumor necrosis factor-induced cell death, suggesting a role for proteases in the signaling and/or activation phase of apoptosis upstream of ceramide formation (G. Ddaibo, et al., J. Exp. Med. 185:481-490, 1997).
  • ceramide generation in the resting cell is limited by the restricted accessibility of sphingomyelin for hydrolysis by the cytoplasmic enzyme, sphingomyelinase, and that the plasma membrane phospholipid rearrangement upon induction of apoptosis results not only in surface exposure of PS, but also movement of sphingomyelin from outer to the inner leaflet of the plasma membrane .
  • this invention relates to compositions and methods for controlling cell and tissue susceptibility to apoptosis by modifying the transmembrane movement of the plasma membrane phospholipids between inner and outer leaflets.
  • Transformed cancer cells exhibit the capacity to proliferate through unregulated mitotic division and to resist the normal cycle of senescence and programmed cell death characteristic of most normal untransformed cells. Additionally, malignant cancer cells in vivo exhibit the capacity to evade detection or injury by the body's immune defenses, including cellular killing by cytotoxic T-lymphocytes , humoral killing by antibody and complement, and removal by macrophages and other phagocytic cells of the reticuloendothelial system.
  • Zhou, et. al. identified a conserved EF-hand-like motif in PL scramblase that is required for the Ca 2+ -accelerated transbilayer movement of membrane phospholipids (Zhou, et . al- , Biochemistry 37:2356-2360, 1998), and PL scramblase was shown to undergo a prominent conformational change upon binding Ca 2+ (Stout, et al., Biochemistry 37:14860-14866, 1998).
  • Kasukabe et al. 1997 (T. Kasukabe, et al. , Blood 89:2975-2985, 1997) disclose a gene designated NORl that is down-regulated in transformed murine monocytes relative to amount of mRNA expressed in normal mouse cells and a 5 ' -truncated form of this same transcript
  • TRAl (designated TRAl) that is over-expressed in leukemogenic mouse monocytic cell lines but not found in the non- leukemogenic monocytic cell lines. They suggest that the Tral gene product is associated with the leukemogenesis of monocytic Mm cell lines.
  • the present invention also relates to the role of PL scramblase in promoting clearance of injured or apoptotic cells by the phagocytic cells of the reticuloendothelial system.
  • Needed in the art is a method for modulating the activity or level of phospholipid scramblase within a cell, organ or tissue in which one wishes either to reduce the potential for cell death and resulting clearance of beneficial cells by the scavenger macrophages of the reticuloendothelial system (by decreasing cellular PL scramblase expression or activity) or to promote cell death and cell clearance from the body of undesireable cells (by increasing cellular PL scramblase expression or activity) .
  • Fig. 1A is a comparison of the cDNA and deduced amino acid sequence of human PL scramblase (SEQ ID NOs : 1 and 2) .
  • Fig. IB is a comparison of the cDNA and deduced amino acid sequence of murine PL scramblase (SEQ ID NOs : 3 and 4) .
  • Fig. 2 is a bar graph illustrating immunoprecipitation of erythrocyte PL scramblase.
  • Fig. 3 is a graph of an activity assay of recombinant PL scramblase.
  • Fig. 4 is an immunoblot of PL scramblase in human erythrocytes and platelets.
  • Fig. 5 is a comparison of protein sequences of mouse and human PL scramblase (SEQ ID NOs : 2 and 4) .
  • Fig. 6 is a bar graph of PL scramblase activity as a function of mutational analysis of a putative EF hand loop motif contained in human PL scramblase.
  • Fig. 7 graphs the Ca 2+ dependence of mutant human PL scramblase with amino acid substitutions in putative Ca 2+ binding site.
  • Fig. 8 is a Western blot analysis of PL scramblase protein and corresponding functional assay of PL scramblase activity in various human cell lines.
  • Fig. 9A and B are fluorescence micrographs of GFP-PL scramblase in transformed Raj i cells.
  • Fig. 9A depicts fluorescence of cells expressing GFP;
  • Fig. 9B depicts cells transfected with pEGFP-C2-PL scramblase plasmid and expressing GFP-PL scramblase fusion protein.
  • Fig. 10 is a graph showing that the level of expression of PL scramblase determines plasma membrane sensitivity to intracellular calcium.
  • Fig. 11 is a bar graph illustrating inactivation of PL scramblase by hydroxylamine .
  • Fig. 12 is analysis of metabolically-labeled PL scramblase by SDS-PAGE revealing covalent incorporation of thioester-linked fatty acid.
  • Fig. 13 is analysis by thin layer chromatography demonstrating that PL scramblase contains covalent palmitic acid.
  • Fig. 14A and B is a bar graph illustrating the role of PL scramblase in PS exposure during apoptosis of B- lymphocytes .
  • Fig. 14A compares Raj i and W9 cell lines.
  • Fig. 14B compares individual Raj i cell clones that were stably transfected with either pEGFP-C2 or with pEGFP-PL scramblase and then induced to apoptosis.
  • Fig. 15 is a bar graph illustrating the percent apoptotic cells for Raj i cells, Raj i cells transfected with GFP-PL scramblase fusion protein or GFP vector as a control .
  • the present invention relates to the creation and use of reagents and methods to either inhibit or promote cell injury, cell death, and cell clearance from the body.
  • reagents and methods rely on the properties of phospholipid scramblase (PL scramblase) , a protein that mediates Ca 2+ -dependent transbilayer movement of plasma membrane phospholipids that are involved in the activation of the plasma complement and coagulation cascades and are substrates for cellular enzyme cascades that mediate programmed cell death.
  • PL scramblase phospholipid scramblase
  • the present invention is a method for decreasing the viability of cancer cells or virus- infected cells comprising the step of increasing the amount or activity of PL scramblase in the plasma membrane of the cancer cell, cancerous tissue, or virus- infected cell, thereby promoting the movement of plasma membrane phospholipids between inner and outer plasma membrane leaflets.
  • the increase in PL scramblase is accomplished by activating the promoter of the endogenous PL scramblase gene or by delivering cDNA encoding PL scramblase, provided in a suitable targeting vector to the cell or tissue.
  • cDNA encoding PL scramblase is delivered to the cell or tissue in a targeting vector, where the vector is a retrovirus or adenovirus vector containing the PL scramblase cDNA.
