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MECHANISMS OF MYELOMA CELL GROWTH CONTROL
MONOCLONAL GAMMOPATHIES AND RELATED DISORDERS
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MECHANISMS OF MYELOMA CELL GROWTH CONTROL Diane F. Jelinek, PhD
Multiple myeloma is a progressive and fatal disease characterized by the accumulation of malignant plasma cells in the bone marrow. Normal plasma cells have long been viewed as end-stage cells that have lost the potential for further proliferation. The longevity of these cells has been estimated to range from several days to several weeksz6 Recent evidence, however, raises the possibility that, under some circumstances, plasma cells may survive for a much longer period of time.52Myeloma cells differ considerably from normal plasma cells in that the tumor cells retain the capacity for proliferation and their lifespan may exceed that of normal counterpart cells, perhaps as a result of overexpression of several antiapoptotic cellular Although tumor expansion in vivo is critically dependent upon both growth and death rates, this article focuses on several aspects of myeloma cell growth control. THE ROLE OF PROLIFERATION IN B-LYMPHOCYTE DIFFERENTIATION
Before discussing potential regulators of myeloma cell growth, it is essential first to review the role of proliferation in the terminal differentiation of human B lymphocytes into high-rate immunoglobulin-secreting cells. The various stages of normal B-lymphocyte activation, proliferation, and differentiation are summarized in Figure 1. B-lymphocyte activation is initiated by engagement of membrane immunoglobulin. Following receipt of a variety of signals, including direct interaction with T lymphocytes and a number of cytokines, the activated B lymphoblast enters the cell cycle. Although the proliferative phase has often This work was supported by Grants CA62228 and CA64442 from the National Institutes of Health.
From the Department of Immunology, Mayo Clinic and Mayo Foundation, and Mayo Medical School, Rochester, Minnesota HEMATOLOGY/ ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 13 * NUMBER 6 * DECEMBER 1999
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Figure 1. Activation, proliferation, terminal differentiation, and programmed cell death are highly regulated events in normal B-lymphocyte maturation. The large dotted arrows emphasize the ability of immunoglobulin-secretingplasmablasts to proliferate.
been viewed as separate and independent from the differentiative phase, for the purposes of this article it is important to emphasize that immunoglobulinsecreting plasmablasts are also capable of continued proliferati~n.~~ Thus, not only is initial proliferation essential for preparing B lymphocytes to receive a differentiation signal, ongoing proliferation of the plasmablasts appears to be important in increasing the magnitude of the antibody response and in preparing the cell to differentiate into a high-rate immunoglobulin-secreting plasma ce11.56 Although B-cell proliferation is necessary for differentiation, it is also clear that terminal differentiation of B cells into end-stage plasma cells is accompanied by growth arrest. A variety of external and internal signals are probably involved in modifying gene expression in the developing plasma cell so that the end result is a nonproliferative cell with the capacity to secrete high levels of immunoglobulin. The precise mechanisms by which growth potential is lost as a consequence of terminal differentiation remain obscure. It is believed, however, that this loss results from the interplay between cellular signaling and activation of transcription factors important in regulating expression of genes whose products are specific to the differentiated phenotype. Figure 2 graphically displays the relationship between normal B-cell proliferation and differentiation into endstage plasma cells. Because myeloma cells retain proliferative capacity, albeit in many cases this is quite low, the author and colleagues have hypothesized that the genetic events that program the loss of proliferative potential have become uncoupled from the differentiative events that result in development of high-rate immunoglobulin-secreting cells. For example, a number of transcription factors, such as nuclear factor interleukin-6 (NF-IL,-6),13,l4 have been described as playing a role in the terminal differentiationof B lymphocytes. It is plausible that disregulation of such factors at the level of either expression or function could contribute to the proliferative ability of the malignant plasma cell. Of note, the speculative model shown in Figure 2 also suggests that growth cessation of normal plasma
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Onset of end-stage differentiation?
4
Myeloma?
h
h
Figure 2. Model of the relationship between normal B-cell proliferation and differentiation and the hypothesized block of terminal differentiation in myeloma. The model suggests that immunoglobulin secretion rate per cell (dotted line) increases as the proliferative index (heavy solid line) decreases. By contrast, immunoglobulin-secreting myeloma cells retain the ability to proliferate at varying levels.
cells is a gradual process involving several changes in gene expression. In this context, the author and colleagues further speculate that the variable proliferative capacity (labeling index or LI) of myeloma cells observed among patients may similarly be explained if the transforming event occurs at variable places along the differentiation continuum. Given that myeloma cells retain proliferative potential, it becomes important to understand the mechanisms by which a variety of factors can affect the growth of myeloma cells. The difficulty of this task is highlighted by the significant heterogeneity, particularly in cytokine responsiveness, that exists among tumor cells obtained from different patients.', 31, 35, 59
INTERLEUKIN-6
Much of the current understanding of the growth characteristics of myeloma cells in vivo has been obtained by studying myeloma cell lines. Although myeloma cell lines are notoriously difficult to establish, this task has become somewhat easier since the demonstration that the cytokine interleukin (1L)-6 could promote the growth of myeloma cells in ~ i t r oThe . ~ ~biological relevance of this finding has been strengthened by demonstrations that elevated IL-6 levels are directly correlated with tumor burden, bone destruction, and other tumorassociated activities in myeloma patient^.^, 65 Indeed, administration of anti-IL6 antibodies has been shown to alleviate disease ~yrnptorns.~~ Interleukin-6 has also been shown to offer protection against chemotherapy-induced apoptosis.12, 51, Finally, a key role for IL-6 is further underscored by animal studies demonstrating that IL-6 is a crucial requirement for the development of B-cell Q
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neoplasms.u Collectively, these observations suggest that IL-6 plays a pivotal role in myeloma. The IL-6 receptor is a member of the hemopoietin receptor family and consists of two functional chains: (1) an 80-kD IL-&binding molecule (IL6R or gp80) whose expression is induced upon activation, and (2) a constitutively expressed 130-kD signal-transducing chain (g~130).3~ Binding of IL-6 to IL6R triggers IL6R association with and homodimerization of gp130. Although gp130 itself does not display intrinsic tyrosine kinase activity, several nonreceptor tyrosine kinases constitutively associate with the cytoplasmic domain of gp130. Moreover, several functional elements have been identified in the cytoplasmic domain of gp130, that is, box 1, 2, and 3 motifs that underlie the multiple signaling events downstream of receptor ligation." Transcriptional activation of target genes mediated by IL-6 is currently known to result from several signaling pathways, including the ras/MAP kinase (MAPK) and JAK/ STAT pathways downstream of receptor ligation."36 gp130 also serves as the signal transducing component in receptor complexes for leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-11, and cardiotrophin-1.24Not surprisingly, therefore, OSM, LIF, and CNTF have also been reported to support the growth of myeloma cells.19,20, 66 The responsiveness of myeloma cells to these cytokines is more variable, however, probably because the ligand-binding receptor components for these cytokines are not as consistently expressed on myeloma cells as is the IL-&binding gp80 component. Effects of Interleukin-6 on Normal B Lymphocytes and Plasmablasts Compared with Myeloma Cells
As mentioned previously, the gp130 family of cytokines, particularly IL-6, appears to play a key role in the growth control of myeloma cells. Although cytokines that utilize the gp130 signal transduction molecule can trigger myeloma-cell proliferation, the effects of these cytokines on normal B lymphocytes is strikingly distinct. Thus, the author and colleagues have used an in vitro system that allows the generation of large numbers of plasmablasts, that is, proliferating immunoglobulin-secretingB cells, by coculture of normal human B cells with anti-CD3-activatedT cells.25, 56 After 5 days, plasmablasts with proliferative potential can be assessed for cytokine responsiveness. Table 1 demonstrates the effects of IL-6 and oncostatin M on both differentiation and proliferation of normal plasmablasts. As reported previously, IL-6 augments immunoglobulin secretion most effectively when IL-2 is also The author and colleagues reported for the first time the effects of OSM on normal B cellsm;it may be seen that OSM also markedly augments immunoglobulin secretion. By contrast, when these two cytokines are compared with IL-2 for their ability to support growth, both are observed to be completely without effect, even in the presence of IL-2 (Table 1). These results support the conclusion that IL-6, and perhaps other members of this cytokine family, support normal B-cell differentiation but do not support B-cell proliferation. Because of these striking differences between the effects of IL-6 on myeloma cells and on normal B cells or plasmablasts, there is a clear need to understand in greater detail the mechanisms that account for this differential responsiveness. The ability of both normal B cells and myeloma cells to respond to IL-6 and other members of this cytokine family suggests, however, that there may be alterations in gpl30-mediated signal transdudion in tumor cells. Definitive studies have not yet been carried out, but evidence in the literature suggests that the ras/MAPK pathway may be imp~rtant.~, 47 Although
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Table 1. EFFECTS OF INTERLEUKIN-2, INTERLEUKIN-6 AND ONCOSTATIN M ON IMMUNOGLOBULIN SECRETION AND DNA SYNTHESIS BY PREACTIVATED NORMAL PERIPHERAL BLOOD PLASMABLASTS*
DNA Synthesis [3H]-Thymidine Incorporation (cpm x
Total Immunoglobulin (ng Ig/mL) Experiment No.
