Positive News on the Negative Growth Factors ATHANASIUS ANAGNOSTOU," NICHOLAS DAINIAK,~AND ALBERT NAJMAN 'Division of Hematology Brown University Providence, Rhode Island 02912 and Memorial Hospital of Rhode Island Pawtucket, Rhode Island 02860 bDepartments of Medicine and Laboratoty Medicine University of Connecticut Health Center Farmington, Connecticut 06030 'Laboratoire d'Etudes de I'Himatopoikse Facultk de MJdecine St. Antoine 75012 Paris, France
THE HEMATOPOIETIC GROWTH FAmORS AND INHIBITORS The maintenance of a normal number and normal function of mature blood cells depends on the presence in the blood-forming organs of a very small number of hematopoietic stem cells, which can undergo proliferation and differentiation to specific blood cell lineages under the influence of complex and finely tuned networks of humoral regulators and stromal cell reactions. Most of these very early progenitors are in the quiescent, noncycling state (Go),but in response to external signals they can rapidly in preparation for DNA synthesis (S). enter a functional period (GI) An ever-increasing number of glycoproteins,called in general hematopoietic growth factors, colony-stimulating factors (CSF),or interleukins (IL), have been found to enhance the proliferation and differentiation of the various stem cells. Detailed knowledge of the biochemistry, physiology, and pharmacology of these factors has been accumulating rapidly over the past 40 years. A real explosion of new information occurred after the recent purification of these factors, gene cloning, and production of recombinant molecules. For many of them, not only do we now know their chemistry and molecular structure, but we also have identified and quantified their receptors, the type of cells producing them, and the mechanisms of their production and action on target organs. This enthusiastic and productive research on the positive growth factors has also produced an aggressiveand confident biotechnology industry, which is already bringing into clinical use some of these genetically engineered molecules, hoping for a therapeutic revolution comparable to the discovery and clinical use of antibiotics.* By the time of the publication of this volume, three recombinant hematopoietic growth factors will have been approved for clinical use (erythropoietin, granulocyte-colony-stimulating 1
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factor (G-CSF), and granulocyte macrophage-colony-stimulating factor (GM-CSF). Treatment with recombinant erythropoietin has already brought substantial gains in the quality of life of severely anemic patients on hemodialysis. There are high expectations that the colony-stimulating factors will have an equally important impact on cancer chemotherapy by reducing the period of drug-induced neutropenia and thereby allowing the on-schedule administration of full doses of chemotherapy. Similar clinical benefits to patients with cancer who are undergoing chemotherapy could be realized by an alternate strategy: reduction of the period of neutropenia by blocking the entrance of the stem cells into cell cycle and thus protecting them from the toxic side effects of cycle-specific chemotherapeutic drugs.3 In theory one could combine the two methods and (a) suppress the normal stem cell cycling with specific inhibitors before chemotherapy and (b) accelerate the post-chemotherapy peripheral blood cell recovery with an infusion of stimulators of stem cell proliferation. The role of inhibitors in restraining cells from entering the cell cycle and thereby preserving the high proliferative potential of the bone marrow stem cells is only now being appreciated. Suppressor molecules directed at the genes and their encoded products are today being described at a rapid pace. We mention below some of the theoretical ways by which this inhibition of cell cycling can be realized. Some of these methodologies are already being pursued in hematopoietic or non-hematopoietic cells by academic or biotechnology industry researchers: (1) Introduction into marrow progenitor cells of mutated forms of genes
coding for proteins that are targets for cancer drugs4 (2) Replacement of mutated tumor suppressor genes in certain malignancies.5 (3) Down-regulation (reducing the number), transmodulation (reducing the affinity), or blocking the expression of growth factor receptors. The IL-1 inhibitor is such a natural receptor inhibitor.6 (4) Use of soluble extracellular domains of growth factor receptors as ligand blockers or inhibition of growth factor production. ( 5 ) Therapeutic use of high concentrations of growth factors that mayparadoxically-inhibit cells with receptor overexpression.' (6) Use of growth factor fragments or derivatives with inhibitory action. The oxidized dimer of the peptide described in 1982 by Paukovits and Laerum8 stimulates granulopoiesisin vitro, but its reduced form (monomer) inhibits it. (7) Inhibition of specific signal transduction pathways-for example, inhibitors of tyrosine k i n a ~ e sAnd . ~ finally: (8) Use of natural negative regulators of cell cycling. Popularized in 1962, these substances-often referred to early on as "cha1ones"-were thought not only to inhibit cell proliferation without being cytotoxic but also to be tissue specific.1° In this regard, normal human mammary cells have been reported recently to produce mammastatin, a tissue-specific growth inhibitor distinct from transforming growth factor (TGF)-beta." However, progress in hematopoiesis in this research area has been slow because of multiple difficulties in, for example, purifying the regulatory substances, distinguishing suppression of cell proliferation from nonspecific cytotoxicity, and obtaining pure stem cell populations as target cells for such inhibitors. Resting-but not regenerating-rodent marrow stromal cells were reported in 1976 by Lord to produce an inhibitor of CFU-Scycling.12This factor was specific in vivo for
ANAGNOSTOU et al.: NEGATIVE GROWTH FACTORS
3
CFU-S but in vitro only for early-and not more differentiated-progenitor cells. Frindel and Guigon showed in 1977 that fetal calf marrow contained a dialyzable substance that inhibited the post-irradiation cycling of CFU-S.’) It is now known to be a tetrapeptide that protects mice from the cytotoxic effects of ara-C, an S-phase-specific drug. A glycoprotein produced by leukemic, as well as normal, cells and therefore called leukemia-associated inhibitor (LAI) was found to neutralize the activity of GM-CSF and thus reduce the cycling of normal human CFU-GM.14 Other inhibitors of cell proliferation that may afford bone marrow protection include TGF-P, the macrophage inflammatory protein (MIP- la), and the negative regulatory protein (NRP). It has been claimed that MIP-la is the inhibitor of CFU-S cycling reported by Lord in 1976. Some negative regulators are already undergoing clinical trials, as mentioned in this volume in the summary of the workshop entitled “Zn Vivo Studies and Clinical Trials of Negative Regulators.” The discovery, nature, and action of some of the specific negative regulators are described in the recent review of this area by A. Axelrad.Is Also, a detailed listing of negative regulators of erythropoiesis and their possible mechanisms of action is included in this volume in the summary of the workshop “Inhibitors of Erythropoiesis.” We are at the stage of cataloging the many types of negative regulators of hematopoiesis, and this has provided the primary rationale for two international scientific meetings on the topic of negative hematopoietic cell regulators.
