"Science, Faculty of"@en . "Microbiology and Immunology, Department of"@en . "DSpace"@en . "UBCV"@en . "Leitch, Heather Alice"@en . "2008-12-17T23:45:34Z"@en . "1992"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "P30-35 CAMAL is a component of material immunoaffinity enriched from lysates\r\nof myeloid leukemia leucocytes using the monoclonal antibody CAMAL- 1. Reactivity with\r\nCAMAL- 1 is diagnostic of myeloid leukemias. CAMAL- 1 enriched material inhibited\r\nmyelopoiesis by normal progenitor cells in vitro. This study demonstrates that inhibition\r\nis associated with P30-35 CAMAL. Neutrophilic granulocyte colonies were preferentially\r\ninhibited by P30-35 CAMAL while higher concentrations affected all colony types. Nonadherent\r\nprogenitor cell numbers were reduced in P30-35 CAMAL-treated long term\r\ncultures of normal marrow cells whereas adherent cell numbers were increased, suggesting\r\na block in differentiation. In addition, colony formation by murine cells was inhibited by\r\nP30-35 CAMAL. As in cultures of human cells, neutrophilic granulocyte colonies were\r\npreferentially inhibited, indicating that downregulation of normal myelopoiesis by P30-35\r\nCAMAL crosses species barriers, and might be an important regulatory mechanism.\r\nUsing highly enriched P30-35 CAMAL, a stimulatory effect on CML colony\r\nformation was defined. Stimulation of colony formation occurred at low and high\r\nconcentrations of P30-35 CAMAL, but was reduced at intermediate concentrations. At low\r\nconcentrations of P30-35 CAMAL primitive colonies were increased, whereas high\r\nconcentrations affected all colony types. Colony formation by several myeloid leukemiaderived\r\ncell lines was increased by P30-35 CAMAL. P30-35 CAMAL prepared using protease inhibitors lacked effects on colony\r\nformation. Alterations in normal and CML colony formation were similar whether cells\r\nwere preincubated or cocultured with P30-35 CAMAL. Activity was retained in the\r\nsupematant of treated cells, indicating that effects were immediate and irreversible.\r\nAlterations in normal and CML colony formation were fully blocked using either phenyl\r\nmethyl sulfonyl fluoride or a chloro-methyl ketone-linked peptide, both inhibitors of serine\r\nprotease activity, suggesting that the alterations in myelopoiesis require serine protease\r\nactivity. Experiments using characterized neutrophil proteins suggested protease activity in\r\nP30-35 CAMAL preparations which might be unique.\r\nReactivity of the monoclonal antibodies a-P30/35, raised against P30-35 CAMAL,\r\nand CAMAL- 1 was compared. Both antibodies reacted more extensively with CML than\r\nwith normal leucocytes, and cc-P30/35 reacted with several myeloid leukemia-derived cell\r\nlines, suggesting that the antigen recognized by CAMAL- 1 and the component which alters\r\nmyelopoiesis might form an association."@en . "https://circle.library.ubc.ca/rest/handle/2429/3072?expand=metadata"@en . "7221514 bytes"@en . "application/pdf"@en . "CHARACTERIZATION OF THE EFFECTS OF P30-35 CAMAL ONNORMAL AND LEUKEMIC MYELOPOIESISbyHeather Alice LeitchB. Sc. (Hon) 1980A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDoctor of PhilosophyinTHE FACULTY OF GRADUATE STUDIESMicrobiologyWe accept this thesis as conformingto the rquired stanthrdTHE UNIVERSITY OF BRITISH COLUMBIASeptember, 19920 Heather Alice Leitch, 1992Signature(s) removed to protect privacyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________MicrobiologyDepartment ot________________The University of British ColumbiaVancouver, CanadaDate October 15, 1992DE-6 (2/88)Signature(s) removed to protect privacyABSTRACTP30-35 CAMAL is a component of material immunoaffinity enriched from lysatesof myeloid leukemia leucocytes using the monoclonal antibody CAMAL- 1. Reactivity withCAMAL- 1 is diagnostic of myeloid leukemias. CAMAL- 1 enriched material inhibitedmyelopoiesis by normal progenitor cells in vitro. This study demonstrates that inhibitionis associated with P30-35 CAMAL. Neutrophilic granulocyte colonies were preferentiallyinhibited by P30-35 CAMAL while higher concentrations affected all colony types. Non-adherent progenitor cell numbers were reduced in P30-35 CAMAL-treated long termcultures of normal marrow cells whereas adherent cell numbers were increased, suggestinga block in differentiation. In addition, colony formation by murine cells was inhibited byP30-35 CAMAL. As in cultures of human cells, neutrophilic granulocyte colonies werepreferentially inhibited, indicating that downregulation of normal myelopoiesis by P30-35CAMAL crosses species barriers, and might be an important regulatory mechanism.Using highly enriched P30-35 CAMAL, a stimulatory effect on CML colonyformation was defined. Stimulation of colony formation occurred at low and highconcentrations of P30-35 CAMAL, but was reduced at intermediate concentrations. At lowconcentrations of P30-35 CAMAL primitive colonies were increased, whereas highconcentrations affected all colony types. Colony formation by several myeloid leukemia-derived cell lines was increased by P30-35 CAMAL.UP30-35 CAMAL prepared using protease inhibitors lacked effects on colonyformation. Alterations in normal and CML colony formation were similar whether cellswere preincubated or cocultured with P30-35 CAMAL. Activity was retained in thesupematant of treated cells, indicating that effects were immediate and irreversible.Alterations in normal and CML colony formation were fully blocked using either phenylmethyl sulfonyl fluoride or a chloro-methyl ketone-linked peptide, both inhibitors of serineprotease activity, suggesting that the alterations in myelopoiesis require serine proteaseactivity. Experiments using characterized neutrophil proteins suggested protease activity inP30-35 CAMAL preparations which might be unique.Reactivity of the monoclonal antibodies a-P30/35, raised against P30-35 CAMAL,and CAMAL- 1 was compared. Both antibodies reacted more extensively with CML thanwith normal leucocytes, and cc-P30!35 reacted with several myeloid leukemia-derived celllines, suggesting that the antigen recognized by CAMAL- 1 and the component which altersmyelopoiesis might form an association.InTABLE OF CONTENTSPAGEAbstract iiList of Tables xiList of Figures xiiiList of Abbreviations xviiiAcknowledgements xxviiiChapter 1 - Introduction 11.1: Hematopoiesis 11.1.1: Characteristics of stem and progenitor cells, assay systems,and terminology 21.1.2: Colony-stimulating factors, interleukins, cytokines 61.1.3: Cytokines that are noteworthy in hematopoiesis 71.1.4: Signal transduction 161.2: Oncogenesis and leukemogenesis 231.2.1: Leukemia; classification, etiology 261.2.2: The t(9;22) translocation in CML; the Philadelphia chromosomeand P210 bcr-abl 321.2.2.a: Signal transduction by c-abl, bcr, and P210 bcr-abl 34l.2.2.b: Transforming capabilities ofP210 bcr-abl 361.2.2.c: P210 bcr-abl in the suppression of apoptosis 38l.2.2.d: Other characteristics of bcr-abl 40iv1.2.3: Adhesion defects in CML 421.2.4: Alterations of cytokine expression in leukemogenesis 431.2.5: Other abnormalities in CML 451.2.6: Negative regulators of hematopoiesis 461.3: Therapy 541.3.1: Chemotherapy 541.3.2: Allogeneic bone marrow transplantation 561.3.3: Autologous bone marrow transplantation, purging 611.3.4: Cytokines in leukemia therapy 641.3.5: Alterations of adhesive interactions - interferon-a 681.3.6: Lymphokine activated killer cells 681.3.7: Induction of differentiation; retinoic acid in acute promyelocyticleukemia 701.3.8: Immunotoxins 711.3.9: Techniques for targetting bcr-abl 721.3.10: Molecular biological techniques 721.3.11: Positive selection of stem cells 741.4: Biological activities of proteases 771.4.1: Proteases in the function of mature hematopoietic cells 771.4.2: Surprising functions of proteases 781.4.3: Proteases in development 791.4.4: Proteases in cell signalling 791.4.5: Proteases in malignancies, including leukemia 81v1.5: Background on CAMAL .83Chapter2- Chaxacterization ofthe inhibitory effects ofP3O-35 CAMAL onnonnalmyelopoiesis 902.1: Introduction 902.2: Materials and Methods 912.2.1: Purification of P30-35 CAMAL 912.2.1.a: Antibodies 922.2. 1.b: Class identification 942.2.1 .c: Preparation and screening of cz-P30/35 952.2.1.d: Preparation of ascites 972.2.1.e: Purification of immunoglobulins from ascites 982.2. 1.f: Immunoadsorbent column preparation 992.2. 1.g: Protein analysis 1002.2.2: Preparations of P30-35 CAMAL 1012.2.2.a: Sources of P30-35 CAMAL 1012.2.2.b: Cell lysis 1022.2.2.c: Protein purification 1022.2.2.d: Controlprotein 1032.2.3: Hematopoietic progenitor cell assays 1042.2.3.a: Cells 1042.2.3.b: Preparation of conditioned medium and plasma forcolony assays 1052.2.3.c: Colony assays 106vi2.2.3.d: Long-term marrow cultures 1072.3: Results 1092.3.1: Identification of the P30-35 component in CAMAL-1 enrichedmaterial inhibitory to normal colony formation 1092.3.2: Lack of inhibitory effect on CML colony fonnation byP30-35CAMAL 1142.3.3: Preferential targetting of normal CFU-G by P30-35 CAMAL 1172.3.4: Experiments using a-P30/35 enriched P30-35 CAMAL 1212.3.5: Lack of inhibitory activity on normal colony formation by otherproteins in CAMAL-l eluted material 1242.3.6: Evidence that the effects of P30-35 CAMAL on colony formationare not mediated by elastase 1242.3.7: Experiments using fresh progenitor cells 1272.3.8: Apparent reduction in release of cells from the adherent layer inP30-35 CAMAL-treated long-term marrow cultures 1302.4: Discussion 135Chapter 3- The inhibitory effect ofP3O-35 CAMAL on normal myelopoiesiscrosses species barriers; murine assays with P30-35 CAMAL 1453.1: Introduction 1453.2: Materials and Methods 1463.2.1: Cells 1463.2.2: Colony assays 1463.2.3: Spleen colony assay 148vii3.3: Results .1493.3.1: Inhibition of in vitro colony formation by P30-35 CAMAL 1493.3.2: Inhibition of spleen colony formation by P30-35 CAMAL 1543.4: Discussion 157Chapter 4- Characterization ofthe stimulatory effect ofP3O-35 CAMAL oncolony formation by CML progenftor cells andmyeioid leukemia-derivedcell lines 1614.1: Introduction 1614.2: Materials and Methods 1624.2.1: Colony assays 1624.2.2: Cell lines 1634.3: Results 1644.3.1: Defmition of the stimulatory effect of P30-35 CAMAL on colonyformation by CML progenitor cells 1644.3.2: Identification of colony types stimulated by P30-35 CAMAL 1734.3.3: Stimulatory effect of P30-35 CAMAL on colony formation bymyeloid leukemia-derived cell lines 1754.4: Discussion 178Chapter .5- The modulating effects ofP30-35 CAJVIAL on myelopoiesis requfreserine protease activity, which can be blocked by the peptideala-pro-phe-CMK 1875.1: Introduction 187Viii5.2: Materials and Methods .1895.2.1: Experiments in which colony forming cells were preincubatedwithP30-35CAMAL 1895.2.2: Retention of activity on colony formation in the supematant ofP30-35 CAMAL treated cells 1895.2.3: Colony assays with PMSF-treated P30-35 CAMAL 1905.2.4: Enzyme assays with chromogenic peptide substrates and CMKlinked peptides 1915.2.5: Colony assays with CMK peptide treated P30-35 CAMAL 1925.3: Results 1935.3.1: Alterations of colony formation under conditions ofpreincubation with P30-35 CAMAL were similar to effectsin coculture experiments 1935.3.2: Retention of activity on colony formation in the supematant ofP30-35 CAMAL-treated cells 1965.3.3: Colony assays with PMSF-treated P30-35 CAMAL 1965.3.4: Enzyme assays with CMK-peptide treated P30-35 CAMAL 1965.3.5: Colony assays with CMK-peptide treated P30-35 CAMAL 2005.4: Discussion 203Chapter 6- Discussion 2086.1: Overview of results 2086.2: Speculation on possible mechanisms of action of P30-35 CAMAL 2266.3: Future directions 237ix6.3.1: Basic science 2376.3.2: Development of a clinically useful agent 2417: Summary and Conclusions 2448: References 2499: Appendix 1- Differential immunoperoxidase staining ofCML andnonnal cytospin preparations using a-P30/35 2929.1: Introduction 2929.2: Materials andMethods 2939.2.1: Antibodies 2939.2.2: Cell lines 2949.2.3: Preparation of cytospins 2949.2.4: Serum 2959.2.5: Cellstaining 2959.3: Results 2969.4: Discussion 30710: Appendix2- Further evidence that P30-35 CAMAL activity is mediatedby a protein in preparations ofP3O-35 CAMAL which is distinct fromcharacterizedneutrophilproteins 31111. List of publications 3 19xLIST OF TABLESPAGETable I. Summary of the more well known effects and moreinteresting actions of the interleukins, colony-stimulatingfactors, and other cytokines known to influencehematopoiesis. 8Table II. Summary of P30-35 CAMAL inhibitory activity in colonyassays using progenitor cells from five different normalhealthy donors and six preparations of P30-35 CAMALderived from different CML or AML patient specimens. 113Table III. Enrichment of P30-3 5 CAMAL and coenrichment ofinhibitory activity on colony formation by progenitor cellsfrom normal healthy donors. 114Table IV. Summary of inhibitory activity mediated by P30-35 CAMALon colony formation in assays using fresh progenitor cellsfrom six different normal healthy donors. 128Table V. Inhibition by P30-3 5 CAMAL of cell numbers in the non-adherent supernatant compartment of a long-term marrowculture using cells from a normal healthy donor. 131Table VI. Inhibition by P30-35 CAMAL of cell numbers in the non-adherent supernatant compartment of a long-term marrowculture and of the formation of colonies cultured from non-adherent cells, using cells from a normal healthy donor. 132Tabl\u00C3\u00A7 VII. Summary of stimulatory activity mediated by P30-35CAMAL on colony formation using progenitor cells fromeight different patients with CML and three differentpreparations of P30-35 CAMAL. 168Table VIII. Summary of stimulatory activity mediated by P30-3 5CAMAL activity on colony formation using cell linesderived from myeloid leukemias. 176xiTable IX. Immunoperoxidase staining of peripheral blood leucocytesfrom CML patients and normal healthy donors.298Table X. Immunoperoxidase staining of cell lines derived from humanmyeloid leukemias. 302xliLIST OF FIGURESPAGEFigure 1. Identification of the component in CAMAL- 1 eluted materialinhibitory to colony formation by progenitor cells fromnormal healthy donors. a. Silver-stained SDSpolyacrylamide gel of fractions of CAMAL- 1 enrichedmaterial. b. Effect of these fractions on colony formation atday 14. 110Figure 2. Titration of inhibitory activity on colony formation byprogenitor cells from normal healthy donors in two fractionsenriched for P30-35 CAMAL using FPLC gel filtration. 112Figure 3. Activity of P30-35 CAMAL purified from preparative SDSpolyacrylamide gels on colony formation. a. Cultures ofcells from a normal healthy donor. b. A second preparationof P30-3 5 CAMAL, using cells from a normal healthydonor. c. Cultures of cells from a patient with CML. 115Figure 4. Activity of P30-35 CAMAL purified using FPLC gelfiltration on colony formation. a. Cultures of cells from anormal healthy donor. b. Cultures of cells from a patientwith CML. 116Figure 5. Inhibitory activity ofP30-35 CAMAL on the size of coloniescultured from normal cells in two experiments. i. Coloniesfrom control cultures. ii. Colonies from P30-35 CAMALtreated cultures. 118Figure 6. Inhibition by P30-35 CAMAL of CFU-G cultured fromnormal progenitor cells. a. Using 100 ng/ml P30-35CAMAL. b. A second experiment using 50 ngfml P30-35CAMAL 120Figure 7. Silver-stained SDS polyacrylamide gel of P30-35 CAMALprepared using sequential CAMAL-1 and ci-P30/35immunoaffinity chromatography. 122xliiFigure 8. Comparison of activity on colony formation by CAMAL- 1eluted material and material prepared using sequentialCAMAL- 1 and a-P30/35 immunoaffinity chromatography.a. Cultures of cells from a normal healthy donor. b. 123Cultures of cells from a patient with CML.Figure 9. Comparison of P30-35 CAMAL and control protein activityon colony formation by progenitor cells from a normalhealthy donor. a. Control protein from a preparation ofP30-35 CAMAL. b. HSA. 125Figure 10. Inhibition of colony formation by progenitor cells from botha normal healthy donor and from patients with CMLmediated by elastase. 126Figure 11. Two experiments in which P30-35 CAMAL inhibitoryactivity on colony formation by progenitor cells from normalhealthy donors was titrated using fresh progenitor cells. 129Figure 12. Increase in cobblestone areas in the adherent layers of P30-35 CAMAL-treated long-term marrow cultures using bonemarrow cells from normal healthy donors. a. 2 ml cultures.b. 5 ml cultures. i. Control cultures. ii. P30-35 CAMALtreated cultures. 133Figure 13. Comparison of the inhibitory activity of P30-35 CAMAL oncolony formation by normal human and murine progenitorcells. 150Figure 14. Comparison of the inhibitory activity of P30-35 CAMAL oncolony formation by murine progenitor cells underconditions of coculture vs. preincubation. 151Figure 15. Inhibitory activity of P30-35 CAMAL on the size of coloniescultured from murine progenitor cells. 152Figure 16. Inhibitory activity of P30-35 CAMAL on murine CFU-G. 155Figure 17. Inhibitory activity of P30-35 CAMAL on spleen colonyformation. a. Using 100 ngfml P30-35 CAMAL. b.Titration of inhibitory activity. 156Figure 18. Enhancing activity of P30-35 CAMAL on colony formationby progenitor cells from patients with CML. a.Cryopreserved cells cocultured with P30-35 CAMAL. b.Fresh cells preincubated with P30-3 5 CAMAL. 165xivFigure 19. Inhibitory activity of P30-35 CAMAL on colony formationby progenitor cells from a normal healthy donor over thesame concentration range of P30-35 CAMAL at whichenhancement of CML colony formation was observed. 166Figure 20. Lack of enhancing activity by control protein frompreparations of P30-35 CAMAL on colony formation byprogenitor cells from a patient with CML. 169Figure 21. Enhancing activity of P30-35 CAMAL on the size ofcolonies cultured from progenitor cells from patients withCML in two experiments. i. Colonies from controlcultures. i. Colonies from cultures treated with a lowconcentration of P30-35 CAMAL. iii. Colonies fromcultures treated with a high concentration of P30-35CAMAL. 170Figure 22. Identification of colony types in cultures of cells frompatients with CML enhanced by treatment with P30-35CAMAL in two experiments. 174Figure 23. Activity of P30-35 CAMAL on colony formation by two celllines derived from myeloid leukemias. a. Cultures of EM3cells. b. Cultures of HEL cells. 177Figure 24. Effects of P30-35 CAMAL on colony formation underconditions of preincubation with P30-35 CAMAL. a.Cultures of cells from a normal healthy donor. b. Culturesof murine CFU-G. c. Cultures of cells from a patient withCML. d. Cultures of EM3 cells. 195Figure 25. Adsorption studies with P30-35 CAMAL; retention ofactivity on colony formation in the supernatant of P30-35CAMAL-treated cells, a. Cultures of cells from a normalhealthy donor. b. Cultures of murine CFU-G. c. Culturesof cells from a patient with CML. d. Cultures of EM3 cells. 197Figure 26. Blocking effect of PMSF on P30-35 CAMAL-mediatedalterations of colony formation. a. Cultures of cells from anormal healthy donor. b. Cultures of cells from a patientwith CML. 198xvFigure 27. Enzyme assays with P30-35 CAMAL and elastase usingCMK-linked peptides as putative blockers of enzymeactivity, a. Blocking effect of P30-35 CAMAL enzymeactivity but not of elastase enzyme activity using ala-prophe-CMK. b. Blocking effect of elastase and CAMALenzyme activities at different levels using ala-ala-pro-pheCMK. 199Figure 28. Blocking effect of ala-pro-phe-CMK on P30-35 CAMALmediated alterations of colony formation. a. Cultures ofcells from a normal healthy donor. b. cultures of cells froma patient with CML. 201Figure 29. Lack of blocking effect of three other CMK-linked peptideson P30-35 CAMAL-mediated inhibition of colony formationby normal progenitor cells, a. Using ala-ala-pro-val-CMK.b. Using phe-pro-arg-CMK. c. Using gly-gly-phe-CMK. 202Figure 30. Speculative scheme of how components of signaltransduction pathways in normal and CML cells might beaffected by P30-35 CAMAL, resulting in the observedalterations in colony formation. 233Figure 31. Preferential staining by immunoperoxidase of mononuclearcells obtained from patients with CML as compared tonormal healthy donors using a-P30/35. a. CML PBL witha-P30/35. b. CML PBL with CAMAL-1. c. CML PBLwith x-BLV. d. Normal PBL with c&P30/35. e. CML299PBL from a second donor with cc-P30/35.Figure 32. Immunoperoxidase staining of myeloid leukemia-derivedcell lines, a. EM3 with x-P30/35. b. EM3 with CAMAL1. c. EM3 with c&BLV. d. EM2 with a-P30/35. e. HELwith x-P30/35. f. HL6O with c-P30/35. g. HL6O withCAMAL-1. 303Figure 33. Lack of inhibitory activity of myeloblastin, azurocidin, andcathepsin G on colony formation by progenitor cells from anormal healthy donor. a. Conditions of preincubation. b.Conditions of coculture using cathepsin G. 314xviFigure 34. Enzyme assays with P30-35 CAMAL and cathepsin G usingCMK-linked peptides as putative blockers of enzymeactivity, a. Blocking effect of P30-35 CAMAL enzymeactivity but not of cathepsin G enzyme activity using ala-alapro-val-CMK. b. Blocking effect of P30-35 CAMAL andcathepsin G enzyme activities at different levels using alapro-phe-CMK. 316xviiLIST OF ABBREVIATIONSA AbsorbenceAAPF Peptide with the sequence alanine-alanine-proline-phenylalanine (ala-alapro- phe)AAPV Peptide with the sequence alanine-alanine-proline-valine (ala-ala-pro-val)Ab Antibodyabl Protooncogene (c-abl); serine/threonine kinaseABMT Autologous bone marrow transplantationAcSDKP Tetrapeptide inhibitory to hematopoiesisALL Acute lyinphocytic leukemiaAML Acute myeloid leukemiaANLL Acute non-lymphocytic leukemiaAPF Peptide with the sequence alanine-proline-phenylalanine (ala-pro-phe)APL Acute promyelocytic leukemiaAm-C Cytosine ambinosideb Basicb2a2 Fusion gene joining bcr exon 2 to abl exon 2b3a2 Fusion gene joining bcr exon 3 to abl exon 2bas Basophilbcl B cell leukemiabcr Breakpoint cluster region; serine/threonine kinase, GAP for mcbcr-abl Breakpoint cluster regiow\u00E2\u0080\u0099c-abl fusion gene, transcript or proteinBFU-E Burst-forming unit-erythroidbl-CFC Blast colony-forming cellBLV Bovine leukosis virusBM Bone marrowBMT Bone marrow transplantationBP Binding proteinBPD Benzoporphyrin derivativeBSA Bovine serum albuminCa2 Calcium ionCALLA Common acute lymphoblastic leukemia antigenCAM Cellular adhesion moleculeCAMAL Common antigen in myelogenous acute leukemiacAMP Cyclic adenosine monophosphateCD Cluster designationcdc Cell division cycleCFC Colony-forming cellCMK Chloro-methyl ketoneCML Chronic myelogenous leukemiaCMV CytomegalovirusCLL Chronic lymphocytic leukemiaOC Degrees centigradeCFU Colony-forming unitCFU-E Colony-forming unit-erythroidCFU-eo Colony-forming unit-eosinophilCFU-G Colony-forming unit-granulocytexixCFU-GM Colony-forming unit-granulocyte, macrophageCFU-GEMM Colony-forming unit-granulocyte, erythrocyte, macrophage, megakaryocyteCFU-S Colony-forming unit-spleen6\u00C2\u00B0Co Radioactive cobalt-60CO2 Carbon dioxideCSF Colony-stimulating factorCSF- 1 Colony-stimulating factor 1 (M-CSF)d Dayd Distilleddd Deionized, distilledDAB Diaminobenzidine (3 ,4,3\u00E2\u0080\u0099,4\u00E2\u0080\u0099-tetra-aminobiphenyl hydrochoride)DAG Diacyl glycerolDCC Deleted in colorectal carcinomaDIC Differential interference contrast (microscopy)DME Dulbecco\u00E2\u0080\u0099s modified Eagle\u00E2\u0080\u0099s (medium)DMSO DimethylsulfoxideDNA Deoxyribonucleic acidDNase DeoxyribonucleaseDTf DithiothreitolEBV Epstein-Barr virusECM Extracellular matrixEDTA ethylenediamine tetraacetic acidEGF Epidermal growth factorxxELISA Enzyme-linked immunosorbent assayepo ErythropoietinFAB French-American-British classification of leukemiasFACS Flourescence-activated cell sortingFCS Fetal calf serumFe2 Iron ion, ferrous formFe3 Iron ion, ferric formFGF Fibroblast growth factorfms Receptor for M-CSF or CSF-l (c-fms); tyrosinekinasefos Proto-oncogene (c-fos); nuclear transcription factorFPLC Fast protein liquid chromatographyFPR Peptide with the sequence phenylalanine-proline-arginine (phe-pro-arg)FSH Follicle stimulating hormonefyn src related kinaseg Gravityg GramG G proteinG0 Quiescent phase of cell growth cycleG1 First growth phase of cell cycle, prior to DNA synthesisG2 Second growth phase of cell cycle, after DNA synthesisG6PD Glucose-6-phosphate dehydrogenaseGAP GTPase activating proteinG-CSF Granulocyte colony-stimulating factorodGGF Peptide with the sequence glycine-glycine-phenylalanine (gly-gly-phe)G1 Inhibitory a subunit of G proteinGIA Granulocytic inhibitory activityGM-CSF Granulocyte-macrophage colony-stimulating factorgp GlycoproteinG Stimulatory a subunit of G proteinGTP Guanosine triphosphateGVHD Graft versus host diseaseGVL Graft versus leukemiah HoursITA High Affmity4-HC 4-hydroperoxycyclophosphamideHCI Hydrochloric acidITEL Human erythrocytic leukemiaHEPES N-[2-hydroxyethyl] piperazine-N\u00E2\u0080\u0099- [2-ethanesulfonic acid]H-ferritin Heavy chain subunit of acidic isoferritinsHILDA Human interleukin for DA cells (LIF)lILA Human leucocyte associated antigenHLH Helix-loop-helix (nuclear transcription factors)H20 Hydrogen peroxideHOX HomeoboxHP5b Peptide inhibitory to hematopoiesis (pEEDCK, SP1)HPLC High performance liquid chromatographyHPP-CFC High proliferative potential colony-forming cellxxiiHSA Human serum albuminHTLV Human T-lymphotrophic virusI InhibitoryICAM Inter-cellular adhesion moleculeIFN InterferonIg ImmunoglobulinIL Interleukin1P3 inositol triphosphatejun Proto-oncogene (c-jun); nuclear transcription factorkDa Kilodaltonkit Receptor for Steel factor (c-kit)1 LitreIA Low affinityLAI Leukemia associated inhibitorLAK Lymphokine-activated killer cellIck src related kinaseLCM Leucocyte-conditioned mediumHA Leukemia inhibitory activityLW Leukemia inhibitory factor (HILDA)un Cell lineage-specific cell surface antigensIN2 Liquid nitrogenLPS LipopolysaccharideLTCIC Long-term culture initiating cellXXIIILTMC Long-term marrow cultureM FAB classification for AML (Ml through M7)M MitosisM MolarmA MilliampsmAb Monoclonal antibodyM-bcr Major breakpoint cluster region (in P210 bcr-abl of CML)m-bcr Minor breakpoint cluster region (in P190 bcr-abl of ALL)MC-540 Merocyanine-540M-CSF Macrophage colony-stimulating factormdr Multidrag resistanceMeg Megakaryocytemg MilligramMGF Mast cell growth factor (Steel factor, stem cell factor)MHC Major histocompatibility complexmm MinutesMIP Macrophage inflammatory proteinmM Milimolarml MillilitremRNA Messenger ribonucleic acidmyb Proto-oncogene (c-myb); nuclear transcription factormyc Proto-oncogene (c-myc); nuclear transcription factorN NormalNA NitroanilidexxivNa2CO3 Sodium carbonateNaDOC Sodium deoxycholateNBME Normal bone marrow extractNEP Neutrophul endopeptidaseNF Neurofibromatosisng NanogramNGF Nerve growth factorNH4 AmmoniumN terminus Amino terminusNH2 terminal Amino terminalNH4C1 Ammonium chlorideNK Natural killer (cell)OFP Oncofetal proteinOSM Oncostatin Mp Chromosomal short armp ProteinP ProteinPAGE Polyacrylamide gel electropheresisPAl Plasminogen activator inhibitorPBL Peripheral blood leucocytesPBS Phosphate buffered salinePCR Polymerase chain reactionPDGF Platelet-derived growth factorpEEDCK Pentapeptide inhibitory to hematopoiesis (HP5b, SP1)xxvpg PicogramPGE Prostaglandin EPh Philadelphia chromosomePHA Phytohemagglutininphe phenylalaninePT Phosphatidyl inositolPIP2 Phosphatidylinositol phosphatePKA Protein kinase APKC Protein kinase CPMSF Phenyl methyl sulfonyl fluoridePWM Pokeweed mitogenq Chromosomal long armQLT Quadra Logic Technologiesr Receptorr Recombinantrac Protein with ras homologyras Proto-oncogene; tyrosine kinaseRb Retinoblastoma, tumour suppressor geneRGD Peptide sequence arginine-glycine-aspartaterpm Revolutions per minuteRPMI Roswell Park Memorial Institute (medium)s StimulatoryS DNA synthesis phase of cell cycleSCA Stem cell antigenxxviSDS Sodium dodecyl sulphateSE Standard error of the meansec SecondsSH src homology region (SH2 or SH3)SK&F Smith Kline and FrenchSLF Steel factor (Stem cell factoiy\u00E2\u0080\u0099SCF, Mast cell growth factor, MGF, c-kitligand)SP 1 Peptide inhibitory to hematopoiesis (pEEDCK, HP5b)src Protooncogenet Chromosomal translocationTGF Transforming growth factorTNF Tumour necrosis factorU Unitj.ig MicrogramMicrolitreMicrometerVLA Very light antigenxxviiACKNOWLEDGEMENTSMany people, too numerous to mention individually, have supported andencouraged me during the course of the research described in this thesis. I would like tomake special mention of the following people, who have made a particular impact on thesestudies. First and foremost, Dr. Julia Levy, without whose support, guidance, andunfailingly astute input none of these studies would have been possible. Dr. PatriciaLogan, whose careful and consistent work set the stage for this research, who taught memany of the techniques involved, and who introduced me to many of the issues ofhematopoiesis. All the volunteer donors of peripheral blood for progenitor cells,conditioned medium, plasma, and serum, and those who assisted in obtaining clinicalspecimens, Dr. Noel Buskard, and Dr. Armand Keating in particular. The members of mysupervisory committee, Drs. John Schrader, Gerry Weeks, and Tony Warren, for manyhelpful and constructive suggestions, which they will recognize in the form of experimentsdescribed herein. Mr. Shawn Daneshvar, for technical support. Special thanks go to Dr.Catriona Jamieson and to Stephen Yip, for many productive discussions about theory andtechnique, for technical support, and for helping to make my time in the lab so highlyenjoyable, and to Dr. Armand Keating, whose support and encouragement are greatlyappreciated. Finally, to all the people of the Levy Lab and the Department ofMicrobiology, for helping to make my graduate studies such a positive experience.xxviiiCHAPTER 1INTRODUCTION1.1: HematopoiesisCurrent understanding of hematopoiesis is summarized in this section, with anemphasis on myelopoiesis. Included is a summary of current knowledge about thephysiology of primitive stem cells and more committed progenitor cells, cytokines involvedin hematopoiesis, and the role of the extracellular matrix in supporting the survival andregulating the proliferation and differentiation of hematopoietic cells. Culture systems forsupporting and evaluating the survival, growth, and differentiation of hematopoieticprogenitor and stem cells in vitro are described. Current knowledge about mechanisms ofsignal transduction are also summarized.The process of hematopoiesis involves an exquisitely regulated balance betweenself-renewal and terminal differentiation. Mature cells, both erythrocytes and leucocytes,are constantly becoming senescent and must continually be removed and replaced.Neutrophilic granulocytes, for example, have a half-life in the circulation of a mere severalhours (1). The removal and replacement of blood cells takes place on a massive scale; 3.7X 1011 are produced hourly (2). This is achieved through the maturation, ordifferentiation, of primitive pluripotent stem cells, which have high potential for self1renewal, through a series of cell divisions. As the cells divide, they become progressivelymore differentiated, and progressively more restricted in their capacity for self-renewal.Regulation of the process of differentiation and maintenance of the stem cell pooi isachieved through a complex network of interactions between the cells, their solubleproducts, and their immediate environment. The result is the transmission of both positiveand negative regulatory signals to the cell nucleus via intercellular and intracellularpathways, the details of which are currently under intense investigation. Whether a cellwill be induced to remain quiescent, or to proliferate and become terminally differentiated,its lineage of differentiation, and its state of activation are determined by the balance ofsignals acting on it.1.1.1: Chamcteristics ofstem andprogenitor cells, assay systems, and terminology:Classical assays demonstrating colony forming cells or progenitor cells wereinstigated by Till & McCulloch (3). In these studies, lethally irradiated mice were rescuedwith bone marrow from syngeneic donors, resulting in hematopoietic reconstitution, and inthe formation of colonies of hematopoletic cells in the spleen. Examination of thesecolonies revealed that some were composed of one cell type, whereas others werecomposed of several; others still contained cell types of all the hematopoietic lineages.Since the mice were reconstituted with a limited number of cells, it was assumed that eachcolony arose from a single progenitor cell. This result was taken as evidence of theexistence of a primitive pluripotent stem cell, capable of giving rise to all differentiatedhematopoietic cell types, in addition to the existence of progenitor cells already committed2to one lineage or another. These studies have since been repeated using retrovirally markedstem cells, confinning that the pluripotent stem cell does indeed exist (4).Assay systems developed since provide convenient methods for studying stem andprogenitor cells. Colonies of murine and human hematopoletic cells can be grown in vitroin agar or methylcellulose (4). In this way, factois which influence colony formation havebeen assessed, and characteristics of the progenitor cells themselves have been inferredfrom the characteristics of their progeny. A more physiological assay of hematopoiesis isthe long-term marrow culture, or Dexter culture (5). Incubation of marrow cells under theappropriate conditions results in the formation of an adherent layer of stromal cells,consisting of fibroblasts, adipocytes, endothelial cells, and macrophages. These cells arein intimate contact with the primitive hematopoietic cells, and provide a microenvironment,thought to be similar to the microenvironment in the bone marrow, which supports thesurvival and maturation of hematopoietic cells until their release into the circulation.Interaction with the stroma is thought to maintain stem cell numbers and the production ofprogenitor cells committed to the various lineages (6). Contact with the stromal cells isrequired for maintenance of hematopoietic cells; if physically separated from the stroma,the hematopoietic cells die (2). Conditions in long-term cultures are evaluated by removingcells from culture supernatants at weekly intervals, and plating them in a colony assay.Hematopoiesis can be supported by preestablished allogeneic stromal layers, providedthese are irradiated to prevent rejection reactions.Colony assays were used to define the lineage commitment of progenitor cells. Forexample, when bone marrow cells are cultured for colony formation under permissive3conditions, using rich culture medium containing a mixture of cytokines, several colonytypes result. Colonies of the myeloid lineage consisting of more than one cell type includegranulocyte-macrophage colony-forming units or CFU-GM, and granulocyte, erythrocyte,macrophage, megakaryocyte colony forming units, or CFU-GEMM, but not othercombinations of cell types. This is an indication that (neutrophilic) granulocytes andmacrophages are more closely related to each other than to erythrocytes andmegakaryocytes. The formation of single lineage colonies is also supported in theseconditions; these include CFU-M (macrophage), which arises from a macrophage-colonyforming cell or M-CFC. Also included are CFU-G (neutrophilic granulocyte), arising froma G-CFC, CFU-E (erythrocyte) and the more primitive and larger BFU-E (burst-formingunit-erythrocyte), and more rarely, CFU-Meg (megakaryocyte), CFU-eo (eosinophil), andCFU-bas (basophil). Infrequently in normals, and more frequently with leukemicspecimens, colonies of immature blast cells will form, arising from bI-CFC. Colonyassays and Dexter cultures support hematopoiesis by myeloid cells, or myelopoiesis;conditions can be altered to support lymphopoiesis if required.Recently it became evident that the pluripotent CFU-S (colony forming unit-spleen)of the spleen colony assay is not the most primitive hematopoletic cell type present in vivo.By transplanting spleen colonies to secondary irradiated recipients, it became clear thatthese animals could be reconstituted in the short term, but that long term engraftment didnot occur, and conversely, that long-term reconstitution could be obtained without CFU-S(7). Hence, characteristics of yet more primitive cells have been investigated; these includethe high proliferative potential colony forming cell or HPP-CFC (8), and the long termculture initiating cell (LTCIC, reference 9), which is capable of giving rise to Dexter4cultures. By separating populations of bone marrow cells on the basis of physicalcharacteristics such as light scatter (forward and perpendicular, reference 9) and wheatgerm agglutinibility (10), or reactivity with dyes such as rhodamine 123 (9, 11, 12), orHoechst 33342 (13), some of the characteristics of stem and progenitor cells have beendefined. Progenitor cells committed to the various myeloid lineages express lineagespecific markers (linj and the cell surface molecule cluster designation antigen 33(CD33j. More primitive cells, including those committed to the myeloid lineage, areCD3433, whereas the most primitive hematopoletic cells are CD3433iin, and havethe appearance of small lymphocytes (9). Both the LTCIC and the HPP-CFC expressCD34 but have no detectable expression of HLA-DR (9). CD34 cells have also beenshown to give rise to stromal cells of the bone marrow microenvironment (14). CD34 wasrecently cloned; the cDNA encodes a receptor-like 100 kDa transmembrane protein with noobvious homology to other known proteins (15). Two forms are generated by alternativesplicing, which differ in the length of their intracytoplasmic domain. This finding couldhave implications regarding signal transduction by CD34 (16).Headway has also been made into defining the nature of the cell-cell interactions inlong-term cultures. Direct contact between stromal and hematopoietic cells is required forhematopoiesis to occur; in the absence of this contact, the hematopoietic cells rapidly die(2). Foci of hematopoietic cells form in regions covered by blanket cells, which have beenshown to be endothelial cells (17). As these cells mature, they are released into the culturesupernatant by passing through, rather than between, the blanket cells, presumably in thesame manner as they are released from the bone marrow into the circulation (2). Theprecise signals that