Expression of constitutively activated M-Ras in normal murine bone-marrow cells was sufficient to induce the factor-independent, in vitro growth and differentiation of colonies of macrophages and neutrophils, and the generation of immortal lines of factor-independent mast cells, and, upon in vivo injection of the transduced cells, a fatal mastocytosis/mast-cell leukemia. In contrast, expression of constitutively activated H-Ras in bone-marrow cells resulted in the in vitro growth, in the absence of exogenous factors, of colonies that contained only macrophages and of lines of cells resembling dendritic cells, and, upon in vivo injection of the transduced cells, a fatal histiocytosis/monocytic leukemia. Macrophages generated by bone-marrow cells expressing activated M-Ras or activated H-Ras differed morphologically, the latter appearing more activated, a difference abrogated by an inhibitor of Erk activation. Inhibition of either Erk or PI3 kinase blocked the capacity of both activated M-Ras and activated H-Ras to support proliferation and viability. However, inhibition of p38 MAPK activity suppressed proliferation of bone-marrow cells expressing activated H-Ras, but enhanced that of bone-marrow cells expressing activated M-Ras. Thus, expression of either activated M-Ras or H-Ras in normal hematopoietic cells was sufficient for transformation but each resulted in the generation of distinct lineages of cells.
M-Ras or R-Ras-3 is a recently described member of the Ras superfamily of small GTPases (Kimmelman et al., 1997; Matsumoto et al., 1997; Ehrhardt et al., 1999; Louahed et al., 1999; Quilliam et al., 1999). These function as molecular switches that cycle between GTP-bound, active, and GDP-bound, inactive, conformations. This process is regulated positively by guanine nucleotide exchange factors (GEFs) that are activated by extracellular stimuli and catalyse the exchange of bound GDP for GTP, and negatively by GTPase-activating proteins (GAPs) that interact with activated Ras proteins, catalysing hydrolysis of bound GTP back to GDP and thus their inactivation. M-Ras is widely expressed, occurring not only in brain and muscle but also in thymus, spleen and hematopoietic cell lines (Kimmelman et al., 1997; Matsumoto et al., 1997; Ehrhardt et al., 1999). Expression of constitutively active mutants of M-Ras resulted in morphological transformation of NIH-3T3 fibroblasts (Ehrhardt et al., 1999; Quilliam et al., 1999), inhibition of differentiation of C2 myoblasts (Matsumoto et al., 1997; Quilliam et al., 1999), the factor-independent survival and growth of interleukin-3-dependent cell lines (Ehrhardt et al., 1999; Louahed et al., 1999) and the oncogenic transformation of a mammary epithelial cell line (Ward et al., 2004; Zhang et al., 2004). M-Ras shares both upstream regulators and downstream effectors with p21 Ras (Ehrhardt et al., 2002), although it binds only weakly to some key p21 Ras effectors such as Raf-1 and RalGDS (Quilliam et al., 1999; Grill and Schrader, 2002). M-Ras has two downstream effectors that are not shared by H-Ras, RA-GEF-2 and MR-GEF, both of which are GEFs for Rap1 and Rap2 (Rebhun et al., 2000; Gao et al., 2001).
