Department of Biochemistry, St. Jude Children s Research Hospital, Memphis, Tennessee, USA

Transcription

1 Concise Review International Journal of Cell Cloning 7:68-91 (1989) Origins and Properties of Hematopoietic Growth Factor-Dependent Cell Lines James N. file, David Askew Department of Biochemistry, St. Jude Children s Research Hospital, Memphis, Tennessee, USA Key Words. InMrleukin 3 Hematopoietic differentiation Myeloid transformation * Zinc finger proteins Hematopoietic growth factors Signal transduction mechanisms Myeloid cell lines myb Abstract. Studies of the growth regulation, differentiation and transformation of myeloid cells have been greatly facilitated by the availability of a variety of hematopoietic growth factordependent cell lines. These cell lines have been isolated from long-term bone marrow cultures and myeloid tumors using interleukin 3 (IL-3) as a growth factor. Using growth factor-dependent cells, it has been shown that growth regulation by IL-3 involves binding to a high-affinity receptor of 140 Kd and activation of tyrosine phosphorylation. IL-3 binding is associated with a number of cellular responses which are required for maintenance of viability, including induction of transcription of the c-myc and ornithine decarboxylase (ODC) genes. In addition, IL-3 regulates the expression of transcription of the gamma T cell receptor locus. The properties of the IL-3-dependent lines are consistent with the hypothesis that they are transformed in their ability to terminally differentiate. In some of the cell lines, this transformation may terminally differentiate. In other of the cell lines, this transformation may be due to the altered expression of the c-myb gene. In other cell lines, transformation is associated with the activation of the expression of a navel gene, termed Evi-1, of the zinc finger family of transcriptional factors. Comparable transformation of erythroid lineage cells is speculated to be due to the activation of the expression of another navel gene termed spi-1. These studies have emphasized the value of well-characterized hematopoietic gmwth factordependent cell lines in advancing our understanding in the basic biology of myeloid cells. Introduction One of the major advances in the study of the growth and differentiation of hematopoietic cells has been the identification, purification and cloning of a variety of hematopoietic growth factors. The availability of hematopoietic growth factors has allowed the development of in vitro approaches to study normal Correspondence: James N. Ihle, Department of Biochemistry, St. Jude Children s Research Hospital, 332 North Lauderdale, Memphis, TN 38101, USA. Received January 17, 1989; accepted for publication January 17, /89/$2.00/0 oalphamed Press

2 IhleIAskew 69 differentiation. More importantly, the availability of appropriate growth factors has allowed the isolation of a variety of cell lines representing various stages of hematopoietic stem cell differentiation. These cell lines have been invaluable for studying the mechanisms by which hematopoietic growth factors regulate growth. In addition, these cell lines have allowed approaches to better understand the phenotypes of myeloid lineage transformation and to begin to identify the genes which are involved in transformation. Among the hematopoietic growth factors, interleukin 3 (IL-3) has been particularly useful for the establishment of growth factor-dependent cell lines. IL-3 was initially identified by its ability to induce the enzyme 20 alpha hydroxysteroid dehydrogenase (20aSDH), but was later shown to have a variety of effects on hematopoietic lineage cells [l-31. The broad spectrum of biological activities associated with IL-3 is due to its ability to support the proliferation and differentiation of early hematopoietic stem cells. IL-3 is predominantly a T cell-derived glycoprotein of 28 Kd which is produced following antigen or mitogen stimulation [3]. Perhaps the most important exception is the ability of the WEHI-3 myelomonocytic cell line to produce IL-3. The constitutive production of IL-3 by WEHI-3 cells is a consequence of the insertional activation of the IL-3 gene by the integration of the intracisternal A particle in the 5 region of the gene [4, 51. As noted below, conditioned media from the WEHI-3 cell line was an essential component for the establishment of a number of the growth factor-dependent hematopoietic cell lines. Because of the extensive literature dealing with the various hematopoietic growth factors, this review will focus on IL-3 and its use in the establishment of hematopoietic growth factordependent myeloid leukemia cell lines, the mechanisms by which IL-3 supports growth and finally the potential genes involved in the transformation of growth factor-dependent myeloid leukemia cell lines. Origins of Myeloid Lineage Cell Lines As with many cell lineages, the first myeloid lineage cell lines were isolated from tumors. From these early studies, it was clear that relatively few myeloid leukemias could be adapted for tissue culture and that adaption often required multiple in vivo passages. Nevertheless, from the efforts of a number of laboratories, cell lines such as the WEHI-3, M1, HL-60 and K562 were ultimately isolated. These cell lines were independent of the exogenous growth Edctors and had phenotypic properties of immature myeloid cells. The next era in the isolation of myeloid cell lines was due largely to the work of Dexter and Greenberger. Dexter developed in vitro culture conditions which would allow normal myeloid differentiation in the presence of an appropriate hematopoietic microenvironment [6, 71. These studies resulted in the definition of Dexter cultures or long-term bone marrow cultures in which bone marrow