IL-2
Nil
IL-6
OSM
Nil
IL-6
OSM
1
-
1886 5996 1726 10,301
5245 >13,687 >14,882 215,868
2689 >14,000 3105 >19,931
0.2 9.6 0.5 21.3
0.2 10.5 0.6 21.3
0.1 9.5 0.8 22.7
2
+ +
*Highly purified normal human peripheral blood B cells were cocultured with mitomycin C-treated T cells and immobilized CD3 antibodies for 5 days before removal of T cells and reculture of purified preactivated B cells with various cytokines. Total immunoglobulin (IgM, IgG, and IgA) secretion was measured after an additional 5-day incubation. For assay of DNA synthesis, ['HI-thymidine incorporation was determined after 3 days. IL-2 = interleukin-2; IL-6 = interleukin-6; OSM = oncostatin M; - = negative; + = positive.
a number of therapeutic strategies are designed to interrupt extracellular binding of growth factors such as IL-6, it is also important to identify the intracellular events that sustain the aberrant growth of the tumor cells so that these molecules can be targeted as well. In Vivo Sources of Interleukin-6 in Myeloma Patients
As mentioned previously, IL-6 levels have been shown to correlate with tumor burden: This observation calls into question the in vivo source of the elevated IL-6. Although this was once considered a controversial issue, with some studies suggesting that the increased levels of IL-6 derived from paracrine sources and other studies suggesting that the elevated levels of this cytokine resulted from tumor-cell expression of IL-6, review of the literature suggests that both mechanisms are probably involved. Thus, a number of scenarios (summarized in Figure 3) could account for the increased IL-6 levels observed in patients during disease progression. It is important to emphasize that any or all of these mechanisms could be operative at any one time. The simplest model shown in Figure 3 is that of constitutive autocrine IL-6 expression. Although Kawano and colleagues3 were the first to suggest that myeloma cells themselves can express IL-6, others have disputed this suggestion, arguing instead that the IL-6 is expressed by contaminating stromal cells.37,39 The existence of myeloma cell lines expressing IL-6 argues against contamination as the source of IL-6.3l.59 It remains possible, however, that IL-6 expression by the myeloma cell line is not truly constitutive, but rather is induced as a result of cell-to-cell interactions that occur among the tumor cells themselves. Although not all myeloma cells express the IL-6 gene, normal B lymphocytes apparently lose the ability to express this gene on terminal differentiation into plasma cells.67It is possible, therefore, that the cells that do express IL-6 have undergone transformation at an earlier stage of maturation, or that tumor cells in patients exist at a variety of maturational stages, only some of which express IL-6.= At the opposite end of the spectrum is the possibility that IL-6 is constitutively expressed by nonmyeloma cells. Given the complexity of cell-to-cell inter-
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Figure 3. Speculative models of the mechanisms of IL-6 expression in myeloma. In the induced-paracrine and induced-autocrine models, the numbers opposite the arrows depict the hypothesized order of events.
actions in the bone marrow, this possibility is considered unlikely. Instead, a much more attractive mechanism is the induced-paracrine or induced-autocrine model. Evidence in the literature supports both these models. For example, the author and c011eagues~~ and others54have shown that engagement of cell-surface CD40 on myeloma cells results in autocrine IL-6 expression. In other studies, myeloma cells have been shown to induce IL-6 expression in stromal cells in a largely cell-contact-dependent manner?, 57 Although the complete set of molecules that regulate interactions among tumor cells and stromal cells remains to be elucidated, it is clear that the bone marrow provides a unique and optimal environment for the growth of myeloma cells. This environment has been difficult to mimic in vitro, and thus it has been a challenge to study the biology of the malignant plasma cell. A recently described scid-hu model system, however, has the potential to facilitate this analysis greatly.55, INSULIN-LIKE GROWTH FACTOR I
As discussed previously, the qualitative outcome of IL-6 signal transduction in myeloma cells is distinct from that in normal B lymphocytes and plasmablasts. There is considerable interest, therefore, in understanding the mechanisms by whch myeloma cells become growth-responsive to the IL-6 family of cytokines and in identifying potential biologic regulators of this altered response. During the course of such mechanistic studies, the author and colleaguesmand othersI8 demonstrated an important role for insulin-like growth factor (IGF) in the regulation of myeloma cell growth. Figure 4 compares the effects of IGF-I on the IL,-Mependent myeloma-cell line, ANBL-6,2swith the effects of IGF-I on normal B lymphocytes stimulated with the polyclonal activator StuphyZococcus uureus (SA). In contrast with the ability of IGF-I to stimulate ANBL-6 DNA synthesis directly and to augment IL-&stimulated proliferation significantly, IGF-I did not
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Figure 4. A, ANBL-6 myeloma cells were cultured with or without IL-6 and in the presence or absence of varying concentrations of IGF-I. B, Highly purified, normal peripheral blood B cells were stimulated with or without the mitogen SA, in the presence or absence of the indicated cytokines. DNA synthesis was assayed on day 3 for the ANBL-6 experiment and on day 5 for the normal B-cell experiment. Data represent the mean of triplicate values.