THE FIRST SYMPOSIUM ON HEMATOPOIETIC INHIBITORS
The First International Symposium on Inhibitory Factors in the Regulation of Hematopoiesis took place in April 1987 in Paris. It was organized by A. Najman, M. Guigon, and N. C. Gorin of the Department of Hematology, Facult6 de Medecine, St. Antoine (Universit6 Pierre et Marie Curie, Pans) and J-Y. Mary (INSERM U263, Paris VII). That conference was the first large formal gathering of scientists with interest in such diverse inhibitors of hematopoietic cells and their function as interferons, prostaglandins, tumor necrosis factor (TNF), leukemia-associated inhibitor, and the newly described molecules with putative specific suppressive activity on early stem cells. The symposium concluded with the establishment of an International Inhibitor Registry, which has since been kept up-to-date by M. Guigon, W. Paukovits, and H. Broxmeyer. The registry was an attempt to classify inhibitors according to their molecular and biological properties and includes only biomolecular, not pharmaceutical, drugs.l6 The full proceedings of the conference was published by the Institut National de la Sant6 et de la Recherche Medicale (INSERM) and John Libbey Eurotext. Progress reports on the research on inhibitors were given a prominent role at the Annual Meeting of the International Society of Experimental Hematology in Paris in 1989, again organized by the hematology faculty at HBpital Saint Antoine. A discussion of suppressor molecules and their role in the regulation of the immune response was also part of the VIth International Lymphokine Workshop in 1989, in Evian, Savoie, France.
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THE SECOND INTERNATIONAL CONFERENCE ON NEGATIVE REGULATORS OF HEMATOPOIESIS The Second International Conference on Negative Regulators of Hematopoiesis, held in August, 1990 in Providence, Rhode Island under the auspices of Brown University and Memorial Hospital of Rhode Island, was the first such meeting in North America. Meeting organizers were A. Anagnostou, N. Dainiak, M. Guigon, M. Miller, A. Najman, and W.Paukovits, who were assisted by an advisory board composed of A. Axelrad, H. E. Broxmeyer, E. P. Cronkite, D. Metcalf, P. Ralph, F. Ruscetti, A. N. Schechter, and N. S. Young. Principal financial sponsors were Sandoz Pharmaceuticals and the National Institutes of Health; and contributions were received from Ortho Pharmaceutical Corporation, Cetus Corporation, Genetics Institute, Bristol Myers, Chugai-Upjohn, Hoffmann-La Roche, Smith-Kline Beecham Pharmaceuticals, and Biomeasure, Incorporated. The meeting consisted of 23 plenary talks of 25 minutes each, 70 poster presentations, and 12 workshop sessions. The meeting was attended by 136 established investigators (over half of whom were from outside the U.S.A.)and 20 Brown University scientists, which demonstrates the intense scientific interest in negative regulators. Not only has this field of research shown considerable progress, it has also undergone significant changes since the first symposium was held three years ago in Paris. There was less interest in TNF, prostaglandins, and interferons and more in the effects of TGF-beta on hematopoietic stem cells and the immune system. The presentations underscored the pleiotropic actionls of many of these factors, which are either stimulatory or inhibitory depending on the dose, the presence and concentration of other growth factors, and the target cell population. To wit, TGF-6 specifically inhibits proliferation of early stem cells but enhances proliferation of committed stem cells. It was clear that the diverse effects on non-hematological tissues of many of the discussed factors cautioned against their early therapeutic use in humans. The meeting opened with a review by H. Broxmeyer of the in vivo and in vitro actions, and the mechanisms of action, of several types of inhibitors. Lactoferrin and inhibin indirectly reduced production of IL-lb by monocytes and of CSF by mononuclear cells, whereas the acidic isoform of isoferritin directly affected early stem cells. He reported a strong correlation between the inhibitory activity of this isoferritin, peroxidase activity, and the macrophage inflammatory activity (MIP- la). The presentor considered an 8-kDa protein to be the intermediary of the suppressive actions of a number of these molecules. TGF-/? proved to be a molecule ubiquitous not only in most mammalian cell types, but also in the oral and poster presentations of this conference. F. Ruscetti discussed the direct inhibition of early stem cells by TGF-/3 and reported that infusion of TGF-/? into mice produced a rapid suppression of early stem cell cycling and decreased marrow cellularity and peripheral blood leukopenia. T. Bradley also found the inhibitory effects of TGF-6 to be restricted to early progenitor cells. Some found enhancement of growth at low concentrations, while others did not. TGF-/?-induced inhibition can be neutralized by increasing growth factor concentrations, which indicates a dose-related interaction between positive and negative influences on proliferation. This observation is similar to that found using serum as the source of inhibitors. C. Peschle described his experiments with TGF-6 added to populations of “pure” human peripheral blood stem cells obtained by a technique developed in his laboratory. One of the most interesting topics discussed was the progress made on the in vivo, as well as the in vitro, effects of regulatory peptides such as AcSDKP and pEEDCK, putative specific inhibitors of the hematopoietic stem cells and thus potentially useful
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5