The best-studied members of the Ras superfamily are the classical p21 Ras proteins, H-, N- and K-Ras4B and K-Ras4A, which were originally identified as oncogenes. The p21 Ras proteins share 40–55% amino-acid sequence identity with other members of the Ras family of proteins that also includes M-Ras, R-Ras and Tc21 (or R-Ras2), RalA and RalB, and Rap1A, 1B, 2A and 2B. Activating mutations of p21 Ras occur in 25–40% of all human cancers (Bos, 1989). Three effector pathways, involving Raf-1, RalGDS and phosphatidylinositol 3-kinase (PI3K) have been implicated in Ras-induced cellular transformation. The Raf/MEK/Erk pathway has been shown to be essential for cell proliferation (White et al., 1995) and, in mouse NIH 3T3 fibroblasts, its activation is sufficient for transformation (Cowley et al., 1994; White et al., 1995), although this is not the case in human cells, rat cells or in other types of murine cells (Levings et al., 1999; Hamad et al., 2002; Pruitt et al., 2002; Gupta et al., 2003). Instead, activation of the RalGDS/Ral pathway (Urano et al., 1996; Wolthuis et al., 1997; Wolthuis and Bos, 1999) appears sufficient for transformation of human cell lines (Hamad et al., 2002). PI3K (Pacold et al., 2000) and its downstream effector Akt are necessary for p21 Ras-meditated transformation (Rodriguez-Viciana et al., 1997; Sheng et al., 2001). Other effectors of p21 Ras include AF-6, Nore-1, Rin-1 and PKC, but their biological roles are less well-understood (Campbell et al., 1998; Gille and Downward, 1999; Shields et al., 2000; McFall et al., 2001).
Although mutations of p21 Ras, particularly of N-Ras, occur in some 25% of myeloid leukemias (Bos, 1989), relatively little is known about their functional significance. Expression of activated H-Ras in bone-marrow cells has in general not led to the generation of myeloid leukemias but instead to transformation of lymphoid cells (Hawley et al., 1995; Darley et al., 1997, 2002; MacKenzie et al., 1999). Myeloproliferative disorders developed in 60% of mice reconstituted with murine bone-marrow cells expressing activated N-Ras leukemias (MacKenzie et al., 1999). However, these were heterogeneous in terms of their histology and occurred with long latency, suggesting that secondary mutations of other genes were needed to cause disease. There is no information on the ability of other members of the Ras subfamily to transform lymphohemopoietic cells.
Here, we have expressed a constitutively active mutant of M-Ras (Q71L M-Ras) in primary murine bone-marrow cells, and compared it with a constitutively active mutant of H-Ras (G12V H-Ras) in terms of its ability to support the growth, survival and differentiation of hemopoietic cells in the absence of exogenous growth factors, and to generate cells that were leukemogenic when transplanted into mice. We found that, whereas both activated M-Ras and activated H-Ras supported cellular survival and proliferation and the factor-independent formation of colonies of differentiated cells, these differed in their lineages or activation state, reflecting differences in the intracellular signals activated by the two Ras proteins. Moreover, when transplanted into syngeneic mice, bone-marrow cells transduced with activated M-Ras reproducibly generated a lethal mastocytosis and mast cell leukemia, whereas those expressing constitutively active H-Ras gave rise to histiocytosis and monocytic leukemias.
Expression of a constitutively active mutant of M-Ras is sufficient to support the survival and growth of normal bone-marrow cells in the absence of exogenous growth factors
We used the Q71L-activating mutation of murine M-Ras rather than the G22V mutation, as we observed that the former mutant was more strongly activated, with a greater portion in the GTP-bound state (Ehrhardt A and Schrader JW, unpublished data). We transduced bone-marrow cells from Balb/c mice (previously treated with 5-fluorouracil (5-FU) to increase the frequency of stem cell/progenitor cells) with bicistronic retroviral vectors expressing GFP and either Q71L M-Ras or G12V H-Ras, or GFP alone as controls. Both Ras proteins incorporated an epitope from c-Myc at their N-termini to allow accurate comparisons of their relative expression levels. Retrovirally infected bone-marrow cells were selected by culture in the presence of puromycin for 2 days. Immunoblotting of cell lysates of retrovirally infected bone-marrow cells with a monoclonal antibody to the myc-tag demonstrated that activated Q71L M-Ras and G12V H-Ras were expressed at comparable levels (Figure 1a). We then washed the cells and cultured them in either the presence or absence of exogenous growth factors in liquid cultures. In the absence of exogenous growth factors, cells transduced with the vector expressing GFP alone had died by day 3. However, cells transduced with vectors expressing either Q71L M-Ras or G12V H-Ras survived and proliferated rapidly. After 1 week, cultures of bone-marrow cells expressing Q71L M-Ras contained a heterogeneous population of GFP+ cells that included large blasts, small lymphocyte-like cells, neutrophils, monocytes, megakaryocytes and mast cells (Figure 1b). A layer of adherent, spindle-shaped macrophages (discussed below) developed at the bottom of the dishes. Cultures of cells expressing activated H-Ras developed adherent layers of macrophages, with nonadherent cells occurring in clumps. There were intriguing differences between the macrophages developed under the influences of activated M-Ras or H-Ras, which are described in more detail below. These results suggested that expression of either activated M-Ras or H-Ras in hemopoietic stem/progenitor cells supported their survival and proliferation and was sufficient to replace these functions of growth factors.
