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Plasticity in the haemopoietic system
The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242 www.elsevier.com/locate/ijbcb
Review
Plasticity in the haemopoietic system James H. Williams, S. Peter Klinken* Department of Biochemistry, Laboratory for Cancer Medicine, University of Western Australia, Royal Perth Hospital, Level 6, MRF Building, Rear 50 Murray Street, Perth 6000, WA, Australia Received 1 February 1999; accepted 30 April 1999
Cellular dierentiation is the process whereby immature cells develop a functionally mature phenotype. Key decisions concerning the fate of individual cells are made from the earliest stages of development, occur in a highly orchestrated manner and are controlled by complex inter- and intracellular signalling events. Until relatively recently, the underlying paradigm of cellular dierentiation has been that it is a unidirectional process. That is a given cell type is not capable of reverting to an earlier, less dierentiated, phenotype or able to change phenotype entirely. However, the age of molecular biology, together with the detailed analysis of transcriptional regulators have enabled researchers to deliberately over-ride some dierentiation decisions and `reprogram' cells to follow a dierent maturation pathway. These in vitro experiments suggest that cells may retain a certain degree of plasticity even at very advanced stages of their dierentiation programs.
* Corresponding author. Tel.: +61-8-9224-0326; fax: +618-9224-0322. E-mail address: [email protected] (S.P. Klinken)
1. Reprogramming of dierentiation pathways Reprogramming of cells at the molecular level was greatly stimulated by the work of Davis et al. [1] when they demonstrated that expression of a single gene, MyoD1, could convert ®broblasts into myoblasts. MyoD1, therefore, became the ®rst transcription factor to be referred to as a `master gene'. Whilst the concept of single master genes determining cellular dierentiation programs is less in vogue, it is now clear that the relative levels of `tissue speci®c' and ubiquitous transcription factors are crucial. This short review will focus on examples where dierentiation of the haemopoietic system has been changed, or even reversed, and the circumstances that bring about these alterations. Heamopoiesis occurs in four separate locations of the embryo during vertebrate development [2]. The ®rst identi®able haemopoietic cells in the mouse are found in the blood islands of the extraembryonic mesoderm of the yolk sac. Yolk sac-derived heamopoietic precursors are true multipotent haemopoietic stem cells and are capable of repopulating embryonic and neonatal recipients [3]. De®nitive haemopioiesis originates in the para-aortic splanchnopleura, moves to the aortic-gonad-mesonephros region before stem cell activity appears in the foetal liver at late day 10
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 8 1 - 3
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J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
[4,5]. From day 15 onwards, haemopoietic activity occurs in the foetal spleen and bone marrow. As described elsewhere in this issue, regulation of the haemopoietic system is largely achieved through the co-ordinated action of soluble hormones and cytokines which activate signal transduction pathways through speci®c cell surface receptors. These signalling cascades result in the activation of transcription factors which in turn regulate gene expression patterns of target cells. Gradually, the dierentiation potential of the cells is restricted and `irreversibly' committed cells of the eight haemopoietic lineages are produced [6]. A number of recent reports question the irrevocable nature of dierentiation in the haemopoietic system. During the normal progression of blood development, the types of globin genes expressed by red blood cells change as they progress from embryoninc to foetal, then adult erythrocytes. Yolk sac-derived primitive red blood cells produce E-globing chains, while foetal erythrocytes produce g-globin transcripts and adult erythrocytes produce b-globin. Geiger et al. [7] recently demonstrated that adult haemopoietic stem cells transplanted into blasocysts were reprogrammed to display an embryonic phenotype, including expression of embryonic and foetal globin transcripts in the derived erythrocytes. Conversely, foetal and embryonic progenitors produced adult red blood cells that transcribed adult globins when transplanted into adult recipients. This study not only demonstrates the importance of the cellular microenvironment for the process of dierentiation, but clearly demonstrates that there is a degree of plasticity within the normal mammalian haemopoietic system which enables reprogramming to occur. 2. Haemopoietic lineage switching Haemopoietic lineage switching is the phenomenon whereby a transformed cell, apparently committed to one lineage, loses phenotypic markers of that lineage and acquires those of a dierent pathway [8]. Klinken et al. [8] categorically demonstrated lineage switching when B