3 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 70 cells could be used to establish a culture system consisting of stromal cells and non-adherent myeloid progenitor cells [6]. This culture system could be shown to support the differentiation of granulocytes and macrophages over considerable lengths of time in vitro, although the establishment of long-term cell lines was extremely rare. Using the Dexter culture system, Greenberger tried to adapt the system to study the mechanisms by which retroviruses induce leukemia [8, 91. These efforts initially involved examining the effects of infecting Dexter culture systems with retroviruses, which did not result in the isolation of cell lines but which did produce some detectable changes in the growth characteristics of the cultures. However, Greenberger observed that if the infected, non-adherent cells were transferred to WEHI-3-conditioned media, long-term cell lines could be readily established [8]. These lines had the curious property that they retained an absolute requirement for WEHI3conditioned media for growth. These observations were confirmed by Dexter [lo], with the exception that growth factor-dependent cell lines were established without requiring retroviral infection of the cultures. From these studies, a number of cell lines termed Factor Dependent Continuous cell lines, Eaterson Laboratories (FDC-P) were isolated. The lack of a requirement for retroviruses challenged the notion that the cell lines were transformed. From the studies of Greenberger and subsequently those of Dexter, a number of growth factor cell lines were isolated and have been widely distributed. The more common cell lines are listed in 'hble I. In an effort analogous to that of Greenberger, Heard et al. developed a longterm bone marrow culture model for retrovirus-induced leukemias [ll-131. Using Friend murine leukemia virus (MuLV)-infected long-term bone marrow cultures, they were able to identify three stages of culture growth over a one-year period. The first phase was characterized by the proliferation and normal differentiation of granulocyte precursors. The second stage was characterized by the clod growth of immature myelomonocytic cells which were dependent upon stromal cells or factors for growth. The final stage involved the acquisition of autonomous growth properties. From these studies, a series of growth factor-dependent and -independent cell lines were isolated. Unfortunately, detailed studies concerned with the specific growth factors required by the second-stage cells have not been presented. Perhaps the most intriguing IL-3-dependent cell lines described to date have been those isolated by Palacios et al. [14, 151 from bone marrow cells cultured in WEHI-3-conditioned media. These factor-dependent lines have been suggested to represent pro-t (C4, 'hble I) or pro-b cell lines (LIB AgA2, 'lhble I). The lines have no rearrangements of T cell receptor loci or immunoglobulin loci but, with differentiation in vivo, undergo rearrangements and differentiate along either the T cell lineage or the B cell lineage. Among the growth factor-dependent myeloid cell lines which have been identified, only a single example of the isolation of CSF-l-dependent cell lines has been reported [16]. The BACl cells were generated by transfection of SV40

4 ~ Ihlel Askew 71 nble I. Origins of hematopietic growth factordependent cell lines Cell line Conditions of isolation Origin Reference 32-D ~123 B6SUtAcl5 Ro cl 3-1 FDC-PI FDC-P2 NFS-58 NFS-60 DA-1 DA-3 c4 LIB AgA2 BACl BMC-7 M-07 TF-3 TALL-101 AML-193 MY4-11 Long-term marrow cultures Long-term marrow cultures Long-term marrow cultures Long-term marrow cultures Long-term marrow cultures CasBrM MuLV leukemia CasBrM MuLv leukemia MoLV-induced leukemia MoLV-induced leukemia Bone marrow cultures Bone marrow cultures SV-40-infected macrophages Long-term marrow culture Megakaryocytic leukemia Erythroleukemia T cell leukemia Myelomonocytic leukemia B/Myeloid leukemia C3H/HeJ B6.6 CD-1 Swiss DBAI2 DBAI2 (NFSxDBA)Fl (NFSxDBA)Fl BALB/c BALB/c (CBA/NxNIH)Fl MRL/LPR (BALB/cxA.CA)Fl (C57BLxBALBIc)Fl Human Human Human Human Human DNA into splenic adherent cells which were maintained in the presence of L cellconditioned media as a source of CSF-1. The two cell lines which were derived contained SV40 DNA. However, because of the low frequency and long latencies in obtaining lines, the role of the SV40 DNA is unclear. In independent studies, it was demonstrated that primary retrovirus-induced murine myeloid cell leukemias were dependent upon growth factors for proliferation in vitro and that continuous growth factordependent cell lines could be isolated from these tumors [18]. These studies resulted in the isolation of a number of IL-3-dependent lines which have been widely distributed and used for various studies. The NFS series of cell lines were isolated from primary tumors induced by the wild mouse ecotropic virus CasBrM MuLV [18] while the DA series of cell lines were isolated from Moloney leukemia virus-induced leukemias [19]. Several of the more commonly utilized cell lines are listed in Table I. More recent studies have demonstrated that human myeloid leukemias are also largely dependent upon hematopoietic growth factors for proliferation in vitro and, recently, the isolation of several long-term, growth factordependent cell lines has been described. The first description of such lines was from the laboratory of Rmem [23, 241 who reported the isolation of three growth factor-dependent cell lines. Subsequently, Avunzi et ul. obtained an IL-3-dependent leukemic cell line from a megakaryocytic leukemia [20] and Kitumuru obtained an IL-3- dependent cell line from an erythroleukemia [21]. During the isolation of hematopoietic cell lines, a number of important insights were made into the phenotypic properties that might be associated with in

