Vol. 14, No. 8, pp. 689-693, 1990. Printed in Great Britain.
0145--2126/90$3.00 + .00 PergamonPressplc
Leukemia Research
A U T O C R I N E G R O W T H OF LEUKEMIC CELLS CLAUDE JASMIN, VASSILIS GEORGOULIAS, FLORENCE SMADJA-JOFFE, CLAUDE BOUCHEIX, CAROLINE L E BOUSSE-KERDILES, MICHI~LE ALLOUCHE, CHRISTIAN CIBERT a n d BRUNt) AZZARONE
INSERM U 268, H6pital Paul Brousse, 16 av. P.V. Couturier, 94800 Villejuif, France
(Received 15 January 1990. Accepted 16 March 1990) Abstract--Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. This paper describes the stepwise malignant progression of leukemic cells which has been demonstrated in many experimental models of autocrine leukemic growth. In contrast, autocrine growth has not been proven as a major physiopathological mechanism for the growth of leukemic cells in vivo in human myeloid and lymphocytic leukemias. Growth-factor independency of human leukemic cell lines may be due to clonal selection.
Key words: Autocrine growth, paracrine growth, growth-factor, receptor, leukemia, autocrine loop.
its receptor could lead to transformation by a autocrine mechanism. More intriguing is the demonstration that normal smooth muscle cells can produce an IGF-like molecule whose inhibition by monoclonal antibodies results in growth arrest [8]. Receptors can be considered as allosteric enzymes whose action on intracellular metabolic chains is modulated by the receptor's binding to its ligand: i.e. growth factors. Therefore, abnormal growth can be due either to the uncontrolled production of a growth factor in a cell that also synthesizes that growth factor's receptor (public autocrine loop) or to a modification of the growth factor receptor itself. In this latter case the receptor becomes permanently activated and even when the extracellular signal is absent it continues to mimic an enzymatic activity normally associated with the occupied growth factor receptor (private autocrine loop). Most human leukemias and lymphomas are associated with characteristic cytogenetic alterations. In addition, oncogenic retroviruses, such as HTLV1, appear to be etiologically associated with adult human T cell leukemia (ATL) [9-11]; Epstein-Barr virus is also involved in B cell lymphoid malignant transformation. Therefore human leukemias and lymphomas are good candidates for autocrine growth. Recently there has been a great development in the production of gene probes and recombinant purified molecules for many interleukins and CSF, and of monoclonal antibodies that interact with cell surface membrane receptors. Thanks to these valuable tools, it has been possible to analyze growth requirements
1. LEUKEMIAS AND LYMPHOMAS ARE GOOD CANDIDATES FOR AUTOCRINE GROWTH THE BONEmarrow, spleen and lymphoid organs form a network that is intimately associated with hematopoietic precursors [1, 2]. The survival, proliferation and differentiation of hematopoietic (myelopoietic and lymphopoietic) progenitor cells is controlled by a great variety of growth factors called interleukins (IL) and colony stimulating factors (CSF) [3, 4] which interact with their specific receptors. Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. Autocrine signalling is usually confined to pathological conditions; e.g. certain tumor cells synthesize and release growth factors that are required for normal cellular growth and division. These growth factors stimulate the inappropriate, unregulated growth of the tumor itself and can also induce tumor formation. This was first described by Todaro et al. [5-7] for ceils infected by transforming viruses. These authors suggested that co-expression of a growth factor and
Abbreviations: HTLV, human T leukemia virus; ATL, adult T leukemia; GF, growth-factor; GF-R, growth-factor receptor; PDGF, platelet-derived growth-factor; TGF, transforming growth-factor; G-M, granulocytemaerophage; CSF, colony-stimulating factor; IL, interleukin; CLL, chronic lymphocytic leukemia; TNF, tumor necrosis factor; BCGF, B-cell growth factor; E-BV, Epstein-Barr virus. Correspondence to: Dr Claude Jasmin, INSERM U 268, H6pital Paul Brousse, 16 av. P.V. Couturier, 94800 Villejuif, France. 689
