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Haemopoietic growth factors
TIBS 11 - May 1986
207
(1984) FEBS Lett. 178,306-310 7 Horwich, A. L., Kalousek, F., Mellman, I. and Rosenberg, L.E. (1985) EMBO J. 4, 1129-1135 8 Nguyen, M., Argan, C., Lusty, C.J. and Shore, G. C. (1986) J. Biol. Chem. 261,800-805 9 Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L , Takeda, M. and Douglas, M. G. (1986) Z Cell. Biol. 102,523-533 10 Keng, T., Alani, E. and Guarente, L. (1986) Mol. Cell. Biol. 6, 355-364 11 van Loon, A. P. G. M., Brfindli, A. and Schatz, G. (1986) Cell44, 801-812 12 Riezman, H., Hay, R., Witte, C., Nelson, N. and Schatz, G. (1983)EMBOJ. 2, 1113-1118 13 Hurt, E. C., Miiller, U. and Schatz, G. (1985) EMBO J. 4, 3509-3518 14 van Loon, A. P. G. M. and Young, T. E. (1986) EMBOJ. 5,161-165 15 Hurt, E.C., Pesold-Hurt, B., Suda, K., Oppliger, W. and Schatz, G. (1985) EMBOJ.
4, 2061-2068 16 Hare, T., Nakai, M. and Matsubara, H. FEBS Lett. (in press) 17 vanLoon,A. P. G. M. andHurt, E. C. (1986) in Biochemistry and Molecular Biology o f Industrial Yeasts (Stewart, T. G., Russell, I., Klein, R. D. and Hiebsch, R. R., eds) (Uniscience series), CRC Press 18 Harmey, M. A. and Neupert, W. (1985) in The Enzymes of Biological Membranes (Martonosi, A., ed.), Vol. 4, pp. 431-464, Plenum Press 19 van Loon, A. P. G. M., de Groot, R. J,, de Haan, M., Dekker, A. and Grivell, L.A. (1984) EMBOJ. 3, 1039-1043 20 Ohta, S. and Schatz, G. (1984) EMBO 1. 3, 651~57 21 Firgaira, F. A., Hendrick, J. P., Kalousek, F., Kraus, J.P. and Rosenberg, L. E, (1984) Science 226, 1319-1326 22 Yoshida, Y., Hashimoto, T., Kimura, H., Sakakibara, S. and Tagawa, K. (1985)
Haemopoietic growthfactors A. D. Whetton and T. M. Dexter Haemopoiesis is regulated by a number of distinct growth factors, many of which have been purified to homogeneity and their genes cloned. Some intriguing aspects of how these growth factors control the survival, proliferation and development of specific haemopoietic progenitor cells and their role in leukaemogenesis have now been elucidated.
Haemopoiesis Blood cells are produced in the bone marrow and lymphoid organs by a process known as haemopoiesis. Once they are produced, the vast majority of mature cells are destined to live for only a few hours (e.g. granulocytes) or a few weeks (erythrocytes). This 'programmed' death necessitates the constant production of new blood cells throughout life. This is no small undertaking when we consider that, in humans, every hour about 1.1 x 1010erythrocytes come to the end of their lifespan. Apart from bloodcell production under normal 'steady state' haemopoiesis, the haemopoietic system must adapt cellular production to the needs of an organism at any particular time. Under conditions of haemopoietic stress, such as bleeding or infection, the system must retain a considerable degree of flexibility so that more of the appropriate mature cells can be produced - in these cases, erythrocytes or lymphocytes, respectively. Blood cell formation starts from the haemopoietic stem cells which in an A. D. Whetton is at the Department o f Biochemistry and Applied Molecular Biology, University of Manchester Institute o f Science and Technology, PO Box 88, Manchester M60 IQD, UK; T. M. Dexter is at the Experimental Haematology Section, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK. Correspondence to A. D. Whetton.
adult are restricted mainly to the bone marrow. The stem cells have certain properties which distinguish them from other cells: they are pluripotent (they have genetic potential to undergo development into the various blood cell lineages) and they can self-renew (form exact copies of themselves). What determines that 'decision' to self-renew or to differentiate is not known but a consequence of differentiation is seen as the committment of the cells into only one or two cell lineages. Further proliferation leads to a progressively more mature phenotype with a concomitant loss of proliferative potential, the end result being a mature blood cell which has no ability to proliferate (Fig. 1). During normal haemopoiesis stem cells are not usually undergoing mitotic division, but can be rapidly mobilized into proliferation when required. Committed progenitor cells, on the other hand, are a transient population in that they are predominantly dividing, proliferating (and undergoing maturation) to form the mature cell compartment. In the main sites of haemopoiesis in vivo, the marrow and lymphoid organs, the developing haemopoietic cells are found in association with a complex stromal cell network containing diverse cell types. These cells can also support haemopoiesis in vitro, indicating that they may have a key role in blood cell development: a role that involves corn-
Biochem. Biophys. Rex. Commun. 128,775-780 23 Ito, A., Ogishima, T., Ou, W., Omura, T., Aoyagi, H., Lee, S., Mihara, H. and Izumiya, N. (1985) J. Biochem. (Tokyo) 98, 1571-1579 24 Gillespie, L. L., Argan, C., Taneja, A. T., Hodges, R. S., Freeman, K. B. and Shore, G. C. (1986) J. Biol. Chem. 260, 16045--16048 25 Roise, D., Horvath, S.J., Richards, J. H., Tomich, J. M. and Schatz, G. EMBO J. (in press) 26 Rietveld, A., Sijens, P., Verkleij, A. J. and de Kruijff, B. (1983) EMBOJ. 2, 907-913 27 Schleyer, M. and Neupert, W. (1985) Cell 43, 339-350 28 Kellems, R. E., Allison, V. G. and Butow, R. A. (1975) J. CelIBiol. 65, 1-4 29 Maarse, A. C., van Loon, A. P. G. M., Riezman, H., Gregor, I., Schatz, G. and Grivell, L. A. (1984) E M B O Z 3, 2831-2837 30 Young, E. T. and Pilgrim, D. B. (1985) Mol. Cell. Biol. 5, 3024-3034
plex cellular interactionsI and production of diffusible regulatory molecules. The relative importance of soluble mediators in haemopoietic cell growth and development has been difficult to define, especially in the context of the stromal cell environment. However, it was established many years ago that haemopoietic progenitor cells can survive, proliferate and develop in soft agar in the absence of stromal cells, provided that the cultures contained the appropriate growth-stimulating factor 2. These growth factors were present in conditioned media from a variety of normal and leukaemic cell lines or from various tissues, and also from activated T lymphocyte cell populations. Now, however, many of these growth factors have been purified to homogeneity and/or molecularly cloned and their amino acid sequences determined. They can be divided into two main sub-categories in the myeloid system (see Ref. 3 for details of a lymphocytic growth factor), multipotential growth factors and lineage restricted growth factors. Interleukin 3 (IL-3) is an example of a multi-lineage growth factor in that it stimulates the proliferation and development of erythroid, megakaryocytic, eosinophilic, neutrophilic and macrophage progenitor cells, and also the self-renewal and development of multipotent stem cells (see Fig. 1). No human (gene) counterpart to murine IL-3 has yet been isolated, but a multipotent growth factor named pluripoietin has recently been isolated and characterized 4. It also can promote proliferation in several distinct haemopoietic lineages and in multipotent cells. In both human and murine systems, a granulocyte/macrophagecolony stimulating factor (GM-CSF) exists which stimulates granulocyte and macrophage progenitor cell develop-