5 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 72 vitro growth and transformation. In particular, the majority of human and murine myeloid leukemias are dependent upon growth factors for proliferation in vitro, suggesting that transformation does not ordinarily cause an alteration in growth regulation. Growth factor-dependent leukemias show varying abilities to terminally differentiate, suggesting that transformation has primarily affected the ability of normal progenitors to differentiate. An altered ability to differentiate, whether associated with growth factor dependence or independence, does not allow the routine establishment of long-term cell lines. This would suggest that a unique set of transforming events may be associated with the ability to proliferate indefinitely in vitro. This type of transforming event may be similar to the immortalization of fibroblasts for in vitro growth. In summary, it can be hypothesized that three types of transforming events may exist; namely, transforming events which alter the ability of progenitors to terminally differentiate, transforming events which abrogate the requirements for normal growth factors and transforming events which can immortalize cells for long-term growth in vitro. Morphological and Phenotypic Properties of IL-3-Dependent Cell Lines Irrespective of their origin, most murine, IL-3-dependent cell lines show a number of similarities. Morphologically, the most striking feature is the immature phenotype of the cells and the absence of any morphologically mature cells. Some typical examples are shown in Figure 1. Consistent with the morphology, the cell surface phenotypes are those expected from immature cells in myeloid differentiation. The pattern of expression of several lineage-specific markers is indicated in Table II. A number of the IL-3-dependent cell lines express the T cell and early myeloid lineage-associated antigen Thy-1. Indeed, IL-3 has been shown to induce the expression of Thy-1 on normal bone marrow cells [3], The macrophage lineage-specific markers (Mac-2, Mac-3 and c--) are not expressed, while markers which are associated with somewhat early stages of macrophagic (Mac-1) and granulocytic (myeloperoxidase, RB6-8C5) differentiation are occasionally expressed. Phenotypic properties associated with cells which are committed to the T cell lineage, such as rearrangement of T cell receptor loci, expression of Ly-1, Ly-2, "3 or c-lck are not observed. However, all the IL-3-dependent cell lines which we have examined contain transcripts from the non-rearranged T cell receptor y loci, and this expression is specifically dependent upon IL-3 [25]. As noted in 'hble 11, one cell line has also been identified which contains transcripts from the T cell receptor delta locus. The ability of IL-3-dependent cell lines to proliferate in response to other hematopoietic growth factors has been examined, and typical results are summarized in nble 111. It should be noted that there is considerable variation in the responses seen with independently passaged cell lines. For example, considerable variation exists in the response of a particular cell line to a particular growth

6 IhleIAskew 73 Table II. Phenotypic properties of IL-3-dependent myeloid leukemia cell lines Cell Line Lineage marker NFS-58 NFS-60 NFS-78 NFS-107 DA-I DA-3 DA-34 Thy-1 Mac-] Mac-2 Mac-3 Ly-1 Ly-2 RB6-8C5 T3 Myeloperoxidase T cell receptor: TCR /3 RNA TCR y RNA TCR 6 RNA TCR rearrangements c-hck c-lck C-qb c-fms c-kit ND = not determined - -I I+ - ND Table JII. Growth factor responsiveness of IL-3-dependent cell lines Growth factor NFS-58 NFS-60 NFS-78 NFS-107 DA-1 DA-3 DA-34 IL-1 IL-2 IL-3 IL-4 IL-6 G-CSF CM-CSF CSF-1 LIF ND = not determined

7 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 74

8 Ihle/Askew Fig. 1. Examples of IL-3-dependent myeloid cell lines. 75

9 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 76 factor. The basis for this is not known, although the ability to respond to a particular growth factor is a selectable trait. For example, IL-2-dependent variant cell lines have been selected from IL-3-dependent cell lines [26, 271, Mechanism in Growth Regulation The mechanisms by which hematopoietic growth factors regulate growth initially involves growth factor binding to high-affinity receptors. A4ngle class of high-affinity receptors for IL-3 has been shown to be present on IL-3-dependent cells [28, 291. The number of receptors varies with the cell line, but generally is in the order of 1,OOO-l0,OOO receptors per cell. By Scatchard analysis of binding data, most studies have indicated an apparent affinity of 1 X to 1 X lo- [28,30]. The expression of receptors for IL-3 is highly restricted and, in general, receptors have only been found on IL-3-dependent cell lines or factor-independent derivatives obtained from factor-dependent cells [28]. A number of groups have studied the biochemical properties of the IL-3 receptor by using cross-linking reagents [ In all the studies, a major IL-3 cross-linked moiety of approximately Kd has been detected indicating that the major IL-3 binding protein is a Kd protein. In addition, however, larger cross-linked species have been variably detected [30, 321. One study detected a second major cross-linked protein of 60 Kd in addition to a 75 Kd species [32]. The availability of a number of cell lines which require hematopoietic growth factors for growth in vitro has allowed studies to enable us to better understand the role of these factors in regulating growth. Perhaps the first, most important point that comes from these studies is that factors such as IL-3 are more appropriately termed survival or viability factors rather than growth factors. In particular, in the absence of IL-3 all the cell lines examined rapidly lose viability, such that after an initial hour phase, cells may lose viability at the rate of half the population every 2-4 hours. More importantly, it is not yet clear whether IL-3 has any effects on cell cycling. In studies with a number of the cell lines, removal of IL-3 does not detectably alter the rate of cell cycling, but rather only appears to affect the percentage of the cells which can complete cell division. Secondly, there does not appear to be a particular stage of the cell cycle at which the probability of losing viability in the absence of IL-3 is higher than another. Lastly, with the lines examined, exposure to IL-3 during the GI phase of the cell cycle is sufficient to maintain viability. An essential question is whether the requirements of cell lines for IL-3 are similar to those of normal cells. For example, it is conceivable that transformation has affected the mechanisms by which IL-3 regulates cell cycle progression. Unfortunately, there have been relatively few studies to address this question. From the studies which have been done, it is clear that IL-3 is required for the maintenance of the viability of intermediates in myeloid differentiation. In particular, when intermediates are cultured in the absence of IL-3, they rapidly lose viability comparable to the cell lines and do not terminally differentiate. However, it