govern the adhesion, transport, and release of the hematopoietic cells5are not clear, however, several molecules have been shown to be involved in adhesion.These include the proteoglycans of the extracellular matrix, specifically heparan sulfate(18), galactosyl and mannosyl residues on the cell surface glycoproteins of stromal cells,which recognize a 110 kDa membrane homing lectin on hematopoietic cells (19), andCD44, the homing receptor, which recognizes hyaluronate (20, 21). In addition, a 6OkDaprotein from bone marrow extracellular matrix (ECM), haemonectin, which mediatesattachment of cells of the granulocytic lineage has been described (22). Other knowncomponents of the ECM include laminin, vitronectin, and type IV collagen (2). Antibodiesdirected toward classical ECM components such as fibronectin do not block adhesion, nordo peptides containing the adhesion sequence RGD (2). However, one group showed thatthe VLA-4 integrin receptor, expressed on day 12 CFU-S and cells which reconstitutehematopoiesis, attach to the C-terminal heparin-binding fragment of an alternatively splicedform of fibronectin (23). The same group demonstrated that neither type IV collagen norlaminin promoted attachment of day 12 CFU-S. Maturation of hematopoietic cells isassociated with the loss of adhesion molecules, such as ICAM- 1 (CD54, reference 24).1.1.2: Colony-stimulating factors, interleukins, and other cytokines:Original studies with colony assays were performed using conditioned media fromvarious sources, providing a rich mixture of cytokines which support the growth of severalcolony types. Colony-stimulating and inhibiting activities have since been purified andcharacterized from these sources, as well as from the supernatants of cytokine-secreting celllines, and many have been cloned. Their actions and interactions are being investigated by6the addition of recombinant factors to colony assays and to long term cultures, by theireffects in vivo, and by over-expression in animals reconstituted with stem cells infectedwith retroviral constructs encoding cytokine eDNA. Cytokines that influencehematopoiesis are generally derived from a wide variety of sources, and have pleiotropiceffects on a wide variety of cells. There is an extensive body of literature detailing theeffects of these cytokines. Their effects are dependent on the state of the target cell;whether it is expressing the appropriate receptors, its cell lineage and stage ofdifferentiation, the immediate microenvironment, and on which other cytokines are present(25, 26). Moreover, there is considerable overlap in the spectrum of activities of variouscytokines (27). For these reasons, it is difficult to generalize about the biological effects ofindividual cytokines. The more well known effects and more interesting actions of themajor factors known to influence hematopoiesis are summarized in Table 1. This list is byno means exhaustive.1.1.3: Cytokines that are noteworthy in hematopoiesis:Classical cytokines that are known to influence myelopoiesis include interleukin-3(IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin- 1(IL-i), as well as granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF). IL-3 supports the survival and proliferation of primitivecolony forming cells (CFU-GEMM), and can support the survival of these cells in theabsence of a stromal layer (88). It may act as a competence factor, stimulating the cells toleave G0 and rendering them able to respond to other cytokines (28). GM-CSF supports7enhancescytohine&receptorexpression.87-100kDHA,60-O0kDsynergizeswithCSPs,growthfactorforLA,3irnmoooglobuliolymphocytes, augnrntsTcellresponsetodomains,otemberofantigen, endogeooaapyrogen,induceseoperfaosityproductionofacutephaseproteins,radioprotectivegrowthofT,B,&NHcells,aotibodyp55Tacistowaffinity,synthesis, cytokineprodoctionhighaffinityi-p75Tic,solubleformsofreceptor,mamberofsuperfamitystimulates growthofearlyprogenitorg,135,70135ismembercellsaoddifferentiatioatoalllineagesofssperfaotily,soluisleform,shrrsbetesahunitwithGM-fEE,IL-SolfrTgrowthaodactivationoflymphocytes,especiallyB,increasedclassIIexpressionooBTcellseosioophit growthanddifthseotiation,alsobasophil.and BdifferentiationBcellgrowth,supportshyhsidomagrowthisvitro,synergizeswithcytokineaonearlyprogeeitors,differen6atiooofmarinemanocyticleukemiaMl,endogesosspyrogen,acutephaseproteinsBcellgrowth&diffesentiatios,Tcellfunctionschemotacticforneutrophiloenhancescpu-dependentcelanyfosmarionofBPU-Einhibitscytokinesynthesisbynasnocytes,dowomegalateslILAclassllexpression,stimulatesmastcello&progenitors,growth&differentiationofBacutephaseproteins,synergineswithIL)onearlyprogenitors,andsmosgukasyocyteprogenitors, enhancesAbresponsesmaturationofCliTABLELSurnrnssyofthemorewellknowneffectsandmoreinterestingactionsoftheinterleukins,mIssy-stimulatingfactors,andothercytokinesknowntoinfluencehematopoiesi&CIIROMOSOMALCYTOKINEOTHERNAMESFEATtJRESLOCATIONSOURCETARGETS,MAINACrIVflIESRECEPTORFEATURESOTHERREFERENCESINTERLEUKINSmonocytes,neuteophits,endnthelial,fihroblantsactivatedTTcells1L4IL-ia=hemopoietin117-31k1)2ql4IL-2Tcellgrowthfactor(TCGF)14-l7kD4p11-3mrolti-CSP,persistingcellstimulating15-30k])Sq)3-factor(PSI,hematopnieticcellgrowthfactor(HCGF),burstpromotingactivity(BPA),eosinopbil-CSF,rtcellgrowthfactor(MCOF)00IL-4Bcellstisnulatoryfactnr-l(BSF-l)2OkD5q23-32IL-SBCOF-U,Tcellreplacingfactor45k])dirSq31-))(TRIm), eotinophildifferentiationfactor(E)I5)11-6Bcellstimalatoryfactor-2(BSF-2)22k])?p2l11-7l7kDSql2-l)11-8neutenpltilchensotacticfactor(NCP),lOkD4neuteophilactivatingpeptide(NAP)IL-9mostcellgrowthenhantingactivity40k])Sq3l-)SA)IL-IDl8kDIL-it23k])IL-12cytetoxiclymphocytematurationfactor(CLMF)monocytes,endotheist,T,B,fsbroblasts,onsoothmuscle46k])IA,114k])HA(includesp.I\u00C3\u00B3),solubleform,rswmberofsuperfamily50,00,t3OkDabanbunitsharedwithIL-),GM-CSF,member ofanperfumily50,60,75,gpl3Osolublefosm,memberofsuperfamilytransrrsrrsthraneformnofalpho,IL-28,29,30,31,321mbnaturalinhibitor,maybeautocsineinAML,isa3disswnsionalhomonlogueofbFGP29,33,34,35solid-phasechemicalsynthesisef28,29,31,33,entireprotein34,35,36,3728,29,31,33,34.3528,31,35,38homologytoOSM,LIP&0-29,31,34, 35,CSF,receptorshares130kI)39,40subunitwithLIPreceptor,soinhleformofreceptor,receptormemberofsuperfamily31,33,34,35,4131,33,4231,43,44homologoustoEBVgeneBCRFI41,45,46,47,4839,49,50,51Tcellsmanocytes,endotheialTcells52COLONYSTIMUlATINGPACfORSGM-CSPCSP-nM-CSPCSP-1Tcella,endotheial,fibroblssts,smoothmusclemonncytes,neuwopbils,endothelial,fibroblssts,smoothmuscle,preadipocytesmonocytes,neutrophils,endothelial,flbroblastskidneygrowth&diffarentlationofneutsophilo&monocytea,alaseoainophila&basophils,activationofmatsrecellfsoctions,decreaseinseutrophil migrationgrowth&differnotiationof monocytea,activationof maturecellfunctionsincludingpbagocytosis,cltemotaais,ADCCgrowth&differentiationsf0, activation90,150, octoberofofmascrecellfunctions, includingsuperfamilyonidativeburst,cbemotanisinsenponsetostisrarli,synergizeswithIL-3onearlypmgenitorsgrowth&differentiationoferythroid55,100,85,65,progenitorssuperfamilyinducesdifferentiatisnsf111.60,autocrinestimulationinastirAIr4L,transgenicmicebavrmanylesionsmaybeautocrineinAMLM5,strsctsralhomologytoSIP2fornnbydilkmntial splicing6,29,31,33,35,differ by3aminoadds-longer56,58,59lessactive,mistedtoOSM,LIP,116preventsapoptoeloof erytbsoid31,33,34, 35progenitomOThERCY0KINESSteel factor(SLIt)Tumour necrosiscacltectinfactor(TNP)-nTNP-$lympbotoainTronsforminggmwthfactor(TOP)-aTOP-BfibroblastssynergizeswithCSPaonearlystemcellsandinterprogenitors,increassaoelfrenewal, mast cellproductionendogenouspyrogen,acutephaseproteins, cacbexis,septicshock,enhonceaprimitivemlonies, onppressesconnnittedmloniesindoces earlyresponsegenessuchasfos,Jun.cytokines,receptors,boneresorptionregulatescytokinm, earlyresponsegenes,suppressesprimitivecolonies, enhanceslineage-committedmloniesgrowthinhibitory,increasedexpressionofQsseIMHC,diffeaentiation&activationofNKcellsgrowthinhibitory,activationofmonocytes,endothellal cells,Nilcells,increasedclassI &11differontiation,activation,viabilityofeosinopbils,neuirophils, nrnocytes,platelets,neuronsmitogenforstromalcells, competencefactor,inducesM-CSP,angiogenesis,neorotropictrk+p75NGPRTNPreceptorhomologymsltigenefamilyincludesint-2, hsVksproductof Steelloots,homology31,60,61, 62toM-CSP,transmembroneformhasputativeproteolyticcleavagesitewithinMHClocusbetweenclass63,64,65ifi&class I,snnsrrrmhroneform14-35kDSq23-3170-9OkDlpl3-21dionlflde-linkeddinrr2OkDl7qil-2l32-3SkD7q2lG-CSPCSP-3erythropoietin85,84kDaisnrmber ofsuperfamily, solubleformc-fists105,150,Ykinasedomain, Sq23-346,20,30,31,33,34,35,37, 38,53,54,55,566,20,31,33,57stemcellfactor(SCP),kit-ligand,18kBmastcellgrowthfactor(MOP)monocytes,nontrophils,endothelial,fibroblasts,oustcellsTcellsc-kit,prodsctof Wlocus,tyrorinekinasedomain55,75,distinctreceptors,solubleform,NOPreceptorhomology17kBl0kD6p2325kBborneorSgenes,separatehetero-diirrrchromosontsl8kDclust.eron9p:20alphagenes,1beta21-24kBhomo-12dinrr27kBheterodoterSq3l-33Typelinterferon(ifn-uandith-$)Type11interferon(ifn-y)Nervegrowthfactor(NOP)BasicPibroblastgrowthfactor(bFGP)platelets,normalotromallayersnssnocytes (a),fibroblasts(b),otherTcells, Nilcellsfibroblasts,neurons,othermonocyteswithinMllClocus31,65,66FOPfamily,transrrrmbrsne67,68form,Jsxtecriuestimulation69genes lackintrons29,70inducesapoptooioineffectorT29,70,71,72cellsphosphorylationofMAPkinase73,74,75,763dimensionalbonraloguesf11,-28,77,78,79IfS,heparin-binding0LeukemiahumaninterleukinforDAcells58kDdioulfide22q12inbibitooyfactor(IIILDA).differentiationinduriiglinked(LIP)activity(DIA),D-factor,cholinergicdifferentiationfactor(CDF)OncootatinMPlatelet-derivedgrowthfactor(PDGF)28kB22mooocyteo,maintainsemboyoealstemcello&2subunit gpl3OsignalrelatedtoOSM,U-CS?,andIL-31,58,80,81,endotheial,hematopoieticsteincellviability,converter6,maintainostemcelloduring82,83,84,85,86ftbrobluoto,TdifferentiationofrnurineleukemiaMl,tranofectionforgenetherapycellspossibleroleincacbeitia(adenooinedearninaaeprotocol)activateddifferentiationofMl,neuronalreceptorisgpl3OofLIPrelatedtoLIPU-CS?,andIL-631,58,86,87monocytesandTdifferentiation,acutepbarproteinoreceptor, aloobindscellocomplete2subunitLIPreceptorplatelets,synergineswithIL-3andGM-CS?ononchromosomeS79monocytes,earlyprogenitorcefls,enbanceogrowthendothelial,ofBprogenitors&otromalcellofThrnbkcrccolony formation of cells that are slightly more differentiated (CFU-GM). Still moredifferentiated CFU respond to single lineage factors such as G-CSF or M-CSF. Theprogressive responsiveness of more differentiated cells to different cytokines or sets ofcytokines is achieved through sequential expression of receptors for the appropriate factors(6). A hierarchy of receptor expression exists, a phenomenon that has been referred to asreceptor trans-downmodulation. For example, exposure of progenitor cells to IL-3 resultsin the downmodulation of receptors for GM-CSF, M-CSF, and G-CSF, whereas exposureto GM-CSF results in downmodulation of the M-CSF and G-CSF receptors (89).Receptor upregulation can also occur, for example, exposure of cells to IL-i results in theexpression of receptors for many cytokines (30). Cytokines also influence the expressionof other cytokines. As one of numerous examples, IL-i potentiates the response ofprogenitor cells to IL-3 and GM-CSF. This potentiation is mediated at least in part byupregulation of the transcription of the genes for various cytokines by accessory cells inaddition to the upregulation of receptor expression by target cells (28). In addition to thetrans-regulation of cytokines by other cytokines, some factors are capable of upregulatingtheir own expression; these include IL-i, IL-6, tumour necrosis factor-c (TNF-a), andGM-CSF (90, 91). Thus, progenitor cells exhibit a degree of plasticity in their response tocytokines, depending on conditions in their immediate environment. The lineage ofdifferentiation is thought not to be a predetermined property of primitive progenitors cells,but rather is determined by the balance of factors present (92). Many cascades of cytokineaction are currently being defmed in vitro and in vivo. Undoubtedly, common themes andpatterns will emerge. In addition to their complex interactive effects, some cytokines, forexample G-CSF, have been shown to exist in more than one form which differ by severalamino acids, and which may show differences in activity (59).11A cytokine that has generated considerable interest is the recently cloned Steel factor(SLF). SLF is the product of the Steel locus (62, 93). Mutations at this locus result in theSteel phenotype in mice, a phenotype that includes severe macrocytic anemia in addition togerm cell defects, white spotting, and mast cell deficiency. This phenotype is similar tothat seen in W mutants. The receptor for SLF is c-kit, a transmembrane protein withintrinsic tyrosine kinase activity, and the product of the W locus (61, 94). SLF isexpressed by fibroblasts and other cells in a transmembrane form, although a soluble formexists (95). C-kit is expressed by hematopoietic cells (96, 97). SLF potentiates theresponse of a variety of progenitor cells to a variety of cytokines (98). SLF has beenreported to increase the size of colonies in response to several factors (99), and to increasethe secondary plating efficiency of colonies stimulated by IL-3, an indication that the self-renewal capacity of these cells is upregulated (60). This finding has implications for theexpansion of progenitor cell pools for bone marrow transplantation and in the treatment ofneutropenia. SLF also potentiates the development of mast cells (100), hence one of itsmany names, mast cell growth factor, or MGF. Although widely accepted that signalstransduced through the SLF/c-kit pathway have profound effects on hematopolesis, thework of some groups has shown that the presence of wild-type SLF (101) or c-kit (102) isnot strictly required for normal hematopoiesis in some circumstances. The numbers ofhematopoietic stem cells were found to increase during the fetal development of SVS1 mice,which are homozygous for mutations in the Steel gene (101). Similarly, human subjectswith c-kit mutations resulting in null alleles were found to lack hematological abnormalities(102). These results do not exclude the possibility that compensatory pathways may beoperative in these situations.12Classically, cytokines have been thought of as soluble intercellular messengersproduced by stromal and hematopoietic cells which exert their effect on binding to atransmembrane receptor on a nearby cell (paracrine regulation, reference 103). Cytokinesare known to exert their effect in the bone marrow microenvironment at least in partthrough binding to cell surface proteoglycans of the extracellular matrix, which protectsthem from degradation, and facilitates their presentation to hematopoietic cells in localinductive microenviromnents (2, 4, 104, 105, 106). Recently, several soluble cytokineshave been shown to be derived by proteolysis from transmembrane precusors. Theseinclude IL-i (107), TNF-a (64), transforming growth factor-a (TGF-a, reference 68),and SLF (95). The transmembrane forms of these cytokines may bind receptors onadjacent cells, which in some cases has been found to result in signal transduction (108),including activation of receptor kinase activity (109), and activation of proliferation. Thisphenomenon has been referred to as juxtacrine regulation. (67). Moreover, clustering ofadhesion molecules such as integrins, a process that occurs during the formation ofadhesive contacts, was shown to result in protein tyrosine phosphorylation (110). Thus,hematopoiesis appears to be mediated via two tiers of control; one through cell contact, andthe second through the release of and response to soluble factors. The signals required fornormal steady-state hematopoiesis may be regulated by cell-cell contacts, whereas solublecytokines may mediate intercellular signals during periods of stress (28, 111). In additionto the existence of transmembrane cytokines, soluble forms of receptors for several factorshave been described. These include the receptors for interleukins 2 through 8, GM-CSF,interferon-\u00E2\u0080\u0099 (ifn-y) and TNF-a (34, 35). The soluble forms of these receptors may act as13physiological downregulators of cytokine activity by binding excess cytokine (34, 35, 72).Alternatively, they may act as ligands for transmembrane cytokines (112).In addition to their effects on local hematopoiesis, several cytokines are known toexert systemic effects. These include IL-i, TNF-c, IL-6, and IL-li, all of which inducethe production of acute phase proteins by the liver in response to inflammation andinfection (29, 39). IL-i and TNF-a induce fever by acting on the temperature controlcentre in the hypothalamus, (1, 29) and TNF-c\u00E2\u0080\u0099, or cachexin, induces cachexia, a wastingsyndrome in which a \u00E2\u0080\u009Cfutile\u00E2\u0080\u009D glycolytic pathway is induced in muscle and adipose tissue,resulting in the rapid turnover of these tissues (113). TNF-a is also involved in theinduction of septic shock (63).Analysis of the gene structure of several cloned cytokines and their receptors hasresulted in their classification into subsets. Many cytokine receptors have incorporatedcommonly used structural motifs such as immunoglobulin and fibronectin-like domains(34). Some receptors have intrinsic tyrosine kinase activity; these include the SLF receptorc-kit, and the CSF-i/M-CSF receptor, c-fms (96, 114). Others, which are members of ahematopoietic growth factor receptor superfamily, have no kinase domain. Included in thisfamily are the receptors for interleukins 2 (13 chain), 3, 4, 5, 6, 7, GM-CSF, G-CSF, anderythropoietin (34, 35). These receptors are thought to transduce signals via interactionwith non-receptor kinases, similar to the interaction of the src -related kinase Ick with CD4or CD8 of T cells (115, 116). Protein kinases an4 phosphatases are known to be involvedin erythropoietin-mediated signal transduction (117). A potential candidate for the kinaseinvolved in signal transduction from these receptors is the src related c-abl. Proliferative14stimulation of cells through the receptor of this family results in the translocation of acytoplasmic calmodulin binding protein to the nucleus (118). The members of this familyall have a trp-ser-X-trp-ser motif in a section of the protein which is located just outside thecell membrane (27, 34, 35). The significance of these findings is unclear. The receptorsfor some cytokines, for example G-CSF, have been shown to exist in more than one formwhich differ by several amino acids, and which show differences in activity (119).With still other cytokines, it has become clear on expression of recombinant proteinthat additional receptor subunits are required for high affinity binding of ligand. Thereceptors for IL-3, GM-CSF, and IL-5 all share a common 13 subunit (37, 38). Thisprovides an additional mechanism for regulating the actions of these factors, as they mustcompete for possibly limiting concentrations of the 13-subunit in order for high affinitybinding of ligand to occur. It also indicates that several cytokines presumably activatecommon signal transduction pathways, and provides a mechanism by which the samecytokine could mediate its pleiotropic effects; different effects may be mediated via theinteraction of the receptor with different 13 subunits. As a second example of this type ofcontrol, the signal converter for the leukemia inhibitory factor (LIF) receptor, gp 130, alsoacts as a signal converter for IL-6, and is the receptor for oncostatin M (0SM, references85, 87). Moreover, the a subunits for the LIF and IL-6 receptors show considerablestructural homology, as do LIF, IL-6, and G-CSF themselves (58).Although the gene products are unrelated by amino acid sequence, the genes formany cytokines and their receptors are closely linked by chromosomal location, anindication that they may share common modes of regulation (28, 33, 53). The genes15encoding many of the colony-stimulating factors and their receptors are located on the longarm of chromosome 5. The 5q- syndrome, in which regions of this chromosome aredeleted, involves hematological abnormalities and is a common chromosomal abnormalityin AML secondary to therapy (30). The genes for GM-CSF and IL-3 are adjacent and arethought to be functionally linked (6). Moreover, an element common to the upstreamregions of the genes encoding murine GM-CSF, IL-4 and IL-S has been identified (120).Production of cytokines has been noted in some acute leukemias, and it has beensuggested that these may stimulate leukemic cell growth in an autocrine manner. Retroviralgene transfer of cytokine genes into progenitor cells, however, has produced nonneoplastic expansions of cell populations, an indication that autocrine cytokine productionmay aid in expansion of the leukemic cells but is likely not the primary lesion (6, 121).Clinical trials have been undertaken using erythropoietin (epo), G-CSF, GM-CSF,IL-3, M-CSF, IL-i, IL-3, IL-4 and IL-6 for a diverse series of indications, includingacceleration of recovery from myelosuppression following chemotherapy or bone marrowtransplantation for the treatment of leukemia and other disorders (27).1.1.4: Signal transduction:Oncogenesis results from the disruption of signal transduction pathways. Thisdisruption can take place at any step along a signalling pathway, from alteration of thestructure or expression of a growth factor or its receptor, to alteration of a cytoplasmic16signal transducer, to alterations of nuclear factors involved in transcriptional control (122).More than 100 oncogenes have been identified (123); a complete discussion is beyond thescope of this paper. This section gives a brief overview of current understanding of signaltransduction, as an indication of the steps at which leukemogenic changes could potentiallyoccur.Headway has been made in recent years in elucidating the pathways by whichsignals for cellular activation and division are transmitted from cell surface transmembranereceptors to the nucleus, although many aspects of various pathways, and the interactionsbetween pathways, remain unclear. What is clear is that phosphorylation anddephosphorylation of protein substrates are crucial events in controlling the activity ofcellular signal transducing proteins. This is especially true of phosphorylation anddephosphorylation of tyrosine residues, although serine and threonine residues are alsoinvolved. The alteration of activity of these proteins by phosphorylation anddephosphorylation ultimately results in the alteration of the activity or DNA binding state ofnuclear transcription factors or tumour suppressor genes, and consequently the pattern oftranscription and expression of sets of genes regulated by these factors is altered.Many membrane receptors have intrinsic tyrosine kinase activity, these include ckit, the receptor for SLF, and c-fms, the receptor for M-CSF (25). Other tyrosine kinasesare cytoplasmic proteins, which may become translocated to the cell membrane to receiveand pass on signals. More recently, kinases have been located in the cell nucleus. C-abl, aprotein with tyrosine kinase activity, was shown to bind to specific DNA sequences. ItsDNA binding ability is altered depending on its phosphorylation state, which is regulated in17a cell-cycle specific manner (124). The structure, activity, and cellular location of c-abl arealtered in chronic myelogenous leukemia (CML), which has important implications forcellular dysregulation in this disorder. Studies using inhibitors of kinases and ofphosphatases have demonstrated that both kinase and phosphatase activity are required inorder for hematopoietic cells to respond to cytokines (125). In addition, certain signaltransduction pathways appear to be common to different cells, whereas some pathways arepresent in some cells but not in others. For example, transfection of c-fms was shown toenable cells to respond to its ligand CSF- 1, and to rescue them from dependence on SLF(126), an indication that signals transduced by c-fms and c-ki4 the receptor for SLF, usecommon pathways. In another study, transfection of c-fms enabled cells of the myeloidlineage, but not T cells, to respond to CSF-l (127), suggesting that the signal transductionpathway used by c-fms is absent or non-functional in T cells. Both kinases andphosphatases specifically active in hematopoietic cells have recently been identified (128,129, 130), as have some substrates of phosphorylation (131, 132).One of the first signal transduction pathways to be elucidated is that of proteinkinase C (PKC). In this pathway, binding of ligand to receptor results in the activation ofphospholipase C. This enzyme cleaves inositol triphosphate (IP3) to phosphatidylinositolphosphate (PIP2) and diacylglycerol (DAG). PIP2 causes the release of Ca2 fromintracellular stores in the endoplasmic reticulum and mitochondria, resulting in theactivation of protein kinase A (PKA, cyclic AMP-dependent protein kinase). DAG binds tocytoplasmic PKC, resulting in its translocation to the cell membrane and activation of itskinase activity (133). Some of the substrates of PKC have been identified. Although itsfunction is not understood, one of the substrates phosphorylated by PKC is CD34 (134).18Since CD34 is a marker of primitive myelopoietic cells, the phosphorylation of CD34 byprotein kinase C may prove to be an important event in hematopoiesis.Also involved in signal transduction from the cell surface are the receptor-associatedG proteins and the related p2 1r. Binding of ligand to the appropriate receptor bringsabout activation of its associated G protein, and release of the a subunit from the alpha-beta-gamma G protein complex. Release of the a subunit results in alteration of the activityof adenylate cyclase. If the a subunit is a G, or stimulatory subunit, adenylate cyclasebecomes activated, and intracellular levels of cyclic adenosine monophosphate (cAMP)rise, activating cAMP dependent kinases, or protein kinase A. If the a subunit is a G, aninhibitory subunit, the reverse will occur. Recently, a G protein a subunit specificallyexpressed in hematopoietic cells , G-a-16, was identified (135).G proteins depend on the binding of guanosine triphosphate (GTP) for theiractivity. They possess intrinsic GTPase activity, but hydrolysis of GTP is slow. GTPaseactivity is potentiated by GTPase-activating proteins, or GAPs, thus hastening theinactivation of the G protein. It is thought that GAPs may also be regulated by G proteinsas a downstream step in signal transduction (136). A recent finding likely to haveconsiderable impact in the understanding of chronic myelogenous leukemia is that bcr agene product of previously unknown function which is involved in the characteristictranslocation of CML between chromosomes 9 and 22 that results in a bcr-abl fusionprotein, encodes a GTPase activating protein (137, see below).19Common and diverging downstream pathways of signal transduction activated bythe interaction of ligand with cytokine receptors are being elucidated. For example, usingantisense oligodeoxynucleotides to N-ras, it was demonstrated that N-ras is required forcolony formation supported by IL-3, GM-CSF, and M-CSF, but not G-CSF (138).Moreover, ras GAP has been shown to immunoprecipitate with the P210 bcr-abl proteinpresent in cells of chronic myelogenous leukemia, and to be phosphorylated by the kinaseactivity of P210 bcr-abl, a finding which implies that mitogenic signals mediated by p21ras could be altered in bcr-abl positive cells (139). In addition, it has been shown that theM-CSF receptor c-fms, the SLF receptor c-kit, and the platelet-derived growth factor(PDGF) receptor, all of which have intrinsic tyrosine kinase activity, and are activated byautophosphorylation on ligand binding, all form complexes with phosphatidylinositol 3\u00E2\u0080\u0099-kinase (P13-kinase), an indication that they may initiate signalling along a commonpathway. In contrast, the PDGF receptor and c-ki4 but not c-fms, form complexes withphospholipase C-gamma 1, and only the PDGF receptor forms a complex with ras GAP(114). Hence, different receptors appear to initiate similar signalling pathways, but indifferent combinations. Moreover, the PDGF receptor was shown to bind GAP and P13-kinase through different sites, thus activating different signal transduction pathways (140).P13-kinase binds p21 ras in addition to ras GAP, an illustration of the complexity ofinteractions between signalling proteins known to influence each other\u00E2\u0080\u0099s activity (141).Several nuclear protein factors important in the regulation of transcription have beenidentified. These proteins bind each other in a specific manner, which influences theircapacity to bind DNA. Specific DNA recognition sequences have been identified forvarious transcription factors, and located upstream of families of genes, an indication that20regulation of the expression of certain genes has some common features. The nuclearfactors interact with each other via leucine zippers, alpha-helical structures with leucineresidues at intervals of every seventh amino acid. Leucines on the helices of adjacentfactors interact in a staggered conformation and \u00E2\u0080\u009Czip\u00E2\u0080\u009D the proteins together (142).Transcription factors interact with DNA through structures such as zinc fingers, zinc coilsand zinc twists. These were originally identified as structures in which four cysteines ortwo cysteines and two histidines, which could be quite separate by primary structure,become apposed by tertiary structure and incorporate a zinc atom, thus influencing theirDNA binding (143). This has more recently become a somewhat generic term for similarstructures which may incorporate other metals such as copper or iron (123).Examples of known transcription factors are AP- 1, which is a complex of c-fosand c-jwi NFiB, which binds to sequences upstream of immunoglobulin genes as well asothers, and c-myc and c-myb, which are important for progression through the cell cycle.As an example of the complexity of transcriptional control, the transcription of c-fos hasbeen shown to be downregulated by the underphosphorylated, DNA-binding form of theproduct of the Rb gene locus, a ubiquitously expressed tumour suppressor gene which isinactivated in retinoblastoma (144). Phosphorylation of the nuclear Rb is associated withprogression through the cell cycle, and is prevented by TGF-f3 (145), as well as byinterferons and interleukin 6 (146). Transcription of TGF- and IL-6 are in turnsuppressed by Rb (145), and by Rb and the nuclear phosphoprotein p53 (147),respectively. The DNA binding capability of many other nuclear factors is altered by theirphosphorylation state as well. Thus, the activation or repression of specific genes or setsof genes, both in hematopoietic and in non-hematopoietic tissues, is regulated by a series of21complex interactions between numerous gene products, the details ofwhich are currentlybeing elucidated.Many translocations involved in several types of leukemias, particularly the acuteleukemias, involve the dysregulation of nuclear transcription factors (148). For example,the t(15;17) translocation of acute promyelocytic leukemia disrupts the retinoic acidreceptor-a locus. Retinoic acid, in turn, is a negative regulator of AP- 1 responsive genes(149), which mediates transcription induced by phorbol ester tumour promoters (123) andcytokines (150), as well as DNA replication (151, 152). Hence, this translocation disruptsthe ability of the cell to downregulate a specific set of genes involved in growth control.Other types of nuclear factors known to be involved in hematopoiesis include proteinscontaining helix-loop-helix (HLH) domains, such as Id, which inhibits myeloiddifferentiation by sequestering other HLH proteins (153), and homeodomain containingproteins, the products of homeobox (HOX) genes, which are differentially expressedduring terminal differentiation along different hematopoietic lineages (154).The function of cyclins, which are involved in passage through the cell cycle, isalso being clarified. Cyclin levels are known to increase during the cell cycle just prior tothe first growth phase/DNA synthesis (GuS) and second growth phase/mitosis (G2/\u00E2\u0080\u0099M)transitions, by inhibition of specific proteolysis. Injection of cyclin messages intoXenopus oocytes induces entry into M phase (155). Cell division cycle (cdc2) kinasewhich is known to phosphorylate histone Hi and other nuclear proteins such as the tumorsuppressor genes p53 and Rb, and may also phosphorylate RNA polymerase. Cdc2 kinaseis in turn activated by removal of a phosphate group by cdc25 phosphatase (156). Active22cdc2 kinase and a cyclin then associate to form M phase promoting factor, which promotesprogression through the cell cycle. Thus, phosphorylation and dephosphorylation areimportant not only in the activation of mature cell functions, but in the passage of cellsthrough the cell cycle in addition. The growth inhibitory effect of TGF43 is reported to bein part mediated by a decrease in phosphorylation and kinase activity of cdc2 at the G 1/Stransition (157).In summary, activation of positive or negative regulatory signals influencing cellfunction or cycling results in a complex set of interactions between signalling molecules,which act in interrelated cascades at the cell surface, in the cytoplasm, and in the nucleus.These signals ultimately result in alteration of the expression of different sets of genes,which in turn alter cellular functions. Changes in the structure or expression of thesesignalling molecules can lead to the dysregulation of cellular growth, and to oncogenesis.1.2: Oncogenesis and Ieukemogenesis;The clinical features and etiology of myeloid leukemias, with a focus on chronicmyelogenous leukemia (CML), are described in this section. A general discussion ofoncogenesis is included, with a description of the involvement of cellular oncogenes,tumour suppressor genes, and transcription factors. Also included is a description ofcurrent evidence for leukemogenesis by disruption of the balance between self-renewal and23differentiation, with a focus on CML. Evidence that CML is a pluripotent stem celldisorder is described. Current understanding of the translocation between chromosomes 9and 22, which results in the Philadelphia chromosome and P210 bcr-abl fusion product,and the role of bcr-abl in the development of CML, is summarized. Factors involved inthe alteration of hematopoiesis and potentially in leukemogenesis are also described.Included are factors which downregulate normal hematopoiesis, and factors which promotethe outgrowth of myeloid leukemia cells.Oncogenesis results from an uncoupling of the processes that regulate proliferation,terminal differentiation, and cell death. Multiple independent mechanisms are thought toregulate growth and differentiation, and several separate events are needed to subvert thesecontrols and to induce other aspects of transformation (122). In order for a cell to becometransformed, changes in cell regulation must become fixed or heritable, and passed on todaughter cells. Multiple genetic changes are thought to be required for a cell to becomefully transformed. For example, human T lymphotropic virus-I (HTLV- 1), which resultsin adult T cell leukemia, has a latent period in some cases of decades, suggesting thatsufficient time is required to acquire further mutations in order to achieve celltransformation (158). Moreover, progression of tumours to more aggressive forms, whichare in some cases refractory to previously effective therapy, has long been recognized to beassociated with increasingly bizarre mitotic figures and with karyotypic changes (159).These include loss or duplication of chromosomes, and inversions, translocations, anddeletions of various chromosomal segments (160). Solid tumours are thought to becomeinvasive and metastasize as a result of the positive selection of one or more aggressive24subclones, in which a further genetic change has presumably occurred (161). Changeswhich result in increased proliferation can occur in two ways; activation of oncogenes, andinactivation of tumour suppressor genes. Examples of these changes have been found inmany tumour types, in all probability both activation of oncogenes and inactivation oftumour suppressor genes are involved in any particular tumour (159). Moreover,oncogenes have been noted to cooperate in the production of malignant phenotypes (162).This cooperation occurs in many cases between a nuclear oncogene and a cytoplasmiconcogene (159, 163). Alteration of a nuclear oncoprotein results in immortalization,whereas alteration of a cytoplasmic oncoprotein reduces growth factor requirements,induces cell shape changes, and leads to anchorage-independent growth (122).Cellular proto-oncogenes can become activated by several mechanisms; pointmutation, truncation or disruption of coding or of regulatory sequences, or by theintroduction of a strong promoter or enhancer. For example, point mutations of rasproteins have been shown to be involved in various human malignancies (122).Juxtaposition of unaltered c-myc coding sequences near strong immunoglobulin promotersfor lambda, kappa, or heavy chains by chromosomal translocation is a hallmark ofBurkitt\u00E2\u0080\u0099s lymphoma (159). Balanced translocations involving nuclear oncogenes occur inseveral leukemias (159). Truncation of erb-b, the epidermal growth factor receptor, resultsin constitutive activation of receptor kinase activity in the absence of ligand binding, and incell transformation. Similarly, tumour suppressor genes can be inactivated by severalmechanisms. Point mutations, deletions, and rearrangements of p53, a sequence-specificDNA binding phosphoprotein, are involved in several solid tumours, such as cancers of thecolon, lung, breast, ovary, and bladder (164), and are present in many myeloid leukemia25cell lines (165), but in few myeloid leukemia primary cells (166). Unproductive complexesare formed between altered p53 and other regulatory proteins in the nucleus, includingwild-type p53, thus inactivating them in a dominant manner (144, 167). P53 isconstitutively expressed in normal marrow blast populations (168), and is destabilized andlocalized to the nucleus during growth arrest (169), implying that its presence is importantto the