690
C. JASMINet al.
for human and experimental leukemias and lymphomas in the search for autocrine growth of leukemic cells. 2. TESTING OF THE AUTOCRINE GROWTH OF LEUKEMIC CELLS The main method to test for an autocrine mechanism of the neoplasm is the autonomous growth of leukemic cells with long-term or permanent selfrenewal capacity in semi-solid media and/or the establishment of permanent cell-lines in liquid medium in the absence of added CSFs, interleukins or other growth factors. The next step consists of the search for constitutive growth factor (GF) production and/or growth factor receptor (GFR) expression in the transformed cells. Treatment of the malignant cell culture with monoclonal antibodies to GF and GFR must produce a decrease (and even an arrest) of the culture's growth. Addition of the specific GF may or may not stimulate the growth rate of leukemic cells. Finally, transfection of normal cells or of factordependent cell lines with a normal or modified GF or GFR gene may prove the autocrine model. 3. AUTOCRINE MODELS There are some circumstances in which specific cells have been shown to both produce and respond to the same factor: i.e. a platelet-derived growth factor (PDGF)-like factor in a human osteosarcoma [12] and tumor growth factor/3 (TGF/3) in chemically transformed fibroblasts [13]; however, in the latter case, the change in the transformed cells, as compared to the non-transformed parents, is a greatly increased sensitivity to the TG/3 they produce and not an increase in production of TG/3 [14]. This demonstrates that autocrine growth can be the result of qualitative and not only quantitative changes in the GF-GFR interaction. In the case of leukemias and lymphomas, paracrine mechanisms may play an important role in leukemogenesis and autocrine mechanism may often be restricted to subpopulations of leukemic stem cells which have been selected (or even induced) after prolonged growth of the parental cells in vitro in the presence of added GF. For example Morrone et al. [15] have established a cell line from peripheral mononuclear cells of a patient affected by a Sezary syndrome. This line produces and responds to a transferrin-like growth factor. However, the antigenic profile of leukemic cells grown in vitro is dramatically different from that of fresh peripheral blood cells from the same patient and seems to represent a clone derived from a rare
immature leukemic progenitor cell present in the peripheral blood cell population. This subpopulation may have been the more prone to immortalization in vitro. Similar examples of clonal autocrine growth stimulation of a human T-cell lymphoma by interieukin 2 have been previously described [16]. More recent studies show that although leukemic cells from adult T-cell leukemia (ATL) patients [17] and HTLV1 and HTLV2 infected helper T cells [18] constitutively express an excess of IL-2 receptors, growth of leukemic cells in ATL remain IL-2-dependent [19]. Also, we have demonstrated that acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma of T lineage retain IL-2 dependence although they are able to form spontaneous T cell colonies in semi solid medium and to produce a nonIL-2 growth factor [20, 21]. However, Shirakawa et al. [22] have recently reported that the growth of ATL cells freshly obtained from patients was stimulated in a concentration-dependent manner in the presence of recombinant human IL-1 cr and/3. Furthermore, the spontaneous growth of ATL cells was also inhibited by anti-IL-1 cr antibody but not by anti-IL-1/3 antibody. ATL cells exhibited enhanced expression of IL-1 receptors on their surface as detected by the binding of 125I-labeled rlL-1 0¢. These results suggest that IL-1 o: produced by ATL cells stimulates the growth of ATL by an autocrine mechanism. The elevated expression of TGF-/3 gene recently demonstrated in fresh peripheral mononuclear cells from adult T cell leukemia patients [23] is worthy of note and draws attention to GFs other than CSFs and interleukins. The myeloid lineage offers several examples of alterations in receptor genes that account for a triggering of the receptor's kinase activity in spite of the absence of the appropriate ligand. For example, vJkns, an oncogene which encodes a modified form of the receptor for CSF-1 converts a CSF-1 dependent macrophage cell line to factor independence and can make the cell tumorigenic [24]. Similarly, insertion into avian hematopoietic cells of the v - e r b B oncogene which encodes the transmembrane spanning segment of the tyrosine kinase domain of the EGF receptor but lacks extracellular sequences necessary for ligand binding [25, 26], leads to erythroid transformation. Studies of fresh leukemic blasts obtained from either peripheral blood or bone marrow of patients with acute myelogenous leukemia have revealed some cases of autocrine secretion of GF: granulocyte-macrophage (GM)CSF [27], G-CSF and GM-CSF [28] and IL-1/3 [29] have been shown to produce autocrine stimulation of leukemic cells and may be constitutively produced
Autocrine growth of leukemic cells in some patients. It is probable that acute myelogenous leukemia represents a heterogenous disease with diverse underlying mechanisms of transformation. Chronic lymphocytic leukemia (CLL) of B-cell type (B-CLL) is a clonal proliferation of relatively mature-appearing B lymphocytes. A recent study [30] has shown that tumor necrosis factor (TNF) can stimulate, in a dose-dependent manner, the growth of cultured neoplastic B-cells from previously untreated patients with B-CLL. In the case of spontaneous proliferation, neoplastic B-cell growth from two out of three patients was further enhanced by addition of rTNF-cr to the culture medium. However, neither the specific inhibition of growth by anti-TNF-tr and/ or anti-TNF-tr receptor antibodies nor the constitutive production of TNF-or messenger-RNA was demonstrated. In another recent study [31], IL-6 transcript was found in six out of eleven B-CLL patients, while no hybridization was observed in ten cases of ALL of both T and B cell origin. The constitutive expression of IL-6 transcripts was associated with production of a biologically active protein. However, it has not yet been demonstrated that IL6 gene expression is necessary for the neoplastic Bcell growth. One particular case of human B cell lymphoma led to further documentation of autocrine growth of neoplastic B cells [32]. Indeed, a high molecular weight B cell growth-factor (BCGF) of molecular mass 60,000 stimulates the growth of a cell line derived from a patient with large-cell immunoblastic B-cell lymphoma adapted to growth under serumfree conditions. The malignant cells produce and respond to the 60,000mol. wt BCGF. They also express cell surface receptors for this molecule. However, inhibition of growth by anti-BCGF antibodies or by specifically blocking the cell-surface receptors was not performed. In addition, this study was not done on fresh neoplastic cells but on cell lines. Therefore, the autocrine growth of B-CLL has not yet been fully proven. Even if IL-6 and BCGF (low and high molecular weight) are likely to play an important role in the growth of various human Bcell malignancies such as myeloma [33] and EBVtransformed B-cells [34], it is possible that this role is only secondary to other growth control defects of neoplastic B cells. Even if the comparison between spontaneous leukemic transformation and experimental induction of leukemic clones does not show a clear relationship between the two situations, gene manipulation by infection or transfection of target cells is providing important clues concerning autocrine growth. For example bcl-2 is a gene located on chromosome
691
18 which is associated with human follicular lymphoma. Its introduction by a retroviral vector into B cells of transgenic mice provides a distinct survival signal to the cell which may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated [35]. Similar results have been recently obtained in the case of transfection of GM-CSF dependent cell lines by the GM-CSF gene. It was shown that acquisition of true autonomy by transfected cells was a multistep process which could be influenced by the selective pressure for autocrine stimulation maintained on the culture [36]. This stepwise malignant progression has been studied in an IL-3-dependent mouse mast cell line after introduction of the V-H-ras oncogene. As shown by Nair et al. [37], tumor progression was characterized by four distinct phenotypes corresponding to four stages of tumorigenesis: immortalization, immortalized transformed cells characterized by the competence to form V-H-ras-dependent tumors, transformed competent cells with reduced IL-3 requirement and, finally, a fully malignant phenotype marked in vitro by an independence to exogeneous IL-3 and by autocrine IL-3 stimulation. At this stage of transformation, two out of six tumors showed alterations at the 5' region of the IL-3 gene, suggesting that V-H-ras requires complementation by IL-3 gene rearrangements or an alternate event to generate autocrine mastocytomas. Finally, experimental data have also demonstrated that factor-independency can be acquired by hematopoietic cell lines after malignant transformation by Abelson virus without evidence of autocrine mechanism [38]. Indeed, transformed cells become factor-independent and tumorigenic without synthesizing GM-CSF or IL-3 and they display similar numbers of cell surface receptors for these GF as the original factor-dependent cell line. However, even in this case, it is not possible to definitively exclude an autocrine mechanism. Indeed, recent study of normal rat kidney cells which had been transformed by that functional equivalent of PDGF--the v-sis oncogene---showed that autocrine stimulation of the receptor occurred in the intracellular compartments before receptor maturation was complete and before the receptors could have reached the cell surface [39]. Therefore, in such a case of private autocrine loop stimulation, addition of exogenous GF and/or treatment with antisera against the GF does not strongly inhibit cell growth. 4. CONCLUSIONS Exploration of the potential utilities of GF and