10 IhlelAskew 77 has not been determined whether normal myeloid progenitors continue to cycle in the absence of IL-3. The functions that IL-3 might control and which are necessary to maintain viability are not known. One of the first studies to address this question noted that an adenosine triphosphate (ATP) generating system could partially replace the requirement of factor-dependent cells for IL-3 [34,35]. This observation and subsequent studies which demonstrated a rapid decrease in ATP levels following the removal of IL-3 [35, 361 suggested that one effect of IL-3 may be to maintain high ATP levels. The observations that IL-3 stimulates lactic acid production and that IL-3-dependent cells are sensitive to 2-deoxyglucose has lead to the suggestion [35] that IL-3 may maintain ATP levels by its effects on glycolysis, perhaps by increasing hexose transport. The biochemical links between the effects on ATP levels, glycolysis or hexose transport are not currently known. A potential role for protein kinase C (PKC) in IL-3 signal transduction was proposed from the observation that IL-3 stimulation resulted in the translocation of protein kinase C from the cytosolic fraction to the membrane fraction comparable to the response seen when the cells are exposed to phorbol myristic acid (PMA) [37]. Attempts have been made to reproduce these findings with partial success [38] or without success [39]. A role for protein kinase C in IL-3 signal transduction is unlikely, however, because neither PMA nor calcium ionophores alone or in combination can replace the requirement for IL-3 for optimal growth or maintenance of viability [38,40]. Secondly, down-regulation PKC activity by chronic exposure to PMA has no effect on the response to IL-3 [39]. Lastly, the pattern of protein phosphorylation seen in two-dimensional gels following stimulation of cells with IL-3 is completely different from the pattern seen following stimulation with PMA [39,41]. Several studies have also examined the effects of IL-3 binding on phosphatidylinositol turnover and Caz+ flux with variable results. In growth factor-dependent macrophages neither IL-3 nor CSF-1 was found to induce an increase in intracellular Caz+ or to increase phosphatidylinositol turn- Over [42]. In the studies of an IL-3-dependent myeloid cell line, stimulation with IL-3 had no effect on phosphatidylinositol turnover [43]. In contrast, one group has observed an effect of IL-3 on intracellular-free Caz One of the potential mechanisms of growth factor signal transduction is protein phosphorylation. Studies with IL-3-dependent cell lines have examined the pattern of total protein phosphorylation or, as described below, tyrosine phosphorylation. IL-3 has been reported to induce the threonine phosphorylation of a 68 Kd protein [45]. This svbstrate has not been seen in other studies [39, 41, 463. The phosphorylation of a 33 Kd substrate was detected in IL-3-dependent cells when stimulated with IL-3 but not with IL-4 or PMA. The sites of phosphorylation were not determined. The phosphorylation of this substrate has not been detected in other studies [39, 41, 451. A potential role for tyrosine phosphorylation in IL-3 signal transduction was initially proposed by the observation that tyrosine protein kinase containing oncogenes could abrogate the requirement of factor-dependent cells for IL-3 [43,

11 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 78 lsble IV. Cellular substrates of tyrosine phosphorylation Substrate Reference Phosphoamino acids Cellular localization 160 Kd 150 Kd 120 Kd 95 Kd 90 Kd 85 Kd 70 Kd Kd 51 Kd 38 Kd 28 Kd 41 39, , 39 41, ND Tyr, Ser ND ND ND ND Qr, Ser, Thr Tyr, Ser 'Qr, Ser Tyr, Ser ND Cytoplasmic Membrane ND Cytoplasmic Cytoplasmic ND Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic ND ND = not determined 47-51]. Based on these observations, studies examining IL-3-induced tyrosine phosphorylation have been performed. Since tyrosine phosphorylation is a minor component of protein phosphorylation, anti-phosphotyrosine antibodies have been used in Western blot analysis [41,52] or monoclonal antibodies against phosphotyrosine have been used to affinity purify phosphotyrosine-containing proteins [39]. With polyvalent rabbit antisera, Kbymu et al. [52] reported the phosphorylation of a 150 Kd membrane-associated glycoprotein in factor-dependent cells following stimulation with IL-3. The 150 Kd substrate was not detected in cells stimulated with IL-4, GM-CSF, IL-2 or TPA. Using a polyvalent antiserum, Morla et al. [41] demonstrated the phosphorylation of a series of cellular substrates mble IV). Under the conditions of their experiments, they did not detect a membraneassociated 150 Kd substrate, but did observe a cytoplasmic substrate of 160 Kd which was present in two IL-3-responsive cells, but not in two additional lines. They also detected cytoplasmic substrates of 95 Kd, 90 Kd, 70 Kd and 55 Kd which were found in all the cell lines examined. Using monoclonal antibodies to phosphotyrosine, Isfort et al. [39] detected a number of substrates for tyrosine phosphorylation in cells stimulated with IL-3. As indicated in 'bble IV, one of the major substrates was a membrane-associated 150 Kd protein which appears to be the same substrate detected by Kayasu et al. [52]. In addition, there were a number of major (70 Kd, 56 Kd, 38 Kd) and minor (120 Kd, 85 Kd, 51 Kd and 28 Kd) cytoplasmic substrates detected. Wo of these substrates (70 Kd, 56 Kd) appear to be identical to those detected by Morlu et al. [41j. The presence of a 140 Kd membrane-associated protein which is rapidly phosphorylated following IL-3 stimulation [39,52] suggested the possibility that it may be a component of an IL-3 receptor complex containing the 65 Kd protein detected in cross-linking studies. This question has been addressed by determining whether radiolabeled IL-3 becomes associated with a complex which can be iso-