directly stimulate normal B-cell proliferation, nor did it augment the activity of the known B-cell growth factor, IL-2, in the presence or absence of SA. These results suggest that IGF responsiveness may also represent a key difference between normal and malignant B cells. Other investigators have demonstrated that IGF-I may also facilitate myeloma cell longevity through its ability to prevent tumor cells from undergoing apoptosis.61 Insulin-like growth factor I and IGF-I1 have been shown to play a crucial role in the regulation of growth in a wide variety of cell types5,6, 32 Although expression of IGFs is relatively widespread during fetal development, expression of these factors in adults is moderately restricted.16Of interest, a number of cell types residing withn bone marrow are capable of expressing IGF-I in adults.17, 45 The growth-promoting properties of IGFs suggest that inappropriate expression of IGFs may contribute to loss of normal cell growth control. In fact, IGFs have long been recognized as important mitogens in many types of malignancies.6 The IGF-I receptor (IGF-IR) consists of an (Y and a p chain which are synthesized as a single-chain precursor followed by cleavage into the separate chains. The receptor complex is organized as a functional dimer of two cw/p complexes. The (Y subunit is entirely extracellular and is responsible for ligand binding; the p subunit is largely intracellular and is responsible for triggering downstream signaling events.41Because of the important role that IGFs play in development and growth regulation, IGF-IR gene expression is tightly regulated. In general, h g h levels of IGF-IR mRNA are observed embryonically; in adult tissues, however, the levels of IGF-IR mRNA are very low. Importantly, it has been shown that as little as a twofold difference in IGF-IR numbers per cell can qualitatively change the nature of cellular responsiveness from enhanced survival to pr~liferation.~~ In preliminary studies, the author and colleagues (Jelinek et al, unpublished observations) have consistently observed that both chains of the IL-6 receptor complex may be overexpressed in myeloma cells, and that the IGF-IR is overexpressed only in a subset of myeloma cells, particularly in
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Figure 5. Speculative model of the role of the IL-6 and the IGF-I receptor in myeloma cells. Myeloma cell overexpression of the IL-6 receptor is viewed as an initial event that may offer protection from apoptosis, particularly when acting in concert with low levels of insulinlike growth factor (IGF)-I receptor expression. Upon increased expression of the IGF-I-R, myeloma cells are hypothesized to gain direct responsiveness to either IL-6 or IGF.
patients in whom a direct IL-6 proliferative response is observed. These studies suggest the attractive hypothesis that control of both IL-6 and IGF-IR expression may become disregulated in myeloma, resulting in increased protection against apoptosis and a possible increase in the responsiveness of myeloma cells to growth factor. T h s hypothesis is graphically illustrated in Figure 5. In this model, it is hypothesized that increased levels of IL-6 receptors initially delay apoptosis of myeloma cells. After the IGF-IR levels are elevated, it is hypothesized that tumor cells acquire more aggressive growth characteristics. Definitive studies testing this hypothesis are currently in progress. OTHER CYTOKINES WITH GROWTH-REGULATORY EFFECTS ON MYELOMA CELLS
Given the heterogeneity in myeloma, it is perhaps not surprising that a relatively large number of cytokines have been shown to have growth-stimulatory or growth-regulatory effects on myeloma cells. Although an exhaustive review of all of these effects is beyond the scope of this article, several cytokines merit special mention. Myeloma cells have been shown to express receptors for IL-10 and also to respond to this cyt0kine.4~The mechanism of action of IL-10, however, may be indirect, because there is some evidence that IL-10-induced myeloma-cell proliferation may be caused by either the induction of IL-11 responsiveness" or by the induction of an autocrine OSM loop.2oIn contrast with the striking differences between the effects of IL-6 on normal B cells and plasmablasts and on myeloma cells, IL-10 has been shown to support normal B-cell proliferation.8 The response of myeloma cells to IL-10 may therefore represent the exploitation by tumor cells of the proliferative function of a normally expressed receptor, rather than the altered responsiveness that apparently occurs in members of the gp130 family of cytokines.
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Recent studies have also demonstrated the ability of myeloma cells to express an autocrine hepatocyte growth factor (HGF)-c-met receptor ligand This autocrine growth loop was also shown to be present in fresh tumor cells from patients. The overall importance of this pathway in disease progression remains to be explained. Another observation that may prove to have a significant effect on the understanding of myeloma is that the fibroblast growth factor (FGF) receptor may be overexpressed or may contain activating mutations as a result of the translocation of this gene into the immunoglobulin heavy-chain gene 10cus.'~Because FGF is present in the bone marrow, inappropriate or elevated levels of FGF-receptor expression would have biologic consequences if this receptor conveys a proliferative or antiapoptotic signal. Finally, interferon-a (IFN-a) has also been reported to have interesting effects on myeloma cells. Interferon-a differs from the other cytokines that have been described, in that it has been reported to have significant activity either suppressing or promoting growth. Its activity in promoting growth is of great interest, because IFN-a has been commonly used as maintenance treatment for myeloma patients. Despite the relatively extensive clinical use of this cytokine in treatment of myeloma, there are reports suggesting that IFN-a may actually aggravate disease in vivo. Furthermore, some in vitro studies using either fresh cells from patients or established myeloma cell lines have shown that IFN-a may be modestly growth stimulatory for myeloma cells. There is evidence that this effect is caused by the induction of autocrine IL-6.33There is also compelling data, however, that IFN-a stimulation of myeloma-cell proliferation may be independent of autocrine IL-6.29The underlying mechanisms by which myeloma-cell proliferation is stimulated by IFN-a remain to be elucidated, although the author and colleagues have reported that this proliferation may involve differential induction of the p19 cyclin-dependent kinase inhibitor and cyclin D2.* Thus, the KAS-6/1 cell line that is growth-stimulated by IFN-a failed to express the p19 inhibitor on addition of the cytokine. Interferon-a did, however, induce expression of cyclin D2. The author and colleagues have recently demonstrated that IFN-a may stimulate MAPK activation in the KAS-6/ 1 cell line, but not in the IFN-a growth-inhibited cell line ANBL-6.3 The mechanism by which MAPK is strongly activated in KAS-6/1 cells but not in ANBL-6 cells remains unclear but is currently under investigation. It has been suggested that IFNa-mediated growth inhibition occurs as a result of this cytokine's ability to decrease the expression of the IL-6 The author and colleagues were able to demonstrate, however, that IFN-a-reduced expression of IL-6 receptors on both IFN-a-growth-arrested and IFN-a-growth-stimulated myeloma cell lines. Moreover, the decreased IL-6 receptors were still capable of signal transducti0n.2~The growth-inhibitory mechanism, therefore, is more likely related to the ability of IFN-a to induce the expression of cell cycle regulatory proteins.2 Identification of the mechanisms of IFN-a-mediated inhibition or stimulation of myeloma-cell growth may allow development of agents that are more universally effective than IFN-a. SUMMARY
By necessity, this article focuses on only a handful of molecules with demonstrated ability to affect growth of myeloma cells. The heterogeneity in growthfactor responsiveness has made formulation of a uniform hypothesis a daunting challenge. One trait that appears to be consistent among all myeloma patients is the uncoupling of the normally highly integrated relationship between terminal