692
C. JASMINet al.
G F R manipulation is currently in progress. The remarkable success of recombinant o~2 interferon in the treatment of hairy-cell leukemia has demonstrated that trans-down modulation of G F production can be obtained and is of clinical interest. Indeed, it is probable that the therapeutic efficacy of interferon is due to its inhibiting effect either on B C G F [40] and/or T N F [41] which have been found to produce autocrine stimulation of hairy leukemic cells. Clinical trials of various interleukins, cytokines and lymphokines in hematopoietic malignancy will hopefully provide further clinical success. However, we have seen the great complexity of autocrine mechanisms and it is possible that in spontaneous neoplasms, the heterogeneity of the leukemic population may limit the usefulness of biological treatments. Nevertheless, a new era for the treatment of human malignancies may arise from the development of such therapeutic tools as truncated factors capable of competing for receptor-binding and of stimulating down-regulation of receptors, monoclonal antibodies able to block growth factor receptors and gene control by antisense sequences.
Acknowledgements--This work was supported by N.R.B. (Nouvelles Recherches Bio-M6dicales). We are grateful to Mrs Coiette Delteil for expert secretarial assistance.
REFERENCES 1. Dexter T. M., Allen T. P. & Lajhta L. G. (1977) Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. cell. Physiol 91, 335. 2. Dexter T. M., Spooncer E., Simmon P. & Allen T. P. (1984) Long term marrow cultures: an overview of technique and experience. In Long Term Bone Marrow Culture (Wright D. G. & Greenbeyer J. S., Eds) p. 57. Alan Liss, New York. 3. Dexter T. M. & Spooncer E. (1987) Growth and differentiation in the hemopoietic system. Ann. Rev. cell. Biol. 3, 423. 4. Metcalf D. (1977) Hemopoietic Colonies, p. 227. Springer, Berlin. 5. Todaro G. J., De Larco J. E. & Cohen S. (1976) Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells. Nature (Lond.) 264, 26. 6. De Larco J. E. & Todaro G. J. (1978) Growth factors from murine sarcoma virus-transformed cells. Proc. natn. Acad. Sci. U.S.A. 75, 4001. 7. Sporn M. B. & Todaro G. J. (1980) Autocrine secretion and malignant transformation of cells. New Engl. J. Med. 303, 878. 8. Clemmons R. R. & Van Wyk J. J. (1985) Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J. clin. Invest. 75, 1914.
9. Poiesz B., Ruscetti F. W., Gazdar A. F., Bunn P. A., Minna J. D. & Gallo R. C. (1980) Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous Tcell lymphoma. Proc. natn. Acad. Sci., U.S.A. 77, 7415. 10. Hinuma Y., Nagata K., Hanaoka M., Nakai M., Matsumoto T., Kinoshita K. I., Shirakawa S. & Miyoshi I. (1981) Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. natn. Acad. Sci. U.S.A. 78, 6476. I 1. Wong-Staal F. & Gallo R. C. (1985) Human T-lymphotropic retroviruses. Nature 317, 395. 12. Betsholtz C., Westermark B., Ek B. & Heldin C. H. (1984) Co-expression of a PDGF-Iike growth factor and PDGF receptors in a human osteosarcoma cell line: implications for autocrine receptor activation. Cell 39, 447-457. 13. Moses H. L. & Robinson R. A. (1982) Growth factors, growth factor receptors and cell cycle control mechanisms in chemically transformed cells. Fedn. Proc. Fedn Am. Socs exp. Biol. 41, 3008-3011. 14. Moses H. L., Childs C. B., Halper J., Shipley G. D. & Tucker R. F. (1984) In Control o f Cell Growth and Proliferation (Veneziale C. M., Ed.), p. 147. Van Nostrand Reinhold Co., New York. 15. Morrone G., Corbo L., Turco M. C., Pizzano R., De Felice M., Bridges S. & Venuta S. (1988) Transferrinlike autocrine growth factor derived from T-lymphoma cells, that inhibits normal T-cell proliferation. Cancer Res. 48, 3425. 16. Duprez V., Lenoir G. & Dautry-Varsat A. (1985) Autocrine growth stimulation of a human T-cell lymphoma line by interleukin 2. Proc. natn. Acad. Sci. U.S.A. 82, 6932. 17. Hattori T., Uchiyama T., Toibana T., Takatsuki K. & Uchino H. (1981) Surface phenotype of Japanese adult T-cell leukemia cells characterized by monoclonal antibodies. Blood 58, 645. 18. Tsudo M., Uchiyama T., Uchino H. & Yodoi J. (1983) Failure of regulation of Tac antigen/TCGF receptor on adult T-cell leukemia cells by anti-Tac monoclonal antibody. Blood 61, 1014. 19. Maeda M., Arima N., Daitoku Y., Kashihara M., Okamoto H., Uchiyama T., Shirono K., Matsuoka M., Hattori T., Takatsuki K., Ikuta K., Shimizu A., Honjo T. & Yodot J. (1987) Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia. Blood 70, 1407. 20. Georgoulias V., Auclair H. & Jasmin C. (1984) Heterogeneity of peripheral blood T-cell colony-forming cells in patients with T-cell malignancies. Leukemia Res. 8, 1025. 21. Allouche M., Georgoulias V., Varela-Millot C., Itzhaki M., Misset J. L. & Jasmin C. (1989) Prognostic value of spontaneous peripheral blood T-colony forming cells in T-cell acute lymphoblastic leukemia in complete remission: an update evaluation. Br. J. Haemat. 72, 291. 22. Shirakawa F., Tanaka Y., Oda S., Eto S. & Yamashita U. (1989) Autocrine stimulation of interleukin 1o~ in the growth of adult human T-cell leukemia cells. Cancer Res. 49, 1143. 23. Niitsu Y., Urushizaki Y., Koshida Y., Terui K., Mahara K., Kohgo Y. & Urushizaki I. (1988) Expression of TGF-beta gene in adult T cell leukemia. Blood 71,263.