function and cycling of these cells. P53 is the most frequently altered gene in humantumours (144), and transgenic mice homozygous for a p53 null mutation showed increasedsusceptibility to spontaneous tumour formation (170). It was recently found that p53forms a stable covalent linkage with RNA, suggesting that it may be involved in RNAmetabolism (171). Similar to p53, Rb is inactivated in retinoblastoma (144), and is thoughtto be inactivated in megakaryoblastic crisis of CML (172). The observation of consistentkaryotypic involvement in certain malignancies has led to the identification of other cellcycle regulatory genes; an example is the recently cloned DCC (Deleted in ColonCarcinoma, reference 159). DCC was identified by consistent loss of heterozygousity incolon cancers. It has the predicted structure of an integral membrane receptor; or cellularadhesion protein (144); its loss presumably confers a growth advantage on the affectedcells. Progression of colon cancer following loss of DCC is correlated with subsequentinactivation of p53.1.2.1: Leukemia; classification, etiology:Leukemias are classified according to their aggressiveness, and further subdividedby the cell lineage involved. Common to leukemias is a clonal expansion that occurs in26cells with high potential for self-renewal, likely stem or progenitor cells. The precise siteof expansion varies in different cases, accounting for the diverse phenotypes of theleukemias (173). The chronic leukemias include chronic myelogenous leukemia (CML)and chronic lymphocytic leukemia (CLL). CLL is further classified as B-CLL or T-CLL.The chronic leukemias are relatively indolent disorders, which in many cases areasymptomatic, and may be diagnosed on routine blood testing. In other cases, recurrentinfections may occur due to defective functioning of cells of the leukemic clone; this resultsin symptoms such as fatigue and fever. Characteristic of chronic myelogenous leukemia isa partial uncoupling of proliferation and differentiation (174). There is an expansion ofmyeloid progenitors in the bone marrow and peripheral blood. These differentiate,however, into relatively normal neutrophilic granulocytes, which may be defective in somefunctions. For example, a defective respiratory burst is found in the neutrophils of CMLpatients in some cases. This can lead to impaired killing of phagocytosed bacteria, and thusto recurrent infections (175). Additional symptoms may be seen in CML, these includesplenomegaly and bleeding or bruising, due to the suppression of normal myelopoiesis,which leads to decreased platelet counts.The chronic phase of CML lasts for an average of about 3 years; after this time itenters what is referred to as an accelerated phase, followed by blast crisis and conversion toacute leukemia. The blastic transformation of CML involves increasing. resistance totreatment, increasing proliferation, and maturation arrest. About two-thirds of cases ofCML convert to AML (acute myelogenous leukemia, or acute non-lymphocytic leukemia,ANLL), whereas one-third convert to ALL (acute lymphocytic leukemia) or acuteundifferentiated leukemia (176). Further karyotypic changes accompany the conversion of27CML to a more aggressive leukemia, a further indication that leukemogenesis is a multi-step process involving several genetic changes (177). These changes frequently include aduplication of the Philadelphia chromosome (25 to 30%), trisomy 8 (25 to 30%) andisochromosome 17 (20%) (175). Non-random acquired chromosome abnormalities areobserved in other leukemias as well, frequent examples being loss of chromosomes 7 or 5or their long arms (-7 or -7q or -5 or -5q) in AML after treatment with chemotherapy orradiation (178). An accumulation of immature blast cells occurs in the acute leukemias; theimpediment to differentiation thus appears to be more complete. The acute leukemias arerapidly fatal. Average survival without treatment is three to four months (179).The incidence of leukemia is 9 to 10 persons per 100,000 per year in the UnitedStates. About half are acute leukemias, and half are chronic. ALL and AML occur atapproximately equal frequency. CLL occurs at four times the frequency of CML. Themaximum incidence of ALL is in childhood, between ages 2 and 6. AML comprises about20% of childhood leukemias, but is more common in older persons, and the incidenceincreases with age. CLL is rarely seen before mid-life, and the incidence increases withadvancing age. CML occurs at any age, with the peak incidence in mid-life (1, 176).Other leukemia-related disorders include the myeloproliferative syndromes, inwhich clonal growth expansion occurs (1, 176). These disorders include polycythemiarubra vera and essential thrombocythemia. These disorders often progress tomyelofibrosis, in which the marrow is infiltrated by an overabundance of megakaryocyteswhich stimulate the replacement of hematopoietic tissue by fibroblasts and collagen fibers.Myelofibrosis, in turn, often terminates as acute leukemia. The myelodysplastic28syndromes are characterized by abnormal differentiation, and an increased risk oftransformation to AML (1, 176).Causative environmental or predisposing factors have in general not been clearlyidentified, aside from the involvement ofHTLV- 1 in ATL. Exposure to ionizing radiationis thought to result in an increased incidence of CML; a higher incidence of CML ANDAML was reported in Hiroshima and Nagasaki following the atomic bomb explosions(180). Ionizing radiation results in the generation of free radicals which can introducedouble strand DNA breaks and induce reciprocal translocations in cells in the quiescent(G0) or first growth (G1) phases of the cell cycle, as well as chromatid exchanges in thesecond growth phase (G2) and in the phase of DNA synthesis (S, reference 181), inaddition to disrupting the functions of proteins and other nucleic acids (182). Ionizingradiation results in a higher incidence of myeloid than of lymphoid leukemias (183).Generally, in the case of lower level exposure to radiation, such as near power stations, ora nuclear reactor accident such as occurred at Chemobyl, the overall incidence of leukemiais too low and/or records are not accurate enough for appropriate comparisons to be madeand accurate conclusions to be reached (180). However, patients who previously receivedradiation therapy for ankylosing spondylitis, a condition of chronic inflammation in thespine, have a higher incidence of leukemia. Hereditary disorders which are associated withchromosomal abnormalities, genetic instability, or defects in DNA repair, such as Bloom\u00E2\u0080\u0099ssyndrome, Down\u00E2\u0080\u0099s syndrome, and Fanconi\u00E2\u0080\u0099s anemia, result in a higher incidence of acuteleukemia (176). Neurofibromatosis, a hereditary autosomal dominant disorder, isassociated with a higher rate of hematological malignancies. It has been suggested that theG-CSF gene may be involved in leukemic development, as the neurofibromatosis gene29maps to the same chromosomal band as does the G-CSF gene (184). However, thefinding that the NF- 1 gene has ras GAP homology, and is thus highly similar to bcr, islikely to prove to be of importance (122, see below). Exposure to alkylating agents, forexample in the treatment of previous malignancies, results in a higher incidence of leukemiaand lymphoma (1). Chronic benzene exposure is also associated with an increased risk ofdeveloping a hematological malignancy (185).CML is one of the more thoroughly studied human malignancies. This is not onlybecause tissue is relatively readily accessible, but also because it was the first humanmalignancy for which a consistent chromosomal aberration was described. ThePhiladelphia chromosome was described by Nowell and Hungerford in 1960 (186). Itresults from a reciprocal translocation between the long arms of chromosomes 9 and 22[t(9;22)(q34;ql 1)] and is detectable in the blood and bone marrow cells of more than 90%of CML patients. The result of this translocation is the in frame juxtaposition of codingsequences for the 5\u00E2\u0080\u0099 portion of bcr with 3\u00E2\u0080\u0099 sequences of c-abl (187), resulting in theexpression of a bcr-abl fusion protein, P210, the function of which is dysregulated inseveral ways (see below). Progression of CML in a majority of cases is associated withthe acquisition of further karyotypic changes. Molecular studies have revealed that 30-50%of Philadelphia chromosome negative (Ph-) CML have variant translocations resulting inthe bcr-abl fusion product. The remainder are considered \u00E2\u0080\u0098atypical\u00E2\u0080\u0099, and are aheterogeneous group with different cytological features including a lower incidence ofbasophilia and higher degrees of thrombocytopenia (low platelet counts) and anemia, andhigher monocyte counts. Atypical CML responds poorly to chemotherapy, progresses30more quickly to blast transformation, and may constitute a separate clinical entity (175,187, 188, 189).Although CML presents clinically as an outgrowth of neutrophils and theirprogenitors, it is considered to be a stem cell disorder. This has been demonstrated inseveral ways; by analysis of the ratio of glucose-6-phosphate dehydrogenase (G6PD)isoenzymes in various hematopoietic cell lineages as compared to normal tissue, and byobservation of the Philadelphia chromosome, bcr-abl transcripts, and fusion protein incells of all hematopoietic lineages (175, 190, 191). This conclusion is supported by theobservation that CML derived Ph cell lines are capable of differentiating along several celllineages (192). Thus, the genetic lesion occurs in a pluripotent stem cell, but thetransformed phenotype does not appear until the stage at which an expansion of theneutrophil progenitor pool is manifest.In contrast, the genetic defects in many of the acute leukemias are thought to occurin more committed cells, although this is variable. For example, the French-American-British (FAB) classification subdivides AML into seven types, Ml through M7, on thebasis of cytological and clinical criteria, and the predominant pathway of differentiation anddegree of maturation (1, 185). Ml, for example is an undifferentiated leukemia, in whichthe malignant phenotype is manifest in a relatively early cell. M6 and M7, in contrast,show clear characteristics of erythroid and megakaryocyte differentiation, respectively.About 10 to 15% of acute leukemias show characteristics of both myeloid and lymphoidlineage to varying degrees, such as cell surface markers and rearrangements ofimmunoglobulin or T cell receptor genes. Previously, this was thought to be an indication31of \u00E2\u0080\u009Clineage infidelity\u00E2\u0080\u009D, or aberrent expression of markers of one lineage in cells partiallydifferentiated for the other lineage. However, more recently, it has been suggested that thisphenomenon is an indication that the leukemic lesion occurs in a more primitive cell thanpreviously thought, one that is earlier than the myeloid-lymphoid branch point (162, 193,194). In keeping with this idea, it has been shown that cross-lineage rearrangements werefound more commonly in acute leukemias following CML blastic transformation ascompared to de novo cases (195). This is not surprising, since the leukemic defect inCML is known to occur in a very early cell. As cell surface markers and genetic lesions arebecoming more well defined, they are being found to correlate fairly well with thecytological working classification, for example, of the FAB system in AML. Large studieshave been undertaken in order to define cell phenotypes more clearly in these leukemias inorder to facilitate diagnosis, biological understanding, and rational and effective therapy(185).1.2.2: The 9;22 translocation in CML; the Philadelphia chromosome and P210 bcr-abl:The balanced reciprocal translocation between chromosomes 9 and 22[t(9;22)(q34;ql 1)] which results in the expression of P210 bcr-abl is the hallmark ofCML. More than 90% of CML patients show evidence of the Philadelphia chromosome(Ph). Of patients that are Ph-, at least another 5% express a rearranged bcr-abl transcriptwhich is detectable by Northern blot analysis or by reverse transcriptase polymerase chainreaction (PCR). Thus, over 95% of CML patients are bcr-abl , and, as discussed, theremainder may form a separate clinical entity. The t(9;22) translocation in CML results in32juxtaposition of the N-terminal sequences of bcr with downstream sequences of c-abl(196). The breakpoint in the bcr gene occurs within a 5.8 kb region, the major breakpointcluster region, or M-bcr. The resulting fusion protein, P210, incorporates sequences fromthe first two to three exons of bcr depending on the exact location of the break, excludingthe fourth exon, and in some cases the third, and resulting in a b2a2 or b3a2 fusion.Expression of the b2a2 versus b3a2 product has been suggested to correlate with differingclinical features, such as differing cytogenetic abnormalities during the development ofblast crisis (197) and platelet count (198). The breakpoint in c-abl occurs within a 100 kbregion. The regulatory SH3 (src homology 3) domain near the N terminus of c-abl isdeleted in the resulting P210 fusion protein, resulting in upregulation of abi tyrosinekinase activity, which in c-abl is low. A second breakpoint, the minor breakpoint clusterregion, or m-bcr is involved in ALL arising de nova In these leukemias, the first exononly of the bcr gene is incorporated into the fusion protein, P 190, which also exhibitsupregulated tyrosine kinase activity, but to a greater extent than does P210 (199). ALLsarising from pre-established CML express the P210 bcr-abl gene product. Balancedtranslocations can result by double strand DNA breaks in G0 or G1 induced by exposure toionizing radiation, but more commonly occur by chromatid exchanges induced by radiationor chemicals in G2 or S phase. Of interest is the presence of alu sequences near thebreakpoints on chromosomes 9 and 22, suggesting that these sequences may be involved inthe recombination (181). Although CML is rare in children, the bcr-abl rearrangements injuvenile CML have been shown to be similar to those in adult CML (200).331.2.2.a: Signal transduction byc-abi, bcr, and P210 bcr-abl;C-abl is highly conserved, is ubiquitously expressed in mammalian tissues, andhas considerable homology with the src family of tyrosine kinases (196). It has beenshown to be a nuclear protein, the first protein with kinase activity to be demonstrated tolocate to the nucleus (201). Phosphorylation of c-abl is cell-cycle dependent; it isphosphorylated by the cell cycle associated cdc2 kinase on two sites in interphase cells, andon seven in mitotic cells (202), and the hyperphosphorylated form is translocated to thecytoplasm (201). Recently, it was shown that c-abl has sequence-specific DNA bindingcapability (124). Using antisense to c-abl, c-abl expression was shown to be essential forthe formation of CFU-GM and CFU-G by hematopoietic cells, whereas CFU-E and BFUE were unaffected (203). In addition transgenic mice lacking abi activity are severelyaffected, with high perinatal mortality, ranting, and abnormal development of severalorgans (204). These observations suggest that c-abl is important in the development andfunction of many cell types. In addition, TNF-ct and IL-i increase c-abl expression inmarrow stromal cells (205). Expression ofboth abland bcr-abl were found to decrease ondifferentiation of a CML cell line (206). Moreover, c-abl is thought to be regulated by acellular inhibitor; this interaction could be disrupted by the bcr-abl translocation (207).These results strongly suggest that dysregulation of abi in CML is important in theselective expansion of cells of the neutrophilic lineage.Until very recently, little was known about the normal cellular function of bcr. Itwas originally thought to be a passive player in the bcr-abl translocation, with upregulationof abi tyrosine kinase activity occurring by replacement of the c-abl SH3 regulatory34domain. More recently, first exon bcr sequences were shown to specifically activate thebcr-abl tyrosine kinase activity (208). Bcr is now known to have serine/threonine kinaseactivity encoded within its first exon. This is a novel coding structure for a kinase;sequences are usually spread over five to seven exons (209). In addition, bcr has beenshown to encode a GTPase-activating protein (GAP) for p2lrac (137). Little is knownabout the function of the rac proteins, but they are xac -related, so they presumably areinvolved in signal transduction, and they are known to be more highly expressed inmyeloid cells (137). Phagocyte oxygen radical production has been reported to beregulated by mc2 at the level of NADPH oxidase activity, so alteration of mcGAP activityby the bcr-abl translocation may be related to the observed defects in the respiratory burstobserved in CML cells (210). Moreover, it was shown that the rodent homologue of raeencodes a serine-threonine kinase which contains an SH2 domain, and is carried in aretrovirus (211). Intriguingly, one of the known GAPs for p2l is the neurofibromatosisgene product NF-1 (144, recall that there is a higher than normal incidence of hematologicalmalignancies in neurofibromatosis). In addition, the 85 K subunit of phophatidylinositol3-kinase has been shown to have homology to bcr. Thus, evidence is accumulating thatbcr plays an as yet undefined, but likely important, role in signal transduction.It was demonstrated that bcr binds to the SH2 domain of c-abl (212). SH2domains are important in interactions between proteins involved in signalling via tyrosinekinase pathways. The presence of two putative signal transducing activities, GAP andserine/threonine kinase, in addition to its demonstrated binding to the abi SH2 domain, hasled to the suggestion that bcr may be involved at the intersection of cellular signaltransduction pathways (209). C-abl is known to be expressed from the unrearranged allele35in CML cells (213), however, others report that it is undetectable by immunoblotting,implying that it may be expressed at lower levels than in normal cells (214). It is unclearwhether normal bcr is expressed in CML cells. The inactivation or constitutive activationof the putative signal transducing functions of bcr could be expected to have significanteffects on the biological functions of these cells, as could the alterations in abi activity.Long range mapping of the bcr gene demonstrated the presence of three genes withhomology to 3\u00E2\u0080\u0099 sequences in bcr The significance of these genes is unknown (215).1 .2.2.b: Tmnsforming capabilities ofbcr.-abl;The form of c-abl expressed in Abelson murine leukemia virus, v-abl, which is agag-abi fusion protein, is known to be transforming in hematopoietic cells (216). BothP190 and P210 bcr-abl have been demonstrated to have transforming capabilities as well.Introduction of P210 bcr-abl into hematopoletic cell lines caused them to become growthfactor independent and tumorigenic (217). Reconstitution of mice with bone marrow cellsinfected with a retroviral vector carrying P210 bcr-abl resulted in various hematologicalmalignancies, including a CML-like syndrome, macr\u00C3\u00B3phage tumours, and lymphoidmalignancies (218, 219). One group found these tumours to be rarely transplantable, andconcluded that bcr-abl confers a proliferative advantage and that complete transformationinvolves additional genetic changes (218). Other investigators found that transfection ofbone marrow enriched for multipotent progenitor cells with P210 bcr-abl resulted in theformation of in vitro colonies which were responsive to growth factor regulation butsubsequently became growth factor independent. The growth factor independent cells,36however, were not leukemogenic in mice with severe combined immunodeficiency (220).These studies support the concept of leukemogenesis as a multi-step process; acquisitionof P210 bcr-abl appears to confer on a clone the ability to expand in a relatively benignmanner, and further genetic lesions appear to be involved in the acquisition of anaggressive phenotype. Another group found that passage of bone marrow cells from micewith the CML-like syndrome to secondary recipients resulted in induction of one AML,several T-ALLs, and one CML, a situation the authors compare to blast crisis (221). Micereconstituted with P190 or P210 bcr-abl displayed a similar spectrum of hematologicalabnormalities, which were more aggressive in those receiving P190 (222). This result isan indication that the target cells affected by the two fusion proteins are similar.Conversely, mice receiving v-abl transfected cells were reported to display hematologicalabnormalities which differed from those receiving P210 in some respects, such as theinvolvement of other organs, for example the lymph nodes and spleen (223). Interestingly,transfection of P210 bcr-abl into an IL-3 dependent myeloid cell line was found to result inthe phosphorylation of similar sets of proteins as are involved in signal transduction by IL-3, indicating that growth factor independence may be facilitated by bcr-abl (224), andpotentially implicating bcr and/or abi in the transduction of cellular signals stimulated bygrowth factors under normal conditions. In this regard, it is thought that src relatedkinases are involved in the transduction of signals from cytokine receptors lacking intrinsickinase activity, such as the interaction of Ick with CD4 or CD8, or the interaction of fynwith the T cell receptor. Recall that the absence of a kinase domain is characteristic of mostof the CSF receptors, and that abi is a src related kinase; c-abl could be involved in thetransduction of signals from these receptors. This is supported by the observation,37discussed above, that treatment of hematopoietic progenitor cells with antisensedeoxynucleotides to c-abl abrogates the formation of CFU-GM and CFU-G (224).1.2.2 .c: P210 bcr-abl in the suppression ofapoptosis;In normal tissues, self-renewal is balanced by terminal differentiation andprogrammed cell death or apoptosis. Apoptosis is an active process that requires RNAtranscription and protein synthesis (225). It is induced in hormone-dependent tissues onwithdrawal of hormone (226), in HL6O cells during retinoic acid-induced differentiation(227), and in aging normal neutrophils during inflammation (228, 229). The colony-stimulating factors erythropoietin, IL-3, GM-CSF and G-CSF have all been shown tosuppress apoptosis in cells that depend on them for survival (225, 226, 230). MessengerRNAs associated with apoptosis in immature thymocytes have been identified; oneencodes a zinc finger protein, suggesting that it is involved in DNA regulation, and theother encodes an integral membrane protein (231). IL-i has been found to be processedand released during apoptosis, which is presumably a cellular response to cell stress andinjury (232).Clues as to how the balance between self-renewal and apoptosis may becomedysregulated can be found in the functioning of bcl-2 in B cell follicular lymphoma. BcI2 is a protein normally associated with the inner mitochondrial membrane, and is expressedin tissues in which apoptosis accounts for cell turnover, such as germinal centres in lymphnodes and at the base of intestinal crypts (233). Survival of 1L3-dependent cell lines was38supported by the expression of bcl -2 (225, 226, 230). Moreover, stimulation oflymphocytes with mitogens correlates with bcl -2 expression (234). The t( 14; 18)translocation in follicular B cell lymphoma juxtaposes intact bcl -2 coding sequences withthe immunoglobulin heavy chain region, upregulating the expression of bcl-2. Enforcedbcl -2 expression in B-lymphoid cells in transgenic mice resulted in prolonged antibodyresponses and a high incidence of autoimmune disease, suggesting that expression of bcl-2promotes prolonged cell survival (235). Moreover, the latent membrane protein 1 (LMP 1)of Epstein Barr Virus (EBV) was shown to up-regulate be] -2 expression and to protectcells from apoptotic cell death (236). Taken together, these results suggest that bcl -2functions in prolonging the life of cells in which it is expressed.Follicular lymphoma characteristically has a relatively indolent course (1), however,a leukemic phase may arise after some time. The leukemic cells often harbour the t(14; 18)translocation, suggesting that the more aggressive malignancy arose in the clone in whichcell survival was prolonged by bcl-2 (225, 226). Many of the tissues that express bcl -2,such as skin, colon, and breast, have a high incidence of cancer (233). 70% of AML cells,irrespective of FAB classification, were found to express bcl-2, whereas most CML cellswere bcl-2 negative (237). These findings imply that a similar function may be performedby P210 bcr-abl in CML as is performed by be] -2 in follicular lymphoma. Prolongationof the lifetime of a clone of cells by P210 bcr-abl could provide a long lived pool of cells inwhich further genetic changes could accumulate, eventually resulting in a more aggressivemalignancy. Indeed, a recent report demonstrates that apoptosis is in fact suppressed in acell line rendered growth factor independent by transfection of P210 ber-abl; inhibition bytyrphostins inhibitors of bcr-abl kinase activity resulted in apoptosis (238). Prolongation39of the lifetime of the cells in the expanded clone supported by P210 bcr-abl can be viewedas correlating with chronic phase CML. Further genetic changes would then result inaccelerated phase, blast crisis, and the acute leukemias.1 .2.2.d: Other characteristics ofbcr-abi;C-abl was recently demonstrated to have microfilament binding capability, which isupregulated in P210 bcr-abl. Recently, it was reported that activation of abi actin bindingactivity requires bcr N-terminal sequences (239). The actin binding domain is located inthe C-terminus (240). As the Drosophila abi homologue is involved in cell adhesion(241), the authors suggest that mammalian abi may be part of a signal transductionmechanism regulating cell adhesion, and that the bcr-abl translocation may be involved inthe defective adhesion observed in CML. This might occur via phosphorylation ofcytoskeletal proteins involved in the interaction between hematopoietic and stromal cells.However, using antibodies to cross-link cell adhesion molecules, one group found that celladhesion results in the phosphorylation of cellular proteins and presumably in the activationof signal transduction pathways (110). In addition, GPI-anchored cell surface moleculesare known to be complexed to protein tyrosine kinases (242). Furthermore, a defect in aGPI-anchored adhesion molecule is known to be involved in the reduced adhesion of CMLcells to stroma (243). Thus, rather than altered adhesion being a result of altered signaltransduction, altered signal transduction could be a result of altered adhesion. Also ofpotential importance is the observation that pp6O c-src is translocated to the cytoskeleton40during platelet aggregation and in cell transformation (244), reminiscent of the cytoskeletalbinding capacity of c-abl, a protein with considerable src homology.Using a set of bcr-abl constructs, it was shown that upregulation of c-abl kinaseactivity is not due to the removal of SH3 sequences alone, implying an active role for bcrin the increased kinase activity of bcr-abl. Analysis of the bcr promoter demonstratedstructural similarity to the c-abl promoter, suggesting that structural alteration is likely tobe more important that transcriptional dysregulation in conferring transforming ability onbcr-abl (213). In addition, it was recently found that bcr sequences necessary fortransformation by bcr-abl bind to the abi SH2 regulatory domain (212). P210 bcr-ablwas shown to coimmunoprecipitate with ms GAP, which is tyrosine phosphorylated inPh cell lines (139). This finding is significant, since p2lraS is known to be activated byhematopoletic growth factors (115). In addition, P210 bcr-abl was found to form acomplex with wild-type P160 bcr in K562, a CML-derived cell line. P210 was alsoshown to form a complex with the tumour suppressor gene product p53 in the same study(245). This observation is intriguing, since it has been reported that p53 levels increasewith maturation ofhematopoietic cells (246). Moreover, introduction of wild type p53 intoa murine myeloid leukemia cell line, Ml, induced apoptosis (247). Furthermore,transforming viruses such as SV4O, adenovinis, and papillomavirus are known to exerttheir effects by the sequestration of the tumour suppressor genes Rb and p53 intoineffective complexes (144). Hence, bcr-abl may delay differentiation or suppressapoptosis by sequestering p53.411.2.3: Adhesion defects in CML;Defects in the adhesive interactions of hematopoietic cells have been described inCML. These adhesion defects account for the fact that no differences in the proportion ofCD34 cells can be detected between the blood and bone marrow in CML patient material,whereas in normal material, subpopulations of cells expressing CD34 in the peripheralblood are undetectable. In contrast, between 1 to 5% of normal bone marrow cells expressCD34 (248). These results are an indication that immature cells in CML are inappropriatelyreleased into the circulation. CML cells are known to be reduced in their capacity to bind tonormal stromal layers in LTMC (249). Conversely, stromal layers derived from CML cellsare unable to support hematopoiesis by normal cells (250). The numbers ancVor bindingcapacity of bl-CFCs to stromal layers was also reduced in cultures derived from AML cells(251).As mentioned, a phosphatidyl-inositol (P1)-linked cellular adhesion molecule(CAM) has been found to be missing from CML cells (243). This result is interesting sincean abl -related kinase in Drosophila is known to interact with a P1-anchored cellularadhesion molecule (CAM, reference 241). The authors suggest that the CAM in CML maybe a target of the altered bcr-abl kinase. Also, treatment of normal progenitor cells withmedium containing hematopoietic growth factors resulted in decreased adhesion (252).Taken together, these results suggest that growth factors may stimulate the c-abl pathway,resulting in altered adhesion. Upregulated abl kinase activity in the P210 bcr-abl fusionprotein in CML cells may lead to a prolonged signal, resulting in poor adhesion.42Adherence of normal bone marrow cells to stromal layers can be competitivelyinhibited by exogenously added heparan sulfate proteoglycan (18). In other cell systems,heparan sulphate has been found to have anti-proliferative activity, which is associated withthe transfer of heparan sulphate from the cell surface to the nucleus (253). This couldaccount for the relatively quiescent state of adherent hematopoietic progenitor cells. HL6Ocells, derived from a promyelocytic leukemia, have been shown to synthesize and expresson their surface excess amounts of the proteoglycan chondroitin sulphate (254). Moreover,neutrophil adherence in inflammation correlates with shedding of chondroitin sulphate andsynthesis of heparan sulphate (161), and platelet aggregation is inhibited by the expressionof chondroitin sulphate (255). Platelet heparitinase has been shown to be activated byfactors derived from metastatic tumor cells (256). Hence, changes in adherence in CMLcould correlate with changes in proteoglycan expression. Alterations in the adherence ofCML cells could also explain the finding that Ph- hematopoiesis overtakes Phhematopoiesis in long term marrow culture in some cases (257), since additional factorswhich may promote the survival of leukemic cells in the absence of normal adherentinteractions in vivo may not be completely reproduced in this culture system.1.2.4: Altemtions ofcytokine expression in leukemogenesis;Alterations in the expression of cytokines and their receptors have been described inAML. GM-CSF is secreted by AML blasts (258, 259), and it has been suggested thatautocrine stimulation may contribute to the growth of these cells. In contrast, circulatingleukocytes from patients with CML in chronic phase, accelerated phase, blast crisis, and43relapse were found to produce lower colony stimulating activity than cells from CML inremission or normal cells, as evaluated by a two-layered colony assay (260). However,the gene for IL-i was shown to be expressed in blast cells in almost all cases of CML inmyeloid blast crisis, which could initiate a paracrine mechanism of blast cell growth by theinduction of cytokine expression in accessory cells (261). In a separate study, IL- 1, IL-6and LIF were found to be expressed in long term marrow culture adherent layers derivedfrom a majority of patients in myeloid blast crisis, but were undetectable in those inlymphoid blast crisis, suggesting that a paracrine mechanism is indeed operative in thesecases. The authors also suggest that the involvement of stromal cells in growth stimulationin blast crisis may be related to the poor results of marrow transplantation in this phase ofCML (262). In contrast, stromal layers from normals and CML in chronic phase werefound to express G-CSF, and evidence for abnormal autocrine or paracrine mechanisms ofstimulation could not be found (263). Stromal layer expression of G-CSF was commonlylost during blast crisis (264). Involvement of the microenvironment in a paracrine fashionin the development of CML is also suggested by reports of CML developing in donor cellsin patients who had undergone allogeneic BMT (175, 265). Thus, a picture emerges of theoperation of relatively normal cytokine control mechanisms in chronic phase followed byparacrine stimulation in blast crisis, and progression to autocrine stimulation and fullgrowth factor independence in acute phase; this dysregulation could be involved instimulating the growth of the leukemic clone, at least in some cases.There are additional examples of the possible involvement of cytokines inleukemogenesis. The t( 1; 14) translocation in acute pre-B cell leukemia juxtaposes theimmunoglobulin heavy chain locus with IL-3 sequences. Transgenic mice expressing44cytokines (GM-CSF) or mice reconstituted with cytokine-expressing retroviruses showedchanges of a myeloproliferative nature, but did not develop leukemia (54). Introduction ofthe bcr-abl gene into many hematopoietic cell lines rendered them growth factorindependent and tumorigenic (162, 217). It is postulated that the full leukemic phenotyperequires alterations of genes regulating both growth, resulting in myeloproliferation, anddifferentiation, resulting in full transformation.Progression of CML was recently described to be marked by changes in themethylation pattern of the calcitonin gene (266). Abnormal methylation patterns werefound in few patients in chronic phase, but in a majority of cases of accelerated phase orblast crisis, in 90% of cases of acute leukemia (267), as well as in lung cancers,lymphomas, and colonic neoplasms (268, 269). The calcitonin gene is located on the shortarm of chromosome 11(1 ip), a region known to contain tumour suppressor genes such asthat associated with Wilms\u00E2\u0080\u0099 tumour. Loci adjacent to the calcitonin gene show abnormalmethylation patterns as well. Moreover, the authors report abnormally high expression ofthe DNA methyltransferase gene. In addition, different patterns of methylation in themyeloperoxidase gene were reported in AML (270). Undoubtedly more details of themethylation story will emerge in coming months.1.2.5: Other abnormalities in CML;Other abnormalities in CML usually apply to only a minority of cases. Anexception is increased erythropoetin receptor expression on CD-34 cells in CML, which45has been reported to occur in 5 of 5 cases examined (271). A novel receptor tyrosinekinase, axl, was isolated from CML cells and found to be transforming whenoverexpressed. The gene for this kinase was localized to chromosome 19; alterations inchromosome 19, particularly trisomy, are associated with progression to blast crisis in 18%of CML cases (272). Loss of p53 function was demonstrated in 25% of patients in clinicaltransition (144, 273). Activating ms mutations in CML have also been described, but in aminority of cases, and they usually occur late (274). Finally, the mRNA and precursorprotein of ITP-l, a short peptide \u00E2\u0080\u0098defensin\u00E2\u0080\u0099 involved in the formation of voltage-dependention permeability channels in target cell membranes was found to be overexpressed in CML(275). This observation may be related to the defective killing functions of CMLneutrophils observed in some cases (175), and also may provide a convenient marker forCML cells.1.2.6: Negative regulators ofhematopoiesis:It has been recognized for some time that the outgrowth of the leukemic clone inmyeloid leukemias, as in many malignancies, is not due to an inherently faster progressionthrough the cell cycle on the part of the malignant cells (1, 185, 276, 277, 278). In fact, asignificant proportion of the leukemic cells in a population may be quiescent, acharacteristic that makes these cells refractory to treatment with cell cycle-specific agents(185). Myeloid leukemias in some way produce conditions that inhibit both thedevelopment and the function of normal hematopoietic cells. In this way, the leukemicclone gradually gains dominance over normal cell lineages. How this is achieved is not46well understood, but inhibitors of normal hematopoiesis have been described, as well asfactors which support the growth of leukemic cells.Many negative regulators of hematopoiesis are being investigated in the context ofprotecting normal stem cells from cycle specific chemotherapy. These include AcSDKP, atetrapeptide originally isolated from fetal bovine marrow and shown to be a potent inhibitorof normal myelopoiesis (279). The effect of AcSDKP is mediated by prevention of theentry of stem cells in G0 or G1 into S phase. AcSDKP has no effect on leukemicmyelopoiesis (280). It has recently been isolated from human placenta (281). AcSDKPwas shown to increase the survival of mice treated with cytosine arabinoside (Am-C), acycle specific chemotherapeutic agent used in the treatment ofAML, and is entering clinicaltrials for this application (282).The pentapeptide pEEDCK, also known as HP5b or SP 1, is derived fromneutrophilic granulocytes. As a monomer, pEEDCK is suppressive to hematopoiesis andprotects CFU-S from Am-C induced cytotoxicity (283). Oxidation of sulfhydryl