  • the present invention is a method for inducing programmed cell death (apoptosis) of a cell, preferably a mammalian cell, comprising the step of increasing the level of expression of plasma membrane PL scramblase, thereby increasing the movement of plasma membrane phospholipids between membrane leaflets, resulting in the movement of the phospholipid sphingomyelin from outer membrane leaflet to inner leaflet and the increase in production of ceramide from sphingomyelin through the action of the cytoplasmic enzyme sphingomyelinase.
  • the resulting ceramide is known to induce activation of a cascade of cellular enzymes that ultimately lead to apoptotic cell death.
  • the present invention is a method for promoting clearance of a cell from the body by phagocytic uptake into the reticuloendothelial system comprising the step of increasing the level of expression of plasma membrane PL scramblase, thereby increasing cell surface exposure of phosphatidylserine and phosphatidylethanolamine through the movement of these aminophospholipids from inner plasma membrane leaflet to outer plasma membrane leaflet.
  • phosphatidylserine is known to directly promote adhesion and phagocytosis by macrophages and other cells of the reticuloendothelial system whereas phosphatidylethanolamine promotes cell adhesion and phagocytosis by macrophages and other cells of the reticuloendothelial system by promoting complement activation, which opsonizes the cell surface with complement C3b.
  • the present invention is a method of promoting the viability of a mammalian cell, both in in vi tro cell culture and in vivo, comprising the step of decreasing the activity of the plasma membrane PL scramblase, thereby inhibiting movement of plasma membrane phospholipids between inner and outer membrane leaflets.
  • the decrease in plasma membrane PL scramblase is accomplished by either decreasing expression of the endogenous PL scramblase gene or by delivering to the cell an inhibitor of the PL scramblase protein.
  • the expression of PL scramblase is inhibited by delivering to the cell antisense oligonucleotides or antisense cDNA to PL scramblase RNA.
  • a defective form of the PL scramblase protein is expressed in the cell and that mutant PL scramblase inhibits the endogenous PL scramblase .
  • endogenous PL scramblase is inactivated by deacylating essential fatty acids from the protein that are required for normal PL scramblase function in the plasma membrane.
  • the cell By decreasing the activity of plasma membrane PL scramblase, the cell is protected due to decreased hydrolysis of plasma membrane sphingomyelin to ceramide (mediated by cellular sphingomyelinase) , as well as by decreased exposure of the procoagulant and complement-activating aminophospholipids phosphatidylserine and phosphatidylethanolamine at the cell surface.
  • the present invention is a method of preventing the phagocytic removal and clearance of a transfused or transplanted cell by preventing surface exposure of plasma membrane phosphatidylserine by delivering to the cell a mutant phospholipid scramblase that inhibits the endogenous PL scramblase.
  • This scramblase is preferably mutated at a site of post- translational modification, most preferably the site is selected from the group consisting of Asp 273 -Asp 284 , Thr 161 and conserved cysteines of human PL scramblase or the corresponding conserved residues in mouse or other mammalian PL scramblase.
  • a gene construct encoding a mutant phospholipid scramblase is delivered to the cell.
  • the mutant protein itself is delivered.
  • the present invention is a method of diagnosing metatastic and invasive potential or growth potential of a cancer cell or cancerous tissue.
  • This method may comprise the step of analyzing the amount of PL scramblase RNA or protein within a patient's cell sample.
  • the level of PL scramblase R ⁇ A may be measured by in si tu hybridization or Northern blotting with PL scramblase cDNA or by polymerase chain reaction using oligonucleotide primers based on sequence of human PL scramblase RNA .
  • the method may comprise the step of analyzing the sequence of a patient's PL scramblase gene, R ⁇ A or protein for the presence or absence of mutations.
  • This analysis may preferably be selected from the group consisting of restriction fragment length polymorphism, sequencing by RT-PCR, Northern blotting, Western blotting, electrophoretic gels, protease digestion, and other techniques designed to analyze RNA, DNA or protein sequence .
  • the present invention involves the use of a preparation of a plasma membrane phospholipid scramblase ("PL scramblase”) .
  • PL scramblase plasma membrane phospholipid scramblase
  • the protein is approximately 35-37 kD as measured on a 12.5% SDS polyacrylamide gel under reducing conditions.
  • the preparation is a human or a mouse PL scramblase.
  • the PL scramblase comprises SEQ ID NO: 2, representing human PL scramblase, possibly with conservative or functionally equivalent substitutions.
  • the PL scramblase has been modified by the action of mammalian cellular enzymes to covalently incorporate phosphorus at one or more Thr, Ser, or Tyr residues or a fatty acid, preferably palmitate, at a cysteine residue.
  • the present invention may also use a murine cell protein, wherein the protein is a plasma membrane phospholipid scramblase, preferably wherein the protein is approximately 35 kD as measured on a 12.5% SDSpolyacrylamide gel under reducing conditions.
  • the protein is a plasma membrane phospholipid scramblase, preferably wherein the protein is approximately 35 kD as measured on a 12.5% SDSpolyacrylamide gel under reducing conditions.
  • the murine PL scramblase has been modified by the action of mammalian cellular enzymes to covalently incorporate phosphorus at one or more Thr, Ser, or Tyr residues, and a fatty acid, preferably palmitate, at a cysteine residue.
  • the present invention may employ an inhibitor of the PL scramblase activity of PL scramblase.
  • This inhibitor may be an antisense nucleotide derived from the DNA sequence of PL scramblase.
  • the inhibitor is a peptide sequence that is a competitive inhibitor of PL scramblase activity.
  • the inhibitor is an antibody, preferably a monoclonal antibody, raised against PL scramblase.
  • the inhibitor works by modifying or inhibiting the post-translational modifications of the PL scramblase that are disclosed below in the Examples.
  • analysis of the primary PL scramblase sequence reveals a potential site of phosphorylation by protein kinase C or other cellular kinase (Thr 161 ) , potential sites for acylation by fatty acid, and a potential binding site for Ca 2+ ion provided by an EF-hand-like loop spanning residues Asp 273 -Asp 284 . These residues and motifs are conserved in the mouse PL scramblase .
  • the inhibitor is a compound that prevents thioacylation of the protein.
  • a mutant phospholipid scramblase in which cysteine residues have been replaced by alanine or other non-conservative substitution.