1986,ElsevierSciencePublishersB.V,,Amsterdam 0376 5 ~ 7 ~ . ~ - -
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TIBS 1 1 - May 1986
B cells
asop ils
Platelets
Neutrophlls
Macrolo ages
Erythroeytes
Eosinophils
T cells
Pre-B cells
Fig. 1. Murine haemopoiesis. Haemopoiesis begins with the pluripotent haemopoietic stem cell. This cell can either self-renew or undergo commitanent to develop or differentiate into committed progenitor cells. The CFU--S (colony-forming unit-spleen) assay detects cells which belong to the stem cell comparanent but CFU-S may not represent the total stem cellpopulation. CFC-Mix (colony.forming cells-mixed) are cells which in semi-solid media can form multi.lineage colonies, and as such may have some overlap with those cells known as CFU-S. Committed progenitor cellsof several distinct haemopoletic lineages have been identified in vitro, these include the basophil colony-forming cell (Bas-CFC); the megakaryocyte-CFC (Meg-CFC); the granidocyte/maerophage-CFC (GM~FC); the burst-forming unit-erythroid (BFU-e); the more rnao~e erythroid progenitor cell; the colony forming unit-erythroid (CFUI-e): and the eosinophil colony forming cell (Eos-CFC). Also, Pre-B lymphocytic cells and Pre- T lymphocytic cells can be recognized in appropriate assays in vitro. These cells are named colony-forming cells because of their ability to form a clonal colony of mature cells of a given lineage in semi-solid culture medium in vitro2.
ment 5 (Fig. 1 and Table I), eosinophil progenitor cell and also some megakaryocytic cell development (Metcalf, pers. commun.). Other haemopoietic growth factors are restricted in their action to one distinct lineage. For example, erythropoietin promotes only the development of committed erythroid progenitor cells (see Table I). Thus far there is no evidence to indicate that the different growth factors form part of a gene family since there is no apparent sequence homology between the growth factors whose amino acid sequences have been deduced (IL-36, GM-CSF 7, CSF-18 and erythropoieting). The role of haemopoietic growth factors in vivo Although it is apparent that all the haemopoietic growth factors in Table I can support the survival, proliferation and development of the appropriate
target cell in vitro, this does not prove that they have a similar role in vivo. A role in vivo was first demonstrated with erythropoietin: erythroid cell proliferation, haemoglobin accumulation, development and mature red blood cell numbers in the circulation have all been shown to be related to the activities of this growth factor 5. Other haemopoietic growth factors have also been detected in the serum and urine of animals and man, including CSF-1 and GM-CSF. In terms of roles for these molecules in vivo it may be significant that substantial increases in the circulating concentrations of both GM-CSF and CSF-1 are observed in animals treated with bacterial lipopolysaccharides, and this rise correlates with GM progenitor cell proliferation. Although no IL-3 has been detected in vivo, the infusion of exogenous 1L-3 to mice has a profound effect on stem cells, increasing their rate of proliferation
markedly and causing an increase of both stem cells and progenitor cells, especially in the spleen. However, such data do not provide direct evidence of a physiological role in normal haemopoiesis and it is likely that regulation of proliferation and development of haemopoietic cells in vivo is determined by a combination of cell interactions and microenvironmental influences acting in concert with diffusible regulatory molecules. Indeed the major functions of these growth factors may be not in the regulation of proliferation and development but in the activation of mature cell function5. For example, GM-CSF and CSF-1 can stimulate functions such as the production of plasminogen activator and prostaglandin E2 by macrophages, and GM--CSF has been shown to increase parasite killing by macrophages, and killing of neoplastic cells by granulocytes. Such activities promoted by these
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growth factors may be of major importance in vivo. Localized production of such agents at a site of inflammation or infection may play a substantial role in the processes of wound healing and combatting infectious disease.
Receptors for haemopoietic cell growth factors Like all polypeptide growth factors studied to date, the haemopoietic cell growth factors bind to cell surface receptors 10. A major limitation, however, in investigating the nature of these receptors on normal marrow stem cells or progenitor cells, is that these cells represent a minor percentage of the total n u m b e r of marrow cells. For this reason the measurement of receptor numbers and affinities has often been performed on whole marrow (heterogenous) cell populations or on haemopoietic cell lines. Radio-iodinated IL-3, G M - C S F , CSF-1 and G - C S F have all been shown to bind to specific receptors of known relative molecular mass (see Table I); there is no common receptor for all these ligands. A complex set of interactions can occur, however, such that the binding of the multipotential ligand IL-3 can not only down-modulate its own receptors (reduce the n u m b e r available), but
also the receptors for G M - C S F , G - C S F and CSF-111. Also, G M - C S F can downmodulate GM--CSF, G - C S F and CSF-1 receptors whilst G--CSF can downmodulate G - C S F and CSF-1 receptors. This receptor modulation may constitute a means of maintaining fidelity of response within a target cell population to avoid what may be counterproductive signals. The actual numbers of receptors found on the cell surface before any down-modulation events is extremely low. There are about 660 erythropoietin receptors on some target cells with a K a value in the nanomolar region 12 whilst there are IIX~-1000 IL-3, GM--CSF and G - C S F receptors per cell in responsive cell populations with K d values in the picomolar to nanomolar r e # o n (Refs 10, 13 and 14 and Burgess and Dexter, unpublished observations), The CSF-1 receptor is the exception, with around 16 000 (Ref. 10) and up to 73 000 receptors per cell ~5 on mature macrophages. Furthermore, the biological effects of the growth factors erythropoietin, G M CSF and G - C S F can be achieved with receptor occupation in the r e # o n of 1% (erythropoietin t2) to 5-10% ( G M - C S F , G - C S F 1°) suggesting that any transm e m b r a n e signalling event governed by
these receptors can have major effects on the cell at very low levels of receptor occupation.