in protection of marrow injury from chemotherapeutic agents during ex vivo purging. I. Pragnell reviewed his recently published studiesI9of the inhibitory effects of MIP-la on the cycling of early murine bone marrow stem cells and provided evidence for the presence of MIP-la in normal bone marrow, where stem cells are in a resting phase. W. Paukovits provided new details on the leukocyte-derived peptide pyroGlu-GluAsp-Cys-Lys (pEEDCK) and its potential for clinical use. Injection of this peptide rapidly suppressed CFU-GM cycling and the recruitment of CFU-S after ara-C. It prevented the chemotherapy-induced onset of neutropenia, or at least shortened the period of subnormal WBC counts. He suggested that whereas long-term chemotherapy induces exhaustion of pre-CFU-S, this effect is completely prevented by use of the inhibitory monomer, but not by the sthulatory dimer. A. Bogden gave preliminary results on the bone marrow-protective effects of another stem cell inhibitory peptide, N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) shown previously to prevent CFU-S recruitment in ara-C-treated mice. M. Guigon analyzed the in vitro effects of pEEDCK and AcSDKP on both normal and leukemic cells. Tested on hematopoietic cell lines, pEEDCK directly inhibited cells of myelomonocytic origin but did not reduce their sensitivity to ara-C. AcSDKP inhibited the in vitro growth of human progenitor cells (CFU-GM, BFU-E, and CFU-E), although maximal inhibition was never higher than 50%. No inhibitory effect was seen on stem cells from leukemic patients. The effect of AcSDKP reported by Guigon was directed not only to the number of colonies in vitro, but also to the cycling status of the progenitor cells. This latter point is important in understanding the role of this peptide in the protection of bone marrow. Several of the presentations centered on the inhibition of early erythropoietic progenitor cells. A. Axelrad's negative regulatory protein,*O which has now been purified and identified as a 1dkDa protein, was discussed by F. Pluthero. NRP is produced by mouse bone marrow and specifically decreases BFU-E cycling. Its action is countered by IL-3. Pluthero also reported that superoxide dismutase (SOD) inhibits BFU-E but not CFU-E, CFU-GM, or CFU-S. The level of SOD was found to be sufficiently high in normal mouse serum to inhibit BFU-E cycling. A. Yu discussed in detail the prevention by inhibin of the activin-initiated differentiation of erythroid progenitors. N. Dainiak reported on B-lymphocyte membrane-derived growth factors, which include inhibitors of BFU-E, and discussed the role of ambient and accessory cell lipid pools on regulation of hematopoiesis. Our knowledge of the leukemia inhibitory factor (LIF), a highly glycosylated protein produced by a wide variety of cells, was reviewed by D. Gearing. It was originally hoped that LIF would specifically inhibit myeloid leukemia cells by inducing them to differentiate. but LIF is now known to have a variety of in vitro and in vivo actions. The role of the stromal cells on the regulation of hematopoiesis was the theme of many posters and several plenary lectures. D. Zipori reviewed the role of the microenvironment on thymic stem cell proliferation and presented data showing that thymic precursor cells are supported by membrane glycoproteins from stromal cells. A 21-kDa glycoprotein (restrictin) on the surface of stromal cells can arrest the growth of plasmacytoma cells and enhance their apoptosis. C. Eaves presented detailed studies on the clonogenic precursor cell of the long-term bone marrow cell cultures (LTC-IC), the cycling activity of which depends on the relative balance of positive (G-CSF) and negative (TGF-P) growth factors produced by the microenvironment. S. Landreth presents in this volume his working hypothesis for the role of stromal cells on lymphopoiesis. Mechanisms and factors responsible for suppression of immunological cells were discussed by T. Mossmann, J. Kehrl, C. Nathan, and P. Ralph. J. Kehrl reviewed the role of TGF-/3 on the immune system and its in vivo actions, which include increased survival of organ transplants, reduction of severity of collagen-induced arthritis, de-
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crease of NK cells produced by activated lymphocytes, and inhibition of IL-2-induced proliferation and synthesis of IgG and IgM. The endogenous inhibitors of IL-1 and TNF production were discussed by P. Ralph. Production of IL-1 and TNF by lipopolysaccharides (LPS) was inhibited by pretreatment of T cells by TGF-/3 and IL-4 as a result of the activation of an IL-1 receptor antagonist (IL-1-ra). T. Mossman discussed the synthesis by TH-2 cells of a cytokine synthesis inhibitory factor (CSIF), which inhibits the TH- 1 cells and suppresses interferon production after schistosomiasis infection. The area of molecular control of hematopoiesis was the topic of a plenary presentation by A. Gewirtz. He described experiments with c-MYB antisense nucleotides, which, when added at the start of a culture, inhibit the development of progenitor cell colonies. A. Schechter reported the existence of negative controlling regions in the promoter of the @- and q-globin gene. The mechanisms of suppression of hematopoiesis during viral infections was analyzed by N. Young, who reviewed the destruction of CFU-E and BFU-E (but not CFU-GM or CFU-GEM) by the B-19 strain of human parvovirus, causative agent of transient aplastic crises in persons with chronic hemolytic anemias and some cases of pure red cell aplasia. The genome of the virus codes for the cytotoxic nonstructural protein NSP produced in permissive infected cells. Bueren et al. report in this volume that another parvovirus, the minute virus of mice (MVM), demonstrates cytotoxicity for proliferating pluripotent stem cells. A summary of the workshop “Suppression of Hematopoiesis by Viruses,” which includes discussion of the families of viruses that are known or suspected to inhibit hematopoiesis, the relevant animal models used, and the mechanisms-known or putative-of the suppressive action has also been included in this volume. A. Najman discussed the mechanisms of suppression of normal hematopoiesis in acute non-lymphocytic leukemia. Coculture of normal bone marrow cell with leukemic blast resulted in reduced colony formation. This suppression seemed to be due to positive inhibition rather than absence of stimulatory factors and was reduced by immune lysis or differentiation induction of leukemic cells. Possible candidates are acidic isoferritins, TNF-a, leukemia associated inhibitor, or even TGF-/3. A. Soto and C.Sonnenschein eloquently advocated the hypothesis that proliferation is a constitutive property of cells and that suppressor mechanisms, by restraining that proliferation, are thus the principal factors for the maintenance of normal homeostasis. They offered supportive data from their research with estrogen-dependent endometrial cells kept in check by an inhibitory 70-kDa non-glycosylated protein, estrocolyne. J-Y. Mary reminded the participants that the difficulties in experiment design and result interpretation that had dampened the initial enthusiasm in the “chalone” field long ago still exist. He proposed a way out of this experimental minefield involving the use of a standardized procedure and a spreadsheet program for carrying out the appropriate statistical tests.