groupsresults in dimerization of the pentapeptide through the formation of a disulfide bond. As adimer, (pEEDCK)2 is stimulatory to hematopoiesis via stimulation of the production of asynergistic activity by stromal cells (284). A synthetic version of the dimer, SK&F107647 (Smith, Kline and French), has been synthesized, in which the disulfIde bond isreplaced by a dimethylene carbon bridge (285). Interestingly, the sequence EEDCK is partof the effector domain of G alpha proteins, leading to the suggestion that pEEDCK mayinterfere with signal transduction mediated by G alpha proteins (286).47Physiological inhibitors of normal hematopoiesis include transforming growthfactor-3 (TGF-f3), tumour necrosis factor-a (TNF-a), the interferons, and IL-b. TGF-13,like most physiological modulators of hematopoiesis, has pleiotropic effects on progenitorcells depending on their cell lineage, stage of differentiation, immediate microenvironment,and other cytokines present (26). TGF-13 is synthesized by most normal and neoplasticcells, but a major source is the blood platelets. In general, TGF-13 is inhibitory to primitivenormal progenitor cells while stimulating committed progenitors (69, 287). TGF-13 isreported to inhibit leukemic cell growth in addition (287). TNF-a is a 17 kDa cytokineproduced mainly by monocytes. It is released from AML cells (288), and is suppressive tothe proliferation of normal CFU-GM. The effects of TNF-a on colony formation, likethose of TGF-j3, are pleiotropic. In general TNF-a increases the responsiveness ofprimitive cells to other cytokines, while decreasing colony formation in cells of morerestricted lineage (289, 290).Interferon-gamma induces terminal differentiation in human leukemic cells whichare blocked in differentiation by overexpression of c-myc, thus inhibiting leukemic cellproliferation (71). Interferons generally have a suppressive effect on cell growth, which ismediated by the selective inhibition of expression of several mitochondrial gene products(70). The inhibitory effect of interferons, however, seems to be greater on leukemic cellsthan on normal cells, and their use in the treatment of CML is being investigated (291).The suppressive effects both of the interferons and of TNF-a on colony formation arereported to be mediated through direct actions on the progenitor cells. In contrast, thesefactors can stimulate myelopoiesis indirectly by enhancing the production and release of48stimulatory cytokines by accessory cells (292). Moreover, TNF-c has been reported toinduce the expression of receptors for IL-3 and GM-CSF on AML cells, which, in additionto its suppressive effect on normal CFU-GM, could confer a growth advantage onleukemic cells (288). TGF-13, in contrast, has been reported to downmodulate receptorsfor some growth factors on normal cells, which could account for its suppressive activity atleast in part (292).IL- 10 is a cytokine produced by monocytes, which is inhibitory to hematopoiesisthrough inhibition of the synthesis of stimulatory cytokines by monocytes, including IL-i,IL-6, IL-8, TNF-a, GM-CSF, and G-CSF (46). Interestingly, IL-lO has been reported tohave homology to the Epstein-Barr virus (EBV) gene BCRFI, suggesting that EBV mayhave captured this cellular gene and uses it to inhibit the immune response (27).Inhibin and activin are members of a family of proteins which include TGF-3.Inhibin and activin are most well known for their activities on the release of follicle-stimulating hormone (FSH) from pituitary cells, which they inhibit and stimulate,respectively. Inhibin and activin also inhibit and stimulate, respectively, the production ofCFU-GM, BFU-E, and CFU-GEMM through indirect effects which are mediated by Tlymphocytes and/or monocytes (292).Macrophage inflammatory protein-i alpha(MIP-ia or stem cell inhibitor Sd), is amacrophage-derived member of a family of heparin-binding proteins. MIP-lcx issuppressive to colony formation by primitive cells stimulated by optimal concentrations ofcolony-stimulating factors, but enhances colony formation when CSF concentrations are49suboptimal (293, 294). MIP-la is stimulatory to colony formation by more committedprogenitor cells, as are the related proteins MIP-113 and MIP-2. The inhibitory activity ofMIP- 1 a on primitive progenitor cells is direct, and can be blocked by MIP- 13 (292).Primitive hemopoietic colonies were inhibited by an extract from normal marrow (normalbone marrow extract, NBME), which may be identical to MIP-la, or Sc! (295). MIP-la,as well as TGF-13 and TNF-a are reported to be constitutively produced in bone marrowstromal cells, which may constitute a physiological mechanism by which the proliferationofhematopoietic stem cells is downregulated (263, 296).Lactoferrin is an iron-binding glycoprotein derived from neutrophilic granulocyteswhich inhibits hematopoiesis indirectly by decreasing IL-i secretion by monocytes (297).This effect can be overcome by the addition of IL-i, IL-6, or bacterial lipopolysaccaride(LPS). The blocking effect of LPS on lactoferrin-mediated suppression of myeiopoiesis ismediated through formation of a complex between LPS and lactoferrin. This complexbinds preferentially to the LPS receptor versus the lactoferrin.receptor (292). Defects in thequantity and activity of lactoferrin as well as decreased sensitivity to its suppressive effectson colony formation have been reported in patients with CML (292).H-ferritin, the heavy chain subunit of the acidic isoferritins, is suppressive tocolony formation by immature progenitor cells through a direct action on these cells. Theactions of H-ferritin are mediated by its ferroxidase activity, cycling cells require iron forproliferation, which is carried bound to transferrin in the blood in the ferric (Fe3) form.Release from transferrin requires conversion to the ferrous (Fe2) form, which is opposedby the ferroxidase activity of H-ferritin (292). Acidic isoferritin has been reported to50stimulate differentiation of normal neutrophilic granulocyte progenitors (298). Leukemiainhibitory activity (LIA) is identical to acidic isoferritin (299).Prostaglandin E2 (PGE2)has a growth inhibitory effect on normal CFU-GM (300),while myeloid leukemia cells are insensitive to this effect (301). This effect of PGE2 isindirect and is mediated through abrogation of the secretion of GM-CSF, M-CSF and GCSF by macrophages (27). In addition, both PGE1 and PGE2 are directly inhibitory toCFU-M. In contrast, through indirect actions on CD8 T lymphocytes, both PGE1 andPGE2can enhance the proliferation of BFU-E (292).Finally, a less well characterized inhibitor of myelopoiesis produced by normal cellsis a granulopoietic inhibitory activity (GIA) of > 100 kDa, which is produced byunstimulated lymphocytes (302).It has been suggested that the suppressive effects of these inhibitors may bemediated through the induction of a common secondary suppressor molecule. Bonemarrow and spleen cells from mice treated with lactoferrin, H-ferritin, or PGE all release an8 kDa molecule which suppresses colony formation in vitro in a manner similar to MIP- 1 a,but which is distinct from MJP-la. This molecule is being characterized (292).It has been known for some years that myeloid leukemia cells secrete factors thatare inhibitory to normal colony formation (303). In contrast, factors from lymphoidleukemias have been reported to enhance normal colony formation (304). Severalleukemia-derived factors inhibitory to myelopoiesis deserve mention. As discussed, TNF51c\u00E2\u0080\u0099. is secreted by some AML cells, which may confer a growth advantage on the leukemiccells, as TNF-a is inhibitory to normal myelopoiesis in some circumstances. Leukemiaassociated inhibitor (LA!) is a 125 kDa polypeptide which causes reversible suppression ofnormal CFU-GM formation by blocking the cell cycle in S phase. LA! has no effect oncolony formation by leukemic cells (299). A factor isolated from the culture medium ofHL6O cells suppresses activation of normal lymphocytes but does not affect colonyformation by GM-CFC (305). This observation may have implications for the disruptionof immune surveillance in leukemia. A factor in the sera of AML patients was found tosuppress both natural killer cell activity and lectin-induced cellular cytotoxicity (306). Tlymphocytes of AML in remission have been described to inhibit granulopoiesis (307).Finally, one report describes disruption in the ability of normal CFU-S to bind stroma aftertreatment with sera from CML patients (308).Inhibition of normal myelopoiesis is one mechanism by which the leukemic clonecan gain dominance over normal cells; another is stimulation of leukemic myelopoiesisitself. In addition to the paracrine and autocrine mechanisms described in the previoussection, which could promote outgrowth of the leukemic cells, one factor which stimulatesleukemic myelopoiesis deserves mention. Myeloblastin is a recently cloned serine proteasethat was isolated from the azurophilic granules of human neutrophils. It has been shown tobe identical to proteinase 3, the autoantigen in Wegener\u00E2\u0080\u0099s granulomatosis (309). This is adisorder in which neutralizing antibodies are produced against myeloblastin, resulting ingranulomatous lesions. Downregulation of myeloblastin has been shown to result in thegrowth arrest and differentiation of HL6O (310).52In contrast to these inhibitory effects on hematopoiesis, leukemia inhibitory factor(LIF, or human interleukin for DA cells, HILDA) alters hematopoiesis in the oppositedirection. LIF induces the monocytic differentiation of Ml, a murine leukemic cell line. Inaddition, LIF suppresses the differentiation and maintains the proliferation of embryonicstem cells. The use of LIF in promoting the survival of retrovirally transfectedhematopoietic stem cells for gene therapy is under investigation (84). LIF has beenreported to be constitutively expressed by stromal layers in long term culture (262), and tostimulate the development of multipotential progenitor cells (311). Oncostatin M and IL-6,related proteins, have similar actions on Ml cells (85).In addition to these factors, it must be recognized that many cytokines, singly or incombination, exert a variety of effects on cells which could influence outgrowth of theleukemic clone. However, other than the involvement of P210 bcr-abl in CML, and thet( 15; 17) translocation in acute promyelocytic leukemia, few consistent changes have beendescribed which clearly influence leukemogenesis; this is in part due to the greatcomplexity involved in the normal regulation of hematopoiesis, which is far fromcompletely understood. Hence, much work in this area deals with phenomena rather thanprecise information. Deviations from normal, then, have been difficult to characterize.531.3: Therapy;Current therapies for myeloid leukemias are briefly described in this section.Included is a discussion of traditional chemotherapy, as well as investigational therapiessuch as bone marrow transplantation, the use of recombinant cytokines, immunologicallybased therapies, and molecular techniques. The success rate and limitations of allogeneicand autologous bone marrow transplantation are described in addition to techniques andagents under investigation for purging residual leukemic cells from autografts. This sectionis intended to give the reader a sense of the inadequacies of current treatments for myeloidleukemias and the importance of gaining a clearer understanding of how these leukemiasdevelop in order to treat them more effectively.1.3.1: Chemotherapy;The treatment of leukemia with traditional chemotherapy provides little more thansymptomatic control (179). Intensive chemotherapy in acute phase can induce remission,or re-establish chronic phase, but relapse is inevitible in most cases, with the exception ofchildhood ALL, in which permanent remissions are achieved. Long-term survival inchildhood ALL is achieved in approximately 50% of cases using chemotherapy (312).Chemotherapy for AML, however, results in long-term survival in only 15 to 35% of cases(313). Toxicities of chemotherapeutic agents are unpleasant, and in many cases arerelated to cell cycle specific effects. These include disruption of the mucosa of thegastrointestinal tract, and marrow ablation with its accompanying neutropenia and increased54risk of infection, as well as thrombocytopenia (decreased platelet counts) with increasedrisk of bleeding. Difficulties with efficacy such as multi-drug resistance are alsoencountered (314), hence combinations of drugs are often used. In many cases, toxicity isdose limiting (175), and new approaches are being evaluated for improving the delivery ofchemotherapeutic agents. These techniques include delivery via liposomes, which reducestoxicity (315), and delivery of toxins linked to monoclonal antibodies (316). It is unclear,however, whether increasing chemotherapeutic doses in this way will result in increasedcures (317). Many chemotherapeutic agents, for instance, are cell cycle specific, however,leukemic cells do necessarily proliferate more rapidly than normal cells, and substantialproportions of the leukemia cell population may be out of cycle at any given time (278).Cytokines are being used clinically in an attempt to increase the fraction of leukemic cells incycle and render them more sensitive to chemotherapy (see below). \u00E2\u0080\u0098Whether this effectwill result in increased efficacy of chemotherapeutic agents is under evaluation; normalcells might be expected to be forced into cycle and rendered susceptible to cytotoxic effectsas well.Chemotherapeutic agents commonly used in the treatment of CML includebusulfan, a bifunctional alkylating agent, and hydroxyurea, an inhibitor of DNA synthesis(175). Toxicities of these treatments, especially busulfan, include prolonged marrowaplasia and pulmonary damage.One problem with chemotherapy involves the development of resistance. Multidrugresistance (mdr) is mediated by the P-glycoprotein or p170, an energy-dependentmembrane pump which pumps out several structurally unrelated naturally derived cytotoxic55agents such as doxorubicin and etoposide, drugs used in the treatment of acute leukemias(314). The promoter of the mdr-1 gene has been shown to be a target for the c-Ha-ras-1oncogene and the p53 tumour suppressor genes, both associated with tumour progression(318). It has been suggested that targetting P170 with immunotoxins may be useful inpurging resistant cells from the marrow prior to autologous BMT (319). P170 may beinvolved in the secretion of peptides or cellular proteins during normal cell metabolism(320).Most patients achieving complete remission using chemotherapy for myeloidleukemias eventually suffer a relapse. As it has been estimated that patients in remissionharbour a burden of 108 to lO leukemic cells, this is not surprising (185). Long-termsurvival following chemotherapy for AML is only 15 to 35% (313). In CML,chemotherapy does not result in cures (321). For the majority of adult leukemias, thepossibility of cure is currently thought to be limited mainly to allogeneic bone marrowtransplantation (188).1.3.2: Aiogeneic bone marrow tmnsplantation;Of leukemia patients, only about 25% are eligible for allogeneic bone marrowtransplantation (322). Half of patients are considered to be too old; allogeneic BMT iscurrently limited to patients under the age of 50 (323). Best results are achieved withpatients under 30 years of age as the risk of complications, particularly graft versus hostdisease (GVHD) increases with age beyond this point. Five year survival rates following56allogeneic BMT are 50 to 60% for patients under 20 years of age, and only 20 to 30% forthose over 40 (312, 322). Of the half of patients young enough for BMT, an appropriateHLA matched donor will be found for 50%; a related donor will be found for one third ofthese, or 15% of patients overall, and an unrelated donor for one fifth, or 10% overall(179, 324). Patients are treated with high dose myeloablative chemotherapy and often totalbody irradiation, subsequently their hematopoietic and immune system is reconstitutedmostly with transplanted donor cells, thus, they become permanent chimeras. In additionto killing many of the leukemic cells, the conditioning regimen has been reported to aidengraftment by disrupting the bone marrow endothelium, which the engrafting cells musttraverse to reach the marrow (325). Well-known toxicities of total body irradiation (TBI)include gastroenteritis, mucositis, myelosuppression, and alopecia (313).Complications of allogeneic BMT include failure to engraft, and graft versus hostdisease (GVHD). GVITD is an unpleasant complication which manifests in two forms,acute and chronic. Acute GVHD involves inflammatory destraction of epithelial cells in theskin, gastrointestinal tract, and liver, and is mediated by CD8 T lymphocytes. It developswithin the first few weeks post-BMT. Chronic GVHD is characterized by increasedcollagen deposition resulting in fibrosis. It is mediated by CD4 T lymphocytes, anddevelops later, usually after 6 months. It may be preceded by, and be continuous with,acute GVHD (326). Complications of chronic GVHD include epidermal atrophy andcontractures. In an attempt to avoid GVHD, some allogeneic grafts were depleted of Tcells. It was found, however, that although T cell depletion decreased the incidence ofGVHD (20% vs. 40% in non-depleted grafts), the incidence of relapse increased (60% vs.20%) and survival rates decreased (20% vs. 55%) (179). This led to the recognition that a57significant graft versus leukemia (GVL) effect is present in non T cell depleted grafts.Moreover, T cell depletion led to an increased incidence of graft failure or rejection (326).In addition, it is known that there is a higher incidence of relapse in patients receivingtransplanted cells from identical twin donors (188, 239). These observations illustrate theimportance of an anti-leukemia immune effect in successful BMT for CML. GVHD iscurrently treated using immunosuppressive therapy, including steroids, methotrexate, andcyclosporine (312, 327, 328). Recently, the well known mutagen thalidomide has beenused to treat chronic GVHD resistant to conventional therapies (329, 330). Othercomplications following BMT include cytomegalovirus (CMV) infection, either from CMVreactivation or from viral infusion with donor cells, and hepatic toxicity (313).Leukemic relapses occur in 20 to 25% of patients receiving allogeneic BMT overall,and is higher in patients receiving T cell depleted grafts, as discussed (175, 185).Engraftment and the incidence of relapse may be related to the number of donor cellstransplanted. It was recently shown that both the quality and the quantity of stem cells indonor marrow are important for engraftment; significant numbers of host cellsreconstituted long term hematopoiesis in lethally irradiated mice transplanted with lownumbers of syngeneic marrow cells. The syngeneic cells presumably supported survival inthe short term until the host hematopoietic system could recover (10, 13). It may bepossible to exploit this observation for the treatment of leukemias arising in committedprogenitor cells. Low numbers of allogeneic cells could be transplanted in order toreconstitute immunity in the short term, until clones arising from primitive host cells areable to recover. In this way, it may be possible to avoid long term GVTID (10). This kind58of therapy, however, would be unrealistic in CML, since the leukemic defect occurs in avery primitive stem cell.An additional complexity to consider in allogeneic BMT is the fmding that there arestrain-specific differences in the ability ofhematopoietic cells to contribute to short term andlong term reconstitution (331, 332). Moreover, colony formation and DNA synthesis inbone marrow cells were demonstrated to be circadian stage dependent and to show seasonalvariation (333). In addition, successful engraftment in experimental animals was blockedby the administration of anti-class II antibodies (334). Thus, many factors need to beconsidered to maximize the possibility of successful reconstitution, including the number ofcells transplanted, the HLA type of the patient and donor, and, apparently, the time of dayand year at which donor marrow is harvested.Survival rates following allogeneic BMT for the treatment of CML appear tocorrelate to the clinical phase in which the patient underwent transplantation. Two to 3 yearleukemia-free survival rates for patients transplanted in chronic phase are in the range of 40to 70% (175, 188). In contrast, three year survival in patients transplanted in acceleratedphase in one study was 36%, and 12% in acute transformation (188). Moreover, patientsundergoing BMT in accelerated or blastic phase have a very high incidence of leukemiarelapse; 30 to 60% for accelerated phase, and 40 to 90% for blastic phase, as compared to10 to 20% in chronic phase (188, 239). Mortality is strongly correlated with the patient\u00E2\u0080\u0099sage.59Leukemia-free survival at 5 years following allogeneic BMT for the treatment ofCML is 50 to 60% for patients receiving marrow from an HLA-identical sibling. Patientsreceiving marrow from an HLA-matched unrelated donor have a 35% leukemia freesurvival at 3 years, which correlates to the patients\u00E2\u0080\u0099 age and the degree of matchingbetween donor and recipient (239). The three year clinical relapse rate in CML followingallogeneic BMT is 20% overall, while the cytogenetic relapse is up to 35% (175).Cytogenetic studies revealed that recurrence of CML was in the original clones, indicatingthat most failures are due to ineffective eradication of residual leukemic cells.Sources of stem cells other than bone marrow for transplantation are underevaluation. These include peripheral blood; several collections by means of apheresis arethought to be sufficient for transplantation, and the pool of progenitor cells can beexpanded by exposure to recombinant cytokines (335, 336). A series of peripheral blooddonations can be supported by the administration of erythropoietin (337). In addition, theuse of umbilical cord blood, which is highly enriched for progenitor cells is beinginvestigated (338). It is thought that enough stem cells are present in the blood from oneumbilical cord to engraft an adult, particularly when the stem cell pool is expanded usingcytokines (339).Although the toxicities and morbidity of allogeneic BMT are significant, thistherapy at least provides a possibility of cure. For example, BMT performed in firstremission in AML has a 10 year disease free survival rate of 50%. While remissions areinduced with chemotherapy in a majority of cases of AML, the long-term survival rate afterconsolidation treatment, depending on the study, ranges from a mere 10 to 15% (323) to 1560to 35% (313). Unfortunately, allogeneic BMT, as discussed, is limited to a minority ofpatients due to age or lack of a suitable donor, or in other countries, due to its cost.1.3.3: Autologous bone marrow tmnspiantation (ABMT), purging;Patients ineligible for allogeneic BMT may be considered for autologous BMT(ABMT). In ABMT, the advantages of purging leukemic cells from the graft are obvious.For the first 6 months to one year following ABMT many patients harboured cells in whichexpression of the bcr-abl message could be detected, as evaluated by the polymerase chainreaction (PCR), and significant numbers of patients remained Ph by cytogenetic analysis.After this time many progressed to PCR or Ph negativity (265, 340, 341). This is anindication that some leukemic cells can be tolerated and overcome by the body, however, itis not known what levels are dangerous. Limited success in ABMT of unpurged cells hasbeen achieved, due to the apparent greater sensitivity of leukemic cells to cryopreservation,however, the majority ofpatients transplanted in this way suffer leukemic relapse (342).The high rates of leukemic relapse in ABMT for CML may be related to thepossibility that the graft versus leukemia effect is lower with autografted cells than usingallogeneic grafts, which points to the importance of immune effects in controlling theleukemic cells. Although the incidence of GVHD is much lower in ABMT, it does occur ina minority of patients, and this is correlated with an improved prognosis (239). Leukemiafree survival at two years following ABMT for CML is currently a mere 10% (239).61Various agents are being evaluated for their efficacy in purging leukemic cells whilesparing normal cells. CML is a convenient model system for studying the efficacy of theseagents, as residual cells can be detected by PCR analysis for the presence of the bcr-abltranscript. However, CML treated by autologous BMT has a poor prognosis compared toother leukemias, due to the primitive nature of the cell from which it is derived, and thedifficulty in eradicating this cell while sparing enough normal stem cells to reconstitutehematopoiesis. Some specificity has been shown for cells of other leukemias using agentssuch as 4-hydroperxycyclophosphamide (4-HC), the cyclophosphamide derivativemafosfamide (ASTA-Z, reference 343), and Merocyanine 540 (MC-540), aphotoactivatible drug, in combination with light. In addition, hyperthermia is reported toaffect AML CFU-GM to a greater extent than normals (344).4-HC has been used to purge autografts in ANLL. Primitive stem cells, however,are resistant to 4-HC, limiting its use in CML (345). Although it is toxic to normal cells inaddition to malignant cells, a difference in kill of 2 logs was achieved using 4-HC for thetreatment of lymphoma. In one study, the probability of relapse at two years followingABMT for AML in first complete remission was 35% using marrow purged with the 4-HCrelated drug mafosfamide, as compared to 47% using unpurged marrow (346). Using thephotoactivatible drug MC-540 plus light, it was possible to reduce the clonogenicity ofHL6O cells by 4 logs, while one third of normal colony forming cells were spared (347).More recently, promising work has been done using the photoactivatible agentbenzoporphyrin derivative (BPD). BPD has been shown to be taken up preferentially byleukemic cells, and treatment of cells with BPD plus light results in a greater than 4 logreduction in leukemic cells. In contrast to Merocyanine 540, BPD shows no toxic effects62toward colony formation by normal cells at doses which are therapeutic in a murine model,and at some levels appears to be stimulatory to normal colony formation (348).Improvement in survival in BPD-purged grafts has also been demonstrated in a murinemodel using L1210 leukemia cells (349).A variety of novel techniques are being evaluated in order to improve purging forABMT. Cytokines are being tested in combination with various purging andchemotherapeutic regimens in an attempt to force leukemic cells into cycle and improve cellkill. Induction of proliferation in AML cells by GM-CSF or IL-3 has been reported toenhance the cytotoxicity of the cell cycle specific drug cytosine arabinoside (Ara-C,reference 350). Immunomagnetic beads coated with anti-CD 10 antibodies have been usedto purge CD 10 (CALLA) positive common acute lymphoblastic leukemia cells (351).Similar strategies have been used to remove metastatic breast cancer (352) andneuroblastoma cells (313) from autografts. Finally, AML and CML cells were shown tobe differentially sensitive to the inhibitory effects of c-myb antisenseoligodeoxynucleotides, and bcr-abl expressing cells were completely eradicated at levelsthat spared normal progenitor cells (353). Antisense oligodeoxynucleotides may prove tobe useful in purging marrow ex vivo. Difficulties in the evaluation of purging protocolsinclude the inability to detect minimum numbers of residual malignant cells due to thelimitations of the detection procedures (313, see below).There have been reports of the establishment of Ph- hematopoiesis in long-termmarrow cultures derived from CML marrow, and suggestions that patient\u00E2\u0080\u0099s cells can bepurged for autografting in this way (257, 342). However, other groups have reported that63this occurs in a minority of cases, and is dependent on the preconditioning regimen (354).Busulfan has long-term effects on progenitor cells compared to hydroxyurea, which is anobstacle to long-term culture techniques with CML cells (312). In other studies, CFU-GMfrom CML LTMC initiated both from CD34CD33 and CD34CD33 cells were found tobe predominantly clonally derived (355). In addition, the number of CD34CD33- cells,the cells important for long term reconstitution, were found to decline rapidly in LTMC(356). Combination of this method with other methods of purging, however, may be apromising therapeutic alternative in selected cases. Normal cells have also been reported togrow selectively in cultures derived from ALL and ArVIL (355, 357).1.3.4: Cytokines in leukemia thempy;The use of cytokines for the treatment of leukemias is being examined in severalcontexts. These include speeding the recovery ofhematopoiesis following myeloablativetherapy, increasing the fraction of leukemic cells in S phase in order to increase cell kill bychemotherapeutic agents, forcing the differentiation of leukemic cells in an attempt toextinguish the leukemic clone, and enhancing the anti-leukemia immune response (317).Both chemotherapy and BMT are followed by marrow and immune suppression.This includes neutropenia, with an increased risk of infection, as well asthrombocytopenia, or decreased megakaryocytes and hence platelet counts, with increasedrisk ofbleeding. Patients must be kept isolated in sterile rooms until their neutrophil countsare restored to acceptable levels. Half of deaths following BMT are due to infective64complications during severe myelosuppression (358). The use of various recombinantcytokines in speeding restoration of hematopoietic function is under investigation. GMCSF and G-CSF are currently widely used in conjunction with BMT and chemotherapy,and result in fewer neutropenic days post-BMT and a reduction in complications (27, 185,358, 359, 360). GM-CSF has been used to promote recovery after graft rejectionfollowing allogeneic BMT, which occurs in 2% of cases (358). In addition to speedingrecovery of neutrophil counts, G-CSF has also been reported to improve platelet recoveryafter chemotherapy (361). Both GM-CSF and G-CSF also activate the effector functionsof mature neutrophils. Side effects occurring with GM-CSF include fever, myalgias,anorexia, bone pain, fluid retention, pericarditis, and pleural effusions. These effects,however, are generally found to be tolerable. GM-CSF also supports the development ofeosinophils and basophils, which could lead to allergic complications. The systemic sideeffects seen with GM-CSF may be related to its induction of TNF-ci and IL-i (30).Pentoxifylline, a TNF antagonist, when administered in conjunction with GM-CSF, wasreported to decrease the GM-CSF induced pulmonary sequestration of neutrophils and thuspreserve neutrophil migration to sites of infection (362). G-CSF is well tolerated, themajor side effect being bone pain, and occasional reactions at the site of injection (53). GCSF and GM-CSF were both recently approved for use in the United States in conjunctionwith myelosuppressive therapy for non-myeloid malignancies (59).Combination with other cytokines such as IL-i, IL-3, and Steel factor (SLF) maypotentiate the actions of GM-CSF and G-CSF. IL-3 was shown to improve the recoveryof platelets and reticulocytes, as well as neutrophils. IL-3 administered sequentially withGM-CSF acted synergistically to stimulate myelopoiesis (185). IL-i caused a significant65improvement in platelet recovery. It was found to be well tolerated at low doses, althoughorgan toxicities occurred at higher doses (363). SLF has been shown to stimulatehematopoiesis in vivo in primates (364), and in mice in combination with G-CSF, where itincreased the number of cells of all lineages (365). Due to its documented actions on mastcells (100), it seems possible that SLF could increase the risk of allergic reactions andanaphylaxis, however, this has not been reported. Finally, activation of the anti-tumoureffects of macrophages using M-CSF is being investigated (366).Cytokines are being used in conjunction with chemotherapeutic agents in an attemptto increase the cycling of leukemic cells and thus their susceptibility to cycle specificchemotherapy. GM-CSF administered in conjunction with Am-C increased the S phasefraction of AML myeloblasts (367). Similarly, exposure to Am-C in the presence of IL-3,GM-CSF, and G-CSF resulted in preferential kill of leukemic versus normal clonogeniccells (368). In contrast, other groups report that GM-CSF and IL-3 protected AML blastsfrom Ara-C toxicity (369). It has been suggested that the susceptibility of leukemic cells tocytotoxic agents may depend on the fraction of cells undergoing self-renewal vs.differentiation rather than corresponding to the fraction of cells in S phase (370).Leukapheresis was formerly used to decrease the burden of leukemic cells, and isoccasionally still used to prevent vaso-occlusive complications. Like cytokines,leukapheresis is now being used to induce cell cycle changes in order to increase theefficacy of cycle-specific chemotherapy (371).It remains to be determined whether the use of cytokines will increase the risk ofleukemic relapse by preferentially stimulating the proliferation of residual leukemic cells66(27). Although overexpression of cytokine genes when transfected into cellsexperimentally is generally not found to be transforming (54, 59), it is possible thatcytokines may aid in leukemic outgrowth. For example, GM-CSF and IL-3 are known tostimulate the proliferation of AML cells in vitro in at least some cases (55, 185). AMLcells were also found to proliferate in response to SLF in combination with other cytokines(372), and in response to IL-3, GM-CSF, and G-CSF alone or in combination (373).Both of these studies, however, found considerable variation in the response of cells fromdifferent patients to growth stimulation by cytokines. Also, the regrowth of AML cellsstimulated by GM-CSF treatment was found to be reversible on withdrawal of GM-CSF(374). In still other studies, GM-CSF and G-CSF were shown to decrease the growth ofAML cells by increasing differentiation (375). It therefore seems difficult to predict whateffect cytokine therapy will have in a particular patient or in a particular leukemia subtypewithout an increased understanding of the biology and biochemistry of the leukemic cells.Cytokines inhibitory to leukemic cells are also being tested. One group reports longterm inhibition of tumour growth by tumour necrosis factor (TNF) in the absence ofcachexia, indicating a therapeutic window for possible clinical exploitation (376). Inaddition, cytokines inhibitory to normal, but not leukemic cells, such as transforminggrowth factor-13 (TGF-3), are being used in an attempt to protect the normal cells from theeffects of myeloablative therapy (185). Finally, it was shown that the grafting efficiency ofdonor cells in BMT could be improved by treatment of the donor cells with IL-3 and GMCSF (377).671.3.5: Alterations ofadhesive interactions- interferon-a in CML;Interferon-a (ifn-cL) shows selective growth inhibition toward CML CFU-GM ascompared to normal cells (378), and induces complete hematological remissions in amajority of patients with previously untreated CML in chronic phase, often with partial orcomplete suppression of the Ph clone (379). The mechanism of action of this effect isunder investigation, and involves at least in part alterations of the adhesive interactionsbetween hematopoietic and stromal cells. Greater numbers of CML CFU-GM were foundto locate in the adherent layer of ifn-a - treated LTMC, suggesting a reduction in thenumber of CFU-GM in active cycle (291). Of possible relevance is the finding that ifn-areduces phosphorylation of P210 bcr-abl during differentiation of a CML cell line, anindication that the activity of bcr-abl may be affected (380). Class II MHC antigenexpression, which is reduced on CML cells, is enhanced by either ifn-a or ifn-y, whichalso enhance the diminished natural killer (NK) cell activity observed in CML (175).Significant suppression of the malignant clone, however, occurs in only a minority ofpatients using interferon alpha or gamma singly or in combination (381). The use ofinterferons in combination with chemotherapeutic agents is being investigated.1.3.6: Lymphokine activated killer cells;Following the pioneering work of Rosenberg, who used IL-2 and lymphokineactivated killer cells (LAK) cells to treat solid tumours (382), LAK are being generatedtoward CML cells (383). NK cell activity in CML is generally reduced, however,68significant numbers of LAK cells could be generated ex vivo which were cytotoxic toK562, a CML-derived Ph cell line. The LAK cells generated in this way were not derivedfrom the leukemic clone despite considerable contamination of the starting culture withleukemic cells. Selective recognition ofAML cells by LAK appears to be mediated by thedifferential expression of adhesion molecules by normal and leukemic cells (384).Experience with solid tumours, however, showed that it was necessary to administer IL-2to patients in addition to the LAK cells in order to obtain a significant anti-tumour effect.Side effects were sometimes severe, and treatment in many cases had to be discontinued(382). However, this approach may prove to be a useful adjunct to other therapies, forexample in ridding the body of residual leukemic cells following BMT. This approach wasused successfully by one group in managing a variety of leukemias, and the amount oftarget cell lysis was improved by exposing the cells to IL-2 and TNF-cx (385). IL-2administered intravenously has been found to induce the expression of a variety of positiveregulators of hematopoiesis, while failing to induce the expression of negative regulators,hence a net stimulatory effect was obtained (386). Interestingly, however, one of the sideeffects of IL-2/LAK therapy was suppression of hemopoiesis; LAK were found to havecytotoxic activity against normal CFU-GM. This effect was shown to be mediated bysoluble factors, possibly interferon-y or TNF-c, and was abolished by the inhibition ofLAK DNA synthesis using irradiation or hydroxyurea, without affecting cytotoxicitytoward tumour targets (387).691.3.7: Induction ofdifferentiation- retinoic acid in acute promyelocytic leukemia;Another promising avenue of therapy is the induction of differentiation. Aconsistent abnormality in AML M3, or acute promyelocytic leukemia (APL) is the t( 15;! 