The mode of action of haemopoietic growth factors Haemopoietic growth factors are an absolute requirement for the survival, proliferation and development (or self renewal) of their target cell populations when cultured in vitro in the absence of stromal cells; lack of growth factor in these circumstances leads to a rapid loss of progenitor cells. It must be stressed that this is not a withdrawal into a quiescent state, as can be seen in other growth factor systems - when cultured in the absence of the growth factor the cells die. This deterioration of the target cell population in the absence of growth factor adds some complications to the study of haemopoietic growth factor action, but can also be used to advantage by asking what happens when the growth factor is withdrawn and can this effect be reversed by its readdition? Using this approach, the major effect of removing IL-3 from cell lines absolutely dependent on this growth factor for their survival was shown to be a decrease in the primary metabolism of the cell 16. Hexose transport rates fall, glycolysis is reduced,
Table L Haemopoieticgrowthfactors". Growth factor
Ref.
Species
lnterleukin 3
5, 6,10
Murine
Growth factor Mr
23-26 000
GM-CSF
5, 7,10,14
Responsivecelltypes Stem cells CFC-Mix BFU--e GM~CFC Eos42FC Meg~FC Granulocyteprecursorcells Macrophageprecursorcells Erythroid precursorcells Mast cells GM42FC Eos--CFC Meg-CFC Human GM-CFC Mature granulocytes
Receptor M~
50-70 000
Murine
23 000
Human
30 000 25 000 30 000
Sub-populationof GM-CFC Myeloidleukaemiacells?
150 000
Sub-populationof GM-CFC Macrophageprecursorcells Mature macrophages
150 000
51 0130
G~r~'SF
5, 10,13
Murine Human (CSF 13)
CSF-1
5, 8,10,15, 19
Murine Human
-76 000 - 45 000
Haemopoietin1
28
Human
20 000
Murinemultipotentstem cells?
?
Pluripoietin
4
Human
18 000
Human CFC-Mix GM-CFC BFU-e
?
Erythropoietin
5,12, 29
Murine Human
39 000
Murine CFU--e Human CFU-e
?
aThe above list of growth factors can stimulate the survival,proliferationand developmentof the target cells shown. The progenitor cell types are describedin Fig. 1. Variations in relative molecular mass of a given growth factor may be attributed (at least in part) to differentialglycosylationpatterns on the same polypeptide.
210 and there is a steady fall in ATP concentrations within the cell. All of these effects are reversible if IL-3 is added back to the cells before they die. To date no mechanistic details on how other haemopoietic growth factors promote cell survival and proliferation are known. There have been few detailed investigations of the events triggered by occupation of haemopoietic growth factor receptors. Again, most of the available data have been obtained using IL-3 dependent cell lines. IL-3 has been shown to have little effect on cAMP concentrations within target cell populations. However, a possible mechanism for the promotion of survival and proliferation by IL-3 is indicated by the observation that adding IL-3 to growth factor-dependent ceil lines causes translocation and activation of protein kinase C 17. Although this infers that diacylglycerol formation (via increased phosphatidy-linositol lipid metabolism) is stimulated by IL-3 this has still to be conclusively demonstrated. In this respect it is worth bearing in mind the recent work showing an insulin-stimulated formation of diacylglycerol without any apparent increases in polyphosphoinositide breakdownis. At present the mode of action of other haemopoietic growth factors has not been established, except that the CSF-1 receptor has been shown to be a tyrosinespecific protein kinase of Mr 160 which autophosphorylates on addition of CSF-119.
Haemopoietiegrowthfactorsand leukaemogenesis The rigorous control of survival, proliferation and development in the haemopoietic system is sometimes lost, giving rise to leukaemia or hyperplasia. There is now some important evidence relating haemopoietic growth factors to these malignant diseases. Some murine leukaemic cells are absolutely dependent on IL-3 for their survival and proliferation in vitro. Similarly many human leukaemic cells require a stirnulatory activity (which is as yet poorly characterized) to proliferate in vitro. A great deal of evidence in both human and murine systems suggests that the leukaemic cell may in some cases resemble a haemopoietic progenitor cell which displays an imbalance between proliferation and development, such that the facility to self-renew is inappropriately expressed 2° (Fig. 2). Thus, in the case of some hyperplasias or leukaemias it is the response to, as opposed to independence from, haemopoietic growth factors that may be
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I Proliferative Capacity } Fig. 2. A comparison between normal haemopoiesis and leukaemic haemopoiesis. Self-renewal of stem cells, commitanent andproliferation (accompanied by maturation) arefinely balanced in normal haemopoiesis, under the regulation of the bone marrow stromal environment. In the case of a leukaemia, stir-renewal in either the stem or progenitor cell compaaraaU is apparently not controlled. An inappropriately expressed ability to selfrenew at an increased rate in the stem cell compartment or a developmental block imposed upon a committed progenitor cell are thought to be the essential elements in this loss of control. Phenotypically leukaemic cells markedly resemble progenitor cells, thus oncogene expression or some other event may alter the balance o f the developmental process in haemopoiesis and this may in part contribute to a leukaemia.