Autocrine growth of leukemic cells 24. Wheeler E. F., Rettenmier C. W., Look A. T. & Sheer C. J. (1986) The v-fms oncogene induces factor independence and tumorigenicity in a CSF-1 dependent macrophage cell line. Nature 324, 377. 25. Yamomoto T., Nishida T., Miyajima N., Kawai S., Ooi T. & Toyoshima K. (1983) The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35, 71. 26. Ullrich A., Coussens L., Hayflick J. S. Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J., Downward J., Mayes E. L. V., Whitte N., Waterfield M. D. & Beedurg P. H. (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418. 27. Diane C., Griffin Y. & Young D. C. (1986) Autocrine secretion of GM-CSF in acute myeloblastic leukemia. Blood 68, 1178. 28. Murohashi I., Tohda S., Suzuki T., Nagata K., Yamashita Y. & Nara N. (1989) Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia. Blood 74, 35. 29. Sakai K., Hattori T., Matsuoka M., Asou N., Yamamoto S., Sagawa K. & Takatsuki K. (1987) Autocrine stimulation of interleukin 1/3 in acute myelogenous leukemia cells. J. exp. Med. 66, 1597. 30. Digel W., Stefanic M., Schoniger W., Buck C., Raghavachar A., Frickhofen N., Heimpel H. & Porzsolt F. (1989) Tumor necrosis factor induces proliferation of neoplastic B cells from chronic lymphocytic leukemia. Blood 73, 1242. 31. Biondi A., Rossi V., Bassan R., Barbui T., Bettoni S., Sironi M., Mantovani A. & Rambaldi A. (1989) Constitutive expression of the interleukin 6 gene in chronic lymphocytic leukemia. Blood 73, 1279. 32. Chintaman G., Sahasrabuddhe S., Sekhsaria L., Yoshimura L. & Ford R. J. (1989) Isolation and characterization of growth factor(s) from a human B-cell lymphoma. Blood 73, 1149. 33. Kawano M., Hirano T., Matsuda T., Taga T., Horii Y., Iwato K., Asaoku H., Tang B., Tanabe O., Kuramoto A. & Kishimoto T. (1988) Autocrine generation
34.
35. 36.
37.
38.
39. 40.
41.
693
and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332, 83. Muraguchi A., Hirano T., Tang B., Matsuda T., Horii Y., Nakajiama K. & Kishimoto T. (1988) The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J. exp. Med. 167, 332. Vaux D. L., Cory S. & Adams J. M. (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335,440. Laker C., Stocking C., Berghoiz U., Hess N., De Lamarter J. F. & Ostertag W. (1987) Autocrine stimulation after transfer of the granulocyte/macrophage colony-stimulating factor gene and autonomous growth are distinct but interdependent steps in the oncogenic pathway. Proc. natn. Acad. Sci. U.S.A. 84, 8458. Nair A. P. K., Diamantis I. D., Conscience J. F., Kindler V., Hofer P. & Moroni C. (1989) A v-H-rasdependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production. Molec. cell. Biol. 9, 1183. Cook W. D., Metcalf D., Nicola N. A., Burgess A. W. & Walker F. (1985) Malignant transformation of a growth factor-dependent myeloid cell line by Abelson virus without evidence of an autocrine mechanism. Cell 41, 677. Keating, M. T. & Williams L. T. (1988) Autocrine stimulation of intracellular PDGF receptors in v-sistransformed cells. Science 239, 914. Genot E., Leprince C., Richard Y., Petit-Koskas E., Falcoff E., Galanaud P., Sigaux F. & Kolb J. P. (1988) Expression of the B8.7 antigen on hairy cells and relation with the LMW-BCGF response. Personal communication. Cordingley F. T., Hoffbrand A. V., Heslop H. E. Turner M., Bianchi A., Reittie J. E., Vyakarnam A., Meager A. & Brenner M. K. (1988) Tumor necrosis factor as an autocrine tumour growth factor for chronic B-cell malignancies. Lancet i, 969.