12 IhleIAskew 79 lated with monoclonal antibodies against phosphotyrosine These studies have shown that approximately 25% of the iodinated IL-3 does bind in such a complex. Using glycerol gradients and cross-linking approaches, it was further demonstrated that the complex has an apparent molecular weight of 160 Kd and contains only IL-3 and the 140 Kd protein. These studies therefore indicate that the IL-3 receptor may be a 140 Kd membrane tyrosine protein kinas which is rapidly phosphorylated following binding of IL-3 and, therefore, would appear to have the properties of a number of growth factor receptors, including the receptor for CSF-1 [54]. It has been speculated that the major IL-3 binding protein of 65 Kd is a proteolytic degradation product of the 140 Kd protein which retains its membrane localization and ligand binding activity. A role for tyrosine phosphorylation has also been indicated in recent experiments [43], which demonstrated that transfection of IL-3-dependent cells with a vector capable of expressing the tyrosine kinase-containing epidermal growth factor (EGF) receptor made the cells growth-responsive to EGF. In the parental cells, IL-3 had no effect on phosphoinositide turnover, whereas in the transfected cells, EGF stimulation induced a rapid stimulation of phosphoinositide metabolism. In contrast, transfection of cells with v-erbb, an amino-terminal truncated EGF receptor which has constitutive tyrosine kinase activity, abrogated the requirements of any exogenous growth factors. Perhaps the strongest indication of the continual requirement for tyrosine phosphorylation comes from the studies with temperature-sensitive mutants of Abelson-MuLV. It was initially demonstrated that wild-type Abelson-MuLV could abrogate the requirements of IL-3-dependent cells for IL-3 [48, 551. However, these studies did not address the question of whether Abelson-MuLV was required for the establishment of factor-independent cells or whether the constitutive expression of tyrosine protein kinase activity was required for continued factor independence. Using temperature sensitive (ts) mutants, uclng et al. [50] demonstrated that factor-dependent cells required IL-3 for maintenance of viability at the nonpermissive temperature and were growth Ezctor-independent at the permissive temperature. Therefore, continued tyrosine protein kinase activity is required for abrogation of growth factor requirements. Gene Regulation and Maintenance of Viability Perhaps one of the most challenging problems is to determine why the factordependent hematopoietic cells lose viability in the absence of IL-3. Clearly, a variety of cellular functions can be envisioned to be required for viability, whereas a few, some or many of these functions may be dependent upon the events which are initiated following binding of IL-3 to its receptor. Some functions may affect enzyme systems and involve maintaining ATP levels as suggested above. However, it is clear that IL-3 also has an effect on gene transcription and that this plays an important role in the maintenance of viability.

13 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 80 One potential example of the complex sequence of events which is associated with maintenance of viability starts with the ornithine decarboxylase gene (ODC). Initially, it was demonstrated that the specific inhibition of ODC activity dramatically affected the proliferation of IL-3-dependent cell lines [56], indicating that the synthesis of polyamines is important for the growth of hematopoietic cells comparable to other cell types. It was subsequently demonstrated that IL-3 has a dramatic effect on the levels of ODC enzyme activity and of ODC RNA and may be transcriptionally regulating the expression of the ODC gene [57]. It had also been demonstrated that IL-3, like many growth fdctors, regulated the levels of transcripts for the c-myc gene in IL-3-dependent cells [57,58]. Curiously, however, following stimulation of cells with IL-3, ODC RNA appeared somewhat later, leading to the possibility that expression of the c-myc gene might be required for expression of the ODC gene. To address this question, a series of cell lines which had be& infected with retroviruses which constitutively expressed c-myc was examined. As noted below, in spite of the fact that these cells were still IL-3-dependent, the expression of the ODC gene became independent of IL-3. The observation that IL-3 regulated the levels of transcripts for the c-myc protooncogene suggested the possibility that the constitutive expression of c-myc might abrogate the requirement for IL-3. This question has been addressed in several studies [57,59,60] which demonstrated that the constitutive expression of c-myc from retroviral constructs could partially abrogate the requirement of cells for IL-3, but did not allow the establishment of long-term factor-independent cell lines. These results indicate that while IL-3-regulated expression of c-myc is an essential function for maintenance of viability, it is not the only necessary function. The next question is whether tyrosine phosphorylation is involved in the regulation of c-myc expression. This question has been addressed by examining the effects of ts mutants of Abelson on c-myc expression and demonstrating that cells infected with such mutants show a temperature dependence for expression of c-myc [61]. At the non-permissive temperature, IL-3 is required for c-myc expression, while at the permissive temperature, c-myc expression is independent of IL-3. The available data indicate one possible scenario for an effect of IL-3 on the maintenance of viability of hematopoietic cells. In particular, IL-3 binding to its receptor activates tyrosine phosphorylation, resulting in the phosphorylation of a number of cellular substrates. One or more of these substrates is then involved in an unknown mechanism in the regulation of the transcription of the c-myc gene. The expression of c-myc, in turn, affects the expression of the ODC gene, which affects the levels of ODC enzyme activity. Finally, ODC is required for the synthesis of polyamines which are necessary for the continued growth of the cells. This aspect, however, is likely to be only one component of a complex series of events by which hematopoietic cells are provided with all the cellular functions necessary for proliferation and maintenance of viability.