altered. None the less, some models do exist which suggest that growth factor-independence is one possible step in leukaemic transformation. There is evidence from the leukaemic murine cell line WEHI-3B that the IL-3 gene has at some stage come under the influence of a viral promoter sequence, which might explain why these cells actually secrete IL-3, a phenomenon which may have been associated with their initial transformation21. Furthermore, a nonleukaemic IL-3/GM-CSF-dependent cell line has been described22, which, upon transfection with viruses containing the a b l or m y c oncogenes, becomes independent of growth factors for survival and proliferation and also leukaemic23-25. This same cell line can also be transformed to a ieukaemic factor-independent cell when transfected with a virus containingthe gene for GMCSF26. Following transfection and expression of m y c or abl, the growth autonomy is not associated with auto-production of growth factors 22-24or of altered levels of expression of the receptors (Burgess and Dexter, unpublished observations); in other words, expression of m y c or a b l has bypassed the requirement for growth factor. Transfection with the GM--CSF gene, however, has allowed auto-stimulation of growth in the absence of added exogenous growth factors. Superficially, these two pathways to growth autonomy appear to be quite different, but the
biochemical pathways underlying the stimulation of growth are surely overlapping in certain key intracellular events, and these two systems seem to provide suitable models to determine which these events are. So far the role of oncogenes in this process remains obscure and m y c a n d a b l have as yet no clearly ascribed biochemical function. However, data reported recently have provided strong evidence that the v-fins gene product may be homologous to the CSF-1 recepto# 7. Since v-fms can transform cells of the macrophage lineage this may be an analogous situation to that seen with v--erb b which codes for a truncated EGF receptor. Molecular analysis is urgently required to obtain the nucleotide sequences of v-fins and c-fins (the CSF1 receptor?). Clearly many of the biochemical and cell biological events governing haemopoiesis are still poorly understood. But the tools are now available to elucidate how these growth factors control the survival, proliferation and development in normal haemopoietic cells and to determine which events can lead to the loss of growth control and malignancy in the haemopoietic system. Acknowledgements We wish to thank Dr Clare Heyworth for much helpful discussion. Work in the authors' laboratories is supported by the
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Leukaemia Research Fund and the C a n c e r R e s e a r c h C a m p a i g n , T, M. D e x t e r is a fellow o f t h e C a n c e r Research Campaign.
References I Dexter, T. M., Simmons, P., Pumell, R. A., Spooncer, E. and Schofield, R. (1984) inAplastic Anaemia: Stem Cell Biology and Advances in Treatment (Humphries R. K., Young, N. S. and Levine, A. S., eds), pp. 13-28, Alan R. Liss 2 Metcalf, D. (1977) Haemopoietic Colonies In Vitro Cloning of Normal and Leukaemia Cells, Springer Verlag 3 Robb, R. J. (1984) ImmunoL Today 5,204-209 4 Welte, K., Platzer, E., Li, L., Gabrilove, J. L., Levi, E., Mertelsmann, R. and Moore, M.A.S. (1985) Proc. Natl Acad. Sci. USA 82, 15261530 5 Burgess, A. W. and Nicola, N. A. (1983) Growth Factors and Stem Cells, pp. 93-124, Academic Press 6 Fung, M. C., Hapel, A. J., Ymer, S., Cohen, D. R., Johnson, R. M., Campbell, M. D. and Young, I. G. (1984) Nature 307,233-237
7 Gough, N. M., Gough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N. A., Burgess, A. W. and Dunn, A. R. (1984) Nature 309,763--766 8 Kawasaki, E. S., Ladner, M. B., Wang, A. M., Ardsell, J. V., Warren M. K., Coyne, M. Y., Schweikart, V. L., Lee, M. T., Wilson, K. J., Boosman, A., Stanley, E. R., Ralph, P. and Mark, D. F. (1985) Science 230, 291-296 9 Lee-Huang, S. (1984)Proc. NatlAcad. Sci USA 81,2708-2712 10 Metcalf, D. (1985) Science 229, 16-22 11 Walker, F., Nicola, N. A., Metcalf, D. and Burgess A. W. (1985) Cell 43,269-276 12 Krantz, S. B. and Goldwasser, E. (1984) Proc. Natl Acad. Sci. USA 81, 7574--7578 13 Nicola, N. A., Begley, C. G. and Metcalf, D. (1985) Nature 314,625-628 14 Walker, F. and Burgess, A. W. (1985) EMBO J. 4,933-939 15 Stanley, E. R. and Guilbert, L. J. (1981) J. ImmunoL Methods 42,253--284 16 Whetton, A. D., Bazill, G. W. and Dexter, T. M. (1984) EMBOJ. 3,409-413 17 Farrah, W. L , Thomas, T. P. and Anderson, W. B. (1985)Nature 315,235-237 18 Farese, R. V., Davis, J. S., Barnes, D. E., Standaert, M. L., Babischkin, J. S., Hock, R., Rosic, N. K. and Pollet, R. J. (1985) Biochem.
J. 231,269-278 19 Stanley, E. R. and Jubinsky, P. T. (1984) Clin. Haematol. 13,329-347 20 Greaves, M. F. (1982) CancerSurveys 1,189-204 21 Ymer, S., Tucker, Q. J., Sanderson, C. J., Hapel, A. J., Campbell, H. D. and Young, I. G. (1985) Nature 317, 255-258 22 Dexter, T. M., Garland, J., Scott, D., Scolnick, E. and Metcalf, D. (1980) J. Exp. Med. 152, 1036-1047 23 Cook, W. D., Metcalf, D., Nicola, N. A., Burgess, A. W. and Walker, F. (1985) Cell41, 677-683 24 Pierce, J. H., Di Flore, P. P., Aaronson, S. A., Potter, M., Pumphrey, J., Scott, A. andlhle, J. N. (1985) Cell 41,685-693 25 Rapp, U. R., Cleveland, J. L., Brightman, K., Scott, A. and Ihle, J. N. (1985) Nature 317, 434--438 26 Lang, R. A., Metcalf, D., Gough, N. M., Dunn, A. R. and Gonda, T. J. (1985) Cell43, 531-542 27 Sberr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T. and Stanley, E. R. (1985) Cell41,665-676 28 Bartlemez, S. H. and Stanley, E. R. (1985) Z Cell Physiol. 122,370-378 29 Eaves, A. C. and Eaves, C. J. (1984) Clin. Haematol. 13,371-391
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(1984) FEBS Lett. 178,306-310 7 Horwich, A. L., Kalousek, F., Mellman, I. and Rosenberg, L.E. (1985) EMBO J. 4, 1129-1135 8 Nguyen, M., Argan, C., Lusty, C.J. and Shore, G. C. (1986) J. Biol. Chem. 261,800-805 9 Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L , Takeda, M. and Douglas, M. G. (1986) Z Cell. Biol. 102,523-533 10 Keng, T., Alani, E. and Guarente, L. (1986) Mol. Cell. Biol. 6, 355-364 11 van Loon, A. P. G. M., Brfindli, A. and Schatz, G. (1986) Cell44, 801-812 12 Riezman, H., Hay, R., Witte, C., Nelson, N. and Schatz, G. (1983)EMBOJ. 2, 1113-1118 13 Hurt, E. C., Miiller, U. and Schatz, G. (1985) EMBO J. 4, 3509-3518 14 van Loon, A. P. G. M. and Young, T. E. (1986) EMBOJ. 5,161-165 15 Hurt, E.C., Pesold-Hurt, B., Suda, K., Oppliger, W. and Schatz, G. (1985) EMBOJ.