0145--2126/90$3.00 + .00 PergamonPressplc
Leukemia Research
A U T O C R I N E G R O W T H OF LEUKEMIC CELLS CLAUDE JASMIN, VASSILIS GEORGOULIAS, FLORENCE SMADJA-JOFFE, CLAUDE BOUCHEIX, CAROLINE L E BOUSSE-KERDILES, MICHI~LE ALLOUCHE, CHRISTIAN CIBERT a n d BRUNt) AZZARONE
INSERM U 268, H6pital Paul Brousse, 16 av. P.V. Couturier, 94800 Villejuif, France
(Received 15 January 1990. Accepted 16 March 1990) Abstract--Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. This paper describes the stepwise malignant progression of leukemic cells which has been demonstrated in many experimental models of autocrine leukemic growth. In contrast, autocrine growth has not been proven as a major physiopathological mechanism for the growth of leukemic cells in vivo in human myeloid and lymphocytic leukemias. Growth-factor independency of human leukemic cell lines may be due to clonal selection.
Key words: Autocrine growth, paracrine growth, growth-factor, receptor, leukemia, autocrine loop.
its receptor could lead to transformation by a autocrine mechanism. More intriguing is the demonstration that normal smooth muscle cells can produce an IGF-like molecule whose inhibition by monoclonal antibodies results in growth arrest [8]. Receptors can be considered as allosteric enzymes whose action on intracellular metabolic chains is modulated by the receptor's binding to its ligand: i.e. growth factors. Therefore, abnormal growth can be due either to the uncontrolled production of a growth factor in a cell that also synthesizes that growth factor's receptor (public autocrine loop) or to a modification of the growth factor receptor itself. In this latter case the receptor becomes permanently activated and even when the extracellular signal is absent it continues to mimic an enzymatic activity normally associated with the occupied growth factor receptor (private autocrine loop). Most human leukemias and lymphomas are associated with characteristic cytogenetic alterations. In addition, oncogenic retroviruses, such as HTLV1, appear to be etiologically associated with adult human T cell leukemia (ATL) [9-11]; Epstein-Barr virus is also involved in B cell lymphoid malignant transformation. Therefore human leukemias and lymphomas are good candidates for autocrine growth. Recently there has been a great development in the production of gene probes and recombinant purified molecules for many interleukins and CSF, and of monoclonal antibodies that interact with cell surface membrane receptors. Thanks to these valuable tools, it has been possible to analyze growth requirements
1. LEUKEMIAS AND LYMPHOMAS ARE GOOD CANDIDATES FOR AUTOCRINE GROWTH THE BONEmarrow, spleen and lymphoid organs form a network that is intimately associated with hematopoietic precursors [1, 2]. The survival, proliferation and differentiation of hematopoietic (myelopoietic and lymphopoietic) progenitor cells is controlled by a great variety of growth factors called interleukins (IL) and colony stimulating factors (CSF) [3, 4] which interact with their specific receptors. Autocrine growth is a process whereby a cell both secretes and responds to a growth factor. Autocrine signalling is usually confined to pathological conditions; e.g. certain tumor cells synthesize and release growth factors that are required for normal cellular growth and division. These growth factors stimulate the inappropriate, unregulated growth of the tumor itself and can also induce tumor formation. This was first described by Todaro et al. [5-7] for ceils infected by transforming viruses. These authors suggested that co-expression of a growth factor and
Abbreviations: HTLV, human T leukemia virus; ATL, adult T leukemia; GF, growth-factor; GF-R, growth-factor receptor; PDGF, platelet-derived growth-factor; TGF, transforming growth-factor; G-M, granulocytemaerophage; CSF, colony-stimulating factor; IL, interleukin; CLL, chronic lymphocytic leukemia; TNF, tumor necrosis factor; BCGF, B-cell growth factor; E-BV, Epstein-Barr virus. Correspondence to: Dr Claude Jasmin, INSERM U 268, H6pital Paul Brousse, 16 av. P.V. Couturier, 94800 Villejuif, France. 689
690
C. JASMINet al.
for human and experimental leukemias and lymphomas in the search for autocrine growth of leukemic cells. 2. TESTING OF THE AUTOCRINE GROWTH OF LEUKEMIC CELLS The main method to test for an autocrine mechanism of the neoplasm is the autonomous growth of leukemic cells with long-term or permanent selfrenewal capacity in semi-solid media and/or the establishment of permanent cell-lines in liquid medium in the absence of added CSFs, interleukins or other growth factors. The next step consists of the search for constitutive growth factor (GF) production and/or growth factor receptor (GFR) expression in the transformed cells. Treatment of the malignant cell culture with monoclonal antibodies to GF and GFR must produce a decrease (and even an arrest) of the culture's growth. Addition of the specific GF may or may not stimulate the growth rate of leukemic cells. Finally, transfection of normal cells or of factordependent cell lines with a normal or modified GF or GFR gene may prove the autocrine model. 3. AUTOCRINE MODELS There are some circumstances in which specific cells have been shown to both produce and respond to the same factor: i.e. a platelet-derived growth factor (PDGF)-like factor in a human osteosarcoma [12] and tumor growth factor/3 (TGF/3) in chemically transformed fibroblasts [13]; however, in the latter case, the change in the transformed cells, as compared to the non-transformed parents, is a greatly increased sensitivity to the TG/3 they produce and not an increase in production of TG/3 [14]. This demonstrates that autocrine growth can be the result of qualitative and not only quantitative changes in the GF-GFR interaction. In the case of leukemias and lymphomas, paracrine mechanisms may play an important role in leukemogenesis and autocrine mechanism may often be restricted to subpopulations of leukemic stem cells which have been selected (or even induced) after prolonged growth of the parental cells in vitro in the presence of added GF. For example Morrone et al. [15] have established a cell line from peripheral mononuclear cells of a patient affected by a Sezary syndrome. This line produces and responds to a transferrin-like growth factor. However, the antigenic profile of leukemic cells grown in vitro is dramatically different from that of fresh peripheral blood cells from the same patient and seems to represent a clone derived from a rare