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differentiation and loss of growth potential. Thus, the common feature in myeloma may not be the precise cytokines or cell-to-cell interactions that drive tumor-cell growth, but rather the underlying genetic traits that afford the tumor cell the ability to proliferate despite its relatively advanced stage of differentiation. The success of current strategies used to treat myeloma patients has thus far been somewhat limited, and in general, has only modestly prolonged survival. It is clear that successful treatment of this disease will require the development of new therapeutic agents aimed at the biochemical events that sustain the aberrant growth of the tumor cells. The knowledge of cell signaling, gene transcription, and cell growth and differentiation has expanded rapidly, and this information has provided a greater understanding of the cell biology of a variety of malignancies. Application of this information to the study of multiple myeloma, however, has thus far been relatively limited, primarily because the heterogeneity of the disease and the lack of appropriate model systems. Review of the literature, particularly over the last 5 years, reveals a significant number of exciting new findings in this field and the development of new model systems that are certain to yield greater insight into this devastating disease. References 1. Anderson KC, Jones RM, Morimoto C, et al: Response patterns of purified myeloma cells to hematopoietic growth factors. Blood 73:1915, 1989 2. Arora T, Jelinek DF: Differential myeloma cell responsiveness to interferon-a correlates with differential induction of ~ 1 9 " " ~ and cyclin D2 expression. J Biol Chem 273:11799, 1998 3. Arora T, Floyd-Smith G, Espy MJ, et al: Dissociation between IFN-a-induced anti-viral and growth signaling pathways. J Immunol 1623289, 1999 4. Barille S, Collette M, Bataille R, et al: Myeloma cells upregulate interleukin-6 secretion in osteoblastic cells through cell-to-cell contact but downregulate osteocalcin. Blood 86:3151, 1995 5. Baserga R, Rubin R Cell cycle and growth control. Crit Rev Eukaryotic Gene Expr 3:47, 1993 6. Baserga R The insulin-like growth factor I receptor: A key to tumor growth? Cancer Res 55:249, 1995 7. Bataille R, Jourdan M, Zhang XG, et al: Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflection of disease severity in plasma cell dyscrasias. J Clin Invest 84:2008, 1989 8. Benjamin D, Park CD, Sharma V Human B cell interleukin 10. Leuk Lymphoma 12:205, 1994 9. Billadeau D, Jelinek DF, Shah N, et al: Introduction of an activated N-ras oncogene alters the growth characteristics of the interleukin 6-dependent myeloma cell line ANBL-6. Cancer Res 55:3640, 1995 10. Borset M, Hjorth-Hansen H, Seidel C, et al: Hepatocyte growth factor and its receptor c-met in multiple myeloma. Blood 883998, 1996 11. Borset M, Lien E, Espevik T, et al: Concomitant expression of hepatocyte growth factor/scatter factor and the receptor c-MET in human myeloma cell lines. J Biol Chem 271:24655, 1996 12. Chauhan D, Kharbanda S, Ogata A, et al: Interleukind inhibits Fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells. Blood 89227, 1997 13. Chen-Kiang S, Hsu W, Natkunam Y, et ak Nuclear signaling by interleukin-6. Curr Opin Immunol5:124, 1993 14. Chen-Kiang S Regulation of terminal differentiation of human B-cells by IL-6. Curr Top Microbiol Immunol 194:189, 1994
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15. Chesi M, Nardini E, Brents LA, et al: Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 16:260, 1997 16. Cohick WS, Clemmons D R The insulin-like growth factors. AMU Rev Physiol 55:131, 1993 17. Fiorelli G, Orlando C, Benvenuti S, et al: Characterization, regulation, and function of specific cell membrane receptors for insulin-like growth factor I on bone endothelial cells. J Bone Miner Res 9:329, 1994 18. Georgii-Hemming P, Wiklund HJ, Ljunggren 0, et al: Insulin-like growth factor I is a growth and survival factor in human multiple myeloma cell lines. Blood 88:2250, 1996 19. Gu ZJ, Zhang XG, Hallet MM, et al: A ciliary neurotrophic factor-sensitive human myeloma cell line. Exp Hematol 24:1195, 1996 20. Gu ZJ, Costes V, Lu ZY, et al: Interleukin-10 is a growth factor for human myeloma cells by induction of an oncostatin M autocrine loop. Blood 88:3972, 1996 21. Hallek P, Bergsagel PL, Anderson KC: Multiple myeloma: Increasing evidence for a multistep transformation process. Blood 91:3, 1998 22. Hata H, Xiao H, Petrucci MT, et al: Interleukin-6 gene expression in multiple myeloma: A characteristic of immature tumor cells. Blood 81:3357, 1993 23. Hilbert DM, Kopf M, Mock BA, et al: Interleukin 6 is essential for in vivo development of B lineage neoplasms. J Exp Med 182:243, 1995 24. Hirano T, Matsuda T, Nakajima K Signal transduction through gp130 that is shared among the receptors for the interleukin 6 related cytokine subfamily. Stem Cells 12:262, 1994 25. Hirohata S, Jelinek DF, Lipsky PE: T cell dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3. J Immunol 140:3736, 1988 26. Ho F, Lortan JE, MacLennan IC, et al: Distinct short-lived and long-lived antibodyproducing cell populations. Eur J Immunol 16:1297, 1986 27. Jelinek DF, Lipsky PE: The role of B cell proliferation in the generation of immunoglobulin-secreting cells in man. J Immunol 130:2597, 1983 28. Jelinek DF, Ahmann GJ, Greipp PR, et al: Coexistence of aneuploid subclones within a myeloma cell line that exhibits clonal immunoglobulin gene rearrangement: Clinical implications. Cancer Res 535320, 1993 29. Jelinek DF, Aagaard-Tillery KM, Arendt BK, et al: Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression. J Clin Invest 99447, 1997 30. Jelinek DF, Witzig TE, Arendt BK A role for insulin-like growth factor in the regulation of IL-6 responsive human myeloma cell line growth. J Immunol 159:487, 1997 31. Jernberg DF, Pettersson M, Kishimoto T, et al: Heterogeneity in response to interleukin 6 (IL-6), expression of IL-6 and IL-6 receptor mRNA in a panel of established human multiple myeloma cell lines. Leukemia 5255, 1991 32. Jones JI, Clemmons DR: Insulin-like growth factors and their binding proteins: Biological actions. Endocr Rev 16:3, 1995 33. Jourdan M, Zhang XG, Portier M, et al: IFN-alpha induces autocrine production of IL6 in myeloma cell lines. J Immunol 1474402, 1991 34. Kawano M, Hirano T, Matsuda T, et al: Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332:83, 1988 35. King MA, Nelson D S Tumor cell heterogeneity in multiple myeloma: Antigenic, morphologic, and functional studies of cells from blood and bone marrow. Blood 733925, 1989 36. Kishimoto T, Akira S, Taga T: Interleukin-6 and its receptor: A paradigm for cytokines. Nature 258:593, 1992 37. Klein 8, Zhang XG, Jourdan M, et al: Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood 73:517, 1989 38. Klein B, Wijdenes J, Zhang XG, et al: Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia. Blood 78:1198, 1991 39. Klein B, Zhang XG, Lu ZY, et al: Interleukind in multiple myeloma. Blood 85:863, 1995