THE WORKSHOPS The themes of the workshops held (and their chairpersons) included: in vivo studies/ clinical trials of negative regulators (Broxmeyerrnainiak); suppressors of macrophage effector function (Nathan/Ralph); transforming growth factor-beta (Ruscetti); stem cell inhibition (Paukovits/Guigon); lymphocyte suppression/IL-1 (Ralph); erythropoiesis inhibitors (Dainiak/Najman); inhibitors of megakaryocytopoiesis (Levin); viruses
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7
and suppression of hematopoiesis (YoungDavis); cell proliferation: positive and negative controls (Sonnenschein/Soto); molecular biology of gene regulation (Gewirtz/ Schechter); LIF (Gearing); restrictive effects of the microenvironment (Zipori); and classification of negative regulators (PaukovWGuigon: the 3rd International Workshop on Classification of Negative Regulators). Predictably, the interactive evening workshops, each one attended by between 20 to 50 investigators who discussed their own and each others’ work, were thought to be among the most fruitful parts of the conference. And as one would expect in this complex, confusing, but exciting scientific field, the spirited debates and brainstorming of these sessions were remarkable mainly for the important questions raised rather than for the satisfactory answers provided. As yet, we are not at the point where we can see the total picture of these regulatory mechanisms clearly enough to be able to interpret all the fragmentary data. Nevertheless, areas were identified that may constitute starting points for very promising future research. Brief summaries of some of the workshops have been included in this volume in order to give a glimpse of the multiple questions raised by the participants. The conference concluded with a plenary session, which included presentation of the summaries of all the workshops by their chairpersons and open discussion. This publication, made possible through the generous assistance of the New York Academy of Sciences, reflects the merits and disadvantages of any volume based on the proceedings of a scientific conference. Although it encompasses much of the very best work being done today in the field of negative regulators, it does not claim to be comprehensive. We hope, however, that it will serve as a valuable resource for scientists and clinicians who need to know more about this important, fast-moving field in biology, hematology, and cancer treatment.
REFERENCES 1. METCALF,D. 1989. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature 339 27- 30. L. 1986. Clinical role, therapeutic promise of CSFs outlined. Oncology Times 2. DELMONTE, 8: 3-16. 3. GUIGON,M., J-Y. MARY,J. ENOUF& E. FRINDEL. 1982. Protection of mice against lethal doses of 1-beta-D-arabinofuranosylcytosine by pluripotent stem cell inhibitors. Cancer R ~ s .4 2 638-641. 4. BERTINO,J. R. 1990. “Turning the tables”-Making normal marrow resistant to chemotherapy. J. Natl. Cancer Inst. 8 2 1234-1235. 5. BOOKSTEIN, R.,J.-Y. SHEW,P-L. CHEN,P. SCULLY& W-H. LEE. 1990. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 247: 712-715. 6. HANNUM,C. H., C. J. WILCOX,W. P. AREND,G. J. JOSLIN, D. J. DRIPPS, P. L. HEIMDAL, L. G. ARMES, A. SOMMER, S. P. EISENBERG & R. C. THOMPSON.1990. Interleukin-1 receptor antagonist activity of a human interleukin- 1 inhibitor. Nature 343: 336-340. 7. LUPU, R., R. COLOMER,G. ZUGMAIER, J. SARUP,M. SHEPARD,D. SLAMON& M. E. LIPPMAN.1990. Direct interaction of a ligand for the erB2 oncogene product with the EGF receptor and p185erbB2. Science 249 1552-1555. 8. PAUKOVITZ, W. & 0. LAERUM.1982. Isolation and synthesis of a hemoregulatory peptide. Z. Naturforsch. 376 1297-1300. 9. HUDZIAK,R. M., G. D. LEWIS, M. WINGET,B. M. FENDLY,N. M. SHEPARD& A. ULLRICH.1989. pl 85Her2monoclonal antibody has antiproliferative effects in vitro and sensitises human breast tumor cells to tumor necrosis factor. Mol. Cell. Biol. 9 1165- 1172.
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10. IVERSON,D. H. 1981. The chalones. In Handbook of Experimental Pharmacology. R. Baserga, Ed.: 491- 550. Springer-Verlag. Berlin. R. C. CODY& M. S. WICHA.1989. Production of mam11. ERVIN,P. R.. R. C. KAMINSK, mastatin, a tissue specific growth inhibitor by normal human mammary epithelial cells. Science 244: 1585. 12. LORD,B. I., K. J. MORI,E. G. WRIGHT& L. G. LAJTHA.1976. An inhibitor of stem cell proliferation in normal bone marrow. Br. J. Haematol. 3 4 441-445. 13. FRINDEL,E. & M. GUIGON.1977. Inhibition of CFU entry into cycle by a bone marrow extract. Exp. Hematol. 5: 74-76. 14. OLOFSSON,T. & I. OLSSON. 1980. Suppression of normal granulopoiesis in vitro by a leukemia-associated inhibitor (LAI) of acute and chronic leukemia. Blood 5 5 975-982. 1988.The classification ofhematopoietic W., M. GUIGON& H. E. BROXMEYER. 15. PAUKOVITS, inhibitors. Int. J. Cell Cloning 6 76-79. 16. SAUTES,C. & C. W. PIERCE.1989. Suppressor molecules. Lymphokine Res. 8 159-162. 17. SPORN,M. B. & A. B. ROBERTS.1988. Peptide growth factors are multifunctional. Nature 332 217-219. G. J., E. G. WRIGHT,R. HEWICK,S.D. WOLPE,N. M. WILKIE,D. DONALDSON, 18. GRAHAM, S. LORIMORE & I. B. PRAGNELL. 1990. Identification and characterisation of an inhibitor of hematopoietic stem cell proliferation. Nature 344:442-444. 19. AXELRAD,A. A., H. J. CROIZAT& D. ESKINAZI.1981. A washable macromolecule from Fv-2" marrow negatively regulates DNA synthesis in erythropoietic progenitor cells BFU-E. Cell 26 233-244.