7)translocation, which occurs in 70-90% of cases (388, 389). This translocation wasrecently shown to juxtapose the myl locus within the retinoic acid receptor-ct (RAR-ct)coding sequence (388), resulting in a fusion transcript and protein. This finding shedslight on the good clinical response ofpatients with APL to treatment with all-trans-retinoicacid; 37 out of 46 patients were reported to achieve complete hematological remissionsusing this therapy (185). It is thought that high levels of retinoic acid restore relativelynormal function to the abnormal fusion protein (148). Unfortunately, remissions inducedusing retinoic acid are only temporary, however, this is an intellectually gratifying exampleof a correlation between clinical efficacy and biological understanding (163).It has been found that some established treatments in fact act by inducing thedifferentiation of leukemic cells and cell lines; examples are low doses ofmethylprednisone (390), Ara-C, hydroxyurea, interferons (175), and tumour necrosisfactor (391). Others, rather than having directly cytocidal activity, induce programmed celldeath (apoptosis) in the malignant cells (225). GM-CSF and G-CSF have also beenreported to induce the differentiation of AML cells, although in some cases maturation isincomplete and subpopulations of leukemic cells appear to escape the differentiating effect(392, 393). Other compounds that have been reported to induce the differentiation of thePh cell line K562 are the well known mutagens thalidomide and its metabolites (394) andethidium bromide (395). Retinoic acid may be useful in the treatment of CML in70promyelocytic blast crisis (396). 1,25-dihydroxyvitamin D3 is known to cause thedifferentiation of HL6O, a cell line derived from an acute promyelocytic leukemia (397).This effect is accompanied by the dephosphorylation of specific proteins (398), and isthought to involve cAMP-dependent protein kinase (399). Finally, it should be noted thatpart of the therapeutic effect obtained in unpurged transplants of autologous marrow maybe derived from the forced differentiation of leukemic cells by dimethylsulfoxide (DMSO),in which the cells are cryopreserved (313).1.3.8: Immunotoxins;One group has reported the use of an anti-CD33 monoclonal antibody linked to thetoxin ricin for purging in AML. This approach is possible since clonogenic AML cellsexpress CD33 in 80% of cases. The ricin B-chain was blocked, in order to prevent nonspecific binding to galactose residues of cell surface molecules. Using this Ab, a greaterthan 4 log selective kill of clonogenic CD33 AML cells mixed with an excess of normalmarrow cells was achieved (316). When used for purging autografts, sustainedengraftment occurred in all cases, but neutropenia was prolonged due to the removal ofCD33 lineage-committed progenitors (316). It is possible to shorten this period totolerable levels using cytokine therapy (400).711,3.9: Techniques for targetting bcr-abl;Transforming abl proteins, including P210 bcr-abl, P190 bcr-abl, and P160 gagabi were found to have a higher affinity for ATP and synthetic tyrosine containingsubstrates than did c-abl, and for tyrosine kinase blockers of the tyrphostin family. Theseobservations raise the possibility that specific abl kinase inhibitors could be designed thataffect only transforming abl (401). Similarly, it has been possible to induce both theformation of antibodies and T cell immunity directed toward the joining region of P210 bcrabl (402, 403). The significance of these observations is unclear, since bcr-abl is anintracellular protein, and thus presumably inaccessible to immune reactions.1.3.10: Molecular biological techniques;Polymerase chain reaction (PCR) amplification of transcripts is used in diagnosisand in the detection of minimum residual disease following therapy (404, 405, 406, 407,408, 409, 410). One in 10 to 106 tumour cells can be detected using this method whereascytogenetic techniques detect the Ph chromosome at levels of 1 in 10 to 1 in 100 (265,405). Interestingly, a group of patients that were Ph post-BMT had a higher incidence ofsevere chronic GVHD than did Ph- patients, presumably an indication of an active GVLeffect (340). It should be kept in mind that of 1012 nucleated bone marrow and blood cells,106 to 10 leukemic cells could be present below the limits of detection of PCR (411).Similarly, a burden of 1010 to 1011 leukemic cells would be just at the threshold ofdetection of cytogenetic analysis (265). It has been suggested that in some cases72cytogenetic techniques may be more sensitive than PCR for detecting the presence ofresidual CML cells, since the bcr-ablfusion gene may not be continually transcribed (239).Useful PCR-based techniques are being developed for facilitating diagnosis in neoplasia,examples are fluorescence in situ hybridization (FISH), and the detection of mutations inp53 in tumour cells identified on the basis of immunohistochemistry; the cells aresubsequently removed from the slide for molecular analysis (412).Approaches to inhibiting leukemic cell growth based on molecular biologicaltechniques are under investigation. As discussed, antisense oligodeoxynucleotides to cmyb, a phosphorylation dependent DNA binding factor, and the related B-myb, wereshown to inhibit the proliferation of myeloid leukemia cell lines (413) and CML colonyformation (413, 414). Analysis of residual colonies by PCR for bcr-abl message showedgreatly reduced signal, and replating of these colonies resulted in the formation of normalcolonies only (415). Interestingly, the promoter region of the bcr-abl gene has at least oneputative myb binding site (213). Similarly, bcr-abl antisense oligodeoxynucleotidesselectively inhibited CML CFU-GM leaving residual normal colonies (416). Theseobservations are likely to find application in purging for autologous BMT.It may be possible to apply some of these observations to gene therapy. Forexample, the autograft could be transfected with a retroviral construct expressing antisensebcr-abl message, which should selectively target the leukemic clone in CML. To date,gene therapy has been successfully used in the treatment of adenosine deaminasedeficiency, a genetic disorder, in a small number of cases (417). In addition, experimentalanimals have been reconstituted with tumour infiltrating lymphocytes transfected with a73TNF construct. Although these had significant anti-tumour effects, they also showedsignificant systemic toxicity, such as cachexia (113). Genes lost in tumour progressionsuch as p53 or Rb could be replaced by gene therapy (418). Methods by which to increasethe efficiency of gene transfer are being investigated; these include supporting the survivalof primitive hematopoietic stem cells using leukemia inhibitory factor (HF, references 83,84), and stromal feeder layers (419, 420), and increasing the efficacy of gene transferusing liposomes (315). Retroviral transfection of transplanted cells has also been used totrack the source of relapse after ABMT for AML and to evaluate the efficacy of purging(421).1.3.11: Positive selection ofstem cells;Recently, interest has focussed on separating primitive stem cells from the totalleucocyte population on the basis of the expression of CD34. These cells, which comprise1 to 3% of the normal bone marrow cell population, can then be used to reconstitutehematopoiesis following BMT. Purification of CD34 cells proved to be more difficultthan anticipated, due to difficulties in recovering cells without destroying the CD34 antigenor the cells themselves. Initial protocols involved the use of anti-CD34 antibodies coupledto immunomagnetic beads. This required incubation to remove beads from the cells, whichresulted in capping and antigen turnover. Similarly, releasing the cells with proteolyticenzymes resulted in removal of CD34, and homing antigens, from the cell surface.Panning was also used, but resulted in low yields, with significant contamination by CD34cells. Fluorescence activated cell sorting of leucocytes provided pure populations, but was74too slow to isolate the numbers of cells required for transplantation. One group succeededin enriching for CD34 cells using avidin-biotin affinity column chromatography, with 25to 85% yields and 35 to 92% purity (422). Another group enriched for CD34 cells usingimmunomagnetic beads, from which the cells were released by cleavage with aglycoprotease from Pasteurella haemolytica , which recognizes 0-sialoglycan structures.Yield and purity were reported to be 90-95% and 94-98%, respectively. It is reported thatneither the functional competence of the cells nor the levels or distribution of surface CD34were affected (423). Stem cells enriched in this way can be rid of leukemic cells and usedfor transplantation. Transplantation of these cells results in long term engraftment due totheir primitive nature. Several patients have undergone transplantation for breast cancermetastatic to the bone marrow using this method with successful engraftment in 100% ofcases (424).Although it is thought that some leukemic cells can be tolerated by the body withoutresulting in clinical relapse, the CD34 population is an important population in which toensure removal of leukemic cells, since this is the population which supports long-termhematopoietic reconstitution. This is especially true of CML, since the genetic lesion inCML is thought to arise in an earlier stem cell than in most other leukemias. Althoughsome patients with CML showed Ph hematopoiesis following BMT, which became Phover time (265), it is unknown what levels of leukemic cells can be tolerated and overcomeby the body, and the presence of stem cells harbouring the leukemic phenotype clearlyincreases the likelihood of relapse. Positive selection of stem cells may be coupled withone of the purging techniques outlined previously. Since it is a fraction of the totalleucocyte population, fewer leukemic cells would need to be removed from the CD34+75population, and presumably the chances of successful purging of residual cells should beincreased. Promising results from one laboratory indicate that the majority of CD34HLA-DR cells give rise to Ph colonies, whereas CD34HLA-DR- cells give rise to Ph- colonies(425). Positive selection for CD34 followed by negative selection for HLA-DR may thusprove to be a useful protocol for ridding autografts of leukemic cells in autologous BMTfor CML. Unlike protocols which rid the graft of CD33 cells, the period of neutropeniausing CD34 selected cells is not extended, since CFU-GM are included in the CD34population and not depleted from the autograft (422).In summary, several new therapeutic approaches based on improved understandingof leukemia biology are under development and evaluation. However, .many of theseapproaches are complex, invasive, associated with significant toxicity, and, in the case ofcytokine therapy, may increase the risk of leukemic relapse. Moreover, there is someuncertainty as to the optimal use of established therapies; which dose, timing andcombinations are most effective. The mechanism of action of many existing therapies iscurrently being elucidated, and is contributing to an increased understanding of the biologyof leukemic cells (163). The development of new therapies based on increasedunderstanding and targetting of the specific mechanisms involved in maintenance andprogression of the leukemic clone is warranted.761.4: Biological activities ofproteases;Proteases, and serine proteases in particular, have recently been shown to exert avariety of intriguing, and sometimes surprising, biological effects. In addition to theirtraditional role in releasing signal peptides, causing the maturation of other proteins, andtheir well-defined roles in the blood clotting cascade and complement system, proteaseshave been shown to be associated with a variety of tumours, including leukemias, to beimportant in development, and to be involved in the development and functions ofhematopoietic cells.1.4.1: Proteases in the functions ofmature hematopoietic cells;Cytolytic T cells produce a series of granule-associated serine proteases,granzymes, which are involved in antigen-specific cytolysis. (426). Although the specificroles of these proteases are not clear, cytolysis is partially inhibited by inhibitors of serineprotease activity (427, 428). Granzyme A has been shown to localize to the nucleus of thetarget cell, where it cleaves the protein nucleolin, a nucleolar protein involved in thesynthesis and assembly of ribosomes; this results in loss of chromatin structure, activationof an endogenous endonuclease, and programmed cell death (429). In addition, a serineprotease which is cytostatic to a variety of cell lines has been purified from a natural killercell line (430). Mast cell subtypes can be distinguished by the specific serine proteases77present in their granules. The transcription of these proteases is influenced byhematopoietic signalling cytokines (431, 432).Neutrophil proteases are involved in a variety of functions, including thedegradation of connective tissue, which facilitates leucocyte migration to sites of infectionand inflammation. Human leucocyte elastase and cathepsin G cleave connective tissueproteins such as elastin, collagen, and proteoglycans (433). These proteases are able tospecifically cleave their polypeptide inhibitors, which are present in plasma and tissues.Cleavage of the inhibitors inactivates the ability to inhibit the protease, prolonging its half-life, and converting the inhibitor to a chemotactic signal, which results in the influx ofadditional inflammatory cells (434, 435). Neutrophil elastase was shown to inactivatetissue factor pathway inhibitor, regenerating tissue factor activity, thus favouring localcoagulation. Elastase and other enzymes are also known to inactivate several otherinhibitors of coagulation, including antithrombin II, heparin cofactor II, Cl inactivator, andalpha 2-antiplasmin (436). In addition, the neutrophil serine proteases have bactericidalactivity which is independent of their proteolytic capabilities (437, 438). Hence, they aremultifunctional proteins which exert a variety of biological effects through more than onemechanism.1.4.2: Surprising functions ofproteases;Recently, some surprising functions have been described in which the inhibition ofproteolytic activity appears to be involved. Apolipoprotein a, a component of low density78lipoproteins (LDL), has 80% amino acid identity with plasminogen. It is thought thatapolipoprotein a may compete with plasminogen for access to fibrin and to plasminogenactivators; this is consistent with the observation that persons with high LDL levels aresusceptible to atherosclerosis (439). Intriguingly, the amyloid-b protein of extracellularplaques in Alzheimer\u00E2\u0080\u0099s brains was found to contain domains that exhibit sequence identitywith protease inhibitors (440), implicating the involvement of aberrant proteolysis in thedevelopment of Alzheimer\u00E2\u0080\u0099s disease.1.4.3: Proteases in development;Pattern formation in the Drosophila melanogaster embryo in part involves the geneproducts of snake (441) and easter (442); both are serine proteases.1.4.4: Proteases in cell signalling;More importantly, proteolytic activity has been shown to be associated withsignalling in hematopoietic and non-hematopoietic cells. For example, an epidermalgrowth factor binding protein (EGF-BP) has serine protease activity which processes EGFto its active form. In addition, EGF-BP, free of contaminating EGF, potentiates theproliferative response of fibroblasts to EGF (443). The gamma unit of nerve growth factor(NGF), which has serine protease activity, proteolytically processes the beta unit to itsactive form (443). Fibronectin-degrading serine protease activity was found in the heparin79binding domain of human plasma fibronectin (444), suggesting that proteolysis may beinvolved in modulating cell adhesion to the extracellular matrix. Sequence similarity to afamily of serine protease inhibitors was found in a heat shock protein in the endoplasmicreticulum of myoblasts (445), suggesting that the inhibition of proteolysis may be involvedin cellular stress responses. Modification of two isozymes of protein kinase C with aresultant loss of kinase activity in human neutrophils is mediated by serine protease activity(446). In addition; the labile nuclear oncoproteins c-myc, c-los, p13 and E 1A, are rapidlydegraded by the ubiquitin system in vitro (447, 448).In hematopoietic cells, the phorbol ester induced down-modulation of the CSF- 1receptor (449) as well as release of active CSF- 1 from a membrane bound form (107) areboth the results of proteolytic processing. The transmembrane form of Steel factor, whichhas structural homology to CSF- 1 (450), has a putative proteolytic cleavage site in itsextracellular domain (95). The membrane-bound form of CSF- 1 was shown to bebiologically active; it was able to stimulate cells expressing CSF- 1 receptors (108).Protease activity was found to be closely associated with the murine IL-3 receptor (451),and may be involved in its downregulation following IL-3 binding. A serine proteaseinhibitor suppresses the secretion of tumour necrosis factor by peripheral bloodmononuclear cells (112). In addition, TNF has been found to exist as an integralmembrane protein, suggesting that it may by involved in cell-cell adhesion or signalling byinteracting with its receptor on an adjacent cell. The TNF receptor exists in vivo in asoluble as well as a membrane-bound form. The soluble form may act to neutralize solubleTNF, but also may act as a ligand for membrane-bound TNF (112). Downregulation ofGCSF cell-binding capacity can be blocked by inhibitors of protease activity (290). FGF,80IL-i and TGF-3 have all been shown to increase plasminogen activator activity. Both IL-iand TGF-13 are converted to their soluble forms by plasmin. Hence, this protease appearsto be important in influencing hematopoiesis through its actions on cytokines (452).Sequence analysis of the hematopoietic adhesion molecule CD44 indicated that trypsin-likeproteases might cleave within sequences included in the isoforms CD44R1 and CD44R2,generating soluble forms that could mediate adhesive interactions (20). Finally, proteolyticactivity has been shown to be involved in the initiation of signal transduction pathwaysresulting in cell activation at the cell surface, as follows. The serine protease thrombincauses activation ofplatelets, resulting in their aggregation. Platelet activation is mediatedby the thrombin receptor, one of the family of receptors with seven transmembranedomains. Activation of the thrombin receptor requires the serine protease activity ofthrombin, and can be blocked using specific thrombin inhibitors. Receptor activation wasshown to be mediated by the release of a short peptide from the N terminus of the receptor.The newly created N terminus of the receptor then binds to another site on the receptor,resulting in platelet activation and aggregation. Thus, protease activity from theextracellular milieu to the nucleus has been demonstrated to influence cell behaviour (453).1.4.5: Proteases in malignancies, including leukemias;The involvement of proteolysis in the physiology of several malignancies has beendocumented. A serine protease and its associated inhibitor have been found in elevatedlevels in the urine of patients with gynecological cancers (454). In addition, proteaseinhibitors were found to inhibit the growth of transformed murine fibroblast cell lines81(455), and to decrease the frequency of radiation transformation in a murine cell line (456).Proteases have for some time been known to be essential for the metastasis of solidtumours (161). In some cases, this effect may be mediated by familiar cytokines; TGF43was found to induce protease production and invasion by transformed fibrosarcoma cells,while it suppressed protease transcription in non-transformed fibroblasts (457).More important is the demonstrated involvement of proteolytic activity in severalleukemias. The alpha 2-macroglobulin receptor, which binds serine proteases, isexpressed on the cells of a high proportion of monocytic leukemias. This receptor also isknown to bind cytokines such as interleukins 1, 2, and 6, TGF-13 and FGF, leading theinvestigators to speculate that its interactions with both proteases and growth factors mayaffect the turnover of the malignant cells (458). The presence of serum plasminogenactivator inhibitor-2 (PAI-2) was found to be a marker of active leukemias with monocyticcomponents (459). Recently, serine protease activity was shown to be associated with theestrogen receptor when bound to its ligand (460). Intriguingly, in a separate report,estrogen receptors were demonstrated to be expressed in a case of AML M4, as well as inthe cell line HL60. This observation followed reports of spontaneous remission ofAML inpregnancy following parturition, and of the presence of estrogen receptors in multiple casesof CLL, some or which responded to the estrogen receptor blocking drug tamoxifen (461).CALLA (Common Acute Lymphoblastic Leukemia Antigen, also known as CD 10,enkephalinase, and membrane metalloendopeptidase) is a cell surface neutral endopeptidase(NEP) known to be associated with lymphoid leukemias (462). Finally, thedownregulation of a recently cloned serine protease, myeloblastin, was found to cause thegrowth arrest and differentiation of the promyelocytic leukemia cell line HL6O (310).82These examples clearly set precedents for the involvement ofproteases in the development,functioning, and oncogenesis of hematopoietic cells.1.5: Background on \u00E2\u0080\u0098CAMALBackground studies which led to the current investigations are summarized in thissection. The original isolation of \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 (Common Antigen in Myelogenous AcuteLeukemia) from acute non-lymphocytic leukemia (ANLL) leucocytes, and the preparationof anti-CAMAL antibodies is described. Immunoperoxidase studies using anti-CAMALantibodies in which it was demonstrated that the detection of the CAMAL antigen isdiagnostic of myeloid leukemias, and is of prognostic value in these leukemias, aresummarized. Also described are in vitro \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 addition studies using CAMAL- 1enriched preparations of myeloid leukemia cell lysates cultured with normal and CMLprogenitor cells. These studies demonstrated that exposure ofprogenitor cells to CAMAL1 enriched material resulted in alterations of myelopoiesis which could impart a growthadvantage on the leukemic clone. The purpose of the current study was to furthercharacterize and define these effects in order to gain greater insight into the possible role ofCAMAL- 1 enriched material in the events of leukemogenesis, and to facilitate the design ofnew strategies to block or reverse the leukemic progression.83The original isolation of \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 from ANLL leucocytes involved subtractiveprecipitation of normal cellular components from ANLL leucocyte lysates by the sequentialaddition of increasing amounts of rabbit antiseram raised against preparations of poolednormal human leucocytes. Material that remained once precipitation was complete wasused to prepare rabbit anti-CAMAL antisera (463), and to screen monoclonal hybridomasupematants during the preparation of the CAMAL-l antibody (464).The anti-CAMAL antibodies were used for immunophenotyping studies of myeloidleukemias. It was shown by enzyme-linked immunosorbent assay (ELISA), fluorescenceactivated cell sorting analysis (FACS), and an immunoperoxidase slide test that antibodiesraised against \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 preparations reacted to a far greater extent with the mononuclearcells of persons with myelogenous leukemias, both ANLL and CML, than with normalcells or cells of persons with various lymphoproliferative disorders (463, 464, 465). Forexample, using FACS analysis, it was found that the rabbit antibodies reacted with thecells, both peripheral blood leucocytes (PBL) and bone marrow (BM), of 44 out of 45patients with active AML, with 19 out of 19 patients with CML, and with 13 out of 13patients with AML in remission. However, they did not react with any of 14 normals norwith 40 out of 42 patients with a variety of lymphoproliferative disorders (466, 467).Similarly, in an immunoperoxidase slide test using CAMAL- 1, it was found that of cellsfrom patients with ANLL in primary presentation, 35/36 and 2 1/23 were positive (1%staining by cell number, BM and PBL, respectively). In addition, 7/7 PBL from CMLwere positive. In contrast, 0/13 and 0/30 BM and PBL from normals reacted with thisantibody. 1/5 PBL from ALL in primary presentation showed reactivity, as did 1/12 PBLfrom CLL. Average percentages of positive cells were 15.8 \u00C2\u00B1 2.8 and 8.9 \u00C2\u00B1 3.9 for BM84and PBL of ANLL in primary presentation, and 19.3 \u00C2\u00B1 2.6 for CML. In contrast, valueswere 0.3 \u00C2\u00B1 0.1 and 0 for BM and PBL of normals, 0.4 \u00C2\u00B1 0.2 for PBL of ALL, and 0.2 \u00C2\u00B10.1 for CLL PBL (465). Thus, detection of \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 on the bone marrow or peripheralblood cells, as indicated by the reactivity of these cells with the antibody CAMAL- 1,appeared to be indicative of myeloid leukemia. Few other markers common to both chronicand acute myeloid leukemias have been reported. One is oncofetal protein (OFP), acytoplasmic protein of 50 - 55 kDa which locates to the nuclear pores and may be involvedin the transport of mRNA. OFP was detected in HL6O cells using a mAb raised againsthepatoma cells, and is common to all tumour cell types examined (468).The same immunoperoxidase studies indicated that the cells of many patients withANLL in clinical remission continued to express the CAMAL marker (24/25, 11.8 \u00C2\u00B1 1.7for BM; 16/24, 4.9 \u00C2\u00B1 1.4 for PBL). In order to determine whether levels of cells reactivewith the antibody CAMAL- 1 remained consistent over time, a double blind study wasundertaken in which specimens from patients with a variety ofhematological malignancieswere evaluated over a course of three years. It was found that of patients undergoing bonemarrow transplantation for ANLL, 10 of 12 who remained in remission during the studyperiod had \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 bone marrow values of less than 1.0%, as did 10 of 10 patients whodied in remission of causes other than leukemia. In contrast, 6 of 6 patient with ANLLwho relapsed, two of whom died in relapse, had greatly elevated \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 values,ranging from 5 to 100%. In these patients, \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 values were found to becomeelevated up to three months prior to relapse (469). In further studies of patients withANLL undergoing chemotherapy, it was found that patients whose \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 bonemarrow values fell significantly on induction of remission had significantly longer survival85times than those whose \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 values rose or remained the same (6.8 months and 19.2months, respectively, reference 470). These results were taken as an indication thatincreases in detection of the CAMAL antigen on bone marrow cells preceded the recurrenceof clinical symptoms, that levels of the CAMAL antigen were of prognostic value inpredicting survival time, and that the CAMAL antigen might be indicative of a molecularentity involved in the outgrowth of the leukemic clone.The possibility that the entity detected by the antibody CAMAL- 1 was involved inthe outgrowth of myeloid leukemia cells was investigated by the addition of proteinpreparations enriched from lysates of myeloid leukemia cells using CAMAL- 1immunoaffinity chromatography to in vitro colony assays ofprogenitor cells from normalhealthy donors or from patients with CML. These studies showed that colony formationby normal progenitor cells was inhibited by the addition of CAMAL- 1 eluted material, oftenprofoundly. Colony formation by CML progenitor cells, however, was not inhibited bythe addition of the same preparations of CAMAL- 1 enriched material, and in some caseswas stimulated (471). Outgrowth of the leukemic clone, then, could occur in two ways;the suppression of normal myelopoiesis could impart a selective growth advantage onleukemic cells by reducing competition for space and growth requirements in thehematopoietic microenvironment, and the enhancement of leukemic myelopoiesis couldgive the leukemic cells a more direct growth advantage. The purpose of the current studywas to further characterize and define these effects in order to gain greater insight into therole of CAMAL- 1 eluted material in the early events of leukemogenesis, and to facilitate thedesign of new strategies to block or reverse the leukemic progression. As CAMAL- 1eluted material consisted of several protein species by silver-stained SDS-PAGE analysis,86the first step taken was to separate these proteins from each other and determine whichcomponent in these preparations mediated the alterations of myelopoiesis (472, Chapter 2).Studies were undertaken in order to address the biochemical characterization of themarker and in order to further define the activities on colony formation. The biochemicalcharacterization of the marker was the subject of a separate research project, and was thePh.D. research of another graduate student in this laboratory (473). The biologicalcharacterization of the activities on colony formation was the subject of research by theauthor. When these studies were undertaken, it was thought to be probable that theCAMAL marker detected on myeloid leukemia cells by CAMAL- 1 in theimmunoperoxidase assay and the material which mediated the effects on myelopoiesis bynormal and leukemic progenitor cells were equivalent. As work progressed, however, itbecame clear that the marker and the activity were not in fact equivalent entities. In thispaper, the marker on leukemic cells recognized by CAMAL- 1 is referred to as \u00E2\u0080\u0098the CAMALantigen\u00E2\u0080\u0099, \u00E2\u0080\u0098the CAMAL marker\u00E2\u0080\u0099, or \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099. The material shown to mediate the effectson in vitro myelopoiesis is designated \u00E2\u0080\u0098P30-35 CAMAL\u00E2\u0080\u0099, unless otherwise specified.It was recently shown by Western blot analysis that the antibody CAMAL- 1 is notreactive with the P30-35 CAMAL material shown in this study to mediate the effects on invitro myelopoiesis by normal and CML progenitor cells (474). Thus it is likely thatCAMAL-l does notbind directly to P30-35 CAMAL. However, CAMAL-1 is known toenrich for P30-35 CAMAL. This was shown by passing material from lysates of myeloidleukemia cells which fell through a CAMAL-l immunoaffinity column over animmunoaffinity column prepared with a-P30/35, a monoclonal antibody prepared against87highly enriched P30-35 material. No further P30-35 CAMAL material eluted from the a-P30/35 column (474), an indication that the vast majority of P30-35 CAMAL was removedfrom the leukemic cell lysates by the antibody CAMAL- 1. CAMAL- 1 thus enriches forP30-35 CAMAL, which explains its usefulness during P30-35 CAMAL enrichmentprotocols.The enrichment of P30-35 CAMAL by the antibody CAMAL- 1 in the apparentabsence of direct binding is somewhat of a mystery. It is known, however, that CAMAL- 1is reactive with human serum albumin (HSA, reference 474). HSA is known to carry anumber of biologically active molecules, so it is possible that P30-35 CAMAL could itselfbe carried by HSA (reviewed in 475). The monoclonal antibody against the active P30-35CAMAL material, a-P30/35, which was screened for lack of reactivity with HSA, wasraised in order to better address these questions, as well as to facilitate purification of P30-35 CAMAL (Chapter 2). a-P30/35 is known to react by Western blot analysis and byELISA with P30-35 CAMAL, and thus appears to bind directly to P30-35 CAMAL (474,Chapter 2). The similarity in patterns of staining of CML vs. normal nucleated cells byimmunoperoxidase using CAMAL- 1 as compared to a-P30/35 (Appendix 1) suggest anassociation between the CAMAL- 1 reactive CAMAL marker and the P30-3 5 materialrecognized by cx-P30/35. However, no definitive answers are available at this point, andthis matter is the subject of ongoing research (476).For the majority of the studies described in this thesis, P30-35 CAMAL wasenriched in a two-step protocol using immunoadsorbent columns prepared with CAMAL- 1and c - P30/35, since this protocol was shown to result in optimal removal of materials88done using a one-step immunoaffinity protocol with a-P30/35 (474). Recent work hasshown that active material can be obtained using either procedure (Chapter 2, 474,477). Itwas shown that P30-35 CAMAL was highly enriched for inhibitory activity on in vitrocolony formation by normal progenitor cells (Chapter 2), and that stimulatory activity oncolony formation by CML progenitor cells resides in the same P30-35 CAMAL fraction(Chapter 4). Recent information suggests that the inhibitory activity on normal colonyformation might in fact reside in less than ten percent of the P30-35 CAMAL fraction(477). Experiments presented in Chapter 5 suggest the involvement of serine proteaseactivity in the effects on normal and CML myelopoiesis, activity which might be unique(Appendix 2). Further biochemical definition of the entity (or entities) to which theactivities on normal and CML myelopoiesis can be attributed are the subject of ongoingresearch (476).89CHAPTER 2CHARACTERIZATION OF THE INHIBITORY EFFECTS OF P30-35 CAMAL ONNORMAL MYELOPOIESIS2.1: INTRODUCTIONChronic myelogenous leukemia (CML) is a myeloprolifemtive hematopoietic stemcell disorder in which an elevation of neutrophilic granulocytes and their progenitors areobserved in the bone marrow and peripheral blood. How the leukemic clone gainsdominance over normal hematopoietic cells is not well understood, but inhibitors of normalhematopoiesis have been described (Chapter 1). The CAMAL antigen was originallyisolated by elution of ANLL or CML cell lysates from a CAMAL- 1 immunoaffinity columnand shown to inhibit colony formation by normal progenitor cells in vitro. Thisobservation was important, as suppression of normal hematopoiesis could provide anenvironment in which the leukemic clone could gain dominance. As CAMAL- 1 elutedmaterial used for these original experiments consisted of several protein components, theproteins were further separated in order to identify the component to which the inhibition ofnormal myelopoiesis could be attributed. In this chapter evidence that the inhibitory activityon normal colony formation is mediated by the component in CAMAL- 1 eluted materialwhich migrates at 30-35 kilodaltons (kDa) by sodium dodecyl sulphate polyacrylamide gelelectropheresis (SDS-PAGE) analysis is described; this material is referred to as P30-3590CAMAL. In cultures of cells from normal healthy donors, P30-35 CAMAL at lowconcentrations was demonstrated to be profoundly inhibitory to colonies of neutrophilicgranulocytes (CFU-G) and to all colony types at higher levels. In addition, P30-35CAMAL was inhibitory to normal myelopoiesis in long term marrow culture, a conditionwhich more closely approximates the hematopoletic microenvironment.2.2: MATERIALSANDMETHODS2.2.1: Purification ofP3O-35 CAMAL:The major focus of this study was the characterization of the biological activity ofCAMAL-eluted material, with a minor focus on the reactivity of anti-CAMAL and anti-P30/35 antibodies at a cellular level. Separation of the components in CAMAL- 1 elutedmaterial, optimization ofprotein purification, characterization of the protein, and activitiesand characteristics of CAMAL at a subcellular level, and characterization of the reactivitiesof the anti-CAMAL antibodies at a subcellular level were and are the subject of ongoingresearch by another graduate student in this laboratory, as part of a separate project (472,473). Characterization of the biological activity of CAMAL-1 eluted material involved, asan initial step, the identification of the component in CAMAL- 1 eluted preparationsinhibitory to colony formation by progenitor cells from normal healthy donors. Thisinvolved a collaborative effort. CAMAL- 1 eluted material was separated for theexperiments in this initial phase of the study, including experiments described in Figures 191through 8, Figure 9b, and Table II, by Joan Shellard, using FPLC gel filtration orpreparative non-reducing SDS-PAGE to separate the components in CAMAL- 1 elutedmaterial (472). The active P30-35 CAMAL material for all subsequent experiments wasenriched by the author using sequential elution from CAMAL- 1 and a-P30!35immunoadsorbent columns.2.2. l.a: Antibodies;Antibodies used in these studies are all monoclonals. CAMAL- 1 was raised againstoriginal preparations of the CAMAL antigen, which were in turn prepared by subtractivemethods from lysates of cells from patients with myeloid leukemias, as described inChapter 1. The original immunoperoxidase studies which demonstrated that recognition ofCAMAL was diagnostic of myelogenous leukemias were performed using this antibody(465). CAMAL-1 has demonstrated reactivity with proteins other than P30-35 CAMAL. Itwas shown by Coomassie Blue-stained SDS-PAGE, and by immunoprecipitation, that themajor species recognized by CAMAL- 1 is a protein which migrates in the range of 64 - 68kDa by SDS-PAGE (475, 478). Similarly, CAMAL- 1 was shown to be highly reactivewith human serum albumin (HSA) using the enzyme-linked immunosorbent assay (ELISA)technique (474). However, silver-stained SDS-PAGE analysis showed that protein speciesother than the 64- 68 kfla material were present in CAMAL- 1 eluted material (472, Figurela), leading to the experiments in which these components were separated and theinhibitory activity identified.92A new monoclonal antibody was prepared against the 30-35 kDa material inCAMAL- 1 eluted preparations. This was done in order to facilitate purification of P30-35CAMAL, and in order to facilitate investigations into whether the antigen recognized byCAMAL- 1 in the immunoperoxidase test and the inhibitory material might be the sameentity (Appendix 1). This was investigated since rabbit polyclonal antibodies raised againstoriginal preparations of the CAMAL antigen were shown both to recognize the marker onmyeloid leukemia cells (465) and to react with P30-35 CAMAL by Western blot analysis(474). More recently, it was shown by Western blot analysis that the monoclonal antibodyCAMAL- 1, used for the majority of the immunoperoxidase studies, does not react withP30-35 CAMAL (474), however, similarities in cell staining using CAMAL-1 or o-P30/35suggest an association between the CAMAL antigen, and the inhibitory material, P30-35CAMAL (Appendix 1). The possibility of an association between the CAMAL antigen andP30-3 5 CAMAL is supported by the observation that CAMAL- 1 does enrich for the P30-35 CAMAL active material (474).a-P30/35 was raised against highly enriched material which migrated between 30and 35 kfla (< 2% contaminants by silver stained SDS-PAGE analysis). This material waspurified by subjecting CML or AML cell lysates which had been eluted from a CAMAL- 1immunoaffinity column to fast protein liquid chromatography (FPLC) gel filtration or topreparative non-reducing SDS-PAGE as described in the following section. Since theantibody CAMAL- 1 is known to react with HSA, hybridoma supernatants were screeendfor lack of reactivity with HSA, and ct-P30/35 does not react with HSA by ELISA (datanot included). Recently performed reverse phase high performance liquid chromatography(HPLC) separations of P30-35 CAMAL preparations, which were performed after the93completion of this study, have shown that other protein components are present inpreparations purified using -P30/35, components with which x-P30/35 appears to bereactive by Western blot analysis (474); these include elastase, azurocidin, and cathepsinG, but not myeloblastin. There is, however, a protein peak in preparations of P30-35CAMAL which is distinct from the other proteins present, which have been characterized(473, 476, 479). In addition, experiments described in Chapter 5 and in Appendix 2demonstrate clearly that there is an activity in preparations of P30-35 CAMAL whichappears to be distinct from the activities of these other components. This is discussed ingreater detail in sections 2.4, 5.4, 6.1, and in Appendix 2.a-BLV was raised against the major coat protein of bovine leukosis virus at thefacilities of Quadra Logic Technologies (QLT), and was obtained from QLT. ct-BLV wasused as a control antibody for the evaluation of purification protocols and for diagnosticstudies.2.2.1 .b: Class identification;CAMAL-1, ct-P30/35, and cr-BLV were all determined to be of theimmunoglobulin subclass IgG1 by the Ouchterlony technique. 