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40. Kooijman R, Hooghe-Peters EL, Hooghe R Prolactin, growth hormone, and insulinlike growth factor-I in the immune system. Adv Immunol63:377, 1996 41. LeRoith D, Wemer H, Beitner-Johnson D, et al: Molecular and cellular aspects of the insulin-like growth factor I receptor. Endocr Rev 16:143, 1995 42. Lichtenstein A, Tu Y, Fady C, et al: Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol 162248, 1995 43. Lu ZY, Zhang XG, Rodriguez C, et al: Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells. Blood 852521, 1995 44. Lu ZY, Gu ZJ, Zhang XG, et al: Interleukin-10 induces interleukin-11 responsiveness in human myeloma cell lines. FEBS Lett 377515, 1995 45. Middleton J, Amott N, Walsh S, et al: Osteoblasts and osteoclasts in adult human osteophyte tissue express the mRNAs for insulin-like growth factors I and I1 and the type 1 IGF receptor. Bone 16287, 1995 46. Murakami M, Narazaki M, Hibi M, et al: Critical cytoplasmic region of the IL-6 signal transducer, gp130, is conserved in the cytokine receptor family. Proc Natl Acad Sci U S A 88:11349, 1991 47. Neumann C, Zehentmaier G, Danhauser-Riedl S, et al: Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc, and its complex formation with Grb2 in the human multiple myeloma cell line LP-1. Eur J Immunol26379, 1996 48. Ogata A, Chauhan D, Teoh G, et al: IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. J Immunol 159:2212, 1997 49. Rubini M, Hongo A, DAmbrosio C, et al: The IGF-I receptor in mitogenesis and transformation of mouse embryo cells: Role of receptor number. Exp Cell Res 230:284, 1997 50. Schwabe M, Brini AT, Bosco MC, et al: Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains. J Clin Invest 942317, 1994 51. Schwarze MM, Hawley RG: Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin 6-mediated up-regulation of bcl-xL. Cancer Res 55:2262, 1995 52. Slifka MK, Antia R, Whitmire JK, et al: Humoral immunity due to long-lived plasma cells. Immunity 8:363, 1998 53. Splawski JB, McAnally LM, Lipsky PE: IL-2 dependence of the promotion of human B cell differentiation by IL-6 (BSF-2). J Immunol 144:562, 1990 54. Urashima M, Chauhan D, Uchiyama H, et al: CD40 ligand triggered interleukin-6 secretion in multiple myeloma. Blood 85:1903, 1995 55. Urashima M, Chen BP, Chen S, et al: The development of a model for the homing of multiple myeloma cells to human bone marrow. Blood 90:754, 1997 56. Vemino L, McAnally LM, Ramberg J, et al: Generation of nondividing high rate Igsecreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells. J Immunol 148:404, 1992 57. Vidriales MB, Anderson K C Adhesion of multiple myeloma cells to the bone marrow microenvironment: Implications for future therapeutic strategies. Mol Med Today 2:425, 1996 58. Westendorf JJ, Ahmann GJ, Armitage RJ, et a1 CD40 expression in malignant plasma cells: Role in stimulation of autocrine IL-6 secretion by a human myeloma cell line. J Immunol 152493, 1994 59. Westendorf JJ, Ahmann GJ, Greipp PR, et al: Establishment and characterization of three myeloma cell lines that demonstrate variable cytokine responses and ab produce autocrine interleukin-6. Leukemia 10:866, 1996 60. Westendorf JJ, Jelinek DF: Growth regulatory pathways in myeloma: Evidence for autocrine oncostatin M expression. J Immunol 1573081, 1996 61. Xu F, Gardner A, Tu Y, et al: Multiple myeloma cells are protected against dexamethasone-induced apoptosis by insulin-like growth factors. Br J Haematol97429, 1997 62. Xu FH, Sharma S, Gardner A, et al: Interleukin-&induced inhibition of multiple myeloma cell apoptosis: Support for the hypothesis that protection is mediated via inhibition of the JNK/SAPK pathway. Blood 92241,1998 63. Yaccoby S, Barlogie B, Epstein J: Primary myeloma cells growing in SCID-hu mice: A
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64. 65. 66. 67.
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model for studying the biology and treatment of myeloma and its manifestations. Blood 92:2908, 1998 Zhang XG, Klein B, Bataille R Interleukin 6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma. Blood 7411, 1989 B a n g XG, Bataille R, Widjenes J, et al: Interleukin-6 dependence of advanced malignant plasma cell dyscrasias. Cancer 69:1373, 1992 Zhang XG, Gu JJ, Lu ZY, et a1 Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130. J Exp Med 179:1337, 1994 Zubler RH: Key differentiation steps in normal B cells and in myeloma cells. Semin Hematol34 (1 Suppl 1) :13, 1997
Address reprint requests to Diane F. Jelinek, PhD Department of Immunology Mayo Clinic 200 1st Street SW Rochester, MN 55905
0889-8588/99 $8.00
+ .OO
MECHANISMS OF MYELOMA CELL GROWTH CONTROL Diane F. Jelinek, PhD
Multiple myeloma is a progressive and fatal disease characterized by the accumulation of malignant plasma cells in the bone marrow. Normal plasma cells have long been viewed as end-stage cells that have lost the potential for further proliferation. The longevity of these cells has been estimated to range from several days to several weeksz6 Recent evidence, however, raises the possibility that, under some circumstances, plasma cells may survive for a much longer period of time.52Myeloma cells differ considerably from normal plasma cells in that the tumor cells retain the capacity for proliferation and their lifespan may exceed that of normal counterpart cells, perhaps as a result of overexpression of several antiapoptotic cellular Although tumor expansion in vivo is critically dependent upon both growth and death rates, this article focuses on several aspects of myeloma cell growth control. THE ROLE OF PROLIFERATION IN B-LYMPHOCYTE DIFFERENTIATION
Before discussing potential regulators of myeloma cell growth, it is essential first to review the role of proliferation in the terminal differentiation of human B lymphocytes into high-rate immunoglobulin-secreting cells. The various stages of normal B-lymphocyte activation, proliferation, and differentiation are summarized in Figure 1. B-lymphocyte activation is initiated by engagement of membrane immunoglobulin. Following receipt of a variety of signals, including direct interaction with T lymphocytes and a number of cytokines, the activated B lymphoblast enters the cell cycle. Although the proliferative phase has often This work was supported by Grants CA62228 and CA64442 from the National Institutes of Health.
From the Department of Immunology, Mayo Clinic and Mayo Foundation, and Mayo Medical School, Rochester, Minnesota HEMATOLOGY/ ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 13 * NUMBER 6 * DECEMBER 1999
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Figure 1. Activation, proliferation, terminal differentiation, and programmed cell death are highly regulated events in normal B-lymphocyte maturation. The large dotted arrows emphasize the ability of immunoglobulin-secretingplasmablasts to proliferate.
been viewed as separate and independent from the differentiative phase, for the purposes of this article it is important to emphasize that immunoglobulinsecreting plasmablasts are also capable of continued proliferati~n.~~ Thus, not only is initial proliferation essential for preparing B lymphocytes to receive a differentiation signal, ongoing proliferation of the plasmablasts appears to be important in increasing the magnitude of the antibody response and in preparing the cell to differentiate into a high-rate immunoglobulin-secreting plasma ce11.56 Although B-cell proliferation is necessary for differentiation, it is also clear that terminal differentiation of B cells into end-stage plasma cells is accompanied by growth arrest. A variety of external and internal signals are probably involved in modifying gene expression in the developing plasma cell so that the end result is a nonproliferative cell with the capacity to secrete high levels of immunoglobulin. The precise mechanisms by which growth potential is lost as a consequence of terminal differentiation remain obscure. It is believed, however, that this loss results from the interplay between cellular signaling and activation of transcription factors important in regulating expression of genes whose products are specific to the differentiated phenotype. Figure 2 graphically displays the relationship between normal B-cell proliferation and differentiation into endstage plasma cells. Because myeloma cells retain proliferative capacity, albeit in many cases this is quite low, the author and colleagues have hypothesized that the genetic events that program the loss of proliferative potential have become uncoupled from the differentiative events that result in development of high-rate immunoglobulin-secreting cells. For example, a number of transcription factors, such as nuclear factor interleukin-6 (NF-IL,-6),13,l4 have been described as playing a role in the terminal differentiationof B lymphocytes. It is plausible that disregulation of such factors at the level of either expression or function could contribute to the proliferative ability of the malignant plasma cell. Of note, the speculative model shown in Figure 2 also suggests that growth cessation of normal plasma
MECHANISMS OF MYELOMA CELL GROWTH CONTROL
1147
Onset of end-stage differentiation?