  • the present invention is also a method for promoting the viability and circulating lifetime of in vi tro stored leukocytes, lymphocytes, platelets, or red blood cells. This method comprises the steps of adding an inhibitor of PL scramblase activity to the stored blood cells.
  • the present invention is also a method for prolonging survival of transplanted cells, tissues, or organs, comprising the step of adding an inhibitor of PL scramblase activity to an organ perfusate or cell storage medium during in vi tro organ storage.
  • the present invention is also a method for prolonging the survival of transplanted cells, tissues, and organs by genetically engineering the cells to be transplanted so as to reduce their expression of plasma membrane PL scramblase in order to reduce exposure of PS and other thrombogenic and pro-inflammatory phospholipids at the plasma membrane surface.
  • the transplanted cells to be treated are hematopoietic stem cells to be used to repopulate the bone marrow by transfusion into a patient.
  • the present invention is also a method for diagnosing the survivability, metastatic, and invasive potential of a cancer cell or cancerous tissue or virus- infected cell by quantifying the level of cellular expression of PL scramblase and by detecting deletion or loss of function mutations in PL scramblase.
  • this quantitation may take the form of immunoblotting using an antibody to PL scramblase, an ELISA assay using an antibody to PL scramblase, flow cytometric analysis of the binding of monoclonal antibody reactive against the predicted extracellular domain of PL scramblase (residues Ser 310 Trp 318 of sequence disclosed in SEQ ID NO: 2 or the equivalent residue in the conserved region of another PL scramblase) or using oligonucleotides derived from PL scramblase cDNA and the polymerase chain reaction.
  • Northern blotting or in si tu hybridization is performed using cDNA of human PL scramblase as probe.
  • mutations in the PL scramblase gene locus are detected by DNA sequencing of the cDNA product of the polymerase chain reaction and by analysis of restriction length polymorphisms.
  • the quantitation is performed by isolating PL scramblase from tissue biopsy or a patient blood sample, measuring the amount of PL scramblase isolated and comparing the measurement with a control sample. The measurement may be by isolating PL scramblase from a patient tissue biopsy or blood sample and measuring via densitometry the amount of PL scramblase protein electrophoresed in a stained electrophoretic gel.
  • EXAMPLE 1 Cloning of Murine PL scramblase and Identity of a conserveed Motif in Phospholipid Scramblase that is Required for Accelerated Transbilayer Movement of Membrane Phospholipids by Ca 2+ (Zhou, e_t al . , Biochemistry 37:2356-2360, 1998) .
  • Fig. IB human polypeptide
  • human PL scramblase (318 AA) terminates in a short exoplasmic tail
  • murine PL scramblase (307 AA) terminates in the predicted membrane- inserted segment.
  • the aligned polypeptide sequences reveal 65% overall identity, including near identity through 12 residues of an apparent Ca 2+ binding motif
  • Mouse fibroblast 5 '-stretch plus cDNA library and KlenTaq polymerase were obtained from CLONTECH Laboratories.
  • Expressed sequence Tag (EST) clone with GenBankTM accession number gb AA110551 was from American Type Culture Collection (ATCC 977052) .
  • ⁇ - 32 P- dCTP was purchased from Dupont .
  • Random Primed DNA Labeling Kit was from Boehringer Mannheim.
  • Hybond-N Nylon membrane was from Amersham.
  • Expression vector pMAL-C2 amylose resin and all restriction enzymes were from New England Biolabs .
  • Wizard Kit was from Promega.
  • Qiagen Lambda Kit was from Qiagen.
  • PC Egg yolk phosphatidylcholine
  • PS brain phosphatidylserine
  • l-oleoyl-2- [6 (7-nitrobenz-2-oxa-l , 3-diazol-4- yl) amino] caproyl-sn-glycero-3-phosphocholine (NBD-PC) were obtained from Avanti Polar Lipids .
  • Factor Xa was from Haematologic Technologies, and Bio-Beads SM-2 were from BioRad.
  • N-octyl- ⁇ -D-glucopyranoside and Glu-Gly-Arg chloromethyl ketone were from Calbiochem.
  • Sodium dithionite (Sigma) was freshly dissolved in 1 M Tris, pH 10, at a concentration of 1 M.
  • the membranes were first prehybridized in a solution composed of 5X Denhardt , 5X SSC, 1% SDS, and 200 ⁇ g/ml herring sperm DNA for 3 hours at 68°C, and then hybridized in the same solution containing 5 ng/ml 32 P- labeled probe for 16 hours at 68°C.
  • the membranes were washed once with 2X SSC, 0.1% SDS, then three times with 0.1 X SSC, 0.1% SDS for 20 minutes at 65°C, and exposed to X-ray film. Secondary plaque lifts and hybridization were carried out on 8 positive plaques at a density of about 100 plaques/plate.
  • Mouse PL scramblase was expressed as fusion protein with MBP in E. coli TB1 and purified on amylose resin as previously described for human PL scramblase (Q. Zhou, et al. , CL . Biol . Chem. 272:18240- 18244, 1997) .
  • the purified fusion protein was centrifuged at 106,000 x g for 1 hour at 4°C to remove aggregated protein.
  • Reconstitution and Functional Activity of PL Scramblase Reconstitution, removal of MBP, and functional assay of PL scramblase were performed as previously described (F. Basse, et. al.
  • Proteoliposomes labeled with NBD-PC were incubated for 2 hours at 37°C in Tris buffer in the presence or absence of CaCl 2 as indicated in figure legends and diluted 25-fold in Tris buffer containing 4 mM EGTA.
  • Initial fluorescence was recorded (SLM Aminco 8000 spectrofluorimeter ; excitation at 470 nm, emission at 532 nm) , 20 mM dithionite was added, and the fluorescence was continuously monitored for a total of 120 seconds. Scramblase activity was calculated according to the difference in non-quenchable fluorescence observed in presence vs absence of CaCl 2 .
  • Protein concentrations were estimated based upon optical density at 280 nm, using extinction coefficients (M ⁇ crtr 1 ) of 39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PL scramblase-MBP fusion protein) .
  • PL Scramblase Human PL scramblase amino acid residues in EF-hand Ca + -binding motif at positions of Asp 273 , Asp 275 , Phe 277 , He 279 , Phe 281 and Asp 284 were mutated to Ala with oligonucleotide-directed mutagenesis by two rounds of PCR.