lymphoma cell lines derived from Em-myc transgenic mice acquired the phenotype of macrophages following infection with a retrovirus containing the v-raf oncogene. The B-cell-derived macrophages were no longer dependent on 2mercaptoethanol (2-ME) for growth in culture, and had lost expression of B-lineage restricted surface markers Ly5 (B220) and surface immunoglobulin. Simultaneously, the cells became adherent, acquired the macrophage markers Mac-1, 8C5 and F4/80, expressed granulocyte/macrophage-colony stimulating factor (GM-CSF) and were able to phagocytose latex beads. Signi®cantly, the lymphoid origin of each of the macrophage lines was unequivocally demonstrated by the presence of the parental immunoglobulin chain rearrangement. Two other points must be considered in context with this lineage switch. Firstly, as removal of 2-ME from the culture media imposed a powerful selection against stable myc/v-raf expressing B-cells, the shift to the macrophage lineage represented a signi®cant survival advantage. Second, whilst both oncogenes were required for the lineage switch, they were not the only stimulus for the lineage conversion. In almost all instances, the lineage switch was accompanied by the accumulation of gross karyotypic abnormalities. One potential mechanism for the switch, therefore, is that the combined eects of the oncogenes caused chromosomal damage which serendipitously activated genes required to produce a myeloid phenotype. Another example of a lymphoid to macrophage lineage switch was observed by Borzillo et al. [9] when the v-fms oncogene was introduced into D1F9 pre-B cells. The frequency of the switch was very low when cells were cultured in media favouring lymphoid growth. However, when cultures were shifted to media that supported growth of myeloid cells, macrophage clones were readily obtained. Interestingly, an even greater rate of lineage switching was observed when 2-ME was removed from the culture media. Lineage switching of D1F9 cells was also observed when the c-fms proto-oncogene (which encodes the wild-type M-CSF receptor) was expressed and the cells were cultured with
J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
M-CSF [9]. These experiments showed that signalling pathways activated by the M-CSF-receptor in B-lineage cells could activate the normal gamut of genes required for a myeloid phenotype. Having observed the switch of commited Blymphoma cells to the myeloid lineage, Klinken et al. [8] proposed that the two lineages may share a common precursor cell. Independently, Cumano et al. [10] and Hirayama et al. [11] demonstrated that a B-lymphoid/myeloid progenitor did indeed exist. As these two lineages appear closer than previously thought, plasticity in the haemopoietic system may allow myeloid genes to be re-expressed in cells displaying a lymphoid phenotype. Consistent with this notion, the SPGM 1 cell line shows properties of both Blymphoid and macrophage lineages depending on the growth factors present in the culture media [12]. Under normal conditions, the cells display a pre-B phenotype and are dependent on the presence of 2-ME in the culture media. However, following stimulation with IL-3, B-cell markers and phenotype are lost, and the cells acquire a macrophage phenotype, Mac-1 and c-fms mRNA is induced and the cells become adherent in culture. Spontaneous lineage switching in vitro has also been observed when the J2E erythroleukaemic line produced myeloid derivatives [13]. J2E cells were generated by infection of murine haemopoietic precursors from day 12 foetal livers with the v-raf / myc containing J2 retrovirus [14]. The resultant J2E line was immortalised at the proerythroblast stage of dierentiation and represents one of only a few erythroid cell lines that will undergo morphological and biochemical dierentiation in vitro in response to erythropoietin (epo). However, during the course of extended culture, subclones of the J2E line were isolated. Firstly, the J2E-NR (non responder) was identi®ed which had lost the full level of response to epo, but did retain an erythroid phenotype [15]. In addition, adherent cells appeared in cultures of both J2E and J2E-NR lines when the cells were grown in adverse conditions, i.e. severe overgrowth [13]. Several of these were cloned (J2E-m1, m2, m3 and J2E-NR-m1, m2, m3) and
1239
extensive analysis showed that they had lost the characteristic markers of erythroid cells and simultaneously acquired a monocytoid/macrophage phenotype [13]. These myeloid derivatives were shown to have arisen from the J2E and J2E-NR cell lines by the common integration site of the J2 retrovirus. As with the Em-myc B-cell derived macrophages described above, the switch from an erythroid phenotype to a myeloid phenotype was accompanied by a high level of karyotypic abnormality [13]. This common link between the two systems suggests that the raf and myc oncogenes induce sucient genetic instability to activate genes required for myeloid dierentiation. In both instances, the myeloid derivatives appear to have had a signi®cant survival advantage over the parental lines, either in the absence of 2-ME for Em-myc B-cells, or over-crowding for J2E erythroleukaemic cells. 