10 microliter (p1) volumesof CAMAL- 1, a-P30/35 or cs-BLV ascites were loaded into wells cut into 1% agar gelsprepared on 7.5 X 5 cm strips of gel-bond (FMC Bio-Products) fixed onto glass plates.These were prepared by heating agar (Difco) to boiling in the microwave, pipetting 7.5 ml94onto each plate, and allowing it to solidify at room temperature. Antisera (Meloy) tovarious Ig classes and subclasses, including IgG1, IgG, IgG, IgG3, and 1gM wereloaded into adjacent wells (10 il volumes), and gels incubated overnight in a humidatmosphere at 4 degrees centigrade (\u00C2\u00B0C) in order to allow diffusion of antisera to occur.Gels were rinsed with 0.15 M NaC1 to remove soluble proteins, rinsed in distilled water(dH2O), and stained with 0.5% amido black in 5% acetic acid for 1 minute (mm). Gelswere destained with a solution of45% MeOH and 10% acetic acid.2.2.1 .c: Prepanition and screening ofa-P30,\u00E2\u0080\u009935;The fraction in CAMAL- 1 eluted material which migrated at between 30 and 35 kDaby SDS-PAGE analysis was determined to be the component in these preparations whichwas inhibitory to colony formation by n\u00C3\u00A0rmal progenitor cells in vitro. Hence, amonoclonal antibody was raised against this material in order to facilitate purification anddiagnostic studies. Material further separated from CAMAL- 1 immunoaffmity preparationsby FPLC gel filtration or elution from preparative non-reducing SDS polyacrylamide gels,and which corresponded to material that migrated at between 30 to 35 kDa by analyticalSDS-PAGE, was used to immunize Balb/C mice by standard methods. The material usedfor immunization was prepared by another graduate student in this laboratory, and theprocedure is described elsewhere (472). Immune spleen cells were fused with an NS- 1fusion partner by the method of 01 and Herzenberg (480). Cell fusion and cloning wereperformed at the facilities of Quadra Logic Technologies by Herma Neynclorff.95Hybridoma supernatants were screened for reactivity with P30-35 CAMAL materialby enzyme-linked immunosorbent assay (ELISA). P30-35 CAMAL used for screeninghybridoma supernatants was material from lysates ofleucocytes from patients with chronicmyelogenous leukemia (CML) or acute myelogenous leukemia (AML) which eluted from aCAMAL- 1 immunoadsorbent column, and which was further fractionated by FPLC gelfiltration or preparative non-reducing SDS-PAGE. As the major contaminating proteinwith which CAMAL- 1 reacts was shown by ELISA to be human serum albumin (HSA,reference 474), hybridoma supernatants were also screened for lack of reactivity with HSA(Sigma). Cells from wells showing reactivity with P30-35 CAMAL, and lacking reactivitywith HSA were cloned, and supernatants from the clones rescreened.The ELISA procedure is as follows. 96 well ELISA plates (Immunlon 2) werecoated with 1 j.tg/ml of P30-3 5 CAMAL or HSA in 50 mM carbonate coating buffer, pH9.6, in 100 tVwell volumes. Plates were incubated overnight at 4 OC. Buffer and excessprotein were removed by shaking and vigorous washing with PBS-Tween (phosphatebuffered saline containing 0.05% Tween-20). Hybridoma supernatants were added in 100j.il of volumes, and plates were incubated for 60 mm at 3 7\u00C2\u00B0C. Plates were washed withPBS-Tween and alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulins(Jackson) diluted 1:3000 in PBS-Tween was added in 100 l volumes. Plates were againincubated at 37\u00C2\u00B0C for 60 mm, washed, and substrate added (p -nitrophenyl disodium,Sigma, 1 mg/mI in 10% diethanolamine, DEAE, buffer, pH 9.8). Plates were incubatedfor 30 mm to 4 hours at 37\u00C2\u00B0C until visible colour formation occurred. Reactivity wasdetermined by absorbance at 405 nanometers (A405) using a Titertek ELISA reader.Absorbance was compared to controls containing culture media but no cell supernatant.96Three clones were obtained that were reactive with P30-35 CAMAL, and non-reactive withHSA. These were evaluated for their ability to purify P30-35 CAMAL by immunoaffinityas described below.2.2.1 .d: Preparation ofascites;Hybridomas were cultured in Dulbecco\u00E2\u0080\u0099s modified Eagle\u00E2\u0080\u0099s Medium (DME),supplemented with 10% fetal calf serum (FCS). Cells were cryopreserved in liquidnitrogen at a cell density of 3 X 106/milliliter (ml) in 40% media and 50% FCS by volumewith a final concentration of 10% dimethylsulfoxide (DMSO; Sigma, tissue culture grade).DMSO was added dropwise, with gentle shaking, to cells in media containing FCS. 1 to1.5 ml fractions were then frozen in cryovials (Nunc). Initially, cryovials were insulated instyrofoam containers to slow freezing overnight in a at -70\u00C2\u00B0C, after which time they weretransferred to liquid nitrogen (IN2). Later cryopreservation procedures involved freezing ofcells were in liquid nitrogen vapour overnight, following which they were submerged inIN2.Female Balb/C mice between 6 weeks and one year old were injectedintraperitoneally (i.p.) with 0.5 ml pristane (2,6,10, 14-tetramethylpentadecane, Aldrich).Ascites was prepared by i.p. injection of hybridoma cells 5 to 14 days after injection ofpristane. Between 2 to 5 X 106 cells were injected per mouse. Between 5 to 10 mice wereused per group for each ascites preparation. Hybridoma cells from culture, orcryopreserved cells which had been thawed, were used to prepare ascites. Cryopreserved97cells were thawed in a 37\u00C2\u00B0C water bath with gentle shaking, and injected immediately afterthawing. Ascites was collected from mice 7 to 21 days after injection of hybridoma cellsby intraperitoneal placement of a 16 gauge needle (tapped). Mice were tapped one to threetimes before euthanization. Ascites from each hybridoma group was pooled forpurification of immunoglobulins and centrifuged for 10 mm at 5000 rotations per minute(rpm) to remove fibrin clots.2.2.1 .e: Purification ofimmunoglobulins from ascites;Immunoglobulins were initially purified from ascites using hydroxyapatite columnchromatography. 75 ml columns were prepared in degassed 0.01 M sodium phosphatebuffer, pH 6.8. 15 ml volumes of ascftes were diluted 1:2 in the same buffer, and pumpedover the column at a flow rate of 3.5 mVmin. The column was washed with starting bufferto an A280 of less than 0.05, then proteins were eluted with 0.5 M sodium phosphatebuffer, pH 6.8. Protein containing fractions as determined by A280 from column fall-through, and eluted protein, were pooled separately and concentrated by centrifugation at5000 X gravity (g, 8000 rpm on a Sorvall centrifuge using an SS35 rotor) in minicentricons (Amicon) with a molecular weight cutoff of 30 kiJa. Protein concentration wasdetermined by the Lowry assay, and protein content analyzed by silver stained SDSPAGE. Fall-through material consisted mainly of a 66 to 68 kDa protein species, in alllikelihood albumin, and eluted material was enriched for immunoglobulins. Between 5 to25% of eluted proteins appeared to be immunoglobulin heavy and light chain proteins bySDS-PAGE analysis. This corresponded to an approximately eight fold enrichment.98Subsequent purification of antibodies was performed using a goat tx-mouse Igcolumn (Hyclone). 15 ml of ascites was diluted 1:2 in PBS and pumped onto the goat ctmouse Ig column at a flow rate of 0.8 mVmin. The column was washed with PBS to A280<0.05, then eluted with 0.1 normal (N) hydrochloric acid (HCI). The eluated material wasimmediately neutralized by the addition of 1.5 M Tris-HC1 (Tris hydroxymethylaminomethane HC1, Sigma), pH 7.5. Protein containing fractions as determined by A280were pooled. The column eluate was dialyzed against PBS, and concentrated and analyzedas above. The column was immediately neutralized by washing with PBS to pH 7.Greater than 95% of eluted proteins purified by this method appeared to beimmunoglobulin heavy and light chain proteins by SDS-PAGE analysis. Thiscorresponded to an approximately fifty fold enrichment.2.2.1. f: Immunoadsorbent column prepamtion;Purified monoclonal antibodies were dialyzed against 0.01 M Hepes (N-[2-hydroxyethyl] piperazine-N\u00E2\u0080\u0099-[2-ethanesulfonic acid], Sigma) buffer, pH 8.0 (preparedwith degassed deionized distilled water, ddH2O) for coupling to agarose column beads(Bio-Rad Affi-Gel- 10). 20 mg/mI of hydroxyapatite-purified antibodies or 5 mgfml ofanti-mouse Ig purified antibodies were used per ml of beads. All steps were performed at 4\u00C2\u00B0C.Beads were washed in ddH2O, ddH2O was removed, and Ig in coupling buffer added. Anequal volume of Ig solution to bead volume was used. Mixtures were allowed to react for4 hours with gentle shaking, at which time the reaction was stopped by the addition of 0.199ml 1 M glycine ethyl ester, pH 8.0 per ml of gel beads. Beads were packed into Bio-Radcolumns (10 cm X 1.5 cm), washed extensively with PBS, and stored at 4\u00C2\u00B0C in PBScontaining 0.02% NaN2..Antibodies were evaluated for their ability to enrich for P30-35 CAMAL. OneCML cell lysate was divided into fractions and these were purified by immunoaffinity usingvarious columns and combinations of columns, including preabsorption against a columnprepared with the control mAb, a-BLV. Optimal enrichment was obtained using CAMAL1 eluted material further fractionated over a column prepared from one of the threehybridoma antibodies, which was called ct-P30/35, and this protocol was used forsubsequent preparations. The other hybridoma antibodies were not used subsequently.2.2.1 .g: Protein analysis;Protein concentrations were determined using the Micro-Lowry assay (481).Purified mAbs and preparations of P30-35 CAMAL were analyzed by SDS-PAGEaccording to the method of Laemmli (482). 10% polyacrylamide gels were run using theBio-Rad mini-gel system. Protein was diluted in sample buffer containing 2% SDS, 10%glycerol, 0.15 M Tris-HCI pH 6.8 and bromphenol blue with 20 mM dithiothreitol (DTI,Bio-Rad), heated at 70\u00C2\u00B0C for 10 mm, cooled and loaded onto 10% polyacrylamide minigels (7.5 X 10 cm). Gels were run at a constant current of 25 milliamps (mA). Gels weresilver stained as follows. Briefly, gels were fixed in a solution of 50% methanol and 10%100acetic acid, then rehydrated in a solution of 10% methanol and 10% acetic acid, withheating. Gels were washed in dH2O, heated in dH2O containing 30 j.tM DTT, and stainedin a solution of 0.1% silver nitrate in dH2O for 15 minutes at room temperature. Theywere rinsed with dH2O and developed in a solution of 3% sodium bicarbonate (Na2CO3)with 0.037% formaldehyde (Fisher) in dH2O. Development was stopped by the additionof 2.3 M citric acid or acetic acid.2.2.2: Prepantions ofP30-35 CAMAL:2.2.2.a: Sources ofP30-35 CAMAL;Preparations of P30-35 CAMAL were derived from three sources. One preparationwas purified from ANLL leucocytes from a patient who had undergone apheresis to reducethe white blood cell burden. These cells were obtained from the Vancouver GeneralHospital Division of Hematology. One preparation was purified from leucocytes obtainedby apheresis from a patient with CML. These cells were obtained from the TorontoGeneral Hospital by Dr. Armand Keating. The remaining preparations were purified fromCML remission bone marrow buffy coat cells, which are from the fraction enriched forleucocytes, and which includes plasma. These cells were originally intended forautologous transplantation, but the patients had since died. These cells were obtained fromthe Terry Fox Laboratory and the Canadian Red Cross Society. They had originally beenstored in liquid nitrogen, and were subsequently stored at -70\u00C2\u00B0C.1012.2.2.b: Cell lysis;Cells were thawed and mixed with an equal volume of lysis buffer (20 mM TrisHC1 pH 7.5, 150 mM sodium chloride [NaC1], 1.0 mM ethylenediamine tetraacetic acid[EDTA], 1.0% Triton X-l00, 0.5% sodium deoxycholate [NaDOC]), containing 2000units/mi of pancreatic deoxyribonuclease (DNase, Sigma). This mixture was allowed tostir at room temperature for 2 to 3 hours and then at 4\u00C2\u00B0C for an additional 12 hours.Subsequent steps were all performed at 4\u00C2\u00B0C. The lysate was centrifuge at 15000 rpm for30 mm using a Sorvall SS35 rotor, in order to remove insoluble debris.2.2.2.c: Protein purification;Soluble components of cell lysates were affinity purified over CAMAL- 1immunoadsorbent columns prepared as described above. The eluted material was thenfurther purified in one of three ways; by preparative non-reducing SDS-PAGE, by FPLCgel filtration using a Superose- 12 column, or by affinity purification using an cx-P30/35immunoadsorbent column. Protein which was not bound by the cL-P30/35 column, andwhich fell through, was collected, concentrated and used as control protein in biologicalassays.Preparative gels and gel filtration were performed by another student in thislaboratory, and are described elsewhere (472). Briefly, material eiuted from a CAMAL- 1102immunoaffinity column was subjected to electropheresis through a 16 cm 12% SDSpolyacrylamide gel. The separating gel was cut horizontally into 25 mm slices, which wereeluted for analysis by reducing SDS-PAGE in order to determine the molecular weight ofthe protein contained in each fraction. Fractions to be tested in the progenitor cell assaywere dialyzed against PBS and protein content determined by the Micro Lowry assay.Alternatively, CAMAL- 1 eluted material was dialyzed against gel filtration buffer (0.1 Mammonium [NH4]acetate, pH 5.0) and run over a Superose-12 gel filtration column at aflow rate of 0.35 mVmin using FPLC. Individual peak fractions, as monitored by A280were pooled, concentrated and analyzed for protein content by silver-stained reducing SDSPAGE and the Micro Lowry assay. P30-35 containing fractions were dialyzed againstPBS for evaluation ofbiological activity in the colony assay.2.2.2.d: Controlprotein;Protein which fell through the a-P30/35 column was used as control protein inbiological assays. In addition, human serum albumin (HSA,Sigma) was used as a furthercontrol protein in colony assays using normal progenitor cells. N-terminal amino acidsequence suggested that a protein with significant sequence identity to human neutrophilelastase was present in preparations of P30-35 CAMAL (see Discussion), hence the effectof elastase (Calbiochem) on normal and CML progenitors was evaluated in colony assays.1032.2.3: Hematopoieticprogenitor cell assays:2.2.3.a: Cells;Normal leucocytes for colony assays were obtained from the following sources; allleucocytes were obtained with informed consent. Normal peripheral blood leucocytes(PBL) were obtained from normal healthy volunteers. Normal bone marrow was obtainedfrom donors for allogeneic bone marrow transplantation through the Cancer ControlAgency of British Columbia by Dr. Gordon Phillips. Normal marrow was also obtainedfrom persons undergoing thoracic surgery at St. Paul\u00E2\u0080\u0099s Hospital by Drs. Hilton Ling andWilliam MacDonald. CML bone marrow was obtained from the Vancouver GeneralHospital (VGH) Division of Hematology, and CML peripheral blood from the VGHDivision ofHematology and the Munroe Clinic at VGH. Both bone marrow and peripheralblood were collected into tubes containing enough sodium heparin (Fisher) for a minimumof 50U heparin/ml ofbone marrow or blood.Mononuclear cells were separated by centrifugation over Ficoll-Hypaque or Percoll(Pharmacia). This procedure separates the mononuclear cell fraction, includingneutrophils, from erythrocytes and mature neutrophils. Blood or marrow was diluted 1:2to 1:4 in fresh Iscove\u00E2\u0080\u0099s medium (Gibco) which had been warmed to 37\u00C2\u00B0C. Between 7 to10 ml of diluted blood or marrow was carefully layered over 3 ml Ficoll-Hypaque orPercoll (diluted to 1.077 g/ml in sterile PBS) in 15 ml polystyrene tubes and centrifuged at400 X g (1500 rpm) for 12 minutes using a \u00E2\u0080\u009CSilencer\u00E2\u0080\u009D counter-top centrifuge (Fisher). Fatwas removed from the top following centrifugation of bone marrow, using a sterile pasteur104pipette, and was discarded. The mononuclear cells at the interface of the Ficoll-Hypaque orPercoll and media/plasma were recovered by aspiration with a sterile pasteur pipette. Cellswere washed twice and resuspended in fresh Iscove\u00E2\u0080\u0099s medium. Cells were counted inwhite blood cell counting media, which causes the lysis of residual contaminatingerythrocytes (5% acetic acid in PBS with a crystal of methylene blue, sterile filteredthrough a 0.2 j.tm filter). Viability was determined by dye exclusion using 0.2% eosin Y(Fisher). Cells were plated in a colony assay only when cell viability exceeded 98%.Viability was rarely less than 100%.Cells were often cultured fresh in the colony assay, however, at times, particularlyin initial assays, it was necessary to cryopreserve cells for later use. This was done asdescribed above for the hybridoma cells. It was found that greater recovery could beachieved when cells were cryopreserved using higher concentrations of FCS, hence 90%FCS (heat inactivated) was used, with 10% DMSO added dropwise. Cells were thawed ina water bath, as above, and fresh warm Iscoves\u00E2\u0080\u0099s medium containing 20% FCS addeddropwise with gentle shaking to 10 times the frozen volume. Cells were then centrifuged at1000 rpm, washed once in fresh Iscove\u00E2\u0080\u0099s medium with FCS and once in serum-freeIscove\u00E2\u0080\u0099s medium, resuspended in Iscove\u00E2\u0080\u0099s medium, and counted as described above.2.2.3 .b: Preparation ofconditionedmedium andplasma for colonyassays;Phytohemagglutinin-stimulated leucocyte conditioned medium (PHA-LCM) andhuman fasting plasma were obtained from normal healthy volunteers. 500 ml blood was105drawn into a blood bag containing 50U sodium heparin (Fisher) per ml blood. Blood wastransferred to 50 ml Falcon tubes, and allowed to settle for 45 to 90 mm to form a buffycoat layer composed of white blood cells and plasma. The buffy coat layer was removed byaspiration with a pasteur pipette, cells pelleted by centrifugation for 10 mm at 400 X g, andthe plasma set aside. Cells were washed twice in Iscove\u00E2\u0080\u0099s medium, resuspended at adensity of 1X106/ml in Iscove\u00E2\u0080\u0099s medium with 1% PITA by volume (Sigma) and culturedfor 7 days at 37\u00C2\u00B0C, 5% carbon dioxide (C02)in 200 ml flasks. Conditioned medium wascollected into 50 ml Falcon tubes, cells removed by two rounds of centrifugation at 1500rpm for 5 mm, then PITA-LCM was divided into aliquots and frozen at -70\u00C2\u00B0C. Plasma wascollected from the buffy coat layer, and by centrifugation of the remaining fraction at 1500rpm for 20 minutes. Plasma was centrifuged twice at 2500 rpm, filtered through 0.45 j.tmfilters (Millex-HA, Millipore), divided into aliquots, and stored at -70\u00C2\u00B0C. On thawing,plasma was again centrifuged at 2500 rpm for 10 mm in order to remove fibrin clots beforeaddition to colony assays.2.2.3.c: Colony assays;Colony assays were performed according to the method of Messner (483). Freshor previously cryopreserved bone marrow or peripheral blood mononuclear cells wereadded to cultures containing Iscove\u00E2\u0080\u0099s medium, 1% methylcellulose (Fluka AG), 30%human fasting plasma, and 10% PHA-LCM. These conditions supported the formation ofa mixture of colony types, the most primitive of which were CFU-GM. Addition of 1 unitper milliliter (U/mi) erythropoietin (Epo- Conn, Connaught Laboratories), final106concentration, supported the growth of CFU-GEMM in addition. 1 ml cultures were platedin duplicate or quadruplicate in 35 mm tissue culture dishes (Lux), and incubated at 37\u00C2\u00B0C,5% CO2 for 14 days, at which time colonies comprised of 20 cells were scored by visualinspection using an inverted microscope. Cultures were incubated under highly humidconditions by the inclusion of a tray in the incubator brimming with sterile dH2O. Inaddition, duplicates in 35 mm plates were placed in larger 100 mm plates; a third 35 mmplate filled with sterile dH2O was included with each pair of culture plates. Phase contrastphotomicrographs were taken using a Nikon camera on a Zeiss Axiovert invertedmicroscope.Colonies were plucked using a miniature pasteur pipette, placed on glass slides, andspread by gentle air pressure. They were fixed for 10 mm in methanol, stained for 10seconds (sec) with Wright\u00E2\u0080\u0099s Giemsa (Camco Quick Stain, Bridge Chemical Products),rinsed for 20 sec in dH2O, air dried, and examined using a light microscope. The pluckedcolonies examined in the experiments described in Figure 6 were evaluated by Dr. PatriciaLogan at the facilities of Quadra Logic Technologies.2.2.3.d: Long term marrow cultures;Long teim marrow cultures (LTMC) were performed as described by Keating andToor (484), using fresh or cryopreserved normal bone marrow mononuclear cells. Cellswere cultured at a density of 2 X 106 cells/ml in 2 to 5 ml volumes in long term marrow\u00E2\u0080\u00A2 culture media using Corning 24 or 6 well tissue culture plates or 10 ml flasks. LTMCV 107media consisted of McCoy\u00E2\u0080\u0099s medium supplemented with 12.5% horse serum, 12.5% fetalcalf serum, essential (2.5 X from concentrate) and non-essential amino acids vitamins (1 Xfrom concentrate) glutamine (2 mM), sodium pyruvate (1 mM), sodium bicarbonate(0.75% w/vol), penicillin (100 U/mI), streptomycin (100 U/mI), fungizone (all fromGibco), and 0.35 j.tg/ml hydrocortisone (Sigma). Medium was sterile-filtered using a 0.2jifilter and stored at -20\u00C2\u00B0C. Once thawed, medium was stored at 4\u00C2\u00B0C, and used within 2weeks. Cultures were incubated for 7 days at 37\u00C2\u00B0C, 5% C02, following which they wereincubated for the remainder of the culture period at 33\u00C2\u00B0C, 5% CO2. Cultures wereincubated under highly humid conditions. In addition to the inclusion of a tray in theincubator brimming with sterile dH2O, unused wells on culture plates were filled withsterile dH2O containing fungizone (0.04 U/ml). Cultures were fed weekly by removinghalf of the supematant, including cells, and replenishing the culture with fresh medium.P30-35 CAMAL concentration was depleted weekly by feeding the cultures. Supematantcells were washed, resuspended in Iscove\u00E2\u0080\u0099s medium, counted, and plated in amethylcellulose colony assay as described above. Cells were plated at a constant celldensity of 1 X 1 0 cells per 1 ml culture, unless inadequate numbers were recovered fromthe cultures to achieve this cell density. In this case, cells from control or P30-35 CAMALtreated cultures were diluted to the cell density of whichever culture was lowest, so thatcells from control and P30-35 CAMAL-treated cultures were cultured for colony formationat equivalent cell densities. At week 5, adherent layers were sacrificed by trypsinizationusing 0.25% trypsin-EDTA in Iscove\u00E2\u0080\u0099s medium (Gibco-BRL) for 10 mm at 37\u00C2\u00B0C;adherent cells and supernatant cells were cultured separately for colony formation.Cultures were photographed using a Nikon camera and a Zeiss Axiovert invertedmicroscope, using differential interference contrast microscopy (DIC).1082.3: RESULTS2.3.1: Identification ofthe component in CAM4L- 1-enrichedmaterial inhibitory to normalcolonyfonnation;Crude material enriched by CAMAL- 1 immunoaffinity chromatography waspreviouslyshown to be inhibitory to colony formation by normal progenitor cells (471).Since material eluted from CAMAL- 1 immunoadsorbent columns contained a variety ofprotein species, as determined by SDS-PAGE analysis, it was necessary to determinewhich component of CAMAL- 1 eluted material exerted this inhibitory effect on colonyformation. Components in CAMAL- 1 eluted material were further separated using FPLCgel filtration or preparative non-reducing SDS-PAGE, for evaluation in the colony assay.The gel filtration and preparative SDS gel purification procedures have been describedpreviously (472).A silver stained SDS polyacrylamide gel showing the peak fractions resulting froma Superose- 12 gel filtration colunm fractionation of CAMAL- 1 eluated material is shown inFigure Ia (this gel was prepared by Joan Shellard). Figure lb shows the mean number ofday 14 colonies observed when gel filtration fractions were added to CFU-GM assays ofnormal progenitor cells at a protein concentration of 1 j.tg/ml. Only fractions containingmaterial that migrated at 30 to 35 kfla by SDS-PAGE significantly reduced colonyformation; peak 2 (lane c), peak 3, (lane d), and peak 4 (lane e), but not peak 1 (lane b).Lane f is preparative SDS gel-purified P30 CAMAL material, which was used in the109CDCCC)CCzkOa976843131II p.tITT21a b c d e200100\u00E2\u0080\u00A2b \u00C2\u00B0 fraction0FIGURE 1. Identification of the component in CAMAL-1 eluted material inhibitory tocolony formation by progenitor cells from normal healthy donors. A CML cell lysate wasprepared using CAMAL-l immunoaffinity chromatography followed by FPLC gel filtrationor preparative non-reducing SDS-PAGE. a. A silver stained analytical SDSpolyacrylamide gel illustrates protein purification; unfractionated CAMAL-1 eluate, lane a;FPLC fractions corresponding to peaks 1 through 4, lanes b through e respectively; P30-35 CAMAL eluted from a preparative non-reducing SDS gel, lane f. Note that molecularweight standards differ between lane a and lanes b through f. b. The mean number of day14 colonies observed when normal progenitor cells were cocultured with FPLC peakfractions at a protein concentration of 1 .ig/m1 in CFU-GM assays.110experiment labelled \u00E2\u0080\u0098NPBL4/CML6a\u00E2\u0080\u0099, Table II. A titration of FPLC fractions 2 and 3(lanes c and d, Figure la) is illustrated in Figure 2. Inhibitory activity was maintained tolower protein concentrations in fraction 3, which consisted of essentially pure P30-35CAMAL material by SDS-PAGE analysis, than in fraction 2, in which many proteincomponents were present. Table II summarizes many of the experiments performed usingmaterial purified from CAMAL- 1 eluates of CML or AML cell lysates by FPLC gelfiltration or preparative SDS-PAGE. Colony formation was reduced by P30-35 CAMALin these experiments both in number and in size. Inhibition at concentrations of P30-35CAMAL above 1 ,tWml was profound; colony numbers were reduced by 90 to 100%.Inhibition of colony formation at concentrations of P30-35 CAMAL between 35 ng/ml and1 j.igfml was fairly constant at between 30 and 45%; at concentrations of P30-35 below 35ng/ml, a titration of inhibitory activity occurred. Inhibition of normal colony formation byp30-35 CAMAL was a consistent effect; it was seen using six preparations of P30-35CAMAL obtained from different donor sources of leukemic cells on six different sources ofnormal progenitor cells. The enrichment of inhibitory acitivity observed with increasingenrichment of P30-35 CAMAL is described in Table III. Enrichment of P30-35 CAMALfrom CAMAL- 1 eluated material was estimated to result in an approximately 200 foldenrichment of inhibitory activity.111401000FIGURE 2. Titration of inhibitory activity on colony formation by normal progenitor cellsin fractions 2 and 3 (as described in Figure 1). Normal progenitor cells were coculturedwith between 330 and 1 ng/ml of protein from fractions 2 or 3 in a CFU-GM assay; theinhibitory activities of these fractions was compared to sham treated (PBS) controls. Meannumber of control colonies was 60.5 \u00C2\u00B1 1.5.112C.,-3020 -10-0-\u00E2\u0080\u0094.0\u00E2\u0080\u0094\u00E2\u0080\u0094\u00E2\u0080\u0094 fraction 2fraction 3I10 100[protein] (ng/ml)PercentinhibitionbyP30-35CAMALconcentrationNumber ofcontrolNumber ofP30-35(ug/mi)coloniespermononuclear cellsNonnalCAMALmilliliterplatedpermilliliterdonorsource155.01.71.00.60.20.060.0350.010.004culture\u00C2\u00B1SEcultureNBM1CML189162.0\u00C2\u00B13.07.5XioNBM2CML210098444524127.3\u00C2\u00B15.51xio5NBM3AML39546.5\u00C2\u00B16.53X10NPBL4CML42728183.0\u00C2\u00B15.03X105NPBL4CML4a3820b75.0\u00C2\u00B19.03X105NPBL4CML5100129.5\u00C2\u00B12.53X105NPBL4CML6352660.5\u00C2\u00B11.53X10CML6a1710163.5\u00C2\u00B10.53X105372617139.5\u00C2\u00B17.02.5XNPBL4NBM5ciu\u00E2\u0080\u0099aAJP30-35CAMALpreparationswerepurifiedusingCAMAL-1iinmunoaffinitychromatographyandFPLCgelfiltrationexceptthosewiththeasuperscript whichwerepurifiedusingpreparativenon-reducingSDS-PAGE.bsmdt.sf-testanalysisofresultsshowedthatallinhibitorylevelsweresignificant at p<0.05withtheexceptionofb, inwhichp<0.1.cAJIcolonieswereculturedunder conditionsthatsupportedthegrowthofCFU-GM, exceptc,towhicherythropoietinwasadded;theseculturessupportedthegrowthofCFU-GEMM.TABLEII.SummaryofP30-35CAMALinhibitoryactivityincolonyassaysusingprogenitor cellsfromfivedifferentnormalhealthydonorsandsixpreparationsofP30-35CAMALderivedfromdifferentCMLorAMLpatientleucocytespecimens.\u00E2\u0080\u0094TABLE III. Enrichment of P30-35 CAMAL and coenrichment of inhibitory activity on colony formation byprogenitor cells from normal healthy donorsa.Concentration of protein Units of activity per ug of Enrichment of biologicalPreparation effecting 50% inhibition protein\u00E2\u0080\u0099 activityCAMAL-l eluate 20 ug/ml 0.05-FPLC P30-35 enriched 220 ng/inl 4.5 90 xmaterialPure P30-35 (by PAGE 100 ng/nil 10 200 xanalysis)asho are representative data for the purification of P30-3 5 CAMAL and its activity in CFU-GM/CFU-GEMMassays.be unit is defmed as the activity that effects 50% inhibition of CFU-GMJCFU-GEMM in a 1.0 millilitercolony assay.2.3.2: Lack ofinhibitory effect on CML colony formation byP30-35 CAM4L;Since CAMAL- 1 eluated material was observed to inhibit colony formation bynormal progenitor cells, whereas no inhibition of colony formation by CML progenitorcells occurred (471), the effect of material enriched for P30-3 5 CAMAL on colonyformation by CML cells was evaluated in order to verify that the component of interest hadbeen isolated. An experiment in which the effect of P30-35 CAMAL eluted frompreparative gels on colony formation by normal and by CML progenitor cells wascompared is shown in Figure 3. An experiment in which the effect of P30-35 CAMALpurified by FPLC gel filtration on colony formation by CML cells was evaluated is shownin Figure 4. \u00E2\u0080\u0098While it is recognized that these effects are not dramatic, they are not meant tostand on their own, but to be taken in the context of results from many assays using normalprogenitor cells (Table II). It can be seen that, in both instances, whereas colony formation11410080604020r:d.-400Q00z0 0-I-\u00E2\u0080\u0094 _J2 0.4). b. Titration of theinhibitory effect of P30-35 CAMAL on spleen colony formation. p <0.0005 in groupsreceiving cells treated with 33, 11, and 37 ng/ml P30-35 CAMAL, 0.05