immature leukemic progenitor cell present in the peripheral blood cell population. This subpopulation may have been the more prone to immortalization in vitro. Similar examples of clonal autocrine growth stimulation of a human T-cell lymphoma by interieukin 2 have been previously described [16]. More recent studies show that although leukemic cells from adult T-cell leukemia (ATL) patients [17] and HTLV1 and HTLV2 infected helper T cells [18] constitutively express an excess of IL-2 receptors, growth of leukemic cells in ATL remain IL-2-dependent [19]. Also, we have demonstrated that acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma of T lineage retain IL-2 dependence although they are able to form spontaneous T cell colonies in semi solid medium and to produce a nonIL-2 growth factor [20, 21]. However, Shirakawa et al. [22] have recently reported that the growth of ATL cells freshly obtained from patients was stimulated in a concentration-dependent manner in the presence of recombinant human IL-1 cr and/3. Furthermore, the spontaneous growth of ATL cells was also inhibited by anti-IL-1 cr antibody but not by anti-IL-1/3 antibody. ATL cells exhibited enhanced expression of IL-1 receptors on their surface as detected by the binding of 125I-labeled rlL-1 0¢. These results suggest that IL-1 o: produced by ATL cells stimulates the growth of ATL by an autocrine mechanism. The elevated expression of TGF-/3 gene recently demonstrated in fresh peripheral mononuclear cells from adult T cell leukemia patients [23] is worthy of note and draws attention to GFs other than CSFs and interleukins. The myeloid lineage offers several examples of alterations in receptor genes that account for a triggering of the receptor's kinase activity in spite of the absence of the appropriate ligand. For example, vJkns, an oncogene which encodes a modified form of the receptor for CSF-1 converts a CSF-1 dependent macrophage cell line to factor independence and can make the cell tumorigenic [24]. Similarly, insertion into avian hematopoietic cells of the v - e r b B oncogene which encodes the transmembrane spanning segment of the tyrosine kinase domain of the EGF receptor but lacks extracellular sequences necessary for ligand binding [25, 26], leads to erythroid transformation. Studies of fresh leukemic blasts obtained from either peripheral blood or bone marrow of patients with acute myelogenous leukemia have revealed some cases of autocrine secretion of GF: granulocyte-macrophage (GM)CSF [27], G-CSF and GM-CSF [28] and IL-1/3 [29] have been shown to produce autocrine stimulation of leukemic cells and may be constitutively produced
Autocrine growth of leukemic cells in some patients. It is probable that acute myelogenous leukemia represents a heterogenous disease with diverse underlying mechanisms of transformation. Chronic lymphocytic leukemia (CLL) of B-cell type (B-CLL) is a clonal proliferation of relatively mature-appearing B lymphocytes. A recent study [30] has shown that tumor necrosis factor (TNF) can stimulate, in a dose-dependent manner, the growth of cultured neoplastic B-cells from previously untreated patients with B-CLL. In the case of spontaneous proliferation, neoplastic B-cell growth from two out of three patients was further enhanced by addition of rTNF-cr to the culture medium. However, neither the specific inhibition of growth by anti-TNF-tr and/ or anti-TNF-tr receptor antibodies nor the constitutive production of TNF-or messenger-RNA was demonstrated. In another recent study [31], IL-6 transcript was found in six out of eleven B-CLL patients, while no hybridization was observed in ten cases of ALL of both T and B cell origin. The constitutive expression of IL-6 transcripts was associated with production of a biologically active protein. However, it has not yet been demonstrated that IL6 gene expression is necessary for the neoplastic Bcell growth. One particular case of human B cell lymphoma led to further documentation of autocrine growth of neoplastic B cells [32]. Indeed, a high molecular weight B cell growth-factor (BCGF) of molecular mass 60,000 stimulates the growth of a cell line derived from a patient with large-cell immunoblastic B-cell lymphoma adapted to growth under serumfree conditions. The malignant cells produce and respond to the 60,000mol. wt BCGF. They also express cell surface receptors for this molecule. However, inhibition of growth by anti-BCGF antibodies or by specifically blocking the cell-surface receptors was not performed. In addition, this study was not done on fresh neoplastic cells but on cell lines. Therefore, the autocrine growth of B-CLL has not yet been fully proven. Even if IL-6 and BCGF (low and high molecular weight) are likely to play an important role in the growth of various human Bcell malignancies such as myeloma [33] and EBVtransformed B-cells [34], it is possible that this role is only secondary to other growth control defects of neoplastic B cells. Even if the comparison between spontaneous leukemic transformation and experimental induction of leukemic clones does not show a clear relationship between the two situations, gene manipulation by infection or transfection of target cells is providing important clues concerning autocrine growth. For example bcl-2 is a gene located on chromosome
691
18 which is associated with human follicular lymphoma. Its introduction by a retroviral vector into B cells of transgenic mice provides a distinct survival signal to the cell which may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated [35]. Similar results have been recently obtained in the case of transfection of GM-CSF dependent cell lines by the GM-CSF gene. It was shown that acquisition of true autonomy by transfected cells was a multistep process which could be influenced by the selective pressure for autocrine stimulation maintained on the culture [36]. This stepwise malignant progression has been studied in an IL-3-dependent mouse mast cell line after introduction of the V-H-ras oncogene. As shown by Nair et al. [37], tumor progression was characterized by four distinct phenotypes corresponding to four stages of tumorigenesis: immortalization, immortalized transformed cells characterized by the competence to form V-H-ras-dependent tumors, transformed competent cells with reduced IL-3 requirement and, finally, a fully malignant phenotype marked in vitro by an independence to exogeneous IL-3 and by autocrine IL-3 stimulation. At this stage of transformation, two out of six tumors showed alterations at the 5' region of the IL-3 gene, suggesting that V-H-ras requires complementation by IL-3 gene rearrangements or an alternate event to generate autocrine mastocytomas. Finally, experimental data have also demonstrated that factor-independency can be acquired by hematopoietic cell lines after malignant transformation by Abelson virus without evidence of autocrine mechanism [38]. Indeed, transformed cells become factor-independent and tumorigenic without synthesizing GM-CSF or IL-3 and they display similar numbers of cell surface receptors for these GF as the original factor-dependent cell line. However, even in this case, it is not possible to definitively exclude an autocrine mechanism. Indeed, recent study of normal rat kidney cells which had been transformed by that functional equivalent of PDGF--the v-sis oncogene---showed that autocrine stimulation of the receptor occurred in the intracellular compartments before receptor maturation was complete and before the receptors could have reached the cell surface [39]. Therefore, in such a case of private autocrine loop stimulation, addition of exogenous GF and/or treatment with antisera against the GF does not strongly inhibit cell growth. 4. CONCLUSIONS Exploration of the potential utilities of GF and