4, 2061-2068 16 Hare, T., Nakai, M. and Matsubara, H. FEBS Lett. (in press) 17 vanLoon,A. P. G. M. andHurt, E. C. (1986) in Biochemistry and Molecular Biology o f Industrial Yeasts (Stewart, T. G., Russell, I., Klein, R. D. and Hiebsch, R. R., eds) (Uniscience series), CRC Press 18 Harmey, M. A. and Neupert, W. (1985) in The Enzymes of Biological Membranes (Martonosi, A., ed.), Vol. 4, pp. 431-464, Plenum Press 19 van Loon, A. P. G. M., de Groot, R. J,, de Haan, M., Dekker, A. and Grivell, L.A. (1984) EMBOJ. 3, 1039-1043 20 Ohta, S. and Schatz, G. (1984) EMBO 1. 3, 651~57 21 Firgaira, F. A., Hendrick, J. P., Kalousek, F., Kraus, J.P. and Rosenberg, L. E, (1984) Science 226, 1319-1326 22 Yoshida, Y., Hashimoto, T., Kimura, H., Sakakibara, S. and Tagawa, K. (1985)
Haemopoietic growthfactors A. D. Whetton and T. M. Dexter Haemopoiesis is regulated by a number of distinct growth factors, many of which have been purified to homogeneity and their genes cloned. Some intriguing aspects of how these growth factors control the survival, proliferation and development of specific haemopoietic progenitor cells and their role in leukaemogenesis have now been elucidated.
Haemopoiesis Blood cells are produced in the bone marrow and lymphoid organs by a process known as haemopoiesis. Once they are produced, the vast majority of mature cells are destined to live for only a few hours (e.g. granulocytes) or a few weeks (erythrocytes). This 'programmed' death necessitates the constant production of new blood cells throughout life. This is no small undertaking when we consider that, in humans, every hour about 1.1 x 1010erythrocytes come to the end of their lifespan. Apart from bloodcell production under normal 'steady state' haemopoiesis, the haemopoietic system must adapt cellular production to the needs of an organism at any particular time. Under conditions of haemopoietic stress, such as bleeding or infection, the system must retain a considerable degree of flexibility so that more of the appropriate mature cells can be produced - in these cases, erythrocytes or lymphocytes, respectively. Blood cell formation starts from the haemopoietic stem cells which in an A. D. Whetton is at the Department o f Biochemistry and Applied Molecular Biology, University of Manchester Institute o f Science and Technology, PO Box 88, Manchester M60 IQD, UK; T. M. Dexter is at the Experimental Haematology Section, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, UK. Correspondence to A. D. Whetton.
adult are restricted mainly to the bone marrow. The stem cells have certain properties which distinguish them from other cells: they are pluripotent (they have genetic potential to undergo development into the various blood cell lineages) and they can self-renew (form exact copies of themselves). What determines that 'decision' to self-renew or to differentiate is not known but a consequence of differentiation is seen as the committment of the cells into only one or two cell lineages. Further proliferation leads to a progressively more mature phenotype with a concomitant loss of proliferative potential, the end result being a mature blood cell which has no ability to proliferate (Fig. 1). During normal haemopoiesis stem cells are not usually undergoing mitotic division, but can be rapidly mobilized into proliferation when required. Committed progenitor cells, on the other hand, are a transient population in that they are predominantly dividing, proliferating (and undergoing maturation) to form the mature cell compartment. In the main sites of haemopoiesis in vivo, the marrow and lymphoid organs, the developing haemopoietic cells are found in association with a complex stromal cell network containing diverse cell types. These cells can also support haemopoiesis in vitro, indicating that they may have a key role in blood cell development: a role that involves corn-
Biochem. Biophys. Rex. Commun. 128,775-780 23 Ito, A., Ogishima, T., Ou, W., Omura, T., Aoyagi, H., Lee, S., Mihara, H. and Izumiya, N. (1985) J. Biochem. (Tokyo) 98, 1571-1579 24 Gillespie, L. L., Argan, C., Taneja, A. T., Hodges, R. S., Freeman, K. B. and Shore, G. C. (1986) J. Biol. Chem. 260, 16045--16048 25 Roise, D., Horvath, S.J., Richards, J. H., Tomich, J. M. and Schatz, G. EMBO J. (in press) 26 Rietveld, A., Sijens, P., Verkleij, A. J. and de Kruijff, B. (1983) EMBOJ. 2, 907-913 27 Schleyer, M. and Neupert, W. (1985) Cell 43, 339-350 28 Kellems, R. E., Allison, V. G. and Butow, R. A. (1975) J. CelIBiol. 65, 1-4 29 Maarse, A. C., van Loon, A. P. G. M., Riezman, H., Gregor, I., Schatz, G. and Grivell, L. A. (1984) E M B O Z 3, 2831-2837 30 Young, E. T. and Pilgrim, D. B. (1985) Mol. Cell. Biol. 5, 3024-3034
plex cellular interactionsI and production of diffusible regulatory molecules. The relative importance of soluble mediators in haemopoietic cell growth and development has been difficult to define, especially in the context of the stromal cell environment. However, it was established many years ago that haemopoietic progenitor cells can survive, proliferate and develop in soft agar in the absence of stromal cells, provided that the cultures contained the appropriate growth-stimulating factor 2. These growth factors were present in conditioned media from a variety of normal and leukaemic cell lines or from various tissues, and also from activated T lymphocyte cell populations. Now, however, many of these growth factors have been purified to homogeneity and/or molecularly cloned and their amino acid sequences determined. They can be divided into two main sub-categories in the myeloid system (see Ref. 3 for details of a lymphocytic growth factor), multipotential growth factors and lineage restricted growth factors. Interleukin 3 (IL-3) is an example of a multi-lineage growth factor in that it stimulates the proliferation and development of erythroid, megakaryocytic, eosinophilic, neutrophilic and macrophage progenitor cells, and also the self-renewal and development of multipotent stem cells (see Fig. 1). No human (gene) counterpart to murine IL-3 has yet been isolated, but a multipotent growth factor named pluripoietin has recently been isolated and characterized 4. It also can promote proliferation in several distinct haemopoietic lineages and in multipotent cells. In both human and murine systems, a granulocyte/macrophagecolony stimulating factor (GM-CSF) exists which stimulates granulocyte and macrophage progenitor cell develop-
1986,ElsevierSciencePublishersB.V,,Amsterdam 0376 5 ~ 7 ~ . ~ - -