THE HEMATOPOIETIC GROWTH FAmORS AND INHIBITORS The maintenance of a normal number and normal function of mature blood cells depends on the presence in the blood-forming organs of a very small number of hematopoietic stem cells, which can undergo proliferation and differentiation to specific blood cell lineages under the influence of complex and finely tuned networks of humoral regulators and stromal cell reactions. Most of these very early progenitors are in the quiescent, noncycling state (Go),but in response to external signals they can rapidly in preparation for DNA synthesis (S). enter a functional period (GI) An ever-increasing number of glycoproteins,called in general hematopoietic growth factors, colony-stimulating factors (CSF),or interleukins (IL), have been found to enhance the proliferation and differentiation of the various stem cells. Detailed knowledge of the biochemistry, physiology, and pharmacology of these factors has been accumulating rapidly over the past 40 years. A real explosion of new information occurred after the recent purification of these factors, gene cloning, and production of recombinant molecules. For many of them, not only do we now know their chemistry and molecular structure, but we also have identified and quantified their receptors, the type of cells producing them, and the mechanisms of their production and action on target organs. This enthusiastic and productive research on the positive growth factors has also produced an aggressiveand confident biotechnology industry, which is already bringing into clinical use some of these genetically engineered molecules, hoping for a therapeutic revolution comparable to the discovery and clinical use of antibiotics.* By the time of the publication of this volume, three recombinant hematopoietic growth factors will have been approved for clinical use (erythropoietin, granulocyte-colony-stimulating 1
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ANNALS NEW YORK ACADEMY OF SCIENCES
factor (G-CSF), and granulocyte macrophage-colony-stimulating factor (GM-CSF). Treatment with recombinant erythropoietin has already brought substantial gains in the quality of life of severely anemic patients on hemodialysis. There are high expectations that the colony-stimulating factors will have an equally important impact on cancer chemotherapy by reducing the period of drug-induced neutropenia and thereby allowing the on-schedule administration of full doses of chemotherapy. Similar clinical benefits to patients with cancer who are undergoing chemotherapy could be realized by an alternate strategy: reduction of the period of neutropenia by blocking the entrance of the stem cells into cell cycle and thus protecting them from the toxic side effects of cycle-specific chemotherapeutic drugs.3 In theory one could combine the two methods and (a) suppress the normal stem cell cycling with specific inhibitors before chemotherapy and (b) accelerate the post-chemotherapy peripheral blood cell recovery with an infusion of stimulators of stem cell proliferation. The role of inhibitors in restraining cells from entering the cell cycle and thereby preserving the high proliferative potential of the bone marrow stem cells is only now being appreciated. Suppressor molecules directed at the genes and their encoded products are today being described at a rapid pace. We mention below some of the theoretical ways by which this inhibition of cell cycling can be realized. Some of these methodologies are already being pursued in hematopoietic or non-hematopoietic cells by academic or biotechnology industry researchers: (1) Introduction into marrow progenitor cells of mutated forms of genes
coding for proteins that are targets for cancer drugs4 (2) Replacement of mutated tumor suppressor genes in certain malignancies.5 (3) Down-regulation (reducing the number), transmodulation (reducing the affinity), or blocking the expression of growth factor receptors. The IL-1 inhibitor is such a natural receptor inhibitor.6 (4) Use of soluble extracellular domains of growth factor receptors as ligand blockers or inhibition of growth factor production. ( 5 ) Therapeutic use of high concentrations of growth factors that mayparadoxically-inhibit cells with receptor overexpression.' (6) Use of growth factor fragments or derivatives with inhibitory action. The oxidized dimer of the peptide described in 1982 by Paukovits and Laerum8 stimulates granulopoiesisin vitro, but its reduced form (monomer) inhibits it. (7) Inhibition of specific signal transduction pathways-for example, inhibitors of tyrosine k i n a ~ e sAnd . ~ finally: (8) Use of natural negative regulators of cell cycling. Popularized in 1962, these substances-often referred to early on as "cha1ones"-were thought not only to inhibit cell proliferation without being cytotoxic but also to be tissue specific.1° In this regard, normal human mammary cells have been reported recently to produce mammastatin, a tissue-specific growth inhibitor distinct from transforming growth factor (TGF)-beta." However, progress in hematopoiesis in this research area has been slow because of multiple difficulties in, for example, purifying the regulatory substances, distinguishing suppression of cell proliferation from nonspecific cytotoxicity, and obtaining pure stem cell populations as target cells for such inhibitors. Resting-but not regenerating-rodent marrow stromal cells were reported in 1976 by Lord to produce an inhibitor of CFU-Scycling.12This factor was specific in vivo for
ANAGNOSTOU et al.: NEGATIVE GROWTH FACTORS
3
CFU-S but in vitro only for early-and not more differentiated-progenitor cells. Frindel and Guigon showed in 1977 that fetal calf marrow contained a dialyzable substance that inhibited the post-irradiation cycling of CFU-S.’) It is now known to be a tetrapeptide that protects mice from the cytotoxic effects of ara-C, an S-phase-specific drug. A glycoprotein produced by leukemic, as well as normal, cells and therefore called leukemia-associated inhibitor (LAI) was found to neutralize the activity of GM-CSF and thus reduce the cycling of normal human CFU-GM.14 Other inhibitors of cell proliferation that may afford bone marrow protection include TGF-P, the macrophage inflammatory protein (MIP- la), and the negative regulatory protein (NRP). It has been claimed that MIP-la is the inhibitor of CFU-S cycling reported by Lord in 1976. Some negative regulators are already undergoing clinical trials, as mentioned in this volume in the summary of the workshop entitled “Zn Vivo Studies and Clinical Trials of Negative Regulators.” The discovery, nature, and action of some of the specific negative regulators are described in the recent review of this area by A. Axelrad.Is Also, a detailed listing of negative regulators of erythropoiesis and their possible mechanisms of action is included in this volume in the summary of the workshop “Inhibitors of Erythropoiesis.” We are at the stage of cataloging the many types of negative regulators of hematopoiesis, and this has provided the primary rationale for two international scientific meetings on the topic of negative hematopoietic cell regulators.