1 jig/mI) where a more complete shutdown ofnormal myelopoiesis is seen, and complications such as bleeding and fatigue occur due todecreased platelet production, and due to anemia from decreased red blood cell production,respectively.Colony formation by several cell lines derived from myeloid leukemias wasstimulated by treatment with P30-35 CAMAL. These included EM2 and EM3, derivedfrom the leucocytes of the same patient with CML at different stages of leukemicprogression. Colony formation by both of these cell lines was stimulated in a mannersimilar to observed using CML clinical specimens. Colony formation by HL6O, derivedfrom the leucocytes of a patient with acute promyelocytic leukemia, was also stimulated byP30-35 CAMAL. In contrast to EM2 and EM3, a single peak of enhancement occurredover a broad concentration range of P30-35 CAMAL with HL6O, and peak enhancementoccurred at a lower concentration than with the CML-derived cell lines. Colony formationby the cell line IIEL, derived from an erythrocytic leukemia, was not affected by treatmentwith P30-35 CAMAL. It is interesting and possibly significant that the degree ofresponsiveness of those cell lines which responded to treatment with P30-35 CAMALappeared to correlate with the aggressiveness of the leukemia from which the cell lines werederived. Colony formation by EM3, derived when the patient\u00E2\u0080\u0099s CML was in a moreaggressive phase, was enhanced more dramatically than was colony formation by EM2.HL6O, derived from an AML, was stimulated at a lower concentration than were either ofthe CML-derived lines. Thus, different leukemic cell populations appear to respond totreatment with P30-35 CAMAL in non-identical manners, and responsiveness of cells to216the effects of P30-35 CAMAL might be as important as the levels of P30-35 CAMAL invitro or in vivo in determining the outcome of exposure of these cells to P30-35 CAMAL.The similarity in responsiveness to P30-35 CAMAL between the cell lines EM2 and EM3and CML clinical specimens provides a convenient experimental system for future studies.The responsiveness of HL6O to P30-35 CAMAL provides a potentially useful model forthe study of the activity of P30-35 CAMAL on AML cells.Several lines of evidence suggested that protease activity was involved in the effectsof P30-35 CAMAL on colony-forming cells. First, preparations of P30-35 CAMAL inwhich inhibitors of protease activity had been included were found to be inactive in colonyassays. In addition, N-terminal amino acid sequence analysis of preparative gel-purifiedmaterial indicated that a protein with amino acid identity to known serine proteases waspresent (Chapter 2, Appendix 2). Finally, it was observed that the effects of P30-35CAMAL on colony formation were maintained even after P30-35 CAMAL was washedfrom the cells, suggesting that these effects were immediate and maintained even after theP30-35 CAMAL material was removed. For these reasons, the possibility of theinvolvement ofprotease activity in the alterations of in vitro myelopoiesis mediated by P30-35 CAMAL was investigated.Colony formation by normal human cells and by murine progenitor cells wasinhibited to a similar extent whether the cells were cocultured or preincubated with P30-35CAMAL. In addition, colony formation by CML progenitor cells and myeloicl leukemiaderived cell lines was enhanced in a similar manner whether cells were cocultured or217preincubated with P30-35 CAMAL. These results indicate that the effects of P30-35CAMAL on hematopoietic cells and cell lines are immediate and may well take place at thecell surface. This finding was of significance also from a technical standpoint;preincubation of cells with P30-35 CAMAL allowed treatment in small volumes, requiringfar less P30-35 CAMAL material than did coculture experiments.Experiments in which previously untreated cells were incubated with supernatantretrieved from P30-35 CAMAL-treated cells demonstrated that the activities of P30-35CAMAL on colony formation were fully retained in the cell supematant, and not absorbedby the cells. This was demonstrated using normal human cells and murine cells in assaysfor CFU-G; in both cases inhibitory activity on colony formation was fully retained in thesupematant. Similarly, enhancing activity on colony formation by both CML cells and thecell line EM3 was also fully retained in the supematant. These results suggest that theeffects of P30-35 CAMAL on myelopoiesis are exerted at the cell surface, and that theactive component is not taken up by target cells.The observations indicating that the effects of P30-3 5 CAMAL on colony formationare immediate and likely occur at the cell surface are both consistent with the activity of aprotease. Hence, more direct evidence of protease involvement was sought. The effect ofphenyl methyl sulfonyl fluoride (PMSF), an inhibitor of serine protease activity, on P30-35CAMAL-mediated alterations of myelopoiesis was tested. It was found that both theinhibition of normal colony formation and the enhancement of CML colony formationmediated by P30-35 CAMAL were fully blocked by treatment of P30-35 CAMAL withPMSF.218Some progress had been made in this laboratory toward defining the substratepreference of preparations of P30-35 CAMAL using a panel of chromogenic peptidesubstrates. It was found that P30-35 CAMAL preparations recognize the substrate ala-alapro-phe-NA, whereas human neutrophil elastase, a serine protease related to the sequenceobtained from a preparation of P30-35 CAMAL, did not. Both P30-35 CAMAL andelastase recognize the substrate ala-ala-pro-val-NA (474).A peptide highly similar to the putative P30-35 CAMAL substrate, ala-pro-phe,which was linked to a chloro-methyl ketone (CMK) group, was obtained. Chloromethylketones are inhibitors of serine protease activity which form a covalent bond in the activesite of the enzyme. Specificity of blockage using these peptides is mediated by the aminoacid sequence to which the CMK group is linked. In an enzyme assay using chromogenicpeptide substrates, it was demonstrated that the activity of P30-35 CAMAL was blocked byala-pro-phe-CMK, whereas the activity of elastase was not blocked by this peptide.Furthermore, both the inhibition of normal colony formation and the enhancement of CMLcolony formation mediated by P30-35 CAMAL were fully blocked on treatment of P30-35CAMAL with ala-pro-phe-CMK. In contrast, the activity of P30-35 CAMAL on normalcolony formation was not blocked by three other CMK-linked peptides, including thepeptide which blocks elastase activity, ala-ala-pro-val-CMK. These results provide asecond line of evidence that serine protease activity is required for P30-35 CAMALmediated alterations of myelopoiesis. In addition, they demonstrate that the activity of P30-35 CAMAL can be blocked independent of effects on other protease activities, and raise thepossibility that specific blocking of the activity of P30-35 CAMAL using the peptide ala219pro-phe-CMK or other substances might be clinically useful in aiding the restoration ofnormal myelopoietic balance in the treatment of myeloid leukemias.A monoclonal antibody raised against preparations of P30-35 CAMAL, a-P30/35,was shown to react with cytospin preparations of nucleated cells from patients with CML toa far greater extent than with nucleated cells from normal healthy donors. This is a similarpattern of positive staining to that previously observed using CAMAL- 1, a monoclonalantibody raised against original preparations of the CAMAL antigen obtained by subtractivemethods from the lysates of cells from patients with myeloid leukemias; CAMAL- 1 hasdemonstrated reactivity with proteins other than P30-35 CAMAL. It has been shown,however, by ELISA, and more recently by Western blot analysis, that CAMAL- 1 does notreact with P30-35 CAMAL (473). Hence the antigen recognized as diagnostic of CML instudies using CAMAL-1 and the P30-35 CAMAL material that mediates the describedinhibition of normal myelopoiesis and the enhancement of leukemic myelopoiesis do notappear to be identical. However, the similarities in the pattern of staining obtained usingthese two monoclonal antibodies, in addition to the observation that CAMAL- 1 enriches forP30-35 CAMAL from lysates of myeloid leukemia cells, suggest that the entitiesrecognized by CAMAL-1 and ct-P30/35 might form an association.Several cell lines derived from myeloid leukemias were found to react extensivelywith a-P30/35 in the immunoperoxidase test. It is interesting and possibly significant thatthe cell lines shown to react with a-P30/35 by immunoperoxidase, and which thus mightproduce P30-35 CAMAL, also showed enhanced colony formation on treatment with P30-35 CAMAL. The only cell line tested which did not demonstrate enhancement in the220colony assay, HEL, also did not react with x-P30/35 in the immunoperoxidase test. Theseresults raise the possibility that P30-35 CAMAL might be involved in regulatingproliferation and differentiation in an autocrine fashion in these cell lines.Subsequent to the completion of the studies described above, it was recognized thatthe N-terminal amino acid sequence obtained from a preparation of P30-35 CAMAL wasidentical to that of azurocidin (Chapter 2, Appendix 2). The azurocidin sequence was notin the protein data bank at the time that the N-terminal sequence from the P30-35 CAMALpreparation was obtained, but its homology to known serine proteases taken together withthe observations described above influenced the course of the experiments addressingserine protease activity (Chapter 5). Azurocidin was cloned by a group investigating theantibacterial properties of proteases isolated from normal neutrophils. By purification ofproteins from azurophilic granules, this group isolated four proteins which were serineproteases or had serine protease homology. Two were well-known proteases, elastase andcathepsin G (479). One was shown by amino acid sequence to be identical to myeloblastin(309, 495, 496). The fourth, azurocidin, has two important changes; the serine andhistidine of the catalytic triad are both substituted. Consequently, azurocidin has noprotease activity on any chromogenic peptide substrates tested, including several of thosewith which P30-35 CAMAL preparations were shown to react (438, 474).P30-35 CAMAL preparations were fractionated using reverse-phase HPLC as partof the project of another graduate student in this laboratory dealing characterization of P30-35 CAMAL at a subcellular level (474). In these preparations, peaks corresponding to221elastase, cathepsin G, and azurocidin could be identified, in addition to a major peak whichwas unique to preparations of P30-35 CAMAL (474), as compared to preparations derivedfrom normal neutrophils (479). Additional studies were undertaken in order to determinewhether the observed alterations in myelopoiesis could be attributed to the characterizedproteins in these preparations.The results presented in Chapter 5 show clearly that P30-35 CAMAL-mediatedalterations of myelopoiesis require serine protease activity, so the possibility that azurocidinin preparations of P30-35 CAMAL could mediate the observed effects on colony formationseemed unlikely. Elastase is known not to mediate P30-35 CAMAL activity on colonyformation, since, in contrast to P30-35 CAMAL, elastase was shown to be inhibitory tocolony formation by CML progenitor cells, and since preparations of P30-35 CAMALwere screened for lack of this elastase activity. In addition, it was shown that the activityof elastase was not blocked by the peptide ala-pro-phe-CMK at even an 100:1 molarexcess, whereas ala-pro-phe-CMK did fully block P30-35 CAMAL-mediated alterations ofcolony formation by normal progenitor cells and by CML progenitor cells at a 10:1 molarexcess. Since downregulation of myeloblastin was found to cause the growth arrest anddifferentiation of promyelocytic leukemia cells (310), it appeared that the effects of P30-35CAMAL and myeloblastin might be similar. However, myeloblastin, also known asproteinase 3, was found to be essentially absent from P30-35 CAMAL preparations. Inaddition, the chromogenic peptide subtrates recognized by myeloblastin are substantiallydifferent from those recognized by P30-35 CAMAL (438, 474). The effects of azurocidin,myeloblastin, and cathepsin G were evaluated in a colony assay using normal human bonemarrow cells. None of these proteins inhibited colony formation in this assay (Appendix2222). In contrast, recent experiments have demonstrated that protein from the unique peak inpreparations of P30-35 CAMAL was inhibitory to colony formation in cultures of murinecells (477). In addition, protein from this unique peak had activity on the chromogenicpeptide substrate recognized by preparations of P30-35 CAMAL (474). Protein from thispeak is being prepared for N-terminal amino acid analysis.The same peptide substrate recognized by preparations of P30-35 CAMAL is alsorecognized by the serine protease cathepsin G. Although it was shown that colonyformation by normal progenitor cells was not altered by cathepsin G, it remains apossibility that the alterations of colony formation by CML progenitor cells might bemediated by cathepsin G, or that cathepsin G activity might be required to render treatedcells susceptible to alterations in myelopoiesis mediated by other substances in P30-35CAMAL preparations. However, enzyme assays using CMK-linked peptides to block theactivity of P30-35 CAMAL preparations or cathepsin G on the substrate which isrecognized by both, ala-ala-pro-phe-NA, clearly demonstrated the presence of a proteaseactivity in preparations of P30-35 CAMAL which is distinct from that of cathepsin G(Appendix 2). Specifically, the peptide ala-ala-pro-val-CMK fully blocked P30-35CAMAL activity at a CMK:P30-35 CAMAL molar ratio of 10:1 or less, whereas cathepsinG activity was not blocked by this same peptide at a CMK:cathepsin G molar ratio of even100:1 (Figure 34a). This difference in activity cannot be attributed to a block of elastaseactivity, since elastase does not recognize the tchromogenic peptide substrate used toevaluate activity in this assay (474). Similarly, the peptide ala-pro-phe-CMK blocked P30-35 CAMAL activity substantially at a molar ratio of 1:1, whereas cathepsin G activity wasessentially unaffected at this level, and significant activity was retained even at a ratio of22310:1 (Figure 34b). More recently, the unique peak in reverse phase IIPLC-separatedpreparations of P30-35 CAMAL has been shown to have activity on the P30-35CAMAL/cathepsin G substrate (474). It remains a possibility, then, that the alterations ofmyelopoiesis described in this study are mediated by a serine protease which is distinctfrom characterized proteases. Whether the enhancement of colony formation by CML cellsis mediated by cathepsin G or by the unique peak in preparations of P30-35 CAMAL is thesubject of ongoing studies (476).In many systems involving proteolysis, a cascade of zymogen activations occurs,with an altered biological effect as the end result. Proteases activated in a cascade includethe proteases of the blood clotting system (497, 498), the complement system, (499, 500),and apparently the proteases involved in pattern formation in the Drosophila melanogasterembryo (501). It is possible that the presence of more than one protease in P30-35CAMAL preparations is required for the final alterations of in vitro myelopoiesis to occur.Other substances which are inhibitory to normal myelopoiesis have been described;these include leukemia inhibitory activity (LIA), leukemia associated inhibitor (LAI),lactoferrin, acidic isoferritins, macrophage inflammatory protein-la, inhibin, TGF-13,TNF- a, the interferons, interleukin 10, the tetrapeptide Ac-ser-asp-lys-pro, and thepentapeptide pEEDCK (Chapter 1). Substances which alter the growth of leukemic cells,such as the related proteins leukemia inhibitory factor (LIF) and oncostatin M, also havebeen investigated. In addition, there is an enormous volume of literature documenting theactions of various cytokines and combinations of cytokines on normal and malignant224hematopoietic cells. The effects of these cytokines vary depending on several factors, forexample on which other factors are present, on the immediate microenvironment, such aswhether cytokine-producing accessory cells are present which are able to influence targetcells in a paracrine fashion, on whether target cells are undergoing particular adhesiveinteractions, and on the state of dependence of target cells to the cytokine in question, forexample some leukemic cells are thought to be growth factor independent, and mayrespond to factors in an autocrine fashion. Proteases, and serine proteases in particularhave been described which function in or alter the function of hematopoietic cells. Inaddition to the functions of proteases in cytotoxic T lymphocytes, neutrophils, and mastcells, these include, among other examples, protease involvement in the release of TNF-ufrom a membrane-bound form, protease involvement in the release ofbiologically active MCSF from a membrane-bound form, proteolytic downregulation of the M-CSF and G-CSFreceptors, modulation of protein kinase C activity in hematopoietic cells, myeloblastinactivity in promyelocytic leukemia, and CALLA in CLL (Chapter 1). Thus, many events indifferentiation and function in the hematopoietic system involve proteolytic activity. Theinvolvement of proteolysis in alterations of hematopoiesis has become increasingly welldocumented in recent years, and appears to be coming under increasing scientific scrutiny.P30-35 CAMAL appears to be one further example of a proteolytic activity which functionsin the alteration ofhematopoiesis.2256.2: Speculation on possible mechanisms ofaction ofP3O-35 CAMAL;The following ideas about mechanisms by which alterations in myelopoiesis couldbe mediated by P30-35 CAMAL have not been experimentally tested, and are speculative.Although normal and CML progenitor cells responded to treatment with P30-35CAMAL at different concentrations, possible common themes were noted. In the cases ofboth normal and of CML colony formation, one progenitor cell type responded to arelatively lower concentration of P30-35 CAMAL; CML CFU-GEMM were consistentlyaffected at between 4 and 10 ng/ml, and normal CFU-G were affected at between 50 ng/mland 1 j.tgfml. Similarly, at relatively higher concentrations of P30-35 CAMAL, multipleprogenitor cell types were affected in both cases; all CML colony types were increased at100 to 200 ng/ml, whereas all normal colony types were decreased at levels greater than 1j.tg/ml. The effects on one progenitor cell type as compared to multiple progenitor celltypes occurred at a concentration difference of 10 fold or greater in both the CML and thenormal assays. These observations imply that the signal initiated by P30-35 CAMAL in thesingle class of progenitors affected in CML and in normal cells might be similar, as mightthe signal initiated in the multiple progenitor cell types in both cases.These observations raise questions about the substrate(s) or ligand(s) recognized byP30-35 CAMAL on the affected progenitor cells. Several possibilities exist as to thepattern of ligands recognized by P30-3 5 CAMAL under the various treatment conditions.For example, the ligand recognized on a single class of progenitor cells might be similar on226both CML and normal progenitor cells, whereas the ligand recognized on multipleprogenitor, cell types might also be similar on both CML and normal progenitor cells butdifferent from that affected on the single class of progenitor cells in each case. Thedifference in P30-3 5 CAMAL concentration required between CML and normal progenitorcells to effect changes in colony formation in either the case of a single progenitor cell classor multiple progenitor cell classes might be accounted for by a change in regulatorymechanisms in CML cells which increase or stabilize signalling mechanisms initiated byP30-35 CAMALIt seems possible that two ligands are affected by P30-35 CAMAL, at least in thecase of CML progenitor cells, since enhancement of colony formation at an intermediateconcentration of P30-35 CAMAL was reduced or absent. If the same cell surface moleculewere involved in P30-35 CAMAL-mediated enhancement of colony formation in the twoprogenitor cell populations affected, enhancement of primitive colony types at intermediateconcentrations of P30-35 CAMAL would be expected. It is possible, however, that P30-35 CAMAL might interact with the same cell surface molecule in the two cell populations,but activate different signal transduction pathways, resulting in the enhancement ofdifferent colony populations.A stabilizing change to a putative signal transducing portion of a P30-35 CAMALligand, or a signal transducing molecule downstream of a P30-35 CAMAL ligand, couldresult in a continuous signal favouring growth over differentiation in CML progenitor cells.An example from the literature which may be illustrative is that of signal transductioninvolving IL-3. It is thought that proteolytic cleavage of the IL-3 receptor might be227involved in signal transduction mediated by IL-3. Stimulation of IL-3 responsive cells byIL-3 results in the disappearance of a 140 kDa IL-3 binding protein, and the appearance oftwo 68 kDa proteins, each of which can be phosphorylated, one on serine, and the other ontyrosine (451). These phosphoproteins are thought to have signal transducing capabilities,and might be rapidly degraded under normal circumstances as a downregulatorymechanism. It is possible that P30-35 CAMAL might affect a similar cell signallingmechanism; interaction with a cell surface ligand such as a colony-stimulating factor (CSF)receptor could result in intracellular release of a signalling molecule into the cytoplasm,which in normal cells might be rapidly degraded. Under these circumstances, proteolyticcleavage of the cell surface ligand would result in a net loss of the signalling molecule fromthe cell surface. In normal cells, intracellular degradation of the signalling molecule wouldresult in downregulation of the signal transduced. Removal of the ligand from the cellsurface would lead to a loss of responsiveness to stimulatory signals resulting in decreasedcolony formation. If a stabilizing change to the signalling portion of the putative ligandwere to occur in CML cells, however, stimulation of these cells would result in continuedintracellular signalling, due to lack of downregulation, in the absence of further stimulatorysignals, and might be a mechanism involved in the progression of CML from chronic phaseto accelerated phase and blast crisis (Figure 30a). This line of reasoning is consistent withthe observation that one of the hallmarks of full cellular transformation is a loss ofdependence on growth factors (25, 122, 161, 502). This or other models requireexpression of a ligand or substrate for P30-35 CAMAL on CML progenitor cells of alltypes since the growth of all colony types is enhanced by P30-35 CAMAL. A ligand orsubstrate for P30-3 5 CAMAL must be present on all normal progenitor cell types also since228at high concentrations of P30-35 CAMAL colony formation by all types ofprogenitor cellswas inhibited.An alternative explanation for the changes observed in colony formation ontreatment of progenitor cells with P30-35 CAMAL involves differential expression ofligands or substrates for P30-35 CAMAL, resulting in differential sensitivity on the part ofprogenitor cells to the effects of P30-35 CAMAL on myelopoiesis. If aberrant expressionof a P30-35 CAMAL-reactive molecule in CML were the sole cause of enhancement,however, CML CFU-G, like normal CFU-G, would be expected to be downregulated byP30-35 CAMAL. Plucked colony data, however, suggest that CML CFU-G, in addition toother colony types, are increased by concentrations of P30-35 CAMAL of 100 to 200ngfml. Alternatively, CML cells might produce more P30-35 CAMAL than do normalcells, resulting in higher intrinsic culture or serum levels and an apparent increasedsensitivity to exogenously added protein. However, the effect of P30-35 CAMAL oncultures of CML cells was consistent at consistent concentrations of exogenously addedP30-35 CAMAL, despite the fact that the number of presumably P30-35 CAMALproducing colonies varied greatly between experiments. Consistent intrinsic culture levelsof P30-35 CAMAL under these conditions seem unlikely.Insight into how the activation of a signal transduction pathway might be initiated atthe cell surface may be gained through the example of platelet activation by thrombin.Platelet activation and aggregation is blocked by agents which inhibit the serine proteaseactivity of thrombin. The activation signal is transmitted via the thrombin receptor, anintegral membrane protein with seven tmnsmembrane domains. Thrombin has been shown229to release a short peptide from the N terminus of the receptor, allowing the newly created Nterminus to act as a \u00E2\u0080\u009Ctethered ligand\u00E2\u0080\u009D; the new N terminus binds to another site on thereceptor protein and results in platelet activation, as measured by calcium flux, andaggregation (453). It is possible that the protease activity required for the effects of P30-35CAMAL on myelopoiesis might act to initiate signal transduction via a similar mechanism.It is possible that a signal transduction pathway involving bcr and/or abi, or P210bcr-abl, in the case of normal and CML cells respectively, are activated by P30-35CAMAL. In this model, P30-35 CAMAL might activate the same pathway in both normaland CML cells upstream of bcr and/or abi or P210 bcr-abl, resulting in different effectsdownstream. The altered kinase activity and cellular location of P210 bcr-abl could resultin a quantitatively greater or prolonged signal, resulting in a differentiation block, forexample. Since differentiation and proliferation are partially uncoupled in CML, byunknown mechanisms which could conceivably involve bcr-abl, a prolonged stimulatorysignal might shift the balance toward cell proliferation, or away from differentiation,resulting in increased clonogenicity and larger colonies. In contrast, in normal cells, inwhich differentiation and proliferation are coupled, activation of the same pathway mightresult in interaction with unaltered bcr and/or unaltered abi. The net result in this casewould be a signal of normal duration, and a decrease in differentiation/proliferation, leadingto a decrease in colony formation and in colony size. These speculations are consistentwith the observation that higher levels of P30-35 CAMAL are required for observablealterations in myelopoiesis in normal colony-forming cells than in CML cells. A higherconcentration of P30-35 CAMAL would be expected to be required for a significant effectto result in cells in which normal regulatory mechanisms are operative, and signalling is230efficiently downregulated. Conversely, the alterations in kinase activity and cellularlocation of signal transducing proteins such as bcr-abl in CML would be expected toabnormally stabilize and prolong signal transduction, hence lower concentrations and/or ashorter duration of an activating signal would be required in order to obtain an observableeffect.Using antisense oligodeoxynucleotides to ms, it was shown that ms is required forcolony formation supported by IL-3, GM-CSF, and M-CSF, but not by G-CSF (138).Recall that it has been shown that bcr encodes a GTPase activating protein (GAP) for mc.If it is assumed that G-CSF induced colony formation is mediated by the ras -relatedprotein mq partial inactivation of mcGAP activity due to the bcr-abl translocation wouldexplain the increase in cells of the granulocytic lineage seen in CML, since the ability ofbcr/mc GAP to downregulate mc activity would be impaired. P30-35 CAMAL fits intothis picture as follows.In normal cells, at low concentrations of P30-35 CAMAL, CFU-G would beexpected to be targetted if P30-35 CAMAL were to somehow interfere with mc activity,possibly by activating bcr/rac GAP activity. This would result in downregulation of racactivity, thus disrupting the ability of rac to support the formation of CFU-G. (It ispossible, for example, that P30-35 CAMAL induces the ability of bcr to bind to c-abl;recall that it has been documented that bcr binds bcr-abl, see below). In normal cells athigh concentrations of P30-35 CAMAL, this interference might extend to the ras pathwayby virtue of the fact that rac and ras are similar; i.e. ras GAP activity would beupregulated. This would result in downregulation of ras activity, which is required for231colony formation supported by IL-3, GM-CSF and M-CSF. Hence, all colony typesformed by normal cells would be reduced at these concentrations. In contrast, in CMLcells, the ability of P30-35 CAMAL to activate bcr/rac GAP would be disrupted by virtueof the fact that bcr is altered by the bcr-abl translocation. Signals stimulating mc activitywould thus be prolonged (Figure 30b). At low concentrationsof P30-35 CAMAL, P30-35CAMAL treatment of CML cells causes increases in primitive colony types. It would haveto be postulated that rae is involved in signal transduction in more primitive progenitorcells than CFU-G in order for this to occur, which is not unreasonable since bcrab1 isknown to be present in pluripotent stem cells. The inability to activate bcr/mcGAP mightbe expected to result in no net effect on CML colony formation. However, the observedincrease in colony formation might be mediated by the altered cellular location of bcr-abl orits upregulated tyrosine kinase activity. Signalling mechanisms initiated by P30-35CAMAL might result in a change, such as phosphorylation of bcr-abl, which alters thiskinase activity. Or, an increase in the ability of bcr-abl to bind and sequester normalbcr/rac GAP could result, thus further decreasing the ability of the cell to downregulatestimulatory signals mediated through rae, and resulting in increased colony formation.Primitive colony formation might be predominant because the dysregulated bcr-abl activityin primitive cells might be preferentially expressed in these cells, resulting in an expansionin the clone from which leukemic CFU-G arise. At the same time, colony formation innormal clones would be inhibited by virtue of the normal mcGAP activity in these cells. InCML cells at high concentrations of P30-35 CAMAL, P30-35 CAMAL treatment mightinitiate the binding of ras GAP by bcr-abl, resulting in the decreased downregulation of iasactivity, and prolonged stimulatory signals promoting colony formation supported by IL-3,GM-CSF, and M-CSF (ras), and by G-CSF (mc ) In this regard, ms GAP has been232a \u00C3\u00A7I - Normal cellsc\u00E2\u0080\u0094ii.. CML cellsFIGURE 30. Possible mechanisms by which P30-35 CAMAL treatment could affectsignal transduction, resulting in the observed alterations in myelopoiesis. a. Interaction ofP30-35 CAMAL with a substrate (\u00C3\u00A7) at the cell surface results in activation (*) of anintracellular signal transduction pathway. i. In normal cells, the positive signal is rapidlydownregulated by a cellular inhibitor (1). Release of the P30-35 CAMAL substrate fromthe cell surface makes the cells refractory to stimulatory signals, resulting in an overallsuppressive effect. ii. In leukemic cells, a stabilizing change to the signalling molecule (+)results in inefficient downregulation ([) of signals, and in an overall stimulatory effect (!).Removal of the P30-35 CAMAL substrate from the cell surface in these cells isinconsequential. b. Cellular activation by P30-35 CAMAL of a pathway involving rae. i.In normal cells, stimulatory signals transmitted via rac are rapidly downregulated by therac GAP activity of bcr ([). ii. In CML cells, bcr-abl, shown bound to the cytoskeleton,sequesters bcr, allowing prolonged activation of rac (*rac) and a prolonged stimulatorysignal (!). The ras pathway could be affected in a similar manner, as bcr-abl has beenshown to bind ras GAP. In these schemes, \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 refers to P30-35 CAMAL.CAMAL233bCAMALi - Normal cellsCAMALii. CML cell:\u00E2\u0080\u0094\u00E2\u0080\u0094cc234shown to immunoprecipitate with P210 bcr-abl and to be phosphorylated by it, a findingwhich implies that mitogenic signals mediated by p21 ras could be altered by bcr-abl(139). Recall that differences in which colony types were affected by P30-35 CAMALoccurred at approximately 10 fold differences in concentration in the case of both normaland CML cells. A 10 fold increase in P30-35 CAMAL concentration and signals initiatedin this way might be what is required to affect ras activity.The technology to evaluate whether P30-35 CAMAL initiates signal transductionpathways upstream of bcr-abl is currently available. Treating CML cells with antisenseoligodeoxynucleotides to bcr-abl should abrogate the P30-35 CAMAL mediatedstimulation of CML colony formation, if bcr-abl pathways are involved. A block inexpression of the bcr-abl fusion gene might even result in a decrease in colony formationon treatment with P30-35 CAMAL, if the lack of bcr-abl expression restores a \u00E2\u0080\u0098normal\u00E2\u0080\u0099phenotype to these cells. The expression of bcr-abl can be evaluated by the polymerasechain reaction.If P30-35 CAMAL is indeed found to be an upstream signal which actsextracellularly and alters signalling by bcr-abl, this has important therapeutic implicationsin CML. Specific therapies for CML are still in the very experimental stages. Specifictargetting of bcr-abl by antisense oligodeoxynucleotides, for example, requires uptake intothe cells, which must be incubated ex vivo. This is possible so far only in conjunction withbone marrow transplantation, an invasive procedure associated with significant toxicity,mortality, and a significant incidence of leukemic relapse. If the activity of an upstreamextracellular signal affecting bcr-abl could be targetted and blocked, it might be possible to235carry this out in a far less invasive way such as by intravenous injection of a blockingagent.To summarize, the ligands or substrates recognized by P30-35 CAMAL at thesurface of normal and CML progenitor cells are unknown. However, interaction of P30-35 CAMAL with these ligands or substrates might result in activation of cell signalling by amechanism similar to that described for the thrombin receptor, in which proteolyticcleavage unmasks a \u00E2\u0080\u0098tethered ligand\u00E2\u0080\u0099 which causes activation of signalling pathways.Alternatively, it might activate signalling in a manner similar to that described for the IL-3receptor, in which a signalling protein is released from the receptor intracellularly byproteolytic cleavage. Stabilizing changes to regulatory proteins in signalling pathwayscould result in the enhancement of colony formation in CML cells mediated by P30-35CAMAL in contrast to the thhibition of colony formation by normal cells. P30-35 CAMALmight initiate signalling upstream of bcr/rac GAP and ras GAP in normal cells, andupstream of bcr-abl and ms GAP in CML cells. These ideas, while attractive, and whilesupported in part by observations described in this study as well as by data published byother groups, are speculative, and must be tested experimentally before conclusions can bedrawn.2366.3: Future directions;6.3.1: Basic science;Purification procedures are being optimized as part of the research project ofanother graduate student in this laboratory in order to make the purification of P30-35CAMAL less labour intensive yet still reproducible. The apparent similarity in amino acidsequence between that obtained from a preparation of P30-35 CAMAL and elastase, forinstance, made it necessary to ensure that preparations of P30-35 CAMAL were free ofcontaminating elastase activity. This was an issue since elastase has a similar molecularweight to the P30-35 CAMAL entity shown to mediate the described alterations of in vitromyelopoiesis. Most preparations of P30-35 CAMAL were shown to be free of elastaseactivity by virtue of the fact that elastase is inhibitory to colony formation by CMLprogenitor cells (Chapter 2; all preparations of P30-35 CAMAL used in these studies werefree of elastase activity), however screening P30-35 CAMAL preparations for lack ofelastase activity was a time and labour-intensive enterprise.Analysis of reverse phase HPLC separations of P30-3 5 CAMAL preparationsindicates the presence of a protein which might be unique, and which appears to mediateinhibitory activity onmurine colony formation (474, 477). Whether this same materialmediates the enhancement of colony formation by CML progenitor cells remains to bedetermined. This material which is inhibitory to colonies cultures from murine cells hasbeen sent for N-terminal amino acid analysis. Assuming that P30-35 CAMAL proves to be237a unique protein, cloning of the gene from cell lines derived from myeloid leukemias and/orCML clinical specimens would be an obvious asset for further studies. Recombinantprotein might be a convenient source of P30-35 CAMAL for future biological assays. Forexample, the effects of P30-35 CAMAL on myelopoiesis could be examined in greaterdetail using long-term cultures of normal or CML progenitor cells, in one step cultures, orin two step cultures in order to determine the effects of P30-35 CAMAL on myelopoiesisby cells exposed to a preformed adherent layer, and the possible interactive effects of P30-35 CAMAL in conjunction with other cytokines or regulatory factors could be examined.Moreover, the effects of P30-35 CAMAL on the gene expression of other regulatoryfactors could be examined in this system. In addition, it could be determined whether theeffects of P30-35 CAMAL on myelopoiesis are mediated by a direct action on the affectedprogenitor cells, or requirethe presence of accessory cells. This analysis would requiresupport of colony formation by recombinant colony-stimulating factors in cell populationsdepleted of accessory cells.Analysis of the effects of P30-35 CAMAL on the clonogenicity and proliferation ofAML cells was not possible during the course of these studies due to rare access to theseclinical specimens, and due to the fact that a minority of specimens that were obtainedformed colonies in the culture conditions used; they tended to form large numbers of smallcell clusters which were difficult to evaluate. The effect of P30-35 CAMAL on AMLmyelopoiesis could be examined using long term cultures, and access to recombinantprotein would facilitate this. Alternatively, liquid suspension cultures using AML cellshave been described (503), and, although the cloning efficiency is reported to be lower thanthat obtained in methylcellulose cultures (504), these could be pursued. In addition, the238effect of P30-35 CAMAL on cell lines derived from AML leucocytes, such as HL6O, couldbe examined in greater detail.Recombinant protein would also facilitate definition of the reactivities of cx-P30/35at a cellular level. For example, it would be interesting to determine whether therecognition of CML cells by a-P30/35 could be competitively inhibited by the protein ofinterest. In addition, it would be interesting to determine whether incubation of normalcells with this protein increases reactivity with this antibody. This would indicate thepresence ofa ligand or substrate for P30-3 5 CAMAL on the surface of these cells.Molecular probes would allow identification of the cell type(s) which produce P30-35 CAMAL and might shed light on its apparent regulatory role in myelopoiesis. It hasbeen assumed that the major cell population producing P30-35 CAMAL is the leukemicneutrophils and/or their progenitors. However, P30-35 CAMAL could equally well beproduced by other cell types and act in a paracrine fashion on the leukemic cells. Forexample, it is possible that P30-35 CAMAL might be produced by a minority of cells fromnormal bone marrow, and could be a normal regulatory molecule in hematopoiesis whichbecomes dysregulated in CML.One intriguing study would be the examination of CML cell populations using amolecular probe for P30-35 CAMAL, and a probe for the bcr-abl fusion gene product.These probes could be fluorescently labelled to determine whether the P30-35 CAMALproducing cell population is indeed the leukemic population (505). Similarly, obviouslyenlarged CML colonies could be tested by polymerase chain reaction (PCR) for bcr-abl239expression to determine whether, as would be expected on the basis of these studies, theresponsive progenitor cell population is indeed derived from the Philadelphia chromosomepositive clone. Alternatively, leukemic and normal cells could be separated in specimens ofleucocytes obtained from CML patients and the effects of P30-35 CAMAL tested on thesesorted cell populations. In this regard, it has recently been shown that the cell populationwith the surface characteristics CD34DRiin (DR = class II HLA-DR) contain almostexclusively progenitor cells that give rise to colonies which are bcr-abl negative (425).Cells which are CD34DR, conversely, give rise almost exclusively to bcr-abl positivecolonies. Finally, cloning of the putative gene encoding P30-35 CAMAL would enabledetermination of its chromosomal location, and its possible involvement in genetic events inmyeloid leukemias could be evaluated.It might be possible to identify the physiological ligand(s) and/or substrate(s)recognized by P30-3 5 CAMAL by analysis of cells which are susceptible to the effects ofP30-35 CAMAL. For example, it might be possible to analyze two dimensional gels of celllysates of P30-35 CAMAL treated cells using antibodies specific for likely substratecandidates. For example, the group investigating myeloblastin believe its substrate to bethe retinoic acid receptor (310). Western blots could be done using anti-retinoic acidreceptor Abs, or Abs directed to cell surface molecules known to be involved in growthcontrol and differentiation and which are possible candidates as ligands or substrates forP30-35 CAMAL; examples include the IL-3 receptor, c-kit and its ligand Steel factor,CD34, and adhesion molecules. One cell surface molecule which is a possible P30-35CAMAL substrate is the G-CSF receptor. It was recently shown that downregulation ofthe G-CSF receptor can be blocked by inhibitors of serine protease activity (290). This is240an intriguing observation in light of the inhibitory activity of P30-35 CAMAL on normalCFU-G, and one that deserves to be pursued.6.3.2: Development ofa clinically useful agent;Agents which inhibit the activity of specific proteases have been used in a clinicallyrelevent manner. For example, angiotensin converting enzyme (ACE) inhibitors arecommonly used in the treatment of hypertension. Inhibitors are being designed by proteinengineering which inhibit proteases specifically without the untoward effects of otheragents. For example, alpha-i antithrombin has been modified by site-directed mutagenesisto have thrombin blocking activity, making it potentially useful as an anticoagulant, andmaking it possible to avoid the risk ofhemorrhage associated with the use of heparin (506).Other groups are designing protease inhibitors by computer modelling of the threedimensional structure of a particular enzyme based on its primary sequence (507). Hence,blocking the protease activity involved in the alterations of myelopoiesis mediated bypreparations of