14 IhlelAskew 81 Mechanisms in the Transformation of IL-3-Dependent Cells As noted above, the transformation of hematopoietic cells may involve an alteration in growth factor requirements of the cells, an alteration in the ability to terminally differentiate, or an immortalization event which confers on the cells the ability to be continuously passaged in vitro. With the IL-3dependent cell lines which have been isolated from tumors or with IL-3-dependent cell lines isolated from long-term bone marrow cultures, it is relatively easy to study transformation as it affects the growth factor dependence of the cells. Not surprisingly, there are several ways in which growth factor dependency can be abrogated. Conceptionally, the easiest model for the abrogation of growth factor dependence is the acquisition of the ability of the cells to produce their own growth factor resulting in autocrine growth. Indeed, a number of examples have now been found in which IL3dependent cells, when selected for growth factor independence, have been shown to activate the expression of IL-3 [62]. Moreover, several studies have demonstrated that the introduction of retroviral vectors, capable of expressing hematopoietic growth factors to which the cells respond, can abrogate the requirement for exogenously added factors [ A second potential model for abrogating the requirement for growth factors might involve the activation of the expression of a receptor for a growth factor which is either produced by the cells or is present in serum and which can function in place of the activation of the IL-3 receptor. This type of model has not as yet been demonstrated. However, one study [43] has shown that the introduction of the EGF receptor into IL-3-dependent cell lines, can confer on the cells the ability to grow long-term in the presence of EGF, indicating the feasibility of this type of transformation. Another way of abrogating the requirement for IL-3 involves the activation of protein kinases which can bypass the requirement of the cells for the IL-3 signal transduction pathway. This mechanism has been shown in experiments which demonstrated that oncogenes such as Abelson [47,48], STC [67,68], v-fins [69], trk [39] and erbb [43] can abrogate the requirement for IL-3 in a non-autocrine mechanism. In contrast to numerous studies dealing with the transformation of hematopoietic cells as it affects their growth factor requirements, relatively few studies have addressed the mechanisms in the transformation of hematopoietic cells as it affects immortalization or differentiation. In particular, it is clear that some combination of transforming events is required for the induction of myeloid leukemias which have the ability to be established as continuous factor-dependent cell lines in vitro. Similarly, it is evident that there exists a series of events which allows the establishment of IL-3-dependent cell lines from long-term bone marrow cultures. It is equally clear that none of the existing transforming viruses which have been examined will allow the establishment of comparable cell lines from cultures of normal progenitors immediately following infection.

15 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 82 One approach to identifying the types of transforming events associated with the transformation of IL-3-dependent cell lines involves taking advantage of the mechanism by which replication-competent retroviruses induce transformation in vivo and in vitro. In particular, replication-competent retroviruses often induce transformation by inserting near or into cellular genes and altering their expression, thereby causing transformation. Therefore, the potential involvement of known proto-oncogenes can be examined by looking at IL-3-dependent cells for rearrangements of these genes. Alternatively, new cellular proto-oncogenes may be involved and can be identified by cloning sites of viral integrations and determining whether retroviral insertions often occur in a particular region and define a site of common viral integrations. Since viral integrations occur randomly, the observation of a hot spot for integrations might indicate the existence of a gene which, when activated, can result in transformation. This approach has been used to identify a number of new transforming genes in various cell types. IL-3-dependent cell lines, derived from retrovirus-induced leukemias, have been examined for viral insertions in a number of known oncogenes. No rearrangements have been detected in the K-rus, N-ras, H-rus, m s, abl, ers-1, ers-2, N-myc, L-myc, c-myc, c-fms or c-mf proto-oncogenes in approximately 40 IL-3-dependent cell lines. Among the known oncogenes, only rearrangements of the c-myb gene have been detected (NFS-60 cells, nble V). This was of particular interest because the v-myb gene has been shown to cause the transformation of myeloid lineage cells and to specifically alter the ability of the cells to differentiate without altering their growth factor requirements [70, 711. The rearrangement of the c-myb gene involved the integration of a complete CasBrM MuLV retrovirus into a 1.5 Kb EcoRI fragment [72]. This genomic fragment contains the sixth viral-related c-myb exon which encodes sequences found in the middle of c-myb. From the structure of the integration, transcription from the rearranged locus should result in a truncated transcript of approximately 2 Kb which terminates in the 5 viral long terminal repeats (LTRs) at the transcriptional termination site of the virus. As shown in Figure 2, the major transcript detected in NFS-60 cells is a 2 Kb transcript while the normal 4 Kb transcript is present at much lower levels. Since NFS-60 cells retain one normal allele and cells at a comparable stage of differentiation normally express c-myb, the predominance of the truncated transcript indicates that the integrated provirus also effects the levels of transcription of the locus or stability of the RNA. In addition, the major immunoprecipitable c-my6 product in NFS-60 cells has an apparent size of 45 Kd while the normal c-myb gene product is a protein of approximately 90 Kd. In a series of myeloid cell lines [73], viral integrations occurred in the 5 region of the c-myb gene, and the major transcripts from the rearranged locus start in the viral 5 LTRs and splice into the c-myb gene resulting in a 5 deletion [74]. In addition, splicing of the 3 region is altered and results in the introduction of intron sequences in the middle of the gene [75]. The common alteration associated with the activation of the gene is, therefore, a 3 truncation or alteration. IL3dependent myeloid leukemia cell lines have also been examined for viral