THE FIRST SYMPOSIUM ON HEMATOPOIETIC INHIBITORS
The First International Symposium on Inhibitory Factors in the Regulation of Hematopoiesis took place in April 1987 in Paris. It was organized by A. Najman, M. Guigon, and N. C. Gorin of the Department of Hematology, Facult6 de Medecine, St. Antoine (Universit6 Pierre et Marie Curie, Pans) and J-Y. Mary (INSERM U263, Paris VII). That conference was the first large formal gathering of scientists with interest in such diverse inhibitors of hematopoietic cells and their function as interferons, prostaglandins, tumor necrosis factor (TNF), leukemia-associated inhibitor, and the newly described molecules with putative specific suppressive activity on early stem cells. The symposium concluded with the establishment of an International Inhibitor Registry, which has since been kept up-to-date by M. Guigon, W. Paukovits, and H. Broxmeyer. The registry was an attempt to classify inhibitors according to their molecular and biological properties and includes only biomolecular, not pharmaceutical, drugs.l6 The full proceedings of the conference was published by the Institut National de la Sant6 et de la Recherche Medicale (INSERM) and John Libbey Eurotext. Progress reports on the research on inhibitors were given a prominent role at the Annual Meeting of the International Society of Experimental Hematology in Paris in 1989, again organized by the hematology faculty at HBpital Saint Antoine. A discussion of suppressor molecules and their role in the regulation of the immune response was also part of the VIth International Lymphokine Workshop in 1989, in Evian, Savoie, France.
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THE SECOND INTERNATIONAL CONFERENCE ON NEGATIVE REGULATORS OF HEMATOPOIESIS The Second International Conference on Negative Regulators of Hematopoiesis, held in August, 1990 in Providence, Rhode Island under the auspices of Brown University and Memorial Hospital of Rhode Island, was the first such meeting in North America. Meeting organizers were A. Anagnostou, N. Dainiak, M. Guigon, M. Miller, A. Najman, and W.Paukovits, who were assisted by an advisory board composed of A. Axelrad, H. E. Broxmeyer, E. P. Cronkite, D. Metcalf, P. Ralph, F. Ruscetti, A. N. Schechter, and N. S. Young. Principal financial sponsors were Sandoz Pharmaceuticals and the National Institutes of Health; and contributions were received from Ortho Pharmaceutical Corporation, Cetus Corporation, Genetics Institute, Bristol Myers, Chugai-Upjohn, Hoffmann-La Roche, Smith-Kline Beecham Pharmaceuticals, and Biomeasure, Incorporated. The meeting consisted of 23 plenary talks of 25 minutes each, 70 poster presentations, and 12 workshop sessions. The meeting was attended by 136 established investigators (over half of whom were from outside the U.S.A.)and 20 Brown University scientists, which demonstrates the intense scientific interest in negative regulators. Not only has this field of research shown considerable progress, it has also undergone significant changes since the first symposium was held three years ago in Paris. There was less interest in TNF, prostaglandins, and interferons and more in the effects of TGF-beta on hematopoietic stem cells and the immune system. The presentations underscored the pleiotropic actionls of many of these factors, which are either stimulatory or inhibitory depending on the dose, the presence and concentration of other growth factors, and the target cell population. To wit, TGF-6 specifically inhibits proliferation of early stem cells but enhances proliferation of committed stem cells. It was clear that the diverse effects on non-hematological tissues of many of the discussed factors cautioned against their early therapeutic use in humans. The meeting opened with a review by H. Broxmeyer of the in vivo and in vitro actions, and the mechanisms of action, of several types of inhibitors. Lactoferrin and inhibin indirectly reduced production of IL-lb by monocytes and of CSF by mononuclear cells, whereas the acidic isoform of isoferritin directly affected early stem cells. He reported a strong correlation between the inhibitory activity of this isoferritin, peroxidase activity, and the macrophage inflammatory activity (MIP- la). The presentor considered an 8-kDa protein to be the intermediary of the suppressive actions of a number of these molecules. TGF-/? proved to be a molecule ubiquitous not only in most mammalian cell types, but also in the oral and poster presentations of this conference. F. Ruscetti discussed the direct inhibition of early stem cells by TGF-/3 and reported that infusion of TGF-/? into mice produced a rapid suppression of early stem cell cycling and decreased marrow cellularity and peripheral blood leukopenia. T. Bradley also found the inhibitory effects of TGF-6 to be restricted to early progenitor cells. Some found enhancement of growth at low concentrations, while others did not. TGF-/?-induced inhibition can be neutralized by increasing growth factor concentrations, which indicates a dose-related interaction between positive and negative influences on proliferation. This observation is similar to that found using serum as the source of inhibitors. C. Peschle described his experiments with TGF-6 added to populations of “pure” human peripheral blood stem cells obtained by a technique developed in his laboratory. One of the most interesting topics discussed was the progress made on the in vivo, as well as the in vitro, effects of regulatory peptides such as AcSDKP and pEEDCK, putative specific inhibitors of the hematopoietic stem cells and thus potentially useful