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TIBS 1 1 - May 1986
B cells
asop ils
Platelets
Neutrophlls
Macrolo ages
Erythroeytes
Eosinophils
T cells
Pre-B cells
Fig. 1. Murine haemopoiesis. Haemopoiesis begins with the pluripotent haemopoietic stem cell. This cell can either self-renew or undergo commitanent to develop or differentiate into committed progenitor cells. The CFU--S (colony-forming unit-spleen) assay detects cells which belong to the stem cell comparanent but CFU-S may not represent the total stem cellpopulation. CFC-Mix (colony.forming cells-mixed) are cells which in semi-solid media can form multi.lineage colonies, and as such may have some overlap with those cells known as CFU-S. Committed progenitor cellsof several distinct haemopoletic lineages have been identified in vitro, these include the basophil colony-forming cell (Bas-CFC); the megakaryocyte-CFC (Meg-CFC); the granidocyte/maerophage-CFC (GM~FC); the burst-forming unit-erythroid (BFU-e); the more rnao~e erythroid progenitor cell; the colony forming unit-erythroid (CFUI-e): and the eosinophil colony forming cell (Eos-CFC). Also, Pre-B lymphocytic cells and Pre- T lymphocytic cells can be recognized in appropriate assays in vitro. These cells are named colony-forming cells because of their ability to form a clonal colony of mature cells of a given lineage in semi-solid culture medium in vitro2.
ment 5 (Fig. 1 and Table I), eosinophil progenitor cell and also some megakaryocytic cell development (Metcalf, pers. commun.). Other haemopoietic growth factors are restricted in their action to one distinct lineage. For example, erythropoietin promotes only the development of committed erythroid progenitor cells (see Table I). Thus far there is no evidence to indicate that the different growth factors form part of a gene family since there is no apparent sequence homology between the growth factors whose amino acid sequences have been deduced (IL-36, GM-CSF 7, CSF-18 and erythropoieting). The role of haemopoietic growth factors in vivo Although it is apparent that all the haemopoietic growth factors in Table I can support the survival, proliferation and development of the appropriate
target cell in vitro, this does not prove that they have a similar role in vivo. A role in vivo was first demonstrated with erythropoietin: erythroid cell proliferation, haemoglobin accumulation, development and mature red blood cell numbers in the circulation have all been shown to be related to the activities of this growth factor 5. Other haemopoietic growth factors have also been detected in the serum and urine of animals and man, including CSF-1 and GM-CSF. In terms of roles for these molecules in vivo it may be significant that substantial increases in the circulating concentrations of both GM-CSF and CSF-1 are observed in animals treated with bacterial lipopolysaccharides, and this rise correlates with GM progenitor cell proliferation. Although no IL-3 has been detected in vivo, the infusion of exogenous 1L-3 to mice has a profound effect on stem cells, increasing their rate of proliferation
markedly and causing an increase of both stem cells and progenitor cells, especially in the spleen. However, such data do not provide direct evidence of a physiological role in normal haemopoiesis and it is likely that regulation of proliferation and development of haemopoietic cells in vivo is determined by a combination of cell interactions and microenvironmental influences acting in concert with diffusible regulatory molecules. Indeed the major functions of these growth factors may be not in the regulation of proliferation and development but in the activation of mature cell function5. For example, GM-CSF and CSF-1 can stimulate functions such as the production of plasminogen activator and prostaglandin E2 by macrophages, and GM--CSF has been shown to increase parasite killing by macrophages, and killing of neoplastic cells by granulocytes. Such activities promoted by these
T I B S l l - M a y 1986
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growth factors may be of major importance in vivo. Localized production of such agents at a site of inflammation or infection may play a substantial role in the processes of wound healing and combatting infectious disease.
Receptors for haemopoietic cell growth factors Like all polypeptide growth factors studied to date, the haemopoietic cell growth factors bind to cell surface receptors 10. A major limitation, however, in investigating the nature of these receptors on normal marrow stem cells or progenitor cells, is that these cells represent a minor percentage of the total n u m b e r of marrow cells. For this reason the measurement of receptor numbers and affinities has often been performed on whole marrow (heterogenous) cell populations or on haemopoietic cell lines. Radio-iodinated IL-3, G M - C S F , CSF-1 and G - C S F have all been shown to bind to specific receptors of known relative molecular mass (see Table I); there is no common receptor for all these ligands. A complex set of interactions can occur, however, such that the binding of the multipotential ligand IL-3 can not only down-modulate its own receptors (reduce the n u m b e r available), but
also the receptors for G M - C S F , G - C S F and CSF-111. Also, G M - C S F can downmodulate GM--CSF, G - C S F and CSF-1 receptors whilst G--CSF can downmodulate G - C S F and CSF-1 receptors. This receptor modulation may constitute a means of maintaining fidelity of response within a target cell population to avoid what may be counterproductive signals. The actual numbers of receptors found on the cell surface before any down-modulation events is extremely low. There are about 660 erythropoietin receptors on some target cells with a K a value in the nanomolar region 12 whilst there are IIX~-1000 IL-3, GM--CSF and G - C S F receptors per cell in responsive cell populations with K d values in the picomolar to nanomolar r e # o n (Refs 10, 13 and 14 and Burgess and Dexter, unpublished observations), The CSF-1 receptor is the exception, with around 16 000 (Ref. 10) and up to 73 000 receptors per cell ~5 on mature macrophages. Furthermore, the biological effects of the growth factors erythropoietin, G M CSF and G - C S F can be achieved with receptor occupation in the r e # o n of 1% (erythropoietin t2) to 5-10% ( G M - C S F , G - C S F 1°) suggesting that any transm e m b r a n e signalling event governed by
these receptors can have major effects on the cell at very low levels of receptor occupation.