3. Mediators of lineage switching Recent experiments in this laboratory have used a subtractive hybridisation approach to identify genes induced during the J2E erythroid to myeloid lineage switch (Williams et al., submitted for publication). Two known myeloid restricted genes and three novel genes were isolated by this procedure. Constitutive expression of one novel gene (HLS 7) in J2E cells resulted in a morphological change which mimicked the original, spontaneous, lineage switch (Williams et al., submitted for publication). However, this change was not accompanied by the accumulation of gross karyotypic abnormalities, nor a complete loss of erythroid markers and transcription factors. HLS7, therefore, may represent a gene involved in the process of lineage switching. As this gene also supresses erythroid colony formation by non-transformed immature haemopoietic cells and favours myeloid colony formation, it may play a role in lineage commitment. It is interesting to note that the human homologue of HLS7 is myeloid leukemia factor (MLF) 1, which was cloned as the C-terminus of a fusion protein produced by the t(3;5) (q25.1;q34) translocation associated with myelo-
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J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
dysplastic syndrome and acute myeloid leukaemia [16]. GATA-1 and SCL are two genes that are known to in¯uence haemopoietic lineage determination. GATA-1 is a transcription factor expressed primarily by erythroid cells, but also by mast cells, eosinophils and megakaryocytes [17]. It recognises the consensus (A/T)GATA(A/ G) motif found in the promoter and enhancer region of many erythroid genes. Gene targeting experiments have con®rmed the importance of GATA-1 in the generation of erythroid cells [18]. In addition, the potency of this trancription factor as a lineage determining gene has been demonstrated by its ability to reprogram committed cells of other lineages to megakaryocytic and erythroid pathways. Firstly, avian myb-ets transformed myelomonocytic cell lines and v-myc transformed macrophages switch to eosinophils, thromboblasts and erythroblasts upon constitutive expression of GATA-1 [19]. Secondly, murine M1 monoblastoid cells expressing GATA-1 displayed evidence of megakaryocyte and erythroid dierentiation [20]. SCL was originally cloned as the gene involved in the t(1;14) (p33;q11) translocation associated with T-cell acute lymphoblastic leukaemia [21]. Interestingly, the original T-cell lymphoma from which SCL was isolated developed a myeloid phenotype following treatment with 2'-deoxycoformycin [22]. A detailed review of SCL is provided elsewhere in this issue [23]. SCL is pertinent to this article as alterations of the levels of SCL can in¯uence the maturation of erythroid and myeloid cells in vitro. 4. Lineage switching in vivo The examples of in vitro lineage switching discussed above demonstrate that commitment in the haemopoietic system can be reprogrammed by activating alternative lineage determining genes. This can be achieved either by deliberate ectopic expression, or serendipitous chromosomal damage that may activate these genes. Lineage switching also occurs in vivo. Reprogramming of adult haemopoietic stem cells to produce
embryonic and foetal cell types, and visa versa, demonstrates that non-transformed cells can indeed be reprogrammed in vivo [7]. In addition, there are numerous instances where leukaemic patients achieve remission, only to re-present with a leukaemia of a dierent nature [24±26]. The most widely accepted explanation for this phenomenon is the transformation of bi-phenotypic progenitors. This hypothesis states that one lineage preferentially develops into acute leukaemia at the `expense' of the other. Following treatment, the other progenitor develops a survival advantage and becomes malignant. Based on in vitro data, it is likely that some secondary leukaemias result from a genuine lineage switch, induced by the combinated eects of transformation and subsequent chromosomal alterations caused by chemotherapy or radiotherapy. The emergence of myeloid leukaemias, which presented as B- or T-cell lymphomas, bearing clonotypic immunoglobulin or T-cell receptor rearrangements of the parental cell, con®rm the lymphoid origin of these cells [24]. Thus, transformed cells committed to one lineage by undergoing gene rearrangements for that pathway, can present with the phenotype of a dierent cell type. A tragic consequence of these in vivo lineage switches is that the secondary leukeamia is often particularly aggressive, and often results in the death of the patient within a short period of time. 5. Conclusion In conclusion, there is sucient in vitro and in vivo evidence that the mammalian haemopoietic system is not entirely uni-directional and in¯exible in its dierentiation program. As our understanding increases about the way speci®c transcription factors exert their eects at dierent concentrations, and in combination with other transcriptional regulators, so will our ability to selectively over-ride lineage determining decisions. This raises the interesting possibility of deliberately reprogramming transformed cells to another lineage that may be more amenable to dierentiation therapy.
J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