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in protection of marrow injury from chemotherapeutic agents during ex vivo purging. I. Pragnell reviewed his recently published studiesI9of the inhibitory effects of MIP-la on the cycling of early murine bone marrow stem cells and provided evidence for the presence of MIP-la in normal bone marrow, where stem cells are in a resting phase. W. Paukovits provided new details on the leukocyte-derived peptide pyroGlu-GluAsp-Cys-Lys (pEEDCK) and its potential for clinical use. Injection of this peptide rapidly suppressed CFU-GM cycling and the recruitment of CFU-S after ara-C. It prevented the chemotherapy-induced onset of neutropenia, or at least shortened the period of subnormal WBC counts. He suggested that whereas long-term chemotherapy induces exhaustion of pre-CFU-S, this effect is completely prevented by use of the inhibitory monomer, but not by the sthulatory dimer. A. Bogden gave preliminary results on the bone marrow-protective effects of another stem cell inhibitory peptide, N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) shown previously to prevent CFU-S recruitment in ara-C-treated mice. M. Guigon analyzed the in vitro effects of pEEDCK and AcSDKP on both normal and leukemic cells. Tested on hematopoietic cell lines, pEEDCK directly inhibited cells of myelomonocytic origin but did not reduce their sensitivity to ara-C. AcSDKP inhibited the in vitro growth of human progenitor cells (CFU-GM, BFU-E, and CFU-E), although maximal inhibition was never higher than 50%. No inhibitory effect was seen on stem cells from leukemic patients. The effect of AcSDKP reported by Guigon was directed not only to the number of colonies in vitro, but also to the cycling status of the progenitor cells. This latter point is important in understanding the role of this peptide in the protection of bone marrow. Several of the presentations centered on the inhibition of early erythropoietic progenitor cells. A. Axelrad's negative regulatory protein,*O which has now been purified and identified as a 1dkDa protein, was discussed by F. Pluthero. NRP is produced by mouse bone marrow and specifically decreases BFU-E cycling. Its action is countered by IL-3. Pluthero also reported that superoxide dismutase (SOD) inhibits BFU-E but not CFU-E, CFU-GM, or CFU-S. The level of SOD was found to be sufficiently high in normal mouse serum to inhibit BFU-E cycling. A. Yu discussed in detail the prevention by inhibin of the activin-initiated differentiation of erythroid progenitors. N. Dainiak reported on B-lymphocyte membrane-derived growth factors, which include inhibitors of BFU-E, and discussed the role of ambient and accessory cell lipid pools on regulation of hematopoiesis. Our knowledge of the leukemia inhibitory factor (LIF), a highly glycosylated protein produced by a wide variety of cells, was reviewed by D. Gearing. It was originally hoped that LIF would specifically inhibit myeloid leukemia cells by inducing them to differentiate. but LIF is now known to have a variety of in vitro and in vivo actions. The role of the stromal cells on the regulation of hematopoiesis was the theme of many posters and several plenary lectures. D. Zipori reviewed the role of the microenvironment on thymic stem cell proliferation and presented data showing that thymic precursor cells are supported by membrane glycoproteins from stromal cells. A 21-kDa glycoprotein (restrictin) on the surface of stromal cells can arrest the growth of plasmacytoma cells and enhance their apoptosis. C. Eaves presented detailed studies on the clonogenic precursor cell of the long-term bone marrow cell cultures (LTC-IC), the cycling activity of which depends on the relative balance of positive (G-CSF) and negative (TGF-P) growth factors produced by the microenvironment. S. Landreth presents in this volume his working hypothesis for the role of stromal cells on lymphopoiesis. Mechanisms and factors responsible for suppression of immunological cells were discussed by T. Mossmann, J. Kehrl, C. Nathan, and P. Ralph. J. Kehrl reviewed the role of TGF-/3 on the immune system and its in vivo actions, which include increased survival of organ transplants, reduction of severity of collagen-induced arthritis, de-
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crease of NK cells produced by activated lymphocytes, and inhibition of IL-2-induced proliferation and synthesis of IgG and IgM. The endogenous inhibitors of IL-1 and TNF production were discussed by P. Ralph. Production of IL-1 and TNF by lipopolysaccharides (LPS) was inhibited by pretreatment of T cells by TGF-/3 and IL-4 as a result of the activation of an IL-1 receptor antagonist (IL-1-ra). T. Mossman discussed the synthesis by TH-2 cells of a cytokine synthesis inhibitory factor (CSIF), which inhibits the TH- 1 cells and suppresses interferon production after schistosomiasis infection. The area of molecular control of hematopoiesis was the topic of a plenary presentation by A. Gewirtz. He described experiments with c-MYB antisense nucleotides, which, when added at the start of a culture, inhibit the development of progenitor cell colonies. A. Schechter reported the existence of negative controlling regions in the promoter of the @- and q-globin gene. The mechanisms of suppression of hematopoiesis during viral infections was analyzed by N. Young, who reviewed the destruction of CFU-E and BFU-E (but not CFU-GM or CFU-GEM) by the B-19 strain of human parvovirus, causative agent of transient aplastic crises in persons with chronic hemolytic anemias and some cases of pure red cell aplasia. The genome of the virus codes for the cytotoxic nonstructural protein NSP produced in permissive infected cells. Bueren et al. report in this volume that another parvovirus, the minute virus of mice (MVM), demonstrates cytotoxicity for proliferating pluripotent stem cells. A summary of the workshop “Suppression of Hematopoiesis by Viruses,” which includes discussion of the families of viruses that are known or suspected to inhibit hematopoiesis, the relevant animal models used, and the mechanisms-known or putative-of the suppressive action has also been included in this volume. A. Najman discussed the mechanisms of suppression of normal hematopoiesis in acute non-lymphocytic leukemia. Coculture of normal bone marrow cell with leukemic blast resulted in reduced colony formation. This suppression seemed to be due to positive inhibition rather than absence of stimulatory factors and was reduced by immune lysis or differentiation induction of leukemic cells. Possible candidates are acidic isoferritins, TNF-a, leukemia associated inhibitor, or even TGF-/3. A. Soto and C.Sonnenschein eloquently advocated the hypothesis that proliferation is a constitutive property of cells and that suppressor mechanisms, by restraining that proliferation, are thus the principal factors for the maintenance of normal homeostasis. They offered supportive data from their research with estrogen-dependent endometrial cells kept in check by an inhibitory 70-kDa non-glycosylated protein, estrocolyne. J-Y. Mary reminded the participants that the difficulties in experiment design and result interpretation that had dampened the initial enthusiasm in the “chalone” field long ago still exist. He proposed a way out of this experimental minefield involving the use of a standardized procedure and a spreadsheet program for carrying out the appropriate statistical tests.