Activities of both Erk and PI3K are critical for the proliferation of bone-marrow cells expressing constitutively active Q71L M-Ras
Hemopoietic growth factors support the survival and growth of normal hemopoietic cells through a common set of intracellular signals that include activation of Ras proteins (Satoh et al., 1991; Duronio et al., 1992; Ehrhardt et al., 2004), the Erk, JNK and p38 MAP kinases (Welham et al., 1992; Foltz et al., 1997; Foltz and Schrader, 1997) and PI3 kinase (Gold et al., 1994). Moreover, inhibition of the activities of MAP kinases or PI3 kinase by specific chemical compounds inhibited the growth factor-dependent survival and proliferation of hemopoietic cells (Rausch and Marshall, 1999; Karlsson et al., 2003). We therefore asked whether the Erk and PI3K pathways were required for the survival and proliferation of bone-marrow cells expressing activated M-Ras or H-Ras in the absence of exogenous growth factors. To investigate the importance of Erk activity, we blocked its activation using the MEK1/2 inhibitor PD98059. We observed that bone-marrow-derived cells expressing Q71L M-Ras died in the presence of PD98059 (25 μ M). Interestingly, the effects of the same concentration of PD98059 on bone-marrow cells expressing G12V H-Ras were much less severe, with most of the cells surviving (Figure 2a). We postulated that this difference reflected the greater activation of the Erk MAP kinases by activated H-Ras than by activated M-Ras seen in nonhemopoietic cells (Quilliam et al., 1999; Kimmelman et al., 2000). Consistent with these data, levels of activated phospho-Erk were higher in the bone marrow-derived cells expressing G12V H-Ras than in those expressing Q71L M-Ras (Figure 2b). Given that expression of G12V H-Ras induced stronger activation of Erk than did Q71L M-Ras, the weaker effect of PD98059 on the survival of cells expressing activated H-Ras probably reflected the fact that, even after proportionate inhibition of the majority of Erk activity, sufficient remained to support the survival of many cells. Bone-marrow cells expressing either activated M-Ras or activated H-Ras failed to survive in the presence of Ly294002 (Figure 2a), indicating that PI3K activity was critical for the ability of either Q71L M-Ras or G12V H-Ras to support survival. These observations indicate that both Erk and PI3K activities were necessary for the factor-independent proliferation supported by expression of either Q71L M-Ras or G12V H-Ras. They suggest that expression of activated M-Ras, like that of activated H-Ras, was sufficient to provide the necessary levels of activation of Erk and PI3K pathways for the generation of cells including macrophages. However, the higher levels of Erk activation seen with expression of H-Ras led to the constitutive activation of the macrophages.
Differences in the role of p38 MAP kinase in the factor-independent proliferation of bone-marrow cells expressing constitutively active Q71L M-Ras or G12V H-Ras
All true hemopoietic growth factors activate p38 MAP kinase (Foltz et al., 1997), another member of the MAP kinase family, activated in hemopoietic cells downstream of activated H-Ras (Lin et al., 1995; Chen et al., 2000; Grill and Schrader, 2002), and M-Ras (Grill et al. unpublished). We used a specific inhibitor of p38MAPK, SB 203580, to examine the role of p38MAPK. As shown in Figure 2c, SB 203580 (5 μ M) inhibited the factor-independent proliferation of cells expressing G12V H-Ras by about 50%. In contrast, SB 203580 (5 μ M) enhanced threefold the factor-independent proliferation of bone-marrow cells expressing Q71L M-Ras. Thus, p38 MAPK activity had opposing effects in cells expressing activated H-Ras or M-Ras, enhancing growth driven by G12V H-Ras, but suppressing that driven by Q71L M-Ras.