16 Ihlel Askew 83 'Igble V. Proto-oncogenes and common sites of viral integration involved in the transformation of myeloid cells Locus Properties of gene Cell lines with rearrangements CB-1 vim-3) Part of Evi-1 locus DA-3, DA-34 Evi-1 Zinc finger protein DA-1, NFS-58, NFS-78, NFS-60 NFS-48 C-qb Nuclear, DNA binding NFS-60 integrations in the common sites of integration, Fim-1, Fim-2, Evi-1, Evi-2, Mlvi-1, Mlvi-2, Pim-1 and Pim-2. Among these, viral integrations in the Evi-1 locus were initially found in three IL3dependent cell lines (NFS-60, NFS-48, NFS-78) [76], and subsequently, integrations were found outside of the 7 Kb EcoRI fragment which initially defined the Evi-1 locus in two additional lines (DA-1, NFS-58). The consequences of the viral integrations in this locus were examined by looking for transcription from regions near the sites of viral integrations. Using a probe which spanned the integration sites in NFS-60, NFS-78 and NFS-48, low levels of transcripts were detected in NFS-78 cells. This probe was used to isolate cdna clones from a lambda gtlo library made from polya+ RNA from NFS-78 and NFS-58 cells. The structures of two cdna clones were determined by restriction mapping and sequencing and consisted of viral and cellular sequences [77]. Probes for the cellular exon sequences were then used to isolate additional cdna clones. One clone contained no viral sequences and was used to further characterize transcripts from the locus. Using this probe, cell lines containing rearrangements in the Evi-1 locus were shown to express transcripts of 4-5 Kb, while myeloid cell lines without integrations contained no detectable transcripts. The sequence of a full-length clone contained one large open reading frame following a 5 ' non-coding region of approximately 500 base pairs which contained the sequences flanking the viral integration sites. The open reading frame contained a unique sequence which encoded a protein of 120 Kd. Comparison of the predicted amino acid sequence with the protein sequence data bank indicated that the protein contained several repeats of the Zinc finger motif. This structure was initially identified in TFIIIA, a frog transcription regulatory factor which controls the transcription of the ribosomal 5s genes during development and has been shown to be responsible for DNA binding activity [ Subsequently, a wide variety of transcriptional factors have been shown to contain Zinc fingers [81,82], As indicated in Figure 2, the Evi-1 locus gene product contains 10 repeats of a 28-amino acid repeat which is characterized by the placement of the histidines, phenylalanine, leucine and cysteines. Seven of the repeats occur in the amino terminus of the gene where there are six contiguous repeats separated from the first repeat by 25 amino acids. Three contiguous repeats are localized in the carboxyl region near a highly acidic domain which shares limited similarity to an acid do-

17 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 84 Fig. 2. Structure of the Zinc finger repeats within the Evi-1 gene. The amino acid sequences of the 10 finger repeat regions are shown in A. The amino acid positions of the repeats within the two domains of the protein are shown on the left and right. The consensus sequence for the finger regions is shown on the bottom. In B the schematic organization of the predicted protein is shown. The finger repeat regions are indicated by the cross-hatched boxes and correspond to the numbered repeats indicated in A for the two domains. The acidic domain is indicated by the solid boxed area. main in the c-myc gene and is a common feature of transcriptional regulatory proteins. Two IL-3-dependent cell lines were found to contain viral integrations in a common viral integration site termed the CB-1 locus. This locus was initially defined in our laboratory as a common site of integration in IL-3-dependent cell lines [83]. The restriction map of the locus, however, was found to be virtually identical to the restriction map published for thefim-3 locus [84], a common site of Friend MuLV integration in myeloid tumors and myeloid cell lines derived from long-term bone marrow cultures. Genetic mapping of the CB-1 locus localized it to chromosome 3 and showed that it was tightly linked to the Evi-1 locus. In addition, recent studies [85] have mapped thefim-3 locus to chromosome 3 and shown a close linkage to the Evi-1 locus. For these reasons, it is likely that the CB-1 andfim-3 loci define a related region for viral integrations. The close genetic linkage of the CB-1 lfim-3 locus and the Evi-1 locus suggested that these two loci might be associated with the activation of the same transform-

18 IhleIAskew 85 ing gene. However, restriction mapping and Southern blotting experiments with approximately 90 Kd of the CB-ll m-3 locus and 70 Kb of the Evi-1 locus have not indicated a physical linkage. To directly address this possibility, Northern blot analysis was performed with cdna probes of the Evi-1 locus. In these studies, cell lines (DA-3, DA-34) containing rearrangements of the CB-1 locus were found to express the Evi-1 gene product with RNA from myeloid cell lines containing retroviral insertions in the CB-ll m-3 locus and which contained no apparent rearrangements in the Evi-1 gene product, suggesting that retroviral insertions in either region cause activation of the transcription of the same transforming gene. Experiments are currently in progress to determine the physical relationship of the two loci. Spleen focus-forming virus induces an erythroleukemia in which the cells are dependent on erythropoietin for growth and undergo in vitro terminal differentiation normally. When these tumors are transplanted, occasionally continuous tumor cell lines, characterized by the lack of terminal differentiation, can be isolated. These observations suggested that a second transforming event specifically involves an alteration in the ability of the cells to differentiate. This second event has been shown to be associated with viral insertions into a unique common integration site termed spi-1 and the appearance of a new 4 Kb transcript from the locus [86]. The structure of this gene has not been reported. The above examples indicate the potential value of this approach and suggest that over the next several years it should be possible to identify a number of new transforming genes. By using appropriate cell lines, it should also be possible to focus on a specific phenotype of transformation and in particular to focus on genes that might alter differentiation. Ultimately, it will be important to develop retroviral vectors containing potential transforming genes and to specifically determine their effects on normal IL-3-supported differentiation in vitro. From these studies, it should also be possible to begin to identify the genes involved in regulation of normal differentiation. Conclusions A combination of advances have allowed the opportunity for studying the mechanisms by which myeloid cells grow and differentiate. The most important advances have been in the purification and cloning of the growth factors which are required to support growth and differentiation in tissue culture. The availability of the hematopoietic growth factors has, in turn, allowed the isolation of a variety of growth factor-dependent cell lines which can be used to study the mechanisms in growth regulation and the mechanisms in myeloid transformation. Considerable evidence is accumulating to suggest that IL-3 regulates growth through a 140 Kd cell surface binding protein which has many properties in common with the growth factor receptors of the tyrosine protein kinase family. Whether the IL-3 receptor has tyrosine protein kinase activity is not known, nor is it known