4
Myeloma?
h
h
Figure 2. Model of the relationship between normal B-cell proliferation and differentiation and the hypothesized block of terminal differentiation in myeloma. The model suggests that immunoglobulin secretion rate per cell (dotted line) increases as the proliferative index (heavy solid line) decreases. By contrast, immunoglobulin-secreting myeloma cells retain the ability to proliferate at varying levels.
cells is a gradual process involving several changes in gene expression. In this context, the author and colleagues further speculate that the variable proliferative capacity (labeling index or LI) of myeloma cells observed among patients may similarly be explained if the transforming event occurs at variable places along the differentiation continuum. Given that myeloma cells retain proliferative potential, it becomes important to understand the mechanisms by which a variety of factors can affect the growth of myeloma cells. The difficulty of this task is highlighted by the significant heterogeneity, particularly in cytokine responsiveness, that exists among tumor cells obtained from different patients.', 31, 35, 59
INTERLEUKIN-6
Much of the current understanding of the growth characteristics of myeloma cells in vivo has been obtained by studying myeloma cell lines. Although myeloma cell lines are notoriously difficult to establish, this task has become somewhat easier since the demonstration that the cytokine interleukin (1L)-6 could promote the growth of myeloma cells in ~ i t r oThe . ~ ~biological relevance of this finding has been strengthened by demonstrations that elevated IL-6 levels are directly correlated with tumor burden, bone destruction, and other tumorassociated activities in myeloma patient^.^, 65 Indeed, administration of anti-IL6 antibodies has been shown to alleviate disease ~yrnptorns.~~ Interleukin-6 has also been shown to offer protection against chemotherapy-induced apoptosis.12, 51, Finally, a key role for IL-6 is further underscored by animal studies demonstrating that IL-6 is a crucial requirement for the development of B-cell Q
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neoplasms.u Collectively, these observations suggest that IL-6 plays a pivotal role in myeloma. The IL-6 receptor is a member of the hemopoietin receptor family and consists of two functional chains: (1) an 80-kD IL-&binding molecule (IL6R or gp80) whose expression is induced upon activation, and (2) a constitutively expressed 130-kD signal-transducing chain (g~130).3~ Binding of IL-6 to IL6R triggers IL6R association with and homodimerization of gp130. Although gp130 itself does not display intrinsic tyrosine kinase activity, several nonreceptor tyrosine kinases constitutively associate with the cytoplasmic domain of gp130. Moreover, several functional elements have been identified in the cytoplasmic domain of gp130, that is, box 1, 2, and 3 motifs that underlie the multiple signaling events downstream of receptor ligation." Transcriptional activation of target genes mediated by IL-6 is currently known to result from several signaling pathways, including the ras/MAP kinase (MAPK) and JAK/ STAT pathways downstream of receptor ligation."36 gp130 also serves as the signal transducing component in receptor complexes for leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), IL-11, and cardiotrophin-1.24Not surprisingly, therefore, OSM, LIF, and CNTF have also been reported to support the growth of myeloma cells.19,20, 66 The responsiveness of myeloma cells to these cytokines is more variable, however, probably because the ligand-binding receptor components for these cytokines are not as consistently expressed on myeloma cells as is the IL-&binding gp80 component. Effects of Interleukin-6 on Normal B Lymphocytes and Plasmablasts Compared with Myeloma Cells
As mentioned previously, the gp130 family of cytokines, particularly IL-6, appears to play a key role in the growth control of myeloma cells. Although cytokines that utilize the gp130 signal transduction molecule can trigger myeloma-cell proliferation, the effects of these cytokines on normal B lymphocytes is strikingly distinct. Thus, the author and colleagues have used an in vitro system that allows the generation of large numbers of plasmablasts, that is, proliferating immunoglobulin-secretingB cells, by coculture of normal human B cells with anti-CD3-activatedT cells.25, 56 After 5 days, plasmablasts with proliferative potential can be assessed for cytokine responsiveness. Table 1 demonstrates the effects of IL-6 and oncostatin M on both differentiation and proliferation of normal plasmablasts. As reported previously, IL-6 augments immunoglobulin secretion most effectively when IL-2 is also The author and colleagues reported for the first time the effects of OSM on normal B cellsm;it may be seen that OSM also markedly augments immunoglobulin secretion. By contrast, when these two cytokines are compared with IL-2 for their ability to support growth, both are observed to be completely without effect, even in the presence of IL-2 (Table 1). These results support the conclusion that IL-6, and perhaps other members of this cytokine family, support normal B-cell differentiation but do not support B-cell proliferation. Because of these striking differences between the effects of IL-6 on myeloma cells and on normal B cells or plasmablasts, there is a clear need to understand in greater detail the mechanisms that account for this differential responsiveness. The ability of both normal B cells and myeloma cells to respond to IL-6 and other members of this cytokine family suggests, however, that there may be alterations in gpl30-mediated signal transdudion in tumor cells. Definitive studies have not yet been carried out, but evidence in the literature suggests that the ras/MAPK pathway may be imp~rtant.~, 47 Although
MECHANISMS OF MYELOMA CELL GROWTH CONTROL
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Table 1. EFFECTS OF INTERLEUKIN-2, INTERLEUKIN-6 AND ONCOSTATIN M ON IMMUNOGLOBULIN SECRETION AND DNA SYNTHESIS BY PREACTIVATED NORMAL PERIPHERAL BLOOD PLASMABLASTS*
DNA Synthesis [3H]-Thymidine Incorporation (cpm x
Total Immunoglobulin (ng Ig/mL) Experiment No.
IL-2
Nil
IL-6
OSM
Nil
IL-6
OSM
1
-
1886 5996 1726 10,301
5245 >13,687 >14,882 215,868
2689 >14,000 3105 >19,931
0.2 9.6 0.5 21.3
0.2 10.5 0.6 21.3
0.1 9.5 0.8 22.7
2
+ +
*Highly purified normal human peripheral blood B cells were cocultured with mitomycin C-treated T cells and immobilized CD3 antibodies for 5 days before removal of T cells and reculture of purified preactivated B cells with various cytokines. Total immunoglobulin (IgM, IgG, and IgA) secretion was measured after an additional 5-day incubation. For assay of DNA synthesis, ['HI-thymidine incorporation was determined after 3 days. IL-2 = interleukin-2; IL-6 = interleukin-6; OSM = oncostatin M; - = negative; + = positive.