692
C. JASMINet al.
G F R manipulation is currently in progress. The remarkable success of recombinant o~2 interferon in the treatment of hairy-cell leukemia has demonstrated that trans-down modulation of G F production can be obtained and is of clinical interest. Indeed, it is probable that the therapeutic efficacy of interferon is due to its inhibiting effect either on B C G F [40] and/or T N F [41] which have been found to produce autocrine stimulation of hairy leukemic cells. Clinical trials of various interleukins, cytokines and lymphokines in hematopoietic malignancy will hopefully provide further clinical success. However, we have seen the great complexity of autocrine mechanisms and it is possible that in spontaneous neoplasms, the heterogeneity of the leukemic population may limit the usefulness of biological treatments. Nevertheless, a new era for the treatment of human malignancies may arise from the development of such therapeutic tools as truncated factors capable of competing for receptor-binding and of stimulating down-regulation of receptors, monoclonal antibodies able to block growth factor receptors and gene control by antisense sequences.
Acknowledgements--This work was supported by N.R.B. (Nouvelles Recherches Bio-M6dicales). We are grateful to Mrs Coiette Delteil for expert secretarial assistance.
REFERENCES 1. Dexter T. M., Allen T. P. & Lajhta L. G. (1977) Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. cell. Physiol 91, 335. 2. Dexter T. M., Spooncer E., Simmon P. & Allen T. P. (1984) Long term marrow cultures: an overview of technique and experience. In Long Term Bone Marrow Culture (Wright D. G. & Greenbeyer J. S., Eds) p. 57. Alan Liss, New York. 3. Dexter T. M. & Spooncer E. (1987) Growth and differentiation in the hemopoietic system. Ann. Rev. cell. Biol. 3, 423. 4. Metcalf D. (1977) Hemopoietic Colonies, p. 227. Springer, Berlin. 5. Todaro G. J., De Larco J. E. & Cohen S. (1976) Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells. Nature (Lond.) 264, 26. 6. De Larco J. E. & Todaro G. J. (1978) Growth factors from murine sarcoma virus-transformed cells. Proc. natn. Acad. Sci. U.S.A. 75, 4001. 7. Sporn M. B. & Todaro G. J. (1980) Autocrine secretion and malignant transformation of cells. New Engl. J. Med. 303, 878. 8. Clemmons R. R. & Van Wyk J. J. (1985) Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. J. clin. Invest. 75, 1914.
9. Poiesz B., Ruscetti F. W., Gazdar A. F., Bunn P. A., Minna J. D. & Gallo R. C. (1980) Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous Tcell lymphoma. Proc. natn. Acad. Sci., U.S.A. 77, 7415. 10. Hinuma Y., Nagata K., Hanaoka M., Nakai M., Matsumoto T., Kinoshita K. I., Shirakawa S. & Miyoshi I. (1981) Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc. natn. Acad. Sci. U.S.A. 78, 6476. I 1. Wong-Staal F. & Gallo R. C. (1985) Human T-lymphotropic retroviruses. Nature 317, 395. 12. Betsholtz C., Westermark B., Ek B. & Heldin C. H. (1984) Co-expression of a PDGF-Iike growth factor and PDGF receptors in a human osteosarcoma cell line: implications for autocrine receptor activation. Cell 39, 447-457. 13. Moses H. L. & Robinson R. A. (1982) Growth factors, growth factor receptors and cell cycle control mechanisms in chemically transformed cells. Fedn. Proc. Fedn Am. Socs exp. Biol. 41, 3008-3011. 14. Moses H. L., Childs C. B., Halper J., Shipley G. D. & Tucker R. F. (1984) In Control o f Cell Growth and Proliferation (Veneziale C. M., Ed.), p. 147. Van Nostrand Reinhold Co., New York. 15. Morrone G., Corbo L., Turco M. C., Pizzano R., De Felice M., Bridges S. & Venuta S. (1988) Transferrinlike autocrine growth factor derived from T-lymphoma cells, that inhibits normal T-cell proliferation. Cancer Res. 48, 3425. 16. Duprez V., Lenoir G. & Dautry-Varsat A. (1985) Autocrine growth stimulation of a human T-cell lymphoma line by interleukin 2. Proc. natn. Acad. Sci. U.S.A. 82, 6932. 17. Hattori T., Uchiyama T., Toibana T., Takatsuki K. & Uchino H. (1981) Surface phenotype of Japanese adult T-cell leukemia cells characterized by monoclonal antibodies. Blood 58, 645. 18. Tsudo M., Uchiyama T., Uchino H. & Yodoi J. (1983) Failure of regulation of Tac antigen/TCGF receptor on adult T-cell leukemia cells by anti-Tac monoclonal antibody. Blood 61, 1014. 19. Maeda M., Arima N., Daitoku Y., Kashihara M., Okamoto H., Uchiyama T., Shirono K., Matsuoka M., Hattori T., Takatsuki K., Ikuta K., Shimizu A., Honjo T. & Yodot J. (1987) Evidence for the interleukin-2 dependent expansion of leukemic cells in adult T cell leukemia. Blood 70, 1407. 20. Georgoulias V., Auclair H. & Jasmin C. (1984) Heterogeneity of peripheral blood T-cell colony-forming cells in patients with T-cell malignancies. Leukemia Res. 8, 1025. 21. Allouche M., Georgoulias V., Varela-Millot C., Itzhaki M., Misset J. L. & Jasmin C. (1989) Prognostic value of spontaneous peripheral blood T-colony forming cells in T-cell acute lymphoblastic leukemia in complete remission: an update evaluation. Br. J. Haemat. 72, 291. 22. Shirakawa F., Tanaka Y., Oda S., Eto S. & Yamashita U. (1989) Autocrine stimulation of interleukin 1o~ in the growth of adult human T-cell leukemia cells. Cancer Res. 49, 1143. 23. Niitsu Y., Urushizaki Y., Koshida Y., Terui K., Mahara K., Kohgo Y. & Urushizaki I. (1988) Expression of TGF-beta gene in adult T cell leukemia. Blood 71,263.