19 IL-3-Dependent Cell Lines: Growth Regulation and Transformation 86 whether the receptors for the other hematopoietic growth factors which support the proliferation of IL-3-dependent cells function through tyrosine phosphorylation. Within the next several years, it can be speculated that most of the growth factor receptors will be cloned and a detailed comparison can be made between their structure and function in growth regulation. Although the early events in IL-3 signal transduction appear to involve tyrosine phosphorylation, the consequences of this activity to the ultimate regulation of growth or more appropriately the maintenance of viability are not known. It can be speculated that IL-3 can be necessary for a variety of cellular functions any of which, when limiting, might result in cell death. Therefore, it may not be possible to define one essential pathway of signal transduction, but rather it may be necessary to focus on particular sequences of events which might contribute to cell growth. One such series of events is the expression of the ODC gene which clearly involves a number of cellular steps. Presumably, a number of other genes are equally important and will be equally complex in their regulation. Perhaps the most intriguing aspects, however, concern the mechanisms by which hematopoietic cells undergo the intricate sequences of differentiation that can be seen both in vivo and in vitro. Of more immediate accessibility is the question of the types of genes which may be specifically involved in transforming myeloid lineage cells and altering their ability to differentiate. As detailed above, the availability of a number of cell lines which are transformed and no longer undergo differentiation has allowed approaches toward identifying this type of transforming gene. Hopefully by studying this class of genes, it may be possible to gain some insights into the genes which normally control differentiation. With regard to the general area of cell cloning, it can be predicted that in several years a variety of transforming genes will have been identified which can either alter normal differentiation or immortalize myeloid cells for long-term culture in vitro. Thus, just as one can now reproducibly transform myeloid cells and abrogate growth factor dependence, we will be able to infect normal progenitors and arrest differentiation and subsequently transform the cells for long-term growth in vitro. References Me JN, Pepersack L, Rebar L. Regulation of T cell differentiation: in vitro induction of 20 alpha hydroxysteroid dehydrogenase in splenic lymphocytes from athymic mice by a unique lymphokine. J Immunol 1981;126: Ihle JN, Keller J, Oroszlan S, et al. Biological properties of homogenous interleukin-3. I. Demonstration of WHI-3 growth factor activity, mast cell growth factor activity, P-cell stimulating factor activity, colony stimulating factor activity and histamine producing cell stimulating factor activity. J Immunol 1983;131: Ihle JN. The molecular and cellular biology of interleukin-3. In: Cruse JM, Lewis RE Jr, eds. The Year in Immunology. New York: Karger, 1988 (in press).

20 Ihle/ Askew 87 4 Ymer S, Tucker WQ, Sanderson CJ, Hapel AJ, Campbell HD, Young IG. Constitutive synthesis of interleukin-3 by leukaemia cell line WEHI3B is due to retroviral insertion near the gene. Nature 1985; Ymer S, Tucker WQ, Campbell HD, Young IG. Nucleotide sequence of the intracisternal A-particle genome inserted 5 ' to the interleukin-3 gene of the leukemia cell line WEHI3B. Nucleic Acids Res 1986;14: Dexter TM, Allen TD, Lajtha LF. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977;91: Dexter TM, Spooncer E, Schofield R, Lord BI, Simmons P. Haemopoietic stem cells and the problem of self-renewal. Blood Cells 1984;10: Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RI. Demonstration of permanent factor-dependent multipotential (erythroidlneutrophilibasophil) hematopoietic progenitor cell lines. Proc Natl Acad Sci USA l983;80: Greenberger JS, Sakakeeny MA, Davis LM, Moloney WC, Reid D. Biologic properties of factor-independent nonadherent hematopoietic and adherent preadipocyte cell lines derived from continuous bone marrow culture. Leuk Res 1984;8: Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D. Growth of factor-dependent hemopoietic precursor cell lines. J Exp Med 1980;152: Heard JM. Fichelson S, Sola B, et al. Malignant myeloblastic transformation of murine long-term bone marrow cultures by F-MuLV: in vitro reproduction of alongterm leukemogenesis, and investigation of preleukemic events. Int J Cancer 1983; 32~ Heard JM, Fichelson S, Sola B, Martial MA, Varet B, Levy JF! Multistep virus-induced leukemogenesis in vitro: description of a model specifying three steps withiin the myeloblastic malignant process. Mol Cell Biol 1984;4: Heard JM, Sola B, Martial MA, Fichelson S, Gisselbrecht S. Long-term culture of bone marrow-derived preleukemic cells from F-MuLV-infected mice. Blood 1986; 68: Palacios R, Steinmetz M. IL-3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell 1985;41: Palacios R, Kiekr M, Brockhaus M, et al. Molecular, cellular, and functional properties of bone marrow T lymphocyte progenitor clones. J Exp Med 1987;166: Schwarzbaum S, Halpern R, Diamond B. The generation of macrophage-like cell lines by transfection with SV40 origin defective DNA. J Immunol 1984;132: Sakakeeny A, Greenberger JS. Granulopoiesis longevity in continuous bone marrow culhues and fixtordependent cell line generation: significant variation among 28 inbred mouse strains and outbred stocks. JNCI 1982;68: Holmes KL, Palaszynski E, Fredrickson TN, Morse HC III, Me JN. Correlation of cell-surface phenotype with the establishment of interleukin 3dependent cell lines from wild-mouse murine leukemia virus-induced neoplasms. hoc Natl Acad Sci USA 1985 ;82: Ihle JN, Rein A, Mural R. Immunological and virological mechanisms in retrovirusinduced murine leukemogenesis. In: Klein G, ed. Advances in Viral Oncology. Vol. 4. New York: Raven Press, l984: Avanzi GC, Lista P, Giavinazzo B, et al. Selective growth response to L-3 of a human leukaemic cell line with megakaryoblasfic features. Br J Haernatol 1988;69: Kitamura T, Tange T, Chiba S, et al. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3 or erythropoietin. J Cell Physiol 1988 (in press).