  • PL scramblase-pMAL-C2 was selected as template, and the first round of PCR was performed with pairs of a complementary oligonucleotide primer containing the point mutation plus a primer complementary to a site near the ATG initial codon or TAG stop codon.
  • PCR products were purified by Wizard kit.
  • Murine EST clones in GenBank containing putative PL scramblase sequence were identified by a Blast homology search using the human PL scramblase cDNA.
  • a 403 bp Stratagene mouse kidney clone (gb accession number AA110551) with 79% nucleotide sequence identity to human PL scramblase was selected and this clone was used to probe a mouse fibroblast cDNA library.
  • Eight positive clones were identified after two rounds of plaque hybridization. Two of the eight clones were sequenced yielding 1354 bp and 1529 bp, respectively. Alignment revealed 1261 bp of overlapping sequence that spanned an open reading frame of 921 bp and specified a total of 1622 bp of unique cDNA sequence (SEQ ID NO: 3) .
  • SEQ ID NO: 4 represents the open reading frame of the translated sequence of SEQ ID NO : 3 (see Fig. IB) .
  • the overall identity of the mouse and human PL scramblase is 64.8%, with the most divergent sequence generally contained in the N-terminal portion of the polypeptide (Fig. 5) .
  • Fig. 5 the most divergent sequence generally contained in the N-terminal portion of the polypeptide
  • FIG. 5 depicts the alignment of protein sequences of mouse and human PL scramblase. Alignment between mouse (MUR) and human (HUM) PL scramblase was performed by FASTA program using the Smith-Waterman algorithm. (W.R. Pearson and D.J. Lipman, Proc . Natl . Acad. Sci. USA 85:2444-2448, 1988)
  • the human PL scramblase contains an additional nine residues, implying that the short exoplasmic peptide in human PL scramblase is non-essential to function.
  • Homology motifs conserved in both proteins include a potential site for protein kinase C phosphorylation (Thr 159 in mouse, Thr 161 in human) and a potential Ca 2+ -binding EF-hand loop motif adjacent to the transmembrane helix (residue Asp 271 to Asp 282 in mouse and residues Asp 273 to Asp 284 in human) .
  • mouse PL scramblase Functional Activi ty of Recombinant Mouse PL Scramblase .
  • human and mouse proteins were each expressed in E. coli , purified, and reconstituted in proteoliposomes for measurement of PL mobilizing activity.
  • Mouse or human PL scramblase-MBP fusion protein (420 pmoles) was reconstituted into PC/PS liposomes (1 ⁇ mol total PL) , respectively, MBP was removed by digestion of the proteoliposomes with factor Xa, and PL scramblase activity was determined as described under "Experimental Procedures" and plotted as a function of external free [Ca 2+ ] .
  • the results of this experiment indicate that recombinant mouse PL scramblase mediated a Ca 2+ -dependent transbilayer movement of membrane PL with a specific activity and affinity for Ca 2+ indistinguishable from that observed for the recombinant human protein.
  • the C-terminal alpha helix represents a predicted transmembrane segment with a strongly- preferred inside-to-outside orientation, whereas sequence contained within the adjacent 12 -residue acidic loop conforms in-part to a consensus sequence that is characteristic of an EF-hand Ca + -binding loop motif (S. Nakayama and R.H. Kretsinger, Annu . Rev. Biophys . Biomol . Struct. 23:473-507, 1994) .
  • residues in positions 1, 3, 5, 7, 9 and 12 of the loop contribute to octahedral coordination of the Ca 2+ ion, with the residues in position 1 [Asp] , 3 [Asp, Asn, or Ser] and 12 [Asp or Glu] being those most highly conserved.
  • mutant human PL scramblase with Ala substitutions at positions corresponding to residues 1 (Asp 273 ), 3 (Asp 275 ), 5 (Phe 277 ), 7 (He 279 ), 9 (Phe 281 ), and 12 (Asp 284 ) of this putative 12 residue EF-hand loop.
  • Fig. 6 illustrates PL scramblase activity as a function of mutational analysis of putative EF hand loop motif contained in human PL scramblase.
  • Wild-type (WT) and mutant constructs of human PL scramblase were expressed as fusion proteins with MBP in E . coli , purified, and reconstituted in proteoliposomes. After release of MBP by incubation with factor Xa, PL scramblase activity was assessed (see "Experimental Procedures") . For each mutant construct, the residues in human PL scramblase that were replaced by Ala are indicated on the abscissa .
  • PL scramblase activity (ordinate) was measured in presence of 2 mM CaCl 2 , and in each case was normalized to the activity of WT human PL scramblase (11.76 ⁇ 0.44% of total NBD-PC flipped), with correction for the non-specific transbilayer movement of NBD-PC (0.20 ⁇ 0.08% of total NBD-PC flipped) measured in PL vesicles lacking added protein. Error bars indicate mean ⁇ SD of three independent measurements performed with each mutant construct.
  • Fig. 6 illustrates the data of single experiment, representative of two separate experiments so performed. As illustrated by Fig.
  • Fig. 7 illustrates the Ca 2+ -dependence of mutant human PL scramblase.
  • PL scramblase activity of wild-type (WT) and selected mutant constructs of Fig. 6 was determined as described in "Experimental Procedures" and plotted as a function of external free [Ca 2+ ] : WT(*);
  • EXAMPLE 2 Inactivation of Human PL Scramblase by Treatment With the Thiolester Cleaving Reagent, Hydroxylamine (Zhao, e_t al. , Biochemistry 37:6361-6366, 1998) .
  • PL scramblase was purified from human erythrocyte ghost membranes as previously described (F. Basse, e_t al . , supra, 1996; J.G. Stout, et. al . , supra , 1997) .
  • PL scramblase Treatment with Hydroxylamine.
  • PL scramblase was incubated 1 hour at room temperature in 1 M hydroxylamine, 25 mM octylglucoside, 1 M Tris-HCl at pH 7.4.
  • Match control samples of the protein were identically incubated under these conditions, omitting hydroxylamine.
  • samples were dialyzed and reconstituted into PL proteoliposomes for assay of PL scramblase activity.
  • FIG. 11 illustrates inactivation of PL scramblase function by treatment with hydroxylamine.