a number of therapeutic strategies are designed to interrupt extracellular binding of growth factors such as IL-6, it is also important to identify the intracellular events that sustain the aberrant growth of the tumor cells so that these molecules can be targeted as well. In Vivo Sources of Interleukin-6 in Myeloma Patients
As mentioned previously, IL-6 levels have been shown to correlate with tumor burden: This observation calls into question the in vivo source of the elevated IL-6. Although this was once considered a controversial issue, with some studies suggesting that the increased levels of IL-6 derived from paracrine sources and other studies suggesting that the elevated levels of this cytokine resulted from tumor-cell expression of IL-6, review of the literature suggests that both mechanisms are probably involved. Thus, a number of scenarios (summarized in Figure 3) could account for the increased IL-6 levels observed in patients during disease progression. It is important to emphasize that any or all of these mechanisms could be operative at any one time. The simplest model shown in Figure 3 is that of constitutive autocrine IL-6 expression. Although Kawano and colleagues3 were the first to suggest that myeloma cells themselves can express IL-6, others have disputed this suggestion, arguing instead that the IL-6 is expressed by contaminating stromal cells.37,39 The existence of myeloma cell lines expressing IL-6 argues against contamination as the source of IL-6.3l.59 It remains possible, however, that IL-6 expression by the myeloma cell line is not truly constitutive, but rather is induced as a result of cell-to-cell interactions that occur among the tumor cells themselves. Although not all myeloma cells express the IL-6 gene, normal B lymphocytes apparently lose the ability to express this gene on terminal differentiation into plasma cells.67It is possible, therefore, that the cells that do express IL-6 have undergone transformation at an earlier stage of maturation, or that tumor cells in patients exist at a variety of maturational stages, only some of which express IL-6.= At the opposite end of the spectrum is the possibility that IL-6 is constitutively expressed by nonmyeloma cells. Given the complexity of cell-to-cell inter-
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Figure 3. Speculative models of the mechanisms of IL-6 expression in myeloma. In the induced-paracrine and induced-autocrine models, the numbers opposite the arrows depict the hypothesized order of events.
actions in the bone marrow, this possibility is considered unlikely. Instead, a much more attractive mechanism is the induced-paracrine or induced-autocrine model. Evidence in the literature supports both these models. For example, the author and c011eagues~~ and others54have shown that engagement of cell-surface CD40 on myeloma cells results in autocrine IL-6 expression. In other studies, myeloma cells have been shown to induce IL-6 expression in stromal cells in a largely cell-contact-dependent manner?, 57 Although the complete set of molecules that regulate interactions among tumor cells and stromal cells remains to be elucidated, it is clear that the bone marrow provides a unique and optimal environment for the growth of myeloma cells. This environment has been difficult to mimic in vitro, and thus it has been a challenge to study the biology of the malignant plasma cell. A recently described scid-hu model system, however, has the potential to facilitate this analysis greatly.55, INSULIN-LIKE GROWTH FACTOR I
As discussed previously, the qualitative outcome of IL-6 signal transduction in myeloma cells is distinct from that in normal B lymphocytes and plasmablasts. There is considerable interest, therefore, in understanding the mechanisms by whch myeloma cells become growth-responsive to the IL-6 family of cytokines and in identifying potential biologic regulators of this altered response. During the course of such mechanistic studies, the author and colleaguesmand othersI8 demonstrated an important role for insulin-like growth factor (IGF) in the regulation of myeloma cell growth. Figure 4 compares the effects of IGF-I on the IL,-Mependent myeloma-cell line, ANBL-6,2swith the effects of IGF-I on normal B lymphocytes stimulated with the polyclonal activator StuphyZococcus uureus (SA). In contrast with the ability of IGF-I to stimulate ANBL-6 DNA synthesis directly and to augment IL-&stimulated proliferation significantly, IGF-I did not
MECHANISMS OF MYELOMA CELL GROWTH CONTROL
1151
Figure 4. A, ANBL-6 myeloma cells were cultured with or without IL-6 and in the presence or absence of varying concentrations of IGF-I. B, Highly purified, normal peripheral blood B cells were stimulated with or without the mitogen SA, in the presence or absence of the indicated cytokines. DNA synthesis was assayed on day 3 for the ANBL-6 experiment and on day 5 for the normal B-cell experiment. Data represent the mean of triplicate values.
directly stimulate normal B-cell proliferation, nor did it augment the activity of the known B-cell growth factor, IL-2, in the presence or absence of SA. These results suggest that IGF responsiveness may also represent a key difference between normal and malignant B cells. Other investigators have demonstrated that IGF-I may also facilitate myeloma cell longevity through its ability to prevent tumor cells from undergoing apoptosis.61 Insulin-like growth factor I and IGF-I1 have been shown to play a crucial role in the regulation of growth in a wide variety of cell types5,6, 32 Although expression of IGFs is relatively widespread during fetal development, expression of these factors in adults is moderately restricted.16Of interest, a number of cell types residing withn bone marrow are capable of expressing IGF-I in adults.17, 45 The growth-promoting properties of IGFs suggest that inappropriate expression of IGFs may contribute to loss of normal cell growth control. In fact, IGFs have long been recognized as important mitogens in many types of malignancies.6 The IGF-I receptor (IGF-IR) consists of an (Y and a p chain which are synthesized as a single-chain precursor followed by cleavage into the separate chains. The receptor complex is organized as a functional dimer of two cw/p complexes. The (Y subunit is entirely extracellular and is responsible for ligand binding; the p subunit is largely intracellular and is responsible for triggering downstream signaling events.41Because of the important role that IGFs play in development and growth regulation, IGF-IR gene expression is tightly regulated. In general, h g h levels of IGF-IR mRNA are observed embryonically; in adult tissues, however, the levels of IGF-IR mRNA are very low. Importantly, it has been shown that as little as a twofold difference in IGF-IR numbers per cell can qualitatively change the nature of cellular responsiveness from enhanced survival to pr~liferation.~~ In preliminary studies, the author and colleagues (Jelinek et al, unpublished observations) have consistently observed that both chains of the IL-6 receptor complex may be overexpressed in myeloma cells, and that the IGF-IR is overexpressed only in a subset of myeloma cells, particularly in
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Figure 5. Speculative model of the role of the IL-6 and the IGF-I receptor in myeloma cells. Myeloma cell overexpression of the IL-6 receptor is viewed as an initial event that may offer protection from apoptosis, particularly when acting in concert with low levels of insulinlike growth factor (IGF)-I receptor expression. Upon increased expression of the IGF-I-R, myeloma cells are hypothesized to gain direct responsiveness to either IL-6 or IGF.
patients in whom a direct IL-6 proliferative response is observed. These studies suggest the attractive hypothesis that control of both IL-6 and IGF-IR expression may become disregulated in myeloma, resulting in increased protection against apoptosis and a possible increase in the responsiveness of myeloma cells to growth factor. T h s hypothesis is graphically illustrated in Figure 5. In this model, it is hypothesized that increased levels of IL-6 receptors initially delay apoptosis of myeloma cells. After the IGF-IR levels are elevated, it is hypothesized that tumor cells acquire more aggressive growth characteristics. Definitive studies testing this hypothesis are currently in progress. OTHER CYTOKINES WITH GROWTH-REGULATORY EFFECTS ON MYELOMA CELLS
Given the heterogeneity in myeloma, it is perhaps not surprising that a relatively large number of cytokines have been shown to have growth-stimulatory or growth-regulatory effects on myeloma cells. Although an exhaustive review of all of these effects is beyond the scope of this article, several cytokines merit special mention. Myeloma cells have been shown to express receptors for IL-10 and also to respond to this cyt0kine.4~The mechanism of action of IL-10, however, may be indirect, because there is some evidence that IL-10-induced myeloma-cell proliferation may be caused by either the induction of IL-11 responsiveness" or by the induction of an autocrine OSM loop.2oIn contrast with the striking differences between the effects of IL-6 on normal B cells and plasmablasts and on myeloma cells, IL-10 has been shown to support normal B-cell proliferation.8 The response of myeloma cells to IL-10 may therefore represent the exploitation by tumor cells of the proliferative function of a normally expressed receptor, rather than the altered responsiveness that apparently occurs in members of the gp130 family of cytokines.