P30-35 CAMAL might prove to be a realistic goal.Studies demonstrating that P30-35 CAMAL-mediated alterations of myelopoiesiscould be blocked using the peptide ala-pro-phe-CMK, that this effect could be separatedfrom effects on the serine protease elastase, and that the activity of P30-35 CAMAL onmyelopoiesis was not blocked by several other CMK-linked peptides raise the possibilitythat ala-pro-phe-CMK or a similar agent could be clinically useful in the treatment of CML.241A first step in the development of a clinically useful blocker of the activity of P30-35CAMAL is the determination of the optimal substrate for P30-35 CAMAL, and blockingthe activity of P30-35 CAMAL must be shown not to significantly affect other importantprotease activities. As a preliminary evaluation, the potential toxicity of putative blockingagents on primitive progenitor cells should be tested in long-term marrow cultures usingnormal bone marrow cells. If an agent which blocks the activity of P30-3 5 CAMAL is tobe clinically useful, it must be shown to spare primitive normal stem and progenitor cells.If, however, such an agent proves to be toxic to normal stem cells, it might be possible toreduce this toxicity by coupling the agent to a carrier protein. Alternatively carriers whichimprove delivery, and reduce toxicity, such as liposomes, could be used (315).Alternatively, physiological substrates such as inhibitors of proteases found in the plasmacould be altered by protein engineering to a form which is bound but not cleaved by P30-35CAMAL. Such a strategy has been used to block proteases in the treatment ofhypertension(506).Ifputative agents which block the activity of P30-35 CAMAL prove to be non-toxicto normal stem cells, long-term marrow cultures should be done using CML cells in orderto determine whether the blocking agent is inhibitory to the growth of the CML clone. Theeffect on the CML clone can be determined by screening for the presence of the P210 bcrabi transcript by polymerase chain reaction (PCR). This system is already in place in thislaboratory and has been used to evaluate the selective toxicity of benzoporphyrin derivative(BPD) toward Ph progenitors (348). If a P30-35 CAMAL-blocking agent were to befound to differentially inhibit the growth of the Ph clone, it could be used in conjunctionwith purging agents to treat remission marrow intended for autologous bone marrow242transplantation. Or, it is possible that such an agent could be used to control thismalignancy in a less invasive way; by intravenous injection, for example, singly, or inconjunction with chemotherapeutic agents.Should long term marrow culture studies prove promising, toxicity and clearancestudies with agents which block the activity of P30-35 CAMAL can be conducted in mice.Efficacy studies in mice can be conducted using the ex vivo spleen colony assay describedin Chapter 3.243SUMMARY AND CONCLUSIONS;Studies reported in this paper document the effects of P30-35 CAMAL, enrichedfrom lysates of leucocytes obtained from patients with myeloid leukemias, on in vitromyelopoiesis by normal and CML progenitor cells. The inhibition of colony formation bynormal progenitor cells originally noted using crude preparations eluted fromimmunoaffmity columns prepared with the monoclonal antibody CAMAL- 1, which was inturn prepared against protein isolated by substractive methods from lysates of myeloidleukmia cells, was shown to be mediated by the P30-35 material in these preparations.Normal colonies were inhibited in number and in size by this P30-35 CAMAL material,and the effect was preferentially directed toward CFU-G at low concentrations of P30-3 5CAMAL. At high concentrations of P30-35 CAMAL, colony formation by all progenitorcell types was inhibited. Cell numbers in the non-adherent fraction of long term culturesusing normal bone marrow were reduced, but were increased within the adherent layers,indicating that these cells might be blocked in differentiation. The inhibitory effect oncolony formation was also observed in assays using murine progenitor cells. Like inassays of human cells, colonies were inhibited in number and in size. Colony formationwas inhibited to a similar extent in assays of human and murine cells, and the effect titratedover a similar range of P30-35 CAMAL concentration. As was observed in cultures ofhuman cells, the inhibitory effect was directed toward murine CFU-G. In addition, spleencolony formation in an ex vivo assay was reduced. These results are an indication that the244inhibition of normal granulopoiesis mediated by P30-35 CAMAL is conserved acrossspecies barriers, and might be an important regulatory mechanism in myelopoiesis.In contrast, the same P30-35 CAMAL material that was inhibitory to colonyformation by normal progenitor cells enhanced colony formation by CML progenitor cells.This enhancement of colony formation was both in number of colonies and in the size ofcolonies within the P30-35 CAMAL-treated cultures. These effects were diametricallyopposed to the effects observed in cultures of normal progenitor cells. The enhancement ofcolony formation by CML progenitor cells was consistent, and was directed toward twoprogenitor cell populations. At a low concentrations of P30-35 CAMAL, primitive colonytypes were enhanced, whereas at a higher concentration, increases were seen in colonies ofall types. Colony formation by several cell lines derived from the leucocytes of patientswith myeloid leukemias was enhanced in addition.The differential alterations in colony formation by normal and CML progenitor cellsmediated by P30-35 CAMAL are important, since they provide a mechanism by whichleukemic cells might gain a growth advantage over normal cells and become predominant.Suppression of normal myelopoiesis and an often large increase in levels of circulatingleukemic neutrophils and their progenitors are well known features of CML.The observed alterations in colony formation by normal and CML progenitor cellswere demonstrated to require serine protease activity. Experiments in which the P30-35CAMAL-mediated activities on colony formation were maintained after the P30-35CAMAL material was removed from the cells, and were shown to be retained in the245supernatants of treated cells demonstrated that the effects on in vitro myelopoiesis wereimmediate, were not adsorbed by the cells, and likely occurred at the cell surface. P30-35CAMAL-mediated activities on colony formation were blocked by treatment of the P30-35CAMAL material with PMSF, an inhibitor of serine protease activity, and using a peptidefor which material in preparations of P30-35 CAMAL had some selectivity, which waslinked to a chioromethyl ketone group, an inhibitor of serine protease activity. It might bepossible to block this P30-35 CAMAL activity in the treatment of CML using a highlyspecific CMK-linked peptide or other agents which are found to block the activity of P30-35 CAMAL.A monoclonal antibody was raised against highly enriched P30-35 CAMALmaterial, and was used to enrich P30-35 CAMAL from myeloid leukemia cell lysates, andto examine cells in an immunoperoxidase slide test. The immunoperoxidase analysisshowed that nucleated cells derived from patients with CML showed far greater reactivitywith this antibody than did normal cells. This was a similar pattern of reactivity as wasobserved using CAMAL- 1, an antibody with known reactivity with other proteins, and wasan indication that the antigen recognized in the studies using CAMAL- 1 as diagnostic ofmyeloid leukemias might be associated with the P30-35 CAMAL material that was shownto alter colony formation by normal and CML progenitor cells. In addition, several celllines derived from the leucocytes of patients with myeloid leukemias were highly positiveusing this antibody raised against P30-35.Since the completion of these studies, the P30-3 5 CAMAL material was furtherseparated into several constituents using reverse phase HPLC. The inhibitory activity on246normal colony formation was shown not to be mediated by known proteins in thesepreparations, and it was shown using cultures of murine cells that inhibition was mediatedby the only protein peak in these preparations which was unique. One of the contaminatingproteins, the serine protease cathepsin G, has known reactivity with the peptide substratewith which preparations of P30-35 CAMAL are reactive, however, protein in the uniquepeak showed activity on this substrate in addition, and it was shown using CMK-linkedpeptides that a protease activity distinct from that of cathepsin G was present inpreparations of P30-35 CAMAL. While it remains a possiblity that the described activitieson colony formation could require cathepsin G activity in addition to that of othersubstances in preparations of P30-35 CAMAL3 it is also possible that the observedalterations of in vitro myelopoiesis might be mediated by this apparently unique proteaseactivity.In conclusion, the P30-35 CAMAL, prepared from lysates of leucocytes obtainedfrom patients with myeloid leukemias, was shown to mediate an inhibitory effect on colonyformation by normal progenitor cells, which was directed toward CFU-G at lowconcentrations of P30-35 CAMAL, and toward all progenitor cell types at higherconcentrations. A reduction in the number of cells in the non-adherent compartment oflong term marrow cultures also occurred, whereas cell numbers were increased in theadherent compartment, suggesting that the cells were held in the stromal layer and blockedin differentiation. The inhibitory effect on colony formation was maintained in cultures ofmurine bone marrow cells. Similar to the human assays, this effect was directedpreferentially toward CFU-G. CFU-S were also reduced. These results suggest that247inhibition of normal colony formation by P30-35 CAMAL is conserved and might be animportant regulatory mechanism in myelopoiesis. In contrast, the same preparations ofP30-35 CAMAL mediated a stimulatory effect on colony formation by CML progenitorcells, which was directed toward CFU-GEMM at low concentrations of P30-35 CAMAL,and toward all progenitor cell types at higher concentrations of P30-35 CAMAL. Thereduction of colony formation by normal progenitor cells and enhancement of colonyformation by CML progenitor cells provides a mechanism by which the leukemic clone ofcells could gain a growth advantage over normal cells. 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Arrestingtissue invasion of a parasite by protease inhibitors chosen with the aid of computermodeling. 1991; 30: 11221- 11229.291APPENDIX 1DIFFERENTIAL IMMUNOPEROXIDASE STAINING OF CML AND NORMALCYTOSPIN PREPARATIONS USING c,-P30/359.1: INTRODUCTIONImmunoperoxidase staining of cytospin preparations using the monoclonal antibodyCAMAL- 1 was previously shown to be diagnostic of myeloid leukemias (465). Inaddition, the number of positive cells in CML specimens was found to decrease oninduction of clinical remission and an increase in the number of positive cells frequentlypreceded clinical relapse by up to several weeks (470). Patients with lower bone marrowvalues of the CAMAL antigen as determined by CAMAL- 1 immunoperoxidase staining hadsignificantly longer disease free survival than did patients with higher values. Thus, thedetection of the CAMAL antigen was shown to be of prognostic value in these leukemias.The purpose of the studies described in this section was to determine whether the antigenrecognized in the studies using CAMAL-1 was the P30-35 component in material enrichedfrom lysates of myeloid leukemia cells using CAMAL- 1 immunoaffinity chromatography.Since this P30-35 CAMAL material was demonstrated to be inhibitory to normalmyelopoiesis, and stimulatory to leukemic myelopoiesis, these studies were to search forevidence regarding whether or not the CAMAL antigen and the P30-3 5 CAMAL activitywere the same entity. Leucocytes from CML clinical specimens and normal healthy donors292enriched for mononuclear cells were stained in an immunoperoxidase slide test using an a-P30/35 monoclonal antibody raised against highly enriched P30-35 CAMAL material. Thestaining of cell lines derived from myeloid leukemias were also evaluated using thisantibody. Conclusions that can be drawn from these studies, are limited by the recentfmding that preparations of P30-35 CAMAL are not homogeneous (Appendix 2), however,interesting patterns of reactivity with a-P30/35 did result.9.2: MATERIALS AN]) METHODS9.2.1: Antibodies;Monoclonal antibodies used in this study, their preparation, and purification, weredescribed in Chapter 2. Briefly; CAMAL- 1 was raised against original preparations of theCAMAL antigen, which was isolated by subtractive methods from lysates of myeloidleukemia cells as described in Chapter 1, cr-P30/35 was raised and screened against P30-35material further purified from CAMAL- 1 immunoaffinity preparations of CML and orANLL cell lysates by preparative SDS-PAGE or gel filtration, and a-BLV, used as acontrol Ab, was raised against P24, the major coat glycoprotein of the bovine leukosisvirns. a-P30/35 hybridomas were screened by ELISA for positive reactivity with P30-35and negative reactivity with HSA, with which CAMAL- 1 is known to react. All antibodieswere shown to be of the IgG1 subclass by the Ouchterlony technique. Ascites was293prepared from which antibodies were purified by immunoaffinity chromatography using agoat a- mouse Ig immunoadsorbent column, as described in Chapter 2.9.2.2: Cell lines;EM2 and EM3 were derivedfrom a patient with CML, HL6O was derived from anacute promyelocytic leukemia, and IIEL was derived from an erythrocytic leukemia. K562was derived from a CML and is Ph. KG1 was derived from an AML. Cell lines weremaintained at 3 7\u00C2\u00B0C, 5% C02, and split one in five to one in forty every three to four days.EM2 and EM3 were grown in RPMI medium supplemented with 20% FCS. These celllines were split one in five to one in ten. HL60, HEL, K562 and KG1 were grown inDME supplemented with 10% FCS. These cell lines were split one in ten to one in forty.9.2.3: Preparation ofcytospins;Cytospins of freshly separated leucocytes enriched for mononuclear cells, fromnormal or CML peripheral blood, or of cells freshly removed from cell line cultures wereprepared. Separation of leucocytes enriched for mononuclear cells from whole blood wasby Percoll density centrifugation and is described in Chapter 2. Contaminating red bloodcells were removed, when necessary, by lysis with 0.017 M Tris-ammonium chloride(Tris NH4C1), pH 7.2. Cell pellets were resuspended in 1 ml Tris NH4CI, incubated for 5mm at 37\u00C2\u00B0C, and washed once with fresh Iscove\u00E2\u0080\u0099s medium. Cells from cell line cultures294were washed twice in Iscove\u00E2\u0080\u0099s medium in order to remove FCS. Cytospins were preparedfrom cell lines with a minimum viability of 98% as determined by dye exclusion countsperformed using eosin Y. Cells were resuspended at a density of 1 X 106 cell/ml andspun onto glass slides at 5000 rpm (approximately 100 jtl volume, two drops from apasteur pipette per slide) using a cytospin apparatus (Shandon).9.2.4: Serum;Normal human serum was prepared by drawing blood from a healthy volunteer intoa vacutainer tube containing no anticoagulant. Blood was allowed to clot for 30 mm, thenthe clot was removed by centrifugation at 400 X g. The serum was divided into aliquotsand stored at -70\u00C2\u00B0C.9.2.5: Ccli staining,\u00E2\u0080\u0099Cytospins were stained by immunoperoxidase within fourteen days of preparation.Cells were fixed by immersion in methanol with 2% H20(Fisher) for 30 minutes. Slideswere transferred to a staining rack and washed three times with fresh phosphate bufferedsaline (PBS). Slides were incubated for 30 mm at room temperature with the appropriateprimary antibody (30 j.tg/ml in PBS). Slides were again washed three times with PBS,following which they were incubated for 30 minutes with the secondary antibody solution.This consisted of rabbit a-mouse Igs (DAKO) at a 1:100 dilution in PBS containing 4%295normal human serum. Slides were washed three times in PBS, and immersed in a solutionof 0.0013% DAB (diaminobenzidine, 3 ,4,3\u00E2\u0080\u0099,4\u00E2\u0080\u0099-tetra-aminobiphenyl hydrochloride, BDH)with 0.06% H20for 10 minutes. Slides were then immersed in fresh PBS and counter-stained for 90 seconds with hematoxylin (0.5%, Banco, Harris formula, OxfordScientific). Slides were rinsed with gently running tap water for 5 minutes, then allowed toair dry. Dry slides were mounted with Permount (Fisher) and coverslipped.Staining was evaluated using an Olympus microscope. Positive reactivity resultedin a brown precipitate. The percentage of cells staining positive in each preparation wasestimated by counting positive and negative cells. Photomicrographs were taken ofrepresentative preparations.9.3: RESULTSCytospin preparations of mononuclear cell-enriched leucocytes from CML clinicalspecimens showed greater reactivity with o-P30/35 than did cytospins of mononuclear cell-enriched leucocytes from normal healthy donors. This is a similar pattern of reactivity tothat previously described using the CAMAL- 1 monoclonal antibody and reproduced here asa positive control. These results are summarized in Table IX. Photomicrographs ofrepresentative specimens are shown in Figure 31. In individual CML clinical specimens,positive staining was usually found to be more extensive using a-P30/35 than usingCAMAL- 1 (Figure 3 la and b). In all cases, background reactivity as determined using a296non-specific monoclonal antibody of the same subclass, a-BLV, was low (Figure 31c).Although the CML cells appear to stain non-specifically to a greater extent than do normalcells (Figure 3 ic), the level of staining obtained using either a-P30/35 of CAMAL- 1 wasclearly greater than that obtained using the control antibody (Figures 31 a anf b). In eachCML specimen, the amount of staining with control antibody was defmed as \u00E2\u0080\u0098background\u00E2\u0080\u0099,and the number of cells staining to a clearly greater extent than this level was evaluated.The difference in staining between CML and normal cells was greater using a-P30/35 thanusing CAMAL- 1 (Figure 3 la, b, and d, Table IX).Several human cell lines derived from myeloid leukemias showed reactivity with cP30/35. These results are summarized in Table X, and photomicrographs shown in Figure32. The cell lines EM2, EM3, HL6O, K562, and KG1 all stained extensively with aP30/35 (Figure 32a, d, and f, and Table X). In all cases, background reactivityasdetermined using a non-specific monoclonal antibody of the same subclass, a-BLV, waslow (Figure 32c). In addition, the cell line HEL was negative with all antibodies.(Figure297TABLE IX. Immunoperoxidase staining of peripheral blood mononuclear cells from CML patients andnormal healthy donors.PBL specimen % positive cellsCAMAL-1 c-P30/35 a-BLVCML1 <2 >5 <22 >5 >25 <23 >25 >50 <24 <2 >25 <25 >10 >10 <26 >10 >50 <2normal1 >5 <2 <22 >5 <2 <23 >5 <2 <24 >5 <2 <25 <2 <2 <26 <2 >5 <232e). Staining of positive cell lines was far less extensive using CAMAL-1 than using cP30/35, but was above background (Figure 32a and b), with the exception of HL6O, whichreacted to a similar extent using either CAMAL- 1 or ct-P30/35 (Figure 32f and g).298aFIGURE 31. Preferential staining by immunoperoxidase of mononuclear cells obtainedfrom the peripheral blood of patients with CML as compared to normal healthy donorsusing x-P30/35. Cells in panels a through c were derived from the same CML donor, a.CML peripheral blood cells stained using ct-P30/35. b. CML peripheral blood cellsstained using CAMAL-1. c. CML peripheral blood cells stained using the control antibodya-BLV. d. Normal peripheral blood cells stained using a-P30/35. e. Peripheral bloodcells from a second CML donor stained using a-P30/35.299OOE3-_p441J4.2\u00E2\u0080\u00A21\u00E2\u0080\u00A2\u00E2\u0080\u0098:TOE0p0IAVVVVVk)JcI\u00E2\u0080\u0099000 IAVVVVVt\u00E2\u0080\u0099000000-000JcA\u00C3\u00A700(d0C)I/\AAAAAIaFIGURE 32. Immunoperoxidase staining of myeloid leukemia-derived cell lines, a. EM3stained using ct-P30135. b. EM3 stained using CAMAL-l. c. EM3 stained using xBLV. d. EM2 stained using ct-.P30/35. e. HEL stained using a-P30/35. f. HL6Ostained using x-P30/35. g. HL6O stained using CAMAL-l.303bCELb$1-wb4304co.104gfd3069.4: DISCUSSIONIt was of interest to determine whether the antigen recognized in immunoperoxidasestudies using the CAMAL- 1 antibody as diagnostic of myeloid leukemias was the sameP30-35 CAMAL material enriched using CAMAL- 1 immunoaffinity chromatography, andshown mediate an inhibitory effect on colony formation by normal progenitor cells and astimulatory effect on colony formation by CML progenitor cells. That the CAMAL antigenand the P30-35 CAMAL material active on myelopoiesis might be the same entity wasoriginally suggested by the finding that a rabbit polyclonal antibody raised against originalpreparations of the CAMAL antigen, and which was found to preferentially stain cells frompatients with myeloid leukemias (465) was reactive with P30-35 CAMAL by Western blotanalysis (474).A similar pattern of staining was observed using the monoclonal antibodies cP30/35, raised against preparations highly enriched for P30-35 CAMAL, or CAMAL-1,raised against original preparations of the CAMAL antigen; mononuclear cell-enrichedleucocytes from CML clinical specimens generally stained to a greater extent using eitherantibody than did cells from normal healthy donors. This result initially suggested that thetwo antibodies might recognize the same component (P30-3 5 CAMAL) on and within thesecells. However, more recent data indicate that the CAMAL- 1 antibody is not reactive withP30-35 CAMAL by Western blot analysis (474, 476). P30-35 CAMAL and the entityrecognized by CAMAL- 1 in the immunoperoxidase studies thus do not appear to be thesame entity. The similarities in patterns of cell staining obtained using these antibodies,307however, suggest that the CAMAL antigen and P30-35 CAMAL might form a complex, thedifferent components ofwhich might be recognized by the two antibodies.x-P30/35 was found to stain a greater number of cells in CML clinical specimensand cell lines derived from myeloid leukemias than did CAMAL- 1 in many cases. Thisresult suggests that a-P30/35 has greater specificity for myeloid leukemia-derived cellsthan does CAMAL- 1. Recent further separation of preparations of P30-3 5 CAMAL hasshown that other protein species such as elastase, cathepsin G and azurocidin are presentwithin these preparations (Appendix 2). It must be recognized, then, that this antibodymight recognize these proteins within the myeloid leukemia cells. In this regard, recentevidence shows that a-P30-35 is reactive with elastase, azurocidin, and cathepsin G, butnot with myeloblastin, by Western blot analysis (474, 476).Staining by x-P30/35 and CAMAL-l appeared in non-identical cell compartments.For example, CAMAL- 1 was found to stain membranes (465, Figure 3 ib) to a greaterextent than did x-P30/35, which localized mainly to the cytoplasm and perinuclear space(Figure 3 la and e). This result supports the idea that the two antibodies might recognizedifferent proteins. The similarities in patterns of staining, however, suggest that theseproteins might form an association. For example, CAMAL- 1 might preferentiallyrecognize a P30-35 CAMAL substrate or carrier molecule, such as HSA, which is knownto carry many molecules, on the cell surface, whereas a-P30/35 might recognize free P30-35 CAMAL in the cytoplasm or P30-35 CAMAL in the process of being synthesized orpackaged.308Positive staining of cell lines using CAMAL- 1 and cx-P30/35 suggests that theymight produce P30-35 CAMAL. This is consistent with the previously reportedobservation that a rabbit antiserum raised against highly purified P30-35 recognized aprotein in the range of 30 to 35 kDa by Western blot analysis in lysates from the myeloidleukemia-derived cell lines HL6O and K562 (472). It is interesting that the cell lines thatstained positive using a-P30/35 are the same cell lines that responded to P30-35 CAMALmediated enhancement in colony assays. Colony formation by EM2, EM3, and HL60 wasstimulated by P30-35 CAMAL (Chapter 4); these cell lines all stained extensively using a-P30/35. In addition, preliminary results showed that colony formation by K562 and KG1was also stimulated by P30-35 CAMAL (data not included). These cell lines also stainedextensively using a-P30/35. In contrast, colony formation by the cell line HEL was notaffected by treatment with P30-35 CAMAL (chapter 4); this was the only cell line testedthat did not stain using ct-P30/35. Hence,the cell populations that appear to produce P30-35 CAMAL are the same cells that are affected by upregulation of their clonogenicity. Thismight be an indication that P30-35 CAMAL could function as an autocrine regulatorymechanism gone awry in CML.The cell types showing positive reactivity with these antibodies was not evaluated.This was due to the recognition, after the slides were prepared, but before they wereevaluated, that the material against which a-P30/35 was raised consists of a mixture ofproteins (Appendix 2). Subsequent work has shown that a-P30/35 does indeed react withseveral of the proteins known to be present in this mixture by Western blot analysis (474).309It is the opinion of the author that the similarities in pattern of staining of CML and normalcells using the antibodies cs-P30/35 and CAMAL- 1 suggest an association between theCAMAL antigen and the P30-35 CAMAL material which mediates the alterations ofmyelopoiesis described in this paper. Definitive evaluation of cell types which produce orreact with P30-35 CAMAL, however, must await conclusive definition of the entityinvolved in these effects at a biochemical level. The cell types in question can then bedefined using antibodies raised against this material, or using molecular probes.In summary, mononuclear cell-enriched leucocytes from CML patients and severalmyeloid leukemia-derived cell lines stained to a greater extent than did mononuclear cellsfrom normal healthy donors in an immunoperoxidase slide test using a monoclonalantibody raised against P30-35 CAMAL. This is a similar result to that previously reportedusing CAMAL- 1, a monoclonal antibody raised against original crude preparations of theCAMAL antigen, and suggests that the antigen recognized as diagnostic of myeloidleukemias in the studies using CAMAL- 1 might be associated with P30-35 CAMAL, thematerial shown to alter myelopoiesis by normal and CML progenitor cells. Cell lineswhich stained using a-P30/35 were the same cell lines that responded by enhancement ofcolony formation to treatment with P30-35 CAMAL, suggesting that they might produceP30-35 CAMAL and respond to it in an autocrine manner. Conclusions that can be drawnfrom these studies, however, are limited by the recent fmding that preparations of P30-3 5CAMAL, which a-P30/35 was raised against, are not homogeneous, and that this antibodylikely reacts with several components in these preparations (Appendix 2).310APPENDIX 2FURTHER EVIDENCE THAT P30-35 CAMAL ACTIVITY IS MEDIATED BY ACOMPONENT IN PREPARATIONS OF P30-35 CAMAL WHICH IS DISTINCT FROMCHARACTERIZED NEUTROPHIL PROTEINSThe investigations reported up to this point were completed by August, 1991.There was a hiatus of nine months between completion and documentation of the describedstudies. During this time, progress was made with the definition of P30-35 CAMAL at asubcellular level, the project of another graduate student in this laboratory. P30-35CAMAL prepared by enrichment from CML cell lysates using an o-P30/35immunoadsorbent column was separated by reverse phase HPLC and shown to consist ofseveral protein components (476). Three of the peaks obtained in these separationscorresponded to known neutrophil proteins, including cathepsin G, elastase, andazurocidin. A fourth peak, which was the major species in many of the reverse phaseHLPC preparations, was unique to preparations of P30-35 CAMAL as compared topreviously published information on separations of neutrophil proteins (477). Additionalstudies were undertaken in order to determine whether the effects of P30-35 CAMAL onmyelopoiesis by normal and CML progenitor cells might be mediated by characterizedproteins present in these preparations. It should be noted that studies of the biologicalactivities of P30-3 5 CAMAL were largely performed using material enriched from CMLcell lysates by sequential elution from CAMAL- 1 and a-P30/35 immunadsorbent columns,311whereas reverse-phase HPLC separations were performed using material enriched fromCML cell lysates using an cx-P30/35 column only. The relative proportions of neutrophilproteins in material enriched from CML lysates using sequential elution from CAMAL- 1and a-P30/35 columns is currently under investigation (496).Elastase was previously shown to be inhibitory to colony formation by progenitorcells from normal healthy donors. In contrast to P30-35 CAMAL, however, elastase isalso inhibitory to colony formation by progenitor cells from patients with CML (Figure 10,Chapter 2). Since preparations of P30-35 CAMAL were screened to ensure that noinhibitory activity against CML colony formation was present, elastase is in all probability aminor contaminant of the preparations that were used to characterize the activities of P30-35CAMAL reported in this study. Studies using CMK-linked peptides in an attempt to blockthe activity of P30-35 CAMAL on colony formation support this; the elastase blocker alaala-pro-val-CMK did not block the inhibitory activity of P30-35 CAMAL preparations oncolony formation by normal progenitor cells (Figure 27, Chapter 4). This is an indicationthat the inhibition of colony formation by normal progenitor cells on treatment with P30-35CAMAL is not mediated by elastase. Similarly, the enhancement of colony formation byCML progenitor cells on treatment with P30-35 CAMAL cannot be mediated by elastasesince elastase was shown to be inhibitory to colony formation by CML progenitor cells.Myeloblastin, or proteinase 3, was reported to be present in preparations purifiedfrom the azurophilic granules of normal neutrophils (438). The activity of P30-35 CAMALis not likely to be mediated by myeloblastin for several reasons. The activity ofmyeloblastin was evaluated in a colony assay using normal human cells; it was not312inhibitory to colony formation, and, in fact, a slight enhancement of colony formation wasseen (Figure 33a). Enhancement of colony formation by CML progenitor cells is not likelyto be due to myeloblastin since myeloblastin was found to be a very minor component ofreverse phase HPLC-separated preparations of P30-35 CAMAL (<1%). In addition,myeloblastin is known to preferentially recognize chromogenic peptide substrates that aresubstantially different than those which are recognized by preparations of P30-35 CAMAL(438).It was considered to be unlikely that the observed alterations in colony formation bynormal and by CML progenitor cells were due to azurocidin, since azurocidin hassubstitutions in two of the three amino acid residues of the serine protease active site, andhas no protease activity on a panel of chromogenic peptide substrates (438). In contrast,protease activity was shown to be required for the effects on colony formation by bothnormal and CML progenitor cells (Chapter 5). However, the activity of azurocidin wasevaluated in a colony assay using normal human cells nonetheless. Azurocidin was notinhibitory to colony formation by normal progenitor cells, and in fact a slight enhancementof colony formation was observed (Figure 33a). Enhancement of colony formation byCML progenitor cells is not likely to be attributable to azurocidin since protease activity wasshown to be required for this effect as well.Similarly, cathepsin G was not inhibitory to colony formation by normal progenitorcells, at a protein concentration of up to 15 jig/ml (Figure 33 a & b). The possibility,however, that the enhancement of colony formation by CML progenitor cells could bemediated by cathepsin G, cannot yet be ruled out, since cathepsin G is known to recognize313FIGURE 33. Lack of inhibitory activity of myeloblastin, azurocidin, and cathepsin G oncolony formation by progenitor cells from a normal healthy donor. Methylcellulosecultures were used, as described in Chapter 2. a. Conditions of preincubation, asdescribed in Chapters 3 and 4, using myeloblastin, azurocidin, and cathepsin G, ascompared to the activity of a P30-35 CAMAL preparation. Cells were treated with 100ng/ml of protein in each case. In this experiment, \u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099 refers to P30-3 5 CAMAL. b.Conditions of coculture, as described in Chapter 2, using cathepsin G.0000z80 -60 -40 -20 -0III]00r-)0100\u00E2\u0080\u00A250-0--,.001 .01 .1 1 10 100[cathepsin G] (jig/mi)\u00E2\u0080\u00A2-treatmenta314the substrate ala-ala-pro-phe-NA, which is the same substrate with which preparations ofP30-35 CAMAL were found to react. This possibility is currently under investigation(496). It is also possible that the alterations in colony formation by CML progenitor cellsmight be mediated by the action of a non-protease substance in preparations of P30-35CAMAL in conjunction with cathepsin G. For example, cathepsin G might alter stnictureson the cell surface in such a way that they are rendered susceptible to the activities of asecond substance. Enzyme assays using cathepsin G and preparations of P30-35 CAMAL,however, indicate the presence of a protease activity in preparations of P30-35 CAMALwhich appears to be unique. A preparation of P30-35 CAMAL which was fullycharacterized as to its effects on myelopoiesis in all systems described in this paper(inhibition of normal human colony formation, effect on long-term culture of humanmarrow, inhibition of murine CFU-G, stimulation of CML colony formation, andstimulation of colony formation by the CML-derived cell line EM3) was used for thiscomparison with cathepsin G. The activity of P30-35 CAMAL on the substrate ala-ala-pro..phe-NA was fully blocked by the ala-ala-pro-val-CMK peptide in an enzyme assay at aCMK:P30-35 CAMAL molar ratio of 10:1, whereas the activity of cathepsin G on the samesubstrate was not affected by ala-ala-pro-val-CMK at a CMK:cathepsin G molar ratio of100:1 (Figure 34a). This difference in activity cannot be attributed to a blockage of elastaseactivity, since elastase does not react with the chromogenic peptide substrate used toevaluate activity in this experiment (476). Similarly, the activity of P30-35 CAMAL wasblocked by the peptide ala-pro-phe-CMK almost fully at a molar ratio of 1:1, whereas theactivity of cathepsin G using this same CMK-linked peptide as a putative blocker of activitywas only minimally affected at a molar ratio of 1:1, and was not fully blocked even at alevel of 10:1 (Figure 34b). Thus, there appears to be a protease activity in preparations of315150Iix0C000C)0CFIGURE 34. Enzyme assays with P30-3 5 CAMAL (\u00E2\u0080\u0098CAMAL\u00E2\u0080\u0099) and cathepsin G usingCMK-linked peptides as putative blockers of enzyme activity. Enzyme activity wasassessed using the peptide substrate ala-ala-pro-phe-NA. Assays were as described inChapter 5. a. Blocking effect of the activity of P30-35 CAMAL but not of the activity ofcathepsin G using ala-ala-pro-val-CMK (\u00E2\u0080\u0098AAPV\u00E2\u0080\u0099). b. Blocking effect of P30-35 CAMALand cathepsin G activities at different levels using ala-pro-phe-CMK (\u00E2\u0080\u0098APF\u00E2\u0080\u0099).50a 1X lox 100X\u00E2\u0080\u00A2 CAMAL,Q CATHEPSIN,AAPV\u00E2\u0080\u00A2 CAMAL,APFQ CATHEPSIN,APF100806040200b 1X lox lOOXCMK:enzyme molar ratio316P30-35 CAMAL which is unique, and the possibility remains that the enhancement ofcolony formation by CML progenitor cells mediated by P30-35 CAMAL preparationsmight be due to this activity. Other than elastase, cathepsin G, and the unique peak, noother major peaks were present in reverse phase ITPLC separations of P30-35 CAMALpreparations (476, 486).The unique peak in preparations of P30-35 CAMAL, which is distinct from thepeak known to contain cathepsin G, has been shown to cleave the chromogenic peptidesubstrate ala-ala-pro-phe-NA, with which preparations of P30-35 CAMAL are reactive(476). Moreover, it was recently shown that this fraction unique to preparations of P30-35CAMAL is inhibitory to colony formation by murine progenitor cells (486). The inhibitoryactivity of P30-35 CAMAL on normal myelopoiesis, then, appears to be mediated byprotein in this peak. Reverse phase ITPLC-purified material is undergoing amino acidsequence analysis, and further material is currently being purified for evaluation of itsactivity on colony formation by CML progenitor cells (496).In summary, P30-35 CAMAL was separated by reverse-phase HPLC as part of aseparate ongoing study, and was shown to consist of more than one protein species. Manyof these proteins have been characterized; these include elastase, myeloblastin, azurocidin,and cathepsin G. A major peak which was unique to preparations of P30-35 CAMAL andnot found in preparations derived from normal neutrophils was also present in reverse-phase HPLC separated P30-35 CAMAL (477). The activities of P30-35 CAMALpreparations on colony formation by normal and CML progenitor cells could not be317attributed to elastase, mycloblastin, or azurocidin. Similarly, the inbibitory activity of P30-35 CAMAL on colony formation by normal progenitor cells could not be attributed tocathepsin G, and was shown to be mediated by the unique peak in preparations of P30-35CAMAL. The possibility remains that the stimulatory activity on colony formation byCML progenitor cells might be mediated by cathepsin G. However, an enzyme assaywhich compared the activity of a preparation of P30-35 CAMAL to that of cathepsin Gdemonstrated the presence of a protease activity in the P30-35 CAMAL preparation whichappears to be unique, and it remains a possibility that the activity of P30-35 CAMAL oncolony formation by CML progenitor cells is mediated by this fraction.318"@en . "Thesis/Dissertation"@en . "1992-11"@en . "10.14288/1.0086789"@en . "eng"@en . "Microbiology and Immunology"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Characterization of the effects of P30-35 CAMAL on normal and leukemic myelopoiesis"@en . "Text"@en . "http://hdl.handle.net/2429/3072"@en .