References [1] R.L. Davis, H. Weintraub, A.B. Lassar, Expression of a single transfected cDNA converts ®broblasts to myoblasts, Cell 51 (1987) 987±1000. [2] D. Metcalf, M.A.S. Moore, Embryonic aspects of haematopoiesis, in: A. Neuberger, E.L. Tatum (Eds.), Haematopoietic Cells, North-Holland Publishing Company, Amsterdam, London, 1971, pp. 172±271. [3] H. Huang, R. Auerach, Identi®cation and characterization of hematopoietic stem cells of the early mouse embryo, Proceedings of the National Academy of Science USA 90 (1993) 10110±10114. [4] I.E. Godin, J.A. Garcia-Porrero, A. Coutinho, F. Dieterien-Lievre, M.A. Marcos, Para-aortic splanchnopleura form early mouse embryos contains B1a cell progenitors, Nature 364 (1993) 67±70. [5] A.L. Medvinsky, N.L. Samoylina, A.M. MuÈller, Dzierzak, An early pre-liver intraembryonic source of CFU-S in the developing mouse, Nature 364 (1993) 64± 67. [6] D. Metcalf, The molecular control of cell division, dierentiation, commitment and maturation in haemopoietic cells, Nature 339 (1989) 27±30. [7] H. Geiger, S. Sick, C. Bonifer, A.M. MuÈller, Globin gene expression is reprogrammed in chimeras generated by injecting adult hematopoietic stem cells into mouse blastocysts, Cell 93 (1998) 1055±1065. [8] S.P. Klinken, W.S. Alexander, J.M. Adams, Hemopoietic lineage switch: v-raf oncogene converts Em-myc transgenic B-cells into macrophages, Cell 53 (1988) 857±867. [9] G.V. Borzillo, R.A. Ashmun, C.J. Sherr, Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes, Molecular and Cellular Biology 10 (1990) 2703±2714. [10] A. Cumano, C.J. Paige, N.N. Iscove, G. Brady, Bipotential precursors of B cells and macrophages in fetal liver, Nature 356 (1992) 612±615. [11] F. Hirayama, J-P. Shih, A. Awgulewitsch, G.W. Warr, S.C. Clark, M. Ogawa, Clonal proliferation of murine lymphohemopoietic progenitors in culture, Proceedings of the National Academy of Science USA 89 (1992) 5907±5911. [12] M. Martin, A. Strasser, N. Baumgarth, F.M. Cicuttini, K. Welch, E. Salvaris, A.W. Boyd, A novel cellular model (SPGM 1) of switching between the pre-B cell and myelomonocytic lineages, Journal of Immunology 150 (1993) 435±4406. [13] U. Keil, S.J. Bus®eld, T.J. Farr, J. Papadimitriou, A.R. Green, C.G. Begley, S.P. Klinken, Emergence of myeloid cells from cultures of J2E erythroid cells is linked with karyotypic abnormalities, Cell Growth and Dierentiation 6 (1995) 439±448. [14] S.P. Klinken, N.A. Nicola, G.R. Johnson, In vitro-derived leukemic erythroid cell lines indudiced by a rafand myc-containing retrovirus dierentiate in response
[15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
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to erythropoietin, Proceedings of the National Academy of Science USA 85 (1988) 8506±8510. S.P. Klinken, N.A. Nicola, Evolution of a mutant J2E erythroid cell line which does not respond to erythropoietin, Leukemia 4 (1990) 24±28. N. Yoneda-kato, A.T. Look, M.N. Kirsten, M.B. Va,entine, S.C. Raimondi, K.J. Cohen, A.J. Carroll, S.W. Morris, The t(3;5) (q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1, Oncogene 12 (1996) 265±275. S.F. Tsai, D.J. Martin, L.I. Zon, A.D. d'Andrea, G.G. Wong, S.H. Orkin, Cloning of the cDNA for the major DNA-binding protein of the erythroid lineage throigh expression in mammalian cells, Nature 339 (1989) 446± 451. L. Pevny, M.C. Simon, E. Robertson, W.H. Klein, S.F. Tsai, D. d'Agati, S.H. Orkin, Erythroid dierentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1, Nature 349 (1991) 257±260. H. Kulessa, J. Frampton, T. Graf, GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts, Genes and Development 9 (1995) 1250±1262. Y. Yamaguchi, L.I. Zon, S.J. Ackerman, M. Yamamoto, T. Suda, Forced GATA-1 expression in the murine myeloid cell line M1: Induction of c-Mpl expression and megakaryocytic/erythroid dierentiation, Blood 91 (1998) 450±457. C.G. Begley, P.D. Aplan, M.P. Davey, K. Nakahara, K. Tchor, J. Kurtzberg, M.S. Hersh®eld, B.F. Haynes, D.I. Cohen, T.A. Waldmann, I.R. Kirsch, Chromosomal translocation in a human leukemic stemcell line disrupts the T-cell antigen receptor d-chain diversity region and results in a previously unreported fusion transcript, Proceedings of the National Academy of Science USA 86 (1989) 2031±2035. M.S. Hersh®eld, J. Kurtzberg, E. Harden, J.O. Moore, J. Whang-Peng, B.F. Haynes, Conversion of a stem cell leukemia from a T-lymphoid to a myeloid phenotype indiced by the adenosine deaminase inhibitor 2 '-deoxycoformycin, Proceedings of the National Academy of Science USA 81 (1984) 253±257. L.M. Barton, B. Gottgens, A.R. Green, The stem cell leukaemia (SCL) gene: a critical regulator of haemopoietic and vascular development, International Journal of Biochemistry and Cell Biology 31(10) (1999) 1191± 1205. G.A. Gaagnon, C.C. Childs, A. LeMaistre, M. Keating, A. Cork, J.M. Trujillo, K. Nellis, E. Freireich, S.A. Stass, Molecular heterogeneity in acute leukemia lineage switch, Blood 74 (1989) 2088±2095. J. Roman, M.J. de la Torre, P. Andres, J.M. Garcia, A. Torres, Molecular immaturity of the immune receptor genes in a case of acute leukaemia with complete lymphoid lineage switch, European Journal of Haematology 55 (1995) 61±62.
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[26] F. Pane, F. Frigeri, A. Camera, M. Sindona, F. Brighel, V. Martinelli, L. Luciano, C. Selleri, L. del Vecchio, B. Rotoli, F. Salvatori, Complete phenotypic and genoty-
pic lineage switch in a Philadelphia chromosome-positive acute lymphoblastic leukemia, Leukemia 10 (1996) 741±749.