The mode of action of haemopoietic growth factors Haemopoietic growth factors are an absolute requirement for the survival, proliferation and development (or self renewal) of their target cell populations when cultured in vitro in the absence of stromal cells; lack of growth factor in these circumstances leads to a rapid loss of progenitor cells. It must be stressed that this is not a withdrawal into a quiescent state, as can be seen in other growth factor systems - when cultured in the absence of the growth factor the cells die. This deterioration of the target cell population in the absence of growth factor adds some complications to the study of haemopoietic growth factor action, but can also be used to advantage by asking what happens when the growth factor is withdrawn and can this effect be reversed by its readdition? Using this approach, the major effect of removing IL-3 from cell lines absolutely dependent on this growth factor for their survival was shown to be a decrease in the primary metabolism of the cell 16. Hexose transport rates fall, glycolysis is reduced,
Table L Haemopoieticgrowthfactors". Growth factor
Ref.
Species
lnterleukin 3
5, 6,10
Murine
Growth factor Mr
23-26 000
GM-CSF
5, 7,10,14
Responsivecelltypes Stem cells CFC-Mix BFU--e GM~CFC Eos42FC Meg~FC Granulocyteprecursorcells Macrophageprecursorcells Erythroid precursorcells Mast cells GM42FC Eos--CFC Meg-CFC Human GM-CFC Mature granulocytes
Receptor M~
50-70 000
Murine
23 000
Human
30 000 25 000 30 000
Sub-populationof GM-CFC Myeloidleukaemiacells?
150 000
Sub-populationof GM-CFC Macrophageprecursorcells Mature macrophages
150 000
51 0130
G~r~'SF
5, 10,13
Murine Human (CSF 13)
CSF-1
5, 8,10,15, 19
Murine Human
-76 000 - 45 000
Haemopoietin1
28
Human
20 000
Murinemultipotentstem cells?
?
Pluripoietin
4
Human
18 000
Human CFC-Mix GM-CFC BFU-e
?
Erythropoietin
5,12, 29
Murine Human
39 000
Murine CFU--e Human CFU-e
?
aThe above list of growth factors can stimulate the survival,proliferationand developmentof the target cells shown. The progenitor cell types are describedin Fig. 1. Variations in relative molecular mass of a given growth factor may be attributed (at least in part) to differentialglycosylationpatterns on the same polypeptide.
210 and there is a steady fall in ATP concentrations within the cell. All of these effects are reversible if IL-3 is added back to the cells before they die. To date no mechanistic details on how other haemopoietic growth factors promote cell survival and proliferation are known. There have been few detailed investigations of the events triggered by occupation of haemopoietic growth factor receptors. Again, most of the available data have been obtained using IL-3 dependent cell lines. IL-3 has been shown to have little effect on cAMP concentrations within target cell populations. However, a possible mechanism for the promotion of survival and proliferation by IL-3 is indicated by the observation that adding IL-3 to growth factor-dependent ceil lines causes translocation and activation of protein kinase C 17. Although this infers that diacylglycerol formation (via increased phosphatidy-linositol lipid metabolism) is stimulated by IL-3 this has still to be conclusively demonstrated. In this respect it is worth bearing in mind the recent work showing an insulin-stimulated formation of diacylglycerol without any apparent increases in polyphosphoinositide breakdownis. At present the mode of action of other haemopoietic growth factors has not been established, except that the CSF-1 receptor has been shown to be a tyrosinespecific protein kinase of Mr 160 which autophosphorylates on addition of CSF-119.
Haemopoietiegrowthfactorsand leukaemogenesis The rigorous control of survival, proliferation and development in the haemopoietic system is sometimes lost, giving rise to leukaemia or hyperplasia. There is now some important evidence relating haemopoietic growth factors to these malignant diseases. Some murine leukaemic cells are absolutely dependent on IL-3 for their survival and proliferation in vitro. Similarly many human leukaemic cells require a stirnulatory activity (which is as yet poorly characterized) to proliferate in vitro. A great deal of evidence in both human and murine systems suggests that the leukaemic cell may in some cases resemble a haemopoietic progenitor cell which displays an imbalance between proliferation and development, such that the facility to self-renew is inappropriately expressed 2° (Fig. 2). Thus, in the case of some hyperplasias or leukaemias it is the response to, as opposed to independence from, haemopoietic growth factors that may be
TIBS ll-May
Stem Cell Compartment
Progenitor Cell Compartment
1986
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e Capacity
If
~
f
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"
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~ \ ~ ~ ~-~ ( = { (=~ /~._~ ' ~, w )qlIUncontrolledY \ -- ] S'~-'~elf-renewal "~l~self-renewal ~ ))outweighs H a emopoiesis
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I Proliferative Capacity } Fig. 2. A comparison between normal haemopoiesis and leukaemic haemopoiesis. Self-renewal of stem cells, commitanent andproliferation (accompanied by maturation) arefinely balanced in normal haemopoiesis, under the regulation of the bone marrow stromal environment. In the case of a leukaemia, stir-renewal in either the stem or progenitor cell compaaraaU is apparently not controlled. An inappropriately expressed ability to selfrenew at an increased rate in the stem cell compartment or a developmental block imposed upon a committed progenitor cell are thought to be the essential elements in this loss of control. Phenotypically leukaemic cells markedly resemble progenitor cells, thus oncogene expression or some other event may alter the balance o f the developmental process in haemopoiesis and this may in part contribute to a leukaemia.