  • the figure shows loss of functional activity of human erythrocyte PL scramblase due to treatment with the thioester cleaving reagent hydoxylamine .
  • Identical aliquots of human erythrocyte PL scramblase in 50 mM octylglucoside were incubated 1 hour room temperature in either 1 M hydroxylamine, pH 7.2 (solid bar) or 1 M Tris- HCl, pH 7.2 (open bar) .
  • each sample was reconstituted into proteoliposomes and PL scramblase activity measured using NBD-phosphatidylcholine as probe according to methods previously described (Basse, et al . , supra , 1996; Stout, et . al . , supra. 1997; Zhou, et. al . , supra , 1997) .
  • Error bars denote mean ⁇ SD of seven independent experiments. Significance by paired students' t-test, p ⁇ 10 ⁇ 6 . (See Methods, Example 3.)
  • Fig. 12 illustrates that metabolic labeling of PL scramblase with [ 3 H] -palmitate reveals covalent thioester- linked fatty acid.
  • the presence of cysteinyl -linked palmitate in PL scramblase was confirmed by metabolic labeling of pEGFP-PL scramblase-transfected Raj i cells (GFP-PLS) or identically-treated vector-only controls (GFP) with exogenous [ 3 H] -palmitate .
  • Cells were incubated (10 7 cell/ml, 1 hour, 37°C) with 200 ⁇ Ci/ml [ 3 H] -palmitate (Dupont NEN, spec. act.
  • Insoluble material was removed by centrifugation and the supernatants precleared by incubation with 10 ⁇ g/ml normal rabbit IgG and 30 ⁇ l protein A sepharose (Pharmacia) .
  • the GFP and GFP-PLS proteins were then immunoprecipitated overnight with 50 ⁇ g/ml affinity purified rabbit anti-GFP (Clontech) and staph A sepharose.
  • the beads were exhaustively washed with 1% triton X100 and GFP vector control (Lanes 1 and 2) and the GFP-PL scramblase fusion protein (Lanes 3 and 4) each eluted from staph A sepharose into 5% SDS-PAGE sample buffer.
  • UPPER PANEL Western blotting was performed with murine monoclonal antibody against human PL scramblase (anti-PLS) .
  • MIDDLE PANEL Western blotting performed with murine monoclonal antibody against green fluorescent protein (anti-GFP; Clontech) ) .
  • LOWER PANEL shows fluorogram of radioactivity from 3 H.
  • Fig.13 illustrates that TLC analysis of [ 3 H] -fatty acid in PL scramblase.
  • the GFP-PLS protein band at -65 kDa of the previous figure was sliced from the wet gel and the gel soaked in 30% methanol, 10% acetic acid, washed exhaustively with 50% methanol, and then lyophilized. To liberate covalently-bound fatty acids, the dried gel was incubated overnight in 1.5N NaOH. Following acidification with HC1 (pH ⁇ 2) , the released fatty acids (1 ml) were extracted by addition of 3.75 ml chloroform: methanol (1:2 v:v) .
  • Radioactivity liberated from GFP-PLS (lane 2) is compared to matched gel slice from GFP vector control (lane 3), using [ 3 H] -palmitate as standard (standards applied in lanes 1 and 4) . All samples were spotted at position OR; SF indicates position of the solvent front. Data of single experiment.
  • PL scramblase is palmitoylated at one or more cysteine residues.
  • acylation is required for normal function of PL scramblase protein.
  • removal of palmitate through hydrolysis of thioester bonds with hydroxylamine was found to abolish PL scramblase' s phospholipid mobilizing function (Fig. 11).
  • EXAMPLE 3 Plasma Membrane Expression of Phospholipid Scramblase Regulates Ca 2+ Induced Movement of Phosphatidylserine to the Cell Surface: Alteration of Phosphatidylserine Exposure In Human Lymphoblasts Through Stable Transfection with PL Scramblase cDNA.
  • clones expressing GFP-PL scramblase exhibited markedly accelerated movement of PS to the cell surface when compared to A23187 -treated clones expressing GFP with PS movement to the cell surface increasing with amount of rGFP-PL scramblase expressed.
  • OPTI-MEM and geneticin were from Life Technologies (Gaithersburg, MD) .
  • Fetal bovine serum, RPMI 1640, Cell Dissociation Solution, Hank's Balanced Salt Solution (HBSS), Protein A Sepharose-CL4B, leupeptin, and BSA were from Sigma
  • MOLT-4 acute T-cell leukemia Jurkat , Burkitt's lymphoma
  • Raji, and megakaryocytic DAMI were from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 containing 10% fetal bovine serum.
  • EBV-transformed cell line W9 established from peripheral B- lymphocytes of a normal donor was maintained as previously described (H. Kojima, et al. , 1994).
  • Antibodies Anti -GFP: murine monoclonal antibody against green fluorescent protein (GFP) was from CLONTECH Laboratories. Anti -FVa : murine monoclonal antibody V237 reactive against human or bovine factor Va light chain was the generous gift of Dr. Charles T. Esmon (Oklahoma Medical Research Fndn, Oklahoma City, OK) . Anti -PL Scramblase-E306-W318 : Rabbit antibody raised against the carboxyl terminal peptide sequence E306-W318 of human PL scramblase has previously been described (Q. Zhou, et. al . , supra , 1997) .
  • the IgG fraction was isolated on protein A-Sepharose-CL4B and the peptide-reactive antibody purified by affinity chromatography on peptide [Cys] -ESTGSQEQKSGVW (SEQ ID NO:5) coupled to UltraLink Iodoacetyl resin.
  • Plasmid Construction Human PL scramblase cDNA insert was released from plasmid pMAL-C2-PL scramblase (Q. Zhou, e_t al. , supra , 1997) by double cutting with EcoRI and Sail, respectively, and then ligated into pEGFP-C2 vector using the same restriction site.
  • the pEGFP-C2-PL scramblase plasmid was amplified from single clones in E. coli strain Top 10, and the orientation and reading frame of the insert confirmed by sequencing on an ABI DNA Sequencer Model 373 Stretch (Perkin Elmer-Applied Biosystems, Foster City, CA) using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit .
  • Stable transformants exhibiting GFP fluorescence were sorted by flow cytometry (FACStar, Becton-Dickinson Immunocytometry Systems, San Jose, CA) using an FL1 sorting gate.