Review
Plasticity in the haemopoietic system James H. Williams, S. Peter Klinken* Department of Biochemistry, Laboratory for Cancer Medicine, University of Western Australia, Royal Perth Hospital, Level 6, MRF Building, Rear 50 Murray Street, Perth 6000, WA, Australia Received 1 February 1999; accepted 30 April 1999
Cellular dierentiation is the process whereby immature cells develop a functionally mature phenotype. Key decisions concerning the fate of individual cells are made from the earliest stages of development, occur in a highly orchestrated manner and are controlled by complex inter- and intracellular signalling events. Until relatively recently, the underlying paradigm of cellular dierentiation has been that it is a unidirectional process. That is a given cell type is not capable of reverting to an earlier, less dierentiated, phenotype or able to change phenotype entirely. However, the age of molecular biology, together with the detailed analysis of transcriptional regulators have enabled researchers to deliberately over-ride some dierentiation decisions and `reprogram' cells to follow a dierent maturation pathway. These in vitro experiments suggest that cells may retain a certain degree of plasticity even at very advanced stages of their dierentiation programs.
* Corresponding author. Tel.: +61-8-9224-0326; fax: +618-9224-0322. E-mail address: [email protected] (S.P. Klinken)
1. Reprogramming of dierentiation pathways Reprogramming of cells at the molecular level was greatly stimulated by the work of Davis et al. [1] when they demonstrated that expression of a single gene, MyoD1, could convert ®broblasts into myoblasts. MyoD1, therefore, became the ®rst transcription factor to be referred to as a `master gene'. Whilst the concept of single master genes determining cellular dierentiation programs is less in vogue, it is now clear that the relative levels of `tissue speci®c' and ubiquitous transcription factors are crucial. This short review will focus on examples where dierentiation of the haemopoietic system has been changed, or even reversed, and the circumstances that bring about these alterations. Heamopoiesis occurs in four separate locations of the embryo during vertebrate development [2]. The ®rst identi®able haemopoietic cells in the mouse are found in the blood islands of the extraembryonic mesoderm of the yolk sac. Yolk sac-derived heamopoietic precursors are true multipotent haemopoietic stem cells and are capable of repopulating embryonic and neonatal recipients [3]. De®nitive haemopioiesis originates in the para-aortic splanchnopleura, moves to the aortic-gonad-mesonephros region before stem cell activity appears in the foetal liver at late day 10
1357-2725/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 9 ) 0 0 0 8 1 - 3
1238
J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
[4,5]. From day 15 onwards, haemopoietic activity occurs in the foetal spleen and bone marrow. As described elsewhere in this issue, regulation of the haemopoietic system is largely achieved through the co-ordinated action of soluble hormones and cytokines which activate signal transduction pathways through speci®c cell surface receptors. These signalling cascades result in the activation of transcription factors which in turn regulate gene expression patterns of target cells. Gradually, the dierentiation potential of the cells is restricted and `irreversibly' committed cells of the eight haemopoietic lineages are produced [6]. A number of recent reports question the irrevocable nature of dierentiation in the haemopoietic system. During the normal progression of blood development, the types of globin genes expressed by red blood cells change as they progress from embryoninc to foetal, then adult erythrocytes. Yolk sac-derived primitive red blood cells produce E-globing chains, while foetal erythrocytes produce g-globin transcripts and adult erythrocytes produce b-globin. Geiger et al. [7] recently demonstrated that adult haemopoietic stem cells transplanted into blasocysts were reprogrammed to display an embryonic phenotype, including expression of embryonic and foetal globin transcripts in the derived erythrocytes. Conversely, foetal and embryonic progenitors produced adult red blood cells that transcribed adult globins when transplanted into adult recipients. This study not only demonstrates the importance of the cellular microenvironment for the process of dierentiation, but clearly demonstrates that there is a degree of plasticity within the normal mammalian haemopoietic system which enables reprogramming to occur. 2. Haemopoietic lineage switching Haemopoietic lineage switching is the phenomenon whereby a transformed cell, apparently committed to one lineage, loses phenotypic markers of that lineage and acquires those of a dierent pathway [8]. Klinken et al. [8] categorically demonstrated lineage switching when B