Activated Q71L M-Ras or activated G12V H-Ras drive the factor-independent growth and differentiation of morphologically distinct types of macrophages
The adherent cells that were generated in the absence of exogenous growth factors in cultures of cells expressing G12V H-Ras were typical activated macrophages. They had a characteristic ‘fried egg’ appearance, and were round, highly vacuolated, and tightly adherent to the plastic (Figure 3). In contrast, the adherent cells that expressed Q71L M-Ras were spindle-shaped and less well-spread on the plastic. However, they were clearly also macrophages, in that they were Mac-1 positive and readily phagocytosed yeast, in the process becoming activated and assuming an activated appearance closer to that of the macrophages expressing activated H-Ras (data not shown). To test the notion that these differences might relate to the greater levels of Erk activation in cells expressing activated H-Ras as compared with those expressing activated M-Ras, we investigated the effects of addition of PD98059. In control cultures containing only the solvent DMSO (Figure 3), macrophages expressing Q71L M-Ras exhibited their typical, spindle-shaped morphology, but in the presence of PD98059 (25 μ M), they died. In contrast, most macrophages expressing activated G12V H-Ras survived in the same concentration of PD98059. However, they did undergo a marked change in morphology, losing their well-spread, highly vacuolated appearance, and assuming the elongated, spindle-shaped morphology typical of the macrophages expressing Q71L M-Ras. These results suggest that the constitutively activated state of the macrophages expressing activated H-Ras that distinguished them from macrophages expressing activated M-Ras depended on the higher levels of Erk activity stimulated by G12V H-Ras expression.
Expression of activated M-Ras in hemopoietic progenitor cells is sufficient to support their proliferation and differentiation to macrophages and neutrophils
The observation that expression of activated M-Ras or H-Ras in bone-marrow cells resulted in extensive proliferation followed by the generation of differentiated macrophages suggested that the cells affected by expression of activated M- or H-Ras were hemopoietic stem or progenitor cells. We therefore performed colony-forming assays to directly test whether expression of activated M-Ras was sufficient to promote the growth, survival and differentiation of normal hemopoietic progenitor cells. After retroviral transduction and puromycin selection as before, 5000 retrovirally transduced cells were seeded into 1 ml cultures gelified by the inclusion of methylcellulose. Parallel cultures were set up in the presence or absence of a mixture of growth factors (IL-3, IL-6, SLF and GM-CSF). As expected, in the absence of added growth factors, bone-marrow cells expressing wild-type M-Ras or GFP alone failed to form colonies. In contrast, cells expressing either Q71L M-Ras or G12V H-Ras formed colonies. Thus, expression of activated M-Ras, like that of activated H-Ras, was sufficient to permit the survival and growth of colonies from single hemopoietic progenitor cells. Cells expressing G12V H-Ras formed twice as many factor-independent colonies as those expressing Q71L M-Ras (Figure 4a) and the colonies were larger. In the presence of a mixture of growth factors, bone-marrow cells expressing GFP alone, wild-type M-Ras or Q71L M-Ras formed equivalent numbers of colonies, but those expressing G12V H-Ras formed fewer colonies (Figure 4a), suggesting that signals from activated H-Ras suppressed the proliferation of a subset of progenitor cells, or that the combination of signals from activated H-Ras and growth factors was inhibitory.