Autocrine growth of leukemic cells 24. Wheeler E. F., Rettenmier C. W., Look A. T. & Sheer C. J. (1986) The v-fms oncogene induces factor independence and tumorigenicity in a CSF-1 dependent macrophage cell line. Nature 324, 377. 25. Yamomoto T., Nishida T., Miyajima N., Kawai S., Ooi T. & Toyoshima K. (1983) The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35, 71. 26. Ullrich A., Coussens L., Hayflick J. S. Dull T. J., Gray A., Tam A. W., Lee J., Yarden Y., Libermann T. A., Schlessinger J., Downward J., Mayes E. L. V., Whitte N., Waterfield M. D. & Beedurg P. H. (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418. 27. Diane C., Griffin Y. & Young D. C. (1986) Autocrine secretion of GM-CSF in acute myeloblastic leukemia. Blood 68, 1178. 28. Murohashi I., Tohda S., Suzuki T., Nagata K., Yamashita Y. & Nara N. (1989) Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia. Blood 74, 35. 29. Sakai K., Hattori T., Matsuoka M., Asou N., Yamamoto S., Sagawa K. & Takatsuki K. (1987) Autocrine stimulation of interleukin 1/3 in acute myelogenous leukemia cells. J. exp. Med. 66, 1597. 30. Digel W., Stefanic M., Schoniger W., Buck C., Raghavachar A., Frickhofen N., Heimpel H. & Porzsolt F. (1989) Tumor necrosis factor induces proliferation of neoplastic B cells from chronic lymphocytic leukemia. Blood 73, 1242. 31. Biondi A., Rossi V., Bassan R., Barbui T., Bettoni S., Sironi M., Mantovani A. & Rambaldi A. (1989) Constitutive expression of the interleukin 6 gene in chronic lymphocytic leukemia. Blood 73, 1279. 32. Chintaman G., Sahasrabuddhe S., Sekhsaria L., Yoshimura L. & Ford R. J. (1989) Isolation and characterization of growth factor(s) from a human B-cell lymphoma. Blood 73, 1149. 33. Kawano M., Hirano T., Matsuda T., Taga T., Horii Y., Iwato K., Asaoku H., Tang B., Tanabe O., Kuramoto A. & Kishimoto T. (1988) Autocrine generation
34.
35. 36.
37.
38.
39. 40.
41.
693
and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332, 83. Muraguchi A., Hirano T., Tang B., Matsuda T., Horii Y., Nakajiama K. & Kishimoto T. (1988) The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells. J. exp. Med. 167, 332. Vaux D. L., Cory S. & Adams J. M. (1988) Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335,440. Laker C., Stocking C., Berghoiz U., Hess N., De Lamarter J. F. & Ostertag W. (1987) Autocrine stimulation after transfer of the granulocyte/macrophage colony-stimulating factor gene and autonomous growth are distinct but interdependent steps in the oncogenic pathway. Proc. natn. Acad. Sci. U.S.A. 84, 8458. Nair A. P. K., Diamantis I. D., Conscience J. F., Kindler V., Hofer P. & Moroni C. (1989) A v-H-rasdependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production. Molec. cell. Biol. 9, 1183. Cook W. D., Metcalf D., Nicola N. A., Burgess A. W. & Walker F. (1985) Malignant transformation of a growth factor-dependent myeloid cell line by Abelson virus without evidence of an autocrine mechanism. Cell 41, 677. Keating, M. T. & Williams L. T. (1988) Autocrine stimulation of intracellular PDGF receptors in v-sistransformed cells. Science 239, 914. Genot E., Leprince C., Richard Y., Petit-Koskas E., Falcoff E., Galanaud P., Sigaux F. & Kolb J. P. (1988) Expression of the B8.7 antigen on hairy cells and relation with the LMW-BCGF response. Personal communication. Cordingley F. T., Hoffbrand A. V., Heslop H. E. Turner M., Bianchi A., Reittie J. E., Vyakarnam A., Meager A. & Brenner M. K. (1988) Tumor necrosis factor as an autocrine tumour growth factor for chronic B-cell malignancies. Lancet i, 969.