lymphoma cell lines derived from Em-myc transgenic mice acquired the phenotype of macrophages following infection with a retrovirus containing the v-raf oncogene. The B-cell-derived macrophages were no longer dependent on 2mercaptoethanol (2-ME) for growth in culture, and had lost expression of B-lineage restricted surface markers Ly5 (B220) and surface immunoglobulin. Simultaneously, the cells became adherent, acquired the macrophage markers Mac-1, 8C5 and F4/80, expressed granulocyte/macrophage-colony stimulating factor (GM-CSF) and were able to phagocytose latex beads. Signi®cantly, the lymphoid origin of each of the macrophage lines was unequivocally demonstrated by the presence of the parental immunoglobulin chain rearrangement. Two other points must be considered in context with this lineage switch. Firstly, as removal of 2-ME from the culture media imposed a powerful selection against stable myc/v-raf expressing B-cells, the shift to the macrophage lineage represented a signi®cant survival advantage. Second, whilst both oncogenes were required for the lineage switch, they were not the only stimulus for the lineage conversion. In almost all instances, the lineage switch was accompanied by the accumulation of gross karyotypic abnormalities. One potential mechanism for the switch, therefore, is that the combined eects of the oncogenes caused chromosomal damage which serendipitously activated genes required to produce a myeloid phenotype. Another example of a lymphoid to macrophage lineage switch was observed by Borzillo et al. [9] when the v-fms oncogene was introduced into D1F9 pre-B cells. The frequency of the switch was very low when cells were cultured in media favouring lymphoid growth. However, when cultures were shifted to media that supported growth of myeloid cells, macrophage clones were readily obtained. Interestingly, an even greater rate of lineage switching was observed when 2-ME was removed from the culture media. Lineage switching of D1F9 cells was also observed when the c-fms proto-oncogene (which encodes the wild-type M-CSF receptor) was expressed and the cells were cultured with
J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
M-CSF [9]. These experiments showed that signalling pathways activated by the M-CSF-receptor in B-lineage cells could activate the normal gamut of genes required for a myeloid phenotype. Having observed the switch of commited Blymphoma cells to the myeloid lineage, Klinken et al. [8] proposed that the two lineages may share a common precursor cell. Independently, Cumano et al. [10] and Hirayama et al. [11] demonstrated that a B-lymphoid/myeloid progenitor did indeed exist. As these two lineages appear closer than previously thought, plasticity in the haemopoietic system may allow myeloid genes to be re-expressed in cells displaying a lymphoid phenotype. Consistent with this notion, the SPGM 1 cell line shows properties of both Blymphoid and macrophage lineages depending on the growth factors present in the culture media [12]. Under normal conditions, the cells display a pre-B phenotype and are dependent on the presence of 2-ME in the culture media. However, following stimulation with IL-3, B-cell markers and phenotype are lost, and the cells acquire a macrophage phenotype, Mac-1 and c-fms mRNA is induced and the cells become adherent in culture. Spontaneous lineage switching in vitro has also been observed when the J2E erythroleukaemic line produced myeloid derivatives [13]. J2E cells were generated by infection of murine haemopoietic precursors from day 12 foetal livers with the v-raf / myc containing J2 retrovirus [14]. The resultant J2E line was immortalised at the proerythroblast stage of dierentiation and represents one of only a few erythroid cell lines that will undergo morphological and biochemical dierentiation in vitro in response to erythropoietin (epo). However, during the course of extended culture, subclones of the J2E line were isolated. Firstly, the J2E-NR (non responder) was identi®ed which had lost the full level of response to epo, but did retain an erythroid phenotype [15]. In addition, adherent cells appeared in cultures of both J2E and J2E-NR lines when the cells were grown in adverse conditions, i.e. severe overgrowth [13]. Several of these were cloned (J2E-m1, m2, m3 and J2E-NR-m1, m2, m3) and
1239
extensive analysis showed that they had lost the characteristic markers of erythroid cells and simultaneously acquired a monocytoid/macrophage phenotype [13]. These myeloid derivatives were shown to have arisen from the J2E and J2E-NR cell lines by the common integration site of the J2 retrovirus. As with the Em-myc B-cell derived macrophages described above, the switch from an erythroid phenotype to a myeloid phenotype was accompanied by a high level of karyotypic abnormality [13]. This common link between the two systems suggests that the raf and myc oncogenes induce sucient genetic instability to activate genes required for myeloid dierentiation. In both instances, the myeloid derivatives appear to have had a signi®cant survival advantage over the parental lines, either in the absence of 2-ME for Em-myc B-cells, or over-crowding for J2E erythroleukaemic cells. 3. Mediators of lineage switching Recent experiments in this laboratory have used a subtractive hybridisation approach to identify genes induced during the J2E erythroid to myeloid lineage switch (Williams et al., submitted for publication). Two known myeloid restricted genes and three novel genes were isolated by this procedure. Constitutive expression of one novel gene (HLS 7) in J2E cells resulted in a morphological change which mimicked the original, spontaneous, lineage switch (Williams et al., submitted for publication). However, this change was not accompanied by the accumulation of gross karyotypic abnormalities, nor a complete loss of erythroid markers and transcription factors. HLS7, therefore, may represent a gene involved in the process of lineage switching. As this gene also supresses erythroid colony formation by non-transformed immature haemopoietic cells and favours myeloid colony formation, it may play a role in lineage commitment. It is interesting to note that the human homologue of HLS7 is myeloid leukemia factor (MLF) 1, which was cloned as the C-terminus of a fusion protein produced by the t(3;5) (q25.1;q34) translocation associated with myelo-