MECHANISMS OF MYELOMA CELL GROWTH CONTROL
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Recent studies have also demonstrated the ability of myeloma cells to express an autocrine hepatocyte growth factor (HGF)-c-met receptor ligand This autocrine growth loop was also shown to be present in fresh tumor cells from patients. The overall importance of this pathway in disease progression remains to be explained. Another observation that may prove to have a significant effect on the understanding of myeloma is that the fibroblast growth factor (FGF) receptor may be overexpressed or may contain activating mutations as a result of the translocation of this gene into the immunoglobulin heavy-chain gene 10cus.'~Because FGF is present in the bone marrow, inappropriate or elevated levels of FGF-receptor expression would have biologic consequences if this receptor conveys a proliferative or antiapoptotic signal. Finally, interferon-a (IFN-a) has also been reported to have interesting effects on myeloma cells. Interferon-a differs from the other cytokines that have been described, in that it has been reported to have significant activity either suppressing or promoting growth. Its activity in promoting growth is of great interest, because IFN-a has been commonly used as maintenance treatment for myeloma patients. Despite the relatively extensive clinical use of this cytokine in treatment of myeloma, there are reports suggesting that IFN-a may actually aggravate disease in vivo. Furthermore, some in vitro studies using either fresh cells from patients or established myeloma cell lines have shown that IFN-a may be modestly growth stimulatory for myeloma cells. There is evidence that this effect is caused by the induction of autocrine IL-6.33There is also compelling data, however, that IFN-a stimulation of myeloma-cell proliferation may be independent of autocrine IL-6.29The underlying mechanisms by which myeloma-cell proliferation is stimulated by IFN-a remain to be elucidated, although the author and colleagues have reported that this proliferation may involve differential induction of the p19 cyclin-dependent kinase inhibitor and cyclin D2.* Thus, the KAS-6/1 cell line that is growth-stimulated by IFN-a failed to express the p19 inhibitor on addition of the cytokine. Interferon-a did, however, induce expression of cyclin D2. The author and colleagues have recently demonstrated that IFN-a may stimulate MAPK activation in the KAS-6/ 1 cell line, but not in the IFN-a growth-inhibited cell line ANBL-6.3 The mechanism by which MAPK is strongly activated in KAS-6/1 cells but not in ANBL-6 cells remains unclear but is currently under investigation. It has been suggested that IFNa-mediated growth inhibition occurs as a result of this cytokine's ability to decrease the expression of the IL-6 The author and colleagues were able to demonstrate, however, that IFN-a-reduced expression of IL-6 receptors on both IFN-a-growth-arrested and IFN-a-growth-stimulated myeloma cell lines. Moreover, the decreased IL-6 receptors were still capable of signal transducti0n.2~The growth-inhibitory mechanism, therefore, is more likely related to the ability of IFN-a to induce the expression of cell cycle regulatory proteins.2 Identification of the mechanisms of IFN-a-mediated inhibition or stimulation of myeloma-cell growth may allow development of agents that are more universally effective than IFN-a. SUMMARY
By necessity, this article focuses on only a handful of molecules with demonstrated ability to affect growth of myeloma cells. The heterogeneity in growthfactor responsiveness has made formulation of a uniform hypothesis a daunting challenge. One trait that appears to be consistent among all myeloma patients is the uncoupling of the normally highly integrated relationship between terminal
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differentiation and loss of growth potential. Thus, the common feature in myeloma may not be the precise cytokines or cell-to-cell interactions that drive tumor-cell growth, but rather the underlying genetic traits that afford the tumor cell the ability to proliferate despite its relatively advanced stage of differentiation. The success of current strategies used to treat myeloma patients has thus far been somewhat limited, and in general, has only modestly prolonged survival. It is clear that successful treatment of this disease will require the development of new therapeutic agents aimed at the biochemical events that sustain the aberrant growth of the tumor cells. The knowledge of cell signaling, gene transcription, and cell growth and differentiation has expanded rapidly, and this information has provided a greater understanding of the cell biology of a variety of malignancies. Application of this information to the study of multiple myeloma, however, has thus far been relatively limited, primarily because the heterogeneity of the disease and the lack of appropriate model systems. Review of the literature, particularly over the last 5 years, reveals a significant number of exciting new findings in this field and the development of new model systems that are certain to yield greater insight into this devastating disease. References 1. Anderson KC, Jones RM, Morimoto C, et al: Response patterns of purified myeloma cells to hematopoietic growth factors. Blood 73:1915, 1989 2. Arora T, Jelinek DF: Differential myeloma cell responsiveness to interferon-a correlates with differential induction of ~ 1 9 " " ~ and cyclin D2 expression. J Biol Chem 273:11799, 1998 3. Arora T, Floyd-Smith G, Espy MJ, et al: Dissociation between IFN-a-induced anti-viral and growth signaling pathways. J Immunol 1623289, 1999 4. Barille S, Collette M, Bataille R, et al: Myeloma cells upregulate interleukin-6 secretion in osteoblastic cells through cell-to-cell contact but downregulate osteocalcin. Blood 86:3151, 1995 5. Baserga R, Rubin R Cell cycle and growth control. Crit Rev Eukaryotic Gene Expr 3:47, 1993 6. Baserga R The insulin-like growth factor I receptor: A key to tumor growth? Cancer Res 55:249, 1995 7. Bataille R, Jourdan M, Zhang XG, et al: Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflection of disease severity in plasma cell dyscrasias. J Clin Invest 84:2008, 1989 8. Benjamin D, Park CD, Sharma V Human B cell interleukin 10. Leuk Lymphoma 12:205, 1994 9. Billadeau D, Jelinek DF, Shah N, et al: Introduction of an activated N-ras oncogene alters the growth characteristics of the interleukin 6-dependent myeloma cell line ANBL-6. Cancer Res 55:3640, 1995 10. Borset M, Hjorth-Hansen H, Seidel C, et al: Hepatocyte growth factor and its receptor c-met in multiple myeloma. Blood 883998, 1996 11. Borset M, Lien E, Espevik T, et al: Concomitant expression of hepatocyte growth factor/scatter factor and the receptor c-MET in human myeloma cell lines. J Biol Chem 271:24655, 1996 12. Chauhan D, Kharbanda S, Ogata A, et al: Interleukind inhibits Fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells. Blood 89227, 1997 13. Chen-Kiang S, Hsu W, Natkunam Y, et ak Nuclear signaling by interleukin-6. Curr Opin Immunol5:124, 1993 14. Chen-Kiang S Regulation of terminal differentiation of human B-cells by IL-6. Curr Top Microbiol Immunol 194:189, 1994
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Address reprint requests to Diane F. Jelinek, PhD Department of Immunology Mayo Clinic 200 1st Street SW Rochester, MN 55905