  • the FLl-positive cells were dilutionally cloned in 96 well culture plates.
  • pEGFP-PL scramblase transformants expressing the 62 kDa GFP-PL scramblase fusion protein were identified by Western blotting with anti-GFP and with anti-PL-306-W318 antibodies.
  • Western blotting of pEGFP-C2 transformants (without insert) confirmed presence of 27 kDa GFP.
  • Clones expressing various amounts of GFP-PL scramblase were each expanded for functional assay, along with comparable GFP-expressing clones serving as controls.
  • Fluorescence Microscopy Cell clones transfected with pEGPF-C2-PL scramblase or pEGFP-C2 were deposited on glass microscopy slides using a Cytospin 3 (Shandon, Inc., Pittsburgh, PA). Phase contrast and fluorescence microscopy was performed with a ZEISS AXIOSKOP microscope (Carl Zeiss, Inc., Thornwood, NY) equipped for epifluorescence, and images were recorded with a MC100 camera system. The exposure times for photography of fluorescence was 80-200 seconds under automatic control using Kodak Ektachrome 1600 film.
  • Western Blot Analysis Western Blot Analysis .
  • Western blotting of GFP and PL scramblase antigens was performed using 1.5 x 10 6 cells per lane. After washing in HBSS, supernatants were removed, and the cell pellets extracted with 2% (v/v) NP- 40 in 5 mM EDTA, 50 mM benzamidine, 50 mM N-ethyl maleimide, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin in HBSS. After removal of insoluble material (250,000 x g, 30 minutes, 4°C) , the samples were denatured at 100°C in 10% (w/v) SDS sample buffer containing 2% ⁇ -mercaptoethanol .
  • the blocked membrane was incubated with either 10 ⁇ g/ml of rabbit anti-PL scramblase-E306-W318, or 1/10,000 dilution of mouse anti- GFP.
  • the blots were developed with the horseradish peroxidase conjugate of either goat anti-rabbit IgG or goat anti-mouse IgG, respectively, using SuperSignal ULTRA chemiluminescence .
  • Measurement of cell surface PS Calcium ionophore- induced exposure of PS on the surface of all cell lines analyzed was detected by the specific binding of coagulation factor Va (light chain) as previously described (P.J. Sims, e_t al.
  • PS exposed on the cell surface at each time point was detected by incubating (10 minutes, room temperature) 50 ⁇ l of the cell suspension with 10 ⁇ g/ml FVa, followed by 10 ⁇ g/ml anti-FVa, to detect the cell-bound FVa light chain. After staining with 10 ⁇ g/ml Tri -Color conjugated goat anti -mouse IgG (CALTAG Laboratories, Burlingame, CA) , single-cell fluorescence was quantitated by flow cytometry (FL3 channel, FACScan, Becton Dickinson Immunocytometry Systems) .
  • Tri-Color conjugate to detect cell -bound FVa enabled simultaneous measurement of cell-associated GFP fluorescence in cell lines transformed with the pEGFP-C2 expression plasmid (fluorescence of GFP detected in FL1 channel) .
  • cells were stained for bound FVa with FITC-conjugate of goat anti -mouse IgG (FL1 channel) substituting for Tri-Color conjugate, and propidium iodide was detected in FL3 channel .
  • Propidium iodide (0.5 ⁇ g/ml) was added immediately before dilution for flow cytometry. Prothrombinase Assay.
  • Prothrombinase activity of Raji cells was determined by modification of methods previously described for platelets, using the chromogenic thrombin substrate S2238 (P.J. Sims, e_t aL. , supra, 1988) . Briefly, 1 x 10 5 Raji cells (transfected with either pEGFP-C2 or pEGFP-C2-PL scramblase) were suspended in 200 ⁇ l HBSS containing 1% BSA in the presence of 2 nM FVa, 1.4 ⁇ M prothrombin 2.5 mM CaCl 2 , and 4 ⁇ M dansylarginine-N- (3-ethyl-l, 5-pentanediyl) amide (to inhibit feed-back activation by thrombin) , and incubated at 37°C.
  • Fig. 8 depicts western blot analysis of PL scramblase in various human cell lines. Constitutive expression of PL scramblase was analyzed in the human cell lines indicated.
  • Upper Panel Results obtained by Western blotting with antibody specific for PL scramblase carboxyl terminal residues E306-W318 (see Materials & Methods) . Each lane contains the total protein extract of 1.5 x 10 6 cells.
  • Lower Panel Cumulative results of three separate experiments performed as follows: The cells indicated were washed and suspended at 37°C in the presence of 1.2 mM free Ca 2 ", and 2 ⁇ M A23187 was added.
  • Fig. 9 illustrates fluorescence micrographs of GFP-PL scramblase transformed Raji cells. Fluorescence photomicrography of GFP fluorescence expressed in the transformed Raji clones was performed as described in Materials and Methods .
  • Fig 9A shows fluorescence of cells expressing GFP
  • Fig. 9B shows cells transfected with pEGFP-C2-PL scramblase plasmid and expressing GFP-PL scramblase fusion protein.
  • Data of single experiment representative of results obtained for all clones transfected with either pEGFP-C2 or pEGFP-C2-PL scramblase.
  • clones that expressed GFP alone exhibited diffuse fluorescence throughout the cytoplasm, with no obvious staining of the plasma membrane.
  • transformants expressing the GFP-PL scramblase fusion construct showed a marked increase in both the rate and extent that PS became exposed on the cell surface, when compared to either the identically-treated parental Raji cell line or to GFP-expressing clones transformed with pEGFP-C2 vector alone.
  • PL scramblase regulates capaci ty of to mobilize PS to the cell surface .
  • Fig. 8 we analyzed multiple Raji clones that were stably transfected with either GFP-PL scramblase or with GFP vector alone (Fig. 10) .
  • Fig. 10 illustrates that the level of expression of PL scramblase determines plasma membrane sensitivity to intracellular Ca 2+ .
  • GFP fluorescence detected in FL1 channel abscissa
  • numbers of cells that expose PS after 2 minutes incubation with A23187 ordinate
  • these data provide the first indication that the movement of PS and other procoagulant aminophospolipids from plasma membrane inner leaflet to the cell surface can be manipulated by selectively altering the level of expression of a particular cellular protein, either through direct transfection with the PL scramblase cDNA, or potentially, by another intervention affecting cellular expression of functional PL scramblase protein.