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dysplastic syndrome and acute myeloid leukaemia [16]. GATA-1 and SCL are two genes that are known to in¯uence haemopoietic lineage determination. GATA-1 is a transcription factor expressed primarily by erythroid cells, but also by mast cells, eosinophils and megakaryocytes [17]. It recognises the consensus (A/T)GATA(A/ G) motif found in the promoter and enhancer region of many erythroid genes. Gene targeting experiments have con®rmed the importance of GATA-1 in the generation of erythroid cells [18]. In addition, the potency of this trancription factor as a lineage determining gene has been demonstrated by its ability to reprogram committed cells of other lineages to megakaryocytic and erythroid pathways. Firstly, avian myb-ets transformed myelomonocytic cell lines and v-myc transformed macrophages switch to eosinophils, thromboblasts and erythroblasts upon constitutive expression of GATA-1 [19]. Secondly, murine M1 monoblastoid cells expressing GATA-1 displayed evidence of megakaryocyte and erythroid dierentiation [20]. SCL was originally cloned as the gene involved in the t(1;14) (p33;q11) translocation associated with T-cell acute lymphoblastic leukaemia [21]. Interestingly, the original T-cell lymphoma from which SCL was isolated developed a myeloid phenotype following treatment with 2'-deoxycoformycin [22]. A detailed review of SCL is provided elsewhere in this issue [23]. SCL is pertinent to this article as alterations of the levels of SCL can in¯uence the maturation of erythroid and myeloid cells in vitro. 4. Lineage switching in vivo The examples of in vitro lineage switching discussed above demonstrate that commitment in the haemopoietic system can be reprogrammed by activating alternative lineage determining genes. This can be achieved either by deliberate ectopic expression, or serendipitous chromosomal damage that may activate these genes. Lineage switching also occurs in vivo. Reprogramming of adult haemopoietic stem cells to produce
embryonic and foetal cell types, and visa versa, demonstrates that non-transformed cells can indeed be reprogrammed in vivo [7]. In addition, there are numerous instances where leukaemic patients achieve remission, only to re-present with a leukaemia of a dierent nature [24±26]. The most widely accepted explanation for this phenomenon is the transformation of bi-phenotypic progenitors. This hypothesis states that one lineage preferentially develops into acute leukaemia at the `expense' of the other. Following treatment, the other progenitor develops a survival advantage and becomes malignant. Based on in vitro data, it is likely that some secondary leukaemias result from a genuine lineage switch, induced by the combinated eects of transformation and subsequent chromosomal alterations caused by chemotherapy or radiotherapy. The emergence of myeloid leukaemias, which presented as B- or T-cell lymphomas, bearing clonotypic immunoglobulin or T-cell receptor rearrangements of the parental cell, con®rm the lymphoid origin of these cells [24]. Thus, transformed cells committed to one lineage by undergoing gene rearrangements for that pathway, can present with the phenotype of a dierent cell type. A tragic consequence of these in vivo lineage switches is that the secondary leukeamia is often particularly aggressive, and often results in the death of the patient within a short period of time. 5. Conclusion In conclusion, there is sucient in vitro and in vivo evidence that the mammalian haemopoietic system is not entirely uni-directional and in¯exible in its dierentiation program. As our understanding increases about the way speci®c transcription factors exert their eects at dierent concentrations, and in combination with other transcriptional regulators, so will our ability to selectively over-ride lineage determining decisions. This raises the interesting possibility of deliberately reprogramming transformed cells to another lineage that may be more amenable to dierentiation therapy.