As predicted from the results of the liquid cultures, examination of the cells in the colonies that grew as a result of expression of either activated M-Ras or H-Ras revealed macrophages, (Figure 4b). However, in the former case, neutrophils were also present (Figure 4b). Thus, while activated M-Ras and H-Ras shared the ability to promote the survival and growth of hemopoietic progenitor cells and their differentiation to macrophages, only activated M-Ras supported differentiation to neutrophils.
Expression of constitutively active M-Ras in bone-marrow cells reproducibly generates factor-independent mast cell lines
We continued to passage the cultures of bone-marrow cells expressing constitutively active M-Ras or H-Ras proteins to determine whether expression of these proteins promoted immortalization and the emergence of cell lines. When bone-marrow cells expressing constitutively active M-Ras were cultured in the absence of exogenous growth factors, the nonadherent cells outgrew the adherent macrophages, and, by 4 weeks, had given rise to a relatively homogeneous population of cells that had all the hallmarks of mast cells. Thus, they expressed c-Kit and FcɛRI on their surface (Figure 5a) and exhibited cytoplasmic granules (Figure 5a) that were typical of mast cells. In four independent experiments, we obtained similar populations of factor-independent mast cells that continued to grow without apparent limit. In contrast, while parallel cultures of bone-marrow cells expressing GFP alone that were supplemented with a source of IL-3 also gave rise to homogeneous populations of mast cells, as expected, these stopped proliferating after 6–8 weeks and permanent lines were not obtained.
When bone-marrow cells expressing constitutively active H-Ras were cultured in the absence of exogenous growth factors, the dishes became covered within 7 days by adherent layers of activated macrophages (Figure 3). Nonadherent cells tended to form clumps and, in three experiments out of five, passaging of nonadherent cells gave rise to continuously growing lines. These nonadherent cells resembled dendritic cells, exhibiting multiple cytoplasmic processes and low levels of CD11c and MHC Class II molecules (Figure 5b).
The failure of bone-marrow cells expressing activated H-Ras to generate lines of mast cells when cultured in the absence of growth factors was intriguing. This was despite the fact that cultures of bone-marrow cells expressing activated H-Ras did generate mast cells when they were supplemented with a source of IL-3 (Figure 5c). However, we noted that these mast cells expressed much lower levels of GFP than did the macrophages and ‘dendritic’ cells that were generated in parallel cultures that had not been supplemented with a source of IL-3 (data not shown). This observation suggested that, in the presence of IL-3, there had been selection for the generation of mast cells that expressed relatively low levels of the bicistronic mRNA that encoded activated H-Ras and GFP. To investigate directly the levels of exogenous activated H-Ras relative to those of endogenous p21 Ras, we performed immunoblotting with an antibody to p21 Ras. As seen in Figure 5d, levels of exogenous activated H-Ras were markedly lower (about fivefold) in the mast cells grown in the presence of IL-3, than in macrophages or ‘dendritic’ cells generated in its absence. It can also be seen that, whereas in the macrophages or ‘dendritic cells’ the levels of exogenous activated H-Ras were about 5–10-fold higher than those of endogenous p21 Ras, in mast cells grown in the presence of IL-3, the levels of activated H-Ras were only 30–50% of those of the endogenous p21 Ras.
We then asked whether the levels of exogenous activated H-Ras present in the mast cells selected for by growth in IL-3 were sufficient to sustain their viability and growth in the absence of IL-3. When IL-3 was removed, the mast cells died within 96 h, suggesting that the levels of exogenous activated H-Ras present in these cells were insufficient to maintain viability. These observations suggested that, in the presence of IL-3, mast cells that expressed low levels of activated H-Ras and were still dependent on IL-3 for survival had a proliferative advantage over cells that expressed high levels of the activated H-Ras. Collectively, these observations suggest that the reason that bone-marrow cells expressing activated H-Ras did not generate lines of mast cells in the absence of IL-3 was that the levels of activated H-Ras that were required to maintain the viability of mast cells in the absence of IL-3 were incompatible with differentiation to the mast cell lineage. Our results with colony assays and liquid cultures showed that expression of activated M-Ras supported the factor-independent differentiation of macrophages, neutrophils and mast cells. In contrast, the only cells generated in the absence of exogenous IL-3 by bone-marrow cells expressing activated H-Ras were macrophages and cells resembling dendritic cells. However, expression of activated forms of either M-Ras or H-Ras was sufficient to reproducibly lead to the generation of immortal, factor-independent cell lines.