  • PL scramblase Although the transcriptional regulation of the PL scramblase gene remains to be determined, it is of interest to note that such cell or tissue-specific differences in PL scramblase expression has the potential to significantly affect the biological properties of the cell.
  • the content of PL scramblase in human platelet is approximately 10-fold greater than that of the erythrocyte, which is consistent with the respective PS-mobilizing potential and different roles of these two cells in contributing procoagulant membrane surface for thrombin generation during blood clotting (Q. Zhou, et . al. , supra , 1997) .
  • lymphoma-derived cell lines express considerably reduced levels of PL scramblase, and also show distinctly attenuated PS exposure in response to elevated [Ca 2+ ] c , when compared to either peripheral blood leukocytes or to EBV-transforms of normal lymphocytes (Fig. 8) .
  • EXAMPLE 4 Induction of Programmed Cell Death is Related to the Cellular Content of PL Scramblase A. Summary
  • Human cell lines expressing various levels of PL scramblase protein were exposed to agents or conditions of cell culture known to promote apoptosis (programmed cell death) . Analysis of these cell lines revealed that the number of cells induced to undergo apoptosis generally correlated with the level of expression of PL scramblase protein, with those cell lines expressing high levels of PL scramblase being most sensitive to induction of apoptosis while cell lines expressing low levels of PL scramblase being most resistant to induction of apoptosis.
  • the human Burkitts B-lymphoma cell line Raji was found to express low levels of PL scramblase and to be markedly resistant to induction of apoptosis.
  • HBSS Hank's Balanced Salt Solution
  • HBSS Hank's Balanced Salt Solution
  • Protein A Sepharose-CL4B Protein A Sepharose-CL4B
  • leupeptin and BSA were from Sigma Chemical Co. (St. Louis, MO).
  • UltraLink Iodoacetyl resin and SuperSignal ULTRA Chemiluminescence Kit were from Pierce Chemical Co. (Rockford, IL) . All other chemicals were of reagent grade .
  • Antibodi es Anti -GFP : murine monoclonal antibody against green fluorescent protein (GFP) was from CLONTECH Laboratories.
  • Anti -FVa murine monoclonal antibody V237 reactive against human or bovine factor Va light chain was the generous gift of Dr. Charles T. Esmon (Oklahoma Medical Research Fndn, Oklahoma City, OK) .
  • Anti -PL Scramblase-E306-W318 Rabbit antibody raised against the carboxyl terminal peptide sequence E306-W318 of human PL scramblase has previously been described (Q. Zhou, e_t al . , supra , 1997) .
  • the IgG fraction was isolated on protein A-Sepharose-CL4B and the peptide-reactive antibody purified by affinity chromatography on peptide [Cys] -ESTGSQEQKSGVW (SEQ ID NO:5) coupled to UltraLink Iodoacetyl resin.
  • the IgG fraction of anti-human FAS ligand was from Clontech (Sunnyvale, CA) .
  • Human cancer cell lines erythroleukemic HEL, promyelocytic leukemia HL-60, chronic myelogenous leukemia K562, lymphoblastic leukemia MOLT-4, acute T-cell leukemia Jurkat, Burkitt's lymphoma Raji, and megakaryocytic DAMI were from American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 containing 10% fetal bovine serum.
  • EBV-transformed cell line W9 established from peripheral B-lymphocytes of a normal donor was maintained as previously described (H. Kojima, et al., 1994).
  • Plasmid Construction Human PL scramblase cDNA insert was released from plasmid pMAL-C2-PL scramblase (Q. Zhou, et . al. , supr , 1997) by double cutting with EcoRI and Sail, respectively, and then ligated into pEGFP-C2 vector using the same restriction site.
  • the pEGFP-C2-PL scramblase plasmid was amplified from single clones in E. coli strain Top 10, and the orientation and reading frame of the insert confirmed by sequencing on an ABI DNA Sequencer Model 373 Stretch (Perkin Elmer- Applied Biosystems, Foster City, CA) using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit.
  • Electroporator Bio-Rad Laboratories, Hercules, CA set at 450 V, 500°F. After 48 hours in culture, 1.5 mg/ml geneticin was added to the medium and continuously maintained for 4 weeks. Stable transformants exhibiting GFP fluorescence were sorted by flow cytometry (FACStar, Becton-Dickinson Immunocytometry Systems, San Jose, CA) using an FL1 sorting gate. The FL1 -positive cells were dilutionally cloned in 96 well culture plates. pEGFP-PL scramblase transformants expressing the 62 kDa GFP-PL scramblase fusion protein were identified by Western blotting with anti-GFP and with anti-PL-306-W318 antibodies. Western blotting of pEGFP-C2 transformants (without insert) confirmed presence of 27 kDa GFP.
  • Clones expressing various amounts of GFP-PL scramblase were each expanded for functional assay, along with comparable GFP-expressing clones serving as controls.
  • Fluorescence Mi croscopy Cell clones transfected with pEGPF-C2-PL scramblase or pEGFP-C2 were deposited on glass microscopy slides using a Cytospin 3 (Shandon, Inc., Pittsburgh, PA). Phase contrast and fluorescence microscopy was performed with a ZEISS AXIOSKOP microscope (Carl Zeiss, Inc., Thornwood, NY) equipped for epifluorescence, and images were recorded with a MC100 camera system. The exposure times for photography of fluorescence was 80-200 seconds under automatic control using Kodak Ektachrome 1600 film.
  • Western Blot Analysis Western Blot Analysis .
  • Western blotting of GFP and PL scramblase antigens was performed using 1.5 x 10 6 cells per lane. After washing in HBSS, supernatants were removed, and the cell pellets extracted with 2% (v/v) NP- 40 in 5 mM EDTA, 50 mM benzamidine, 50 mM N-ethyl maleimide, 1 mM phenylmethylsulfonyl fluoride, 1 M leupeptin in HBSS. After removal of insoluble material (250,000 x g, 30 minutes, 4°C) , the samples were denatured at 100°C in 10% (w/v) SDS sample buffer containing 2% ⁇ -mercaptoethanol .
  • the blocked membrane was incubated with either 10 ⁇ g/ml of rabbit anti-PL scramblase-E306-W318, or 1/10,000 dilution of mouse anti- GFP.