altered. None the less, some models do exist which suggest that growth factor-independence is one possible step in leukaemic transformation. There is evidence from the leukaemic murine cell line WEHI-3B that the IL-3 gene has at some stage come under the influence of a viral promoter sequence, which might explain why these cells actually secrete IL-3, a phenomenon which may have been associated with their initial transformation21. Furthermore, a nonleukaemic IL-3/GM-CSF-dependent cell line has been described22, which, upon transfection with viruses containing the a b l or m y c oncogenes, becomes independent of growth factors for survival and proliferation and also leukaemic23-25. This same cell line can also be transformed to a ieukaemic factor-independent cell when transfected with a virus containingthe gene for GMCSF26. Following transfection and expression of m y c or abl, the growth autonomy is not associated with auto-production of growth factors 22-24or of altered levels of expression of the receptors (Burgess and Dexter, unpublished observations); in other words, expression of m y c or a b l has bypassed the requirement for growth factor. Transfection with the GM--CSF gene, however, has allowed auto-stimulation of growth in the absence of added exogenous growth factors. Superficially, these two pathways to growth autonomy appear to be quite different, but the
biochemical pathways underlying the stimulation of growth are surely overlapping in certain key intracellular events, and these two systems seem to provide suitable models to determine which these events are. So far the role of oncogenes in this process remains obscure and m y c a n d a b l have as yet no clearly ascribed biochemical function. However, data reported recently have provided strong evidence that the v-fins gene product may be homologous to the CSF-1 recepto# 7. Since v-fms can transform cells of the macrophage lineage this may be an analogous situation to that seen with v--erb b which codes for a truncated EGF receptor. Molecular analysis is urgently required to obtain the nucleotide sequences of v-fins and c-fins (the CSF1 receptor?). Clearly many of the biochemical and cell biological events governing haemopoiesis are still poorly understood. But the tools are now available to elucidate how these growth factors control the survival, proliferation and development in normal haemopoietic cells and to determine which events can lead to the loss of growth control and malignancy in the haemopoietic system. Acknowledgements We wish to thank Dr Clare Heyworth for much helpful discussion. Work in the authors' laboratories is supported by the
T I B S 1 1 - M a y 1986
211
Leukaemia Research Fund and the C a n c e r R e s e a r c h C a m p a i g n , T, M. D e x t e r is a fellow o f t h e C a n c e r Research Campaign.
References I Dexter, T. M., Simmons, P., Pumell, R. A., Spooncer, E. and Schofield, R. (1984) inAplastic Anaemia: Stem Cell Biology and Advances in Treatment (Humphries R. K., Young, N. S. and Levine, A. S., eds), pp. 13-28, Alan R. Liss 2 Metcalf, D. (1977) Haemopoietic Colonies In Vitro Cloning of Normal and Leukaemia Cells, Springer Verlag 3 Robb, R. J. (1984) ImmunoL Today 5,204-209 4 Welte, K., Platzer, E., Li, L., Gabrilove, J. L., Levi, E., Mertelsmann, R. and Moore, M.A.S. (1985) Proc. Natl Acad. Sci. USA 82, 15261530 5 Burgess, A. W. and Nicola, N. A. (1983) Growth Factors and Stem Cells, pp. 93-124, Academic Press 6 Fung, M. C., Hapel, A. J., Ymer, S., Cohen, D. R., Johnson, R. M., Campbell, M. D. and Young, I. G. (1984) Nature 307,233-237
7 Gough, N. M., Gough, J., Metcalf, D., Kelso, A., Grail, D., Nicola, N. A., Burgess, A. W. and Dunn, A. R. (1984) Nature 309,763--766 8 Kawasaki, E. S., Ladner, M. B., Wang, A. M., Ardsell, J. V., Warren M. K., Coyne, M. Y., Schweikart, V. L., Lee, M. T., Wilson, K. J., Boosman, A., Stanley, E. R., Ralph, P. and Mark, D. F. (1985) Science 230, 291-296 9 Lee-Huang, S. (1984)Proc. NatlAcad. Sci USA 81,2708-2712 10 Metcalf, D. (1985) Science 229, 16-22 11 Walker, F., Nicola, N. A., Metcalf, D. and Burgess A. W. (1985) Cell 43,269-276 12 Krantz, S. B. and Goldwasser, E. (1984) Proc. Natl Acad. Sci. USA 81, 7574--7578 13 Nicola, N. A., Begley, C. G. and Metcalf, D. (1985) Nature 314,625-628 14 Walker, F. and Burgess, A. W. (1985) EMBO J. 4,933-939 15 Stanley, E. R. and Guilbert, L. J. (1981) J. ImmunoL Methods 42,253--284 16 Whetton, A. D., Bazill, G. W. and Dexter, T. M. (1984) EMBOJ. 3,409-413 17 Farrah, W. L , Thomas, T. P. and Anderson, W. B. (1985)Nature 315,235-237 18 Farese, R. V., Davis, J. S., Barnes, D. E., Standaert, M. L., Babischkin, J. S., Hock, R., Rosic, N. K. and Pollet, R. J. (1985) Biochem.
J. 231,269-278 19 Stanley, E. R. and Jubinsky, P. T. (1984) Clin. Haematol. 13,329-347 20 Greaves, M. F. (1982) CancerSurveys 1,189-204 21 Ymer, S., Tucker, Q. J., Sanderson, C. J., Hapel, A. J., Campbell, H. D. and Young, I. G. (1985) Nature 317, 255-258 22 Dexter, T. M., Garland, J., Scott, D., Scolnick, E. and Metcalf, D. (1980) J. Exp. Med. 152, 1036-1047 23 Cook, W. D., Metcalf, D., Nicola, N. A., Burgess, A. W. and Walker, F. (1985) Cell41, 677-683 24 Pierce, J. H., Di Flore, P. P., Aaronson, S. A., Potter, M., Pumphrey, J., Scott, A. andlhle, J. N. (1985) Cell 41,685-693 25 Rapp, U. R., Cleveland, J. L., Brightman, K., Scott, A. and Ihle, J. N. (1985) Nature 317, 434--438 26 Lang, R. A., Metcalf, D., Gough, N. M., Dunn, A. R. and Gonda, T. J. (1985) Cell43, 531-542 27 Sberr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T. and Stanley, E. R. (1985) Cell41,665-676 28 Bartlemez, S. H. and Stanley, E. R. (1985) Z Cell Physiol. 122,370-378 29 Eaves, A. C. and Eaves, C. J. (1984) Clin. Haematol. 13,371-391
HUMAN DESIGNER JEANOTYPE
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I 1,3
4
11
~ 13
14
15
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16
17
21
22
Courtesy o f D a v i d S. Roos, D e p a r t m e n t o f Biological Sciences, Stanford University, California 94305, U S A .
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