THE WORKSHOPS The themes of the workshops held (and their chairpersons) included: in vivo studies/ clinical trials of negative regulators (Broxmeyerrnainiak); suppressors of macrophage effector function (Nathan/Ralph); transforming growth factor-beta (Ruscetti); stem cell inhibition (Paukovits/Guigon); lymphocyte suppression/IL-1 (Ralph); erythropoiesis inhibitors (Dainiak/Najman); inhibitors of megakaryocytopoiesis (Levin); viruses
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and suppression of hematopoiesis (YoungDavis); cell proliferation: positive and negative controls (Sonnenschein/Soto); molecular biology of gene regulation (Gewirtz/ Schechter); LIF (Gearing); restrictive effects of the microenvironment (Zipori); and classification of negative regulators (PaukovWGuigon: the 3rd International Workshop on Classification of Negative Regulators). Predictably, the interactive evening workshops, each one attended by between 20 to 50 investigators who discussed their own and each others’ work, were thought to be among the most fruitful parts of the conference. And as one would expect in this complex, confusing, but exciting scientific field, the spirited debates and brainstorming of these sessions were remarkable mainly for the important questions raised rather than for the satisfactory answers provided. As yet, we are not at the point where we can see the total picture of these regulatory mechanisms clearly enough to be able to interpret all the fragmentary data. Nevertheless, areas were identified that may constitute starting points for very promising future research. Brief summaries of some of the workshops have been included in this volume in order to give a glimpse of the multiple questions raised by the participants. The conference concluded with a plenary session, which included presentation of the summaries of all the workshops by their chairpersons and open discussion. This publication, made possible through the generous assistance of the New York Academy of Sciences, reflects the merits and disadvantages of any volume based on the proceedings of a scientific conference. Although it encompasses much of the very best work being done today in the field of negative regulators, it does not claim to be comprehensive. We hope, however, that it will serve as a valuable resource for scientists and clinicians who need to know more about this important, fast-moving field in biology, hematology, and cancer treatment.
REFERENCES 1. METCALF,D. 1989. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature 339 27- 30. L. 1986. Clinical role, therapeutic promise of CSFs outlined. Oncology Times 2. DELMONTE, 8: 3-16. 3. GUIGON,M., J-Y. MARY,J. ENOUF& E. FRINDEL. 1982. Protection of mice against lethal doses of 1-beta-D-arabinofuranosylcytosine by pluripotent stem cell inhibitors. Cancer R ~ s .4 2 638-641. 4. BERTINO,J. R. 1990. “Turning the tables”-Making normal marrow resistant to chemotherapy. J. Natl. Cancer Inst. 8 2 1234-1235. 5. BOOKSTEIN, R.,J.-Y. SHEW,P-L. CHEN,P. SCULLY& W-H. LEE. 1990. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 247: 712-715. 6. HANNUM,C. H., C. J. WILCOX,W. P. AREND,G. J. JOSLIN, D. J. DRIPPS, P. L. HEIMDAL, L. G. ARMES, A. SOMMER, S. P. EISENBERG & R. C. THOMPSON.1990. Interleukin-1 receptor antagonist activity of a human interleukin- 1 inhibitor. Nature 343: 336-340. 7. LUPU, R., R. COLOMER,G. ZUGMAIER, J. SARUP,M. SHEPARD,D. SLAMON& M. E. LIPPMAN.1990. Direct interaction of a ligand for the erB2 oncogene product with the EGF receptor and p185erbB2. Science 249 1552-1555. 8. PAUKOVITZ, W. & 0. LAERUM.1982. Isolation and synthesis of a hemoregulatory peptide. Z. Naturforsch. 376 1297-1300. 9. HUDZIAK,R. M., G. D. LEWIS, M. WINGET,B. M. FENDLY,N. M. SHEPARD& A. ULLRICH.1989. pl 85Her2monoclonal antibody has antiproliferative effects in vitro and sensitises human breast tumor cells to tumor necrosis factor. Mol. Cell. Biol. 9 1165- 1172.
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10. IVERSON,D. H. 1981. The chalones. In Handbook of Experimental Pharmacology. R. Baserga, Ed.: 491- 550. Springer-Verlag. Berlin. R. C. CODY& M. S. WICHA.1989. Production of mam11. ERVIN,P. R.. R. C. KAMINSK, mastatin, a tissue specific growth inhibitor by normal human mammary epithelial cells. Science 244: 1585. 12. LORD,B. I., K. J. MORI,E. G. WRIGHT& L. G. LAJTHA.1976. An inhibitor of stem cell proliferation in normal bone marrow. Br. J. Haematol. 3 4 441-445. 13. FRINDEL,E. & M. GUIGON.1977. Inhibition of CFU entry into cycle by a bone marrow extract. Exp. Hematol. 5: 74-76. 14. OLOFSSON,T. & I. OLSSON. 1980. Suppression of normal granulopoiesis in vitro by a leukemia-associated inhibitor (LAI) of acute and chronic leukemia. Blood 5 5 975-982. 1988.The classification ofhematopoietic W., M. GUIGON& H. E. BROXMEYER. 15. PAUKOVITS, inhibitors. Int. J. Cell Cloning 6 76-79. 16. SAUTES,C. & C. W. PIERCE.1989. Suppressor molecules. Lymphokine Res. 8 159-162. 17. SPORN,M. B. & A. B. ROBERTS.1988. Peptide growth factors are multifunctional. Nature 332 217-219. G. J., E. G. WRIGHT,R. HEWICK,S.D. WOLPE,N. M. WILKIE,D. DONALDSON, 18. GRAHAM, S. LORIMORE & I. B. PRAGNELL. 1990. Identification and characterisation of an inhibitor of hematopoietic stem cell proliferation. Nature 344:442-444. 19. AXELRAD,A. A., H. J. CROIZAT& D. ESKINAZI.1981. A washable macromolecule from Fv-2" marrow negatively regulates DNA synthesis in erythropoietic progenitor cells BFU-E. Cell 26 233-244.