  • the blots were developed with the horseradish peroxidase conjugate of either goat anti-rabbit IgG or goat anti -mouse IgG, respectively, using SuperSignal ULTRA chemiluminescence .
  • Apoptosis was induced either by (1) addition of 60 ⁇ M etoposide to the cell culture (2) by removal of fetal bovine serum from the culture medium (serum starvation) .
  • Cells were maintained in sterile culture flasks in 37°C incubator under each of these conditions for 72 hours, during which cell aliquots were obtained for assay (times indicated in figure legends) .
  • Assays for Apoptosis The induction of apoptosis was detected by (1) egress of PS to the cell surface (2) activation of cellular caspase enzymes (3) cleavage of nuclear DNA. Egress of PS to the cell surface was measured by the binding of Factor Va (light chain) or by the binding of FITC-annexin V, detected by flow cytometry.
  • Cleavage of nuclear DNA was measured either by a decrease in propidium iodide staining of the nucleus, according to published methods (ref) , or by the measured incorporation of FITC-dUTP into 5 ' -ends of chromosomal DNA, catalyzed by the enzyme terminal deoxyribose transferase (TUNEL Assay Kit, Clontech) according to the manufacturer's protocol.
  • fluorescence was detected by flow cytometry (FACScan, Becton Dickinson) , using the FL1 channel to detect FITC or the FL3 channel to detect propidium iodide staining of the nucleus. Forward and side scatter gates were set to exclude cell debris.
  • A23187 (0 or 2 ⁇ M final concentration) was added from 1 mM stock solution in DMSO, and at times indicated in figure legends, the reaction was stopped by addition of 6 mM EGTA.
  • PS exposed on the cell surface at each time point was detected by incubating (10 minutes, room temperature) 50 ⁇ l of the cell suspension with 10 ⁇ g/ml FVa, followed by 10 ⁇ g/ml anti-FVa, to detect the cell- bound FVa light chain.
  • Tri- Color conjugated goat anti-mouse IgG (CALTAG Laboratories, Burlingame, CA)
  • single-cell fluorescence was quantitated by flow cytometry (FL3 channel, FACScan, Becton Dickinson Immunocytometry Systems) .
  • Use of Tri- Color conjugate to detect cell -bound FVa enabled simultaneous measurement of cell-associated GFP fluorescence in cell lines transformed with the pEGFP-C2 expression plasmid (fluorescence of GFP detected in FL1 channel) .
  • Fig. 14 illustrates the role of PL scramblase in PS exposure during apoptosis of B-lymphoblasts .
  • PS exposure (measured with Fva light chain) in human B-lymphoblast cell lines during apoptosis induced by one day incubation in reduced serum (1% fetal bovine serum; solid bars) .
  • Open bars represent identically matched controls maintained continuously in 10% serum.
  • Panel A compares the Raji (low endogenous PL scramblase expression) and W9 (high PL scramblase expression) cell lines.
  • PL scramblase promotes intracellular events that result in apoptotic cell death.
  • the GFP-PL scramblase-transfected Raji cell lines to monitor fragmentation of nuclear DNA in response to known apoptotic stimuli.
  • the transfected cells were induced to apoptosis by either culture under serum starvation conditions or by challenge with etoposide. Nuclear degradation was monitored by TUNEL assay (Clontech) and data analyzed as per cent of all cells induced to DNA fragmentation.
  • Fig. 15 illustrates transfection with cDNA encoding PL scramblase increases apoptotic response of human B- lymphoma line Raji.
  • Raji cell lines were stably transfected with plasmid to express either GFP-PL scramblase fusion protein (GFP-PLS; hatched bars) or pGFP vector as control (GFP; solid bars) and analyzed under conditions of induced apoptosis in cell culture. Also shown are data for non-transfected parental cell line Raji (Raji; open bars) . Each cell line was incubated overnight under either serum-starved conditions (0% serum) or in presence of 60 ⁇ M etoposide (Etoposide) .
  • Control refers to identical cells maintained under normal culture conditions of 10% fetal bovine serum. Apoptosis was assessed by degradation of nuclear DNA, as measured by TUNEL assay (Clontech) and is expressed as percent of total cells induced to undergo nuclear fragmentation. Data of a single experiment, representative of at least five similar experiments so performed.

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Abstract

La présente invention concerne une méthode d'accroissement de la viabilité des cellules ou des tissus mammifères, consistant à inhiber l'expression de la PL-scramblase native dans les cellules ou les tissus. Dans un autre mode de réalisation, l'invention concerne une méthode de réduction du potentiel métastatique et invasif ainsi que du potentiel de viabilité des cellules cancéreuses, des tissus cancéreux, ou des cellules infectées par virus, par l'augmentation de l'expression ou l'activité de la protéine PL-scramblase dans les cellules ou les tissus en question.
PCT/US1999/001087 1998-01-20 1999-01-19 Methodes et compositions permettant de modifier la sensibilite des tissus face aux lesions immunitaires, a la mort cellulaire programmee et a la clairance par le systeme reticulo-endothelial WO1999036536A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081495A1 (fr) * 2001-04-05 2002-10-17 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la scramblase i phospholipidique
WO2002074346A3 (fr) * 2001-03-16 2004-02-12 Nst Neurosurvival Technologies Procede de ciblage de composes chimiques sur des cellules et compositions pharmaceutiques utilisees dans ce procede
WO2003048324A3 (fr) * 2001-12-04 2004-07-01 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la phospholipide scramblase 3

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US6172210B1 (en) * 1996-04-02 2001-01-09 Blood Center Research Foundation DNA encoding phospholipid scramblase
US6204035B1 (en) * 1996-04-02 2001-03-20 The Blood Center Research Foundation Methods and compositions to alter the cell surface expression of phosphatidylserine and other clot-promoting plasma membrane phospholipids

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074346A3 (fr) * 2001-03-16 2004-02-12 Nst Neurosurvival Technologies Procede de ciblage de composes chimiques sur des cellules et compositions pharmaceutiques utilisees dans ce procede
WO2002081495A1 (fr) * 2001-04-05 2002-10-17 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de la scramblase i phospholipidique
WO2003048324A3 (fr) * 2001-12-04 2004-07-01 Isis Pharmaceuticals Inc Modulation antisens de l'expression de la phospholipide scramblase 3

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