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References [1] R.L. Davis, H. Weintraub, A.B. Lassar, Expression of a single transfected cDNA converts ®broblasts to myoblasts, Cell 51 (1987) 987±1000. [2] D. Metcalf, M.A.S. Moore, Embryonic aspects of haematopoiesis, in: A. Neuberger, E.L. Tatum (Eds.), Haematopoietic Cells, North-Holland Publishing Company, Amsterdam, London, 1971, pp. 172±271. [3] H. Huang, R. Auerach, Identi®cation and characterization of hematopoietic stem cells of the early mouse embryo, Proceedings of the National Academy of Science USA 90 (1993) 10110±10114. [4] I.E. Godin, J.A. Garcia-Porrero, A. Coutinho, F. Dieterien-Lievre, M.A. Marcos, Para-aortic splanchnopleura form early mouse embryos contains B1a cell progenitors, Nature 364 (1993) 67±70. [5] A.L. Medvinsky, N.L. Samoylina, A.M. MuÈller, Dzierzak, An early pre-liver intraembryonic source of CFU-S in the developing mouse, Nature 364 (1993) 64± 67. [6] D. Metcalf, The molecular control of cell division, dierentiation, commitment and maturation in haemopoietic cells, Nature 339 (1989) 27±30. [7] H. Geiger, S. Sick, C. Bonifer, A.M. MuÈller, Globin gene expression is reprogrammed in chimeras generated by injecting adult hematopoietic stem cells into mouse blastocysts, Cell 93 (1998) 1055±1065. [8] S.P. Klinken, W.S. Alexander, J.M. Adams, Hemopoietic lineage switch: v-raf oncogene converts Em-myc transgenic B-cells into macrophages, Cell 53 (1988) 857±867. [9] G.V. Borzillo, R.A. Ashmun, C.J. Sherr, Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes, Molecular and Cellular Biology 10 (1990) 2703±2714. [10] A. Cumano, C.J. Paige, N.N. Iscove, G. Brady, Bipotential precursors of B cells and macrophages in fetal liver, Nature 356 (1992) 612±615. [11] F. Hirayama, J-P. Shih, A. Awgulewitsch, G.W. Warr, S.C. Clark, M. Ogawa, Clonal proliferation of murine lymphohemopoietic progenitors in culture, Proceedings of the National Academy of Science USA 89 (1992) 5907±5911. [12] M. Martin, A. Strasser, N. Baumgarth, F.M. Cicuttini, K. Welch, E. Salvaris, A.W. Boyd, A novel cellular model (SPGM 1) of switching between the pre-B cell and myelomonocytic lineages, Journal of Immunology 150 (1993) 435±4406. [13] U. Keil, S.J. Bus®eld, T.J. Farr, J. Papadimitriou, A.R. Green, C.G. Begley, S.P. Klinken, Emergence of myeloid cells from cultures of J2E erythroid cells is linked with karyotypic abnormalities, Cell Growth and Dierentiation 6 (1995) 439±448. [14] S.P. Klinken, N.A. Nicola, G.R. Johnson, In vitro-derived leukemic erythroid cell lines indudiced by a rafand myc-containing retrovirus dierentiate in response
[15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
1241
to erythropoietin, Proceedings of the National Academy of Science USA 85 (1988) 8506±8510. S.P. Klinken, N.A. Nicola, Evolution of a mutant J2E erythroid cell line which does not respond to erythropoietin, Leukemia 4 (1990) 24±28. N. Yoneda-kato, A.T. Look, M.N. Kirsten, M.B. Va,entine, S.C. Raimondi, K.J. Cohen, A.J. Carroll, S.W. Morris, The t(3;5) (q25.1;q34) of myelodysplastic syndrome and acute myeloid leukemia produces a novel fusion gene, NPM-MLF1, Oncogene 12 (1996) 265±275. S.F. Tsai, D.J. Martin, L.I. Zon, A.D. d'Andrea, G.G. Wong, S.H. Orkin, Cloning of the cDNA for the major DNA-binding protein of the erythroid lineage throigh expression in mammalian cells, Nature 339 (1989) 446± 451. L. Pevny, M.C. Simon, E. Robertson, W.H. Klein, S.F. Tsai, D. d'Agati, S.H. Orkin, Erythroid dierentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1, Nature 349 (1991) 257±260. H. Kulessa, J. Frampton, T. Graf, GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts, Genes and Development 9 (1995) 1250±1262. Y. Yamaguchi, L.I. Zon, S.J. Ackerman, M. Yamamoto, T. Suda, Forced GATA-1 expression in the murine myeloid cell line M1: Induction of c-Mpl expression and megakaryocytic/erythroid dierentiation, Blood 91 (1998) 450±457. C.G. Begley, P.D. Aplan, M.P. Davey, K. Nakahara, K. Tchor, J. Kurtzberg, M.S. Hersh®eld, B.F. Haynes, D.I. Cohen, T.A. Waldmann, I.R. Kirsch, Chromosomal translocation in a human leukemic stemcell line disrupts the T-cell antigen receptor d-chain diversity region and results in a previously unreported fusion transcript, Proceedings of the National Academy of Science USA 86 (1989) 2031±2035. M.S. Hersh®eld, J. Kurtzberg, E. Harden, J.O. Moore, J. Whang-Peng, B.F. Haynes, Conversion of a stem cell leukemia from a T-lymphoid to a myeloid phenotype indiced by the adenosine deaminase inhibitor 2 '-deoxycoformycin, Proceedings of the National Academy of Science USA 81 (1984) 253±257. L.M. Barton, B. Gottgens, A.R. Green, The stem cell leukaemia (SCL) gene: a critical regulator of haemopoietic and vascular development, International Journal of Biochemistry and Cell Biology 31(10) (1999) 1191± 1205. G.A. Gaagnon, C.C. Childs, A. LeMaistre, M. Keating, A. Cork, J.M. Trujillo, K. Nellis, E. Freireich, S.A. Stass, Molecular heterogeneity in acute leukemia lineage switch, Blood 74 (1989) 2088±2095. J. Roman, M.J. de la Torre, P. Andres, J.M. Garcia, A. Torres, Molecular immaturity of the immune receptor genes in a case of acute leukaemia with complete lymphoid lineage switch, European Journal of Haematology 55 (1995) 61±62.
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J.H. Williams, S.P. Klinken / The International Journal of Biochemistry & Cell Biology 31 (1999) 1237±1242
[26] F. Pane, F. Frigeri, A. Camera, M. Sindona, F. Brighel, V. Martinelli, L. Luciano, C. Selleri, L. del Vecchio, B. Rotoli, F. Salvatori, Complete phenotypic and genoty-
pic lineage switch in a Philadelphia chromosome-positive acute lymphoblastic leukemia, Leukemia 10 (1996) 741±749.