Expression of activated H-Ras, but not activated M-Ras, results in the autocrine secretion of IL-3
The expression of activated H-Ras in a mouse mast cell line PB-3c was reported to result in the autocrine production of IL-3 (Andrejauskas and Moroni, 1989; Nair et al., 1989). Therefore, to test whether expression of activated M-Ras or H-Ras in bone-marrow cells resulted in production of IL-3, we investigated the production of IL-3 by cells transformed with activated M-Ras (Figure 5a) or activated H-Ras (Figure 5b). We used the ability to support the growth and survival of the IL-3-dependent cell line BaF/3 cells as a sensitive assay for the presence of IL-3 in medium conditioned by cells expressing either activated M-Ras or H-Ras. We observed that the dendritic cells expressing activated H-Ras released a factor that diffused through a cell-impermeable membrane and supported the growth of BaF/3 cells in the absence of exogenous IL-3. In contrast, BaF/3 cells failed to survive when cocultured in the same system with the same number of mast cells expressing activated M-Ras (Figure 6a). Moreover, Baf/3 cells failed to survive when cocultured with mast cells expressing activated M-Ras (data not shown). As shown in Figure 6b, medium conditioned by dendritic cells expressing G12V H-Ras supported the growth of Baf/3 cells in a dose-dependent manner. In contrast, medium from cultures of mast cells expressing Q71L M-Ras did not. To confirm that the factor that supported the growth of the Baf/3 cells was IL-3, we tested the effects of inclusion of specific antibodies that effectively neutralized IL-3 (Figure 6c). The presence of these antibodies blocked completely the ability of medium conditioned by cells expressing G12V H-Ras to support the proliferation of BaF/3 cells, confirming that activity present in the conditioned medium produced by activated H-Ras-transformed cells was IL-3 (Figure 6b).
Consistent with the observation that cultures of bone-marrow cells expressing activated H-Ras generated IL-3, we observed that when we cultured cells expressing activated H-Ras in the absence of exogenous IL-3 but at high cell densities, we observed the emergence of a small sub-population of cells that exhibited low levels of GFP expression. Flow cytometric analyses revealed that these cells had the phenotype of mast cells, expressing c-Kit and the Fc receptor for IgE. Histochemical analyses confirmed that a small subpopulation of cells with the toluidine-blue-positive granules of mast cells was present (data not shown).
Transplantation of bone-marrow cells expressing constitutively active M-Ras or H-Ras gives rise to distinct neoplastic diseases
Our in vitro experiments demonstrated that expression of activated M-Ras or H-Ras was sufficient to abrogate the dependence of hemopoietic progenitor cells on hemopoietic growth factors for growth, survival and differentiation, as well as promoting the emergence of continuous, factor-independent cell lines. This suggested that, in vivo, bone-marrow stem or progenitor cells that expressed activated M-Ras might give rise to a neoplastic disease.
To investigate this possibility, we infected bone-marrow cells with vectors expressing GFP alone or GFP plus constitutively active M-Ras or H-Ras, selected for retrovirally transduced cells by culture for 48 h in the presence of puromycin, and transplanted the cells into groups of sublethally irradiated syngeneic mice. Mice injected intravenously with 1 × 106 cells expressing GFP alone remained healthy. In contrast, all mice injected with 1 × 106 bone-marrow cells expressing activated M-Ras or H-Ras developed obvious signs of morbidity, although the kinetics and nature of the diseases differed in each case.
Mice injected with cells expressing activated M-Ras became moribund 6–8 weeks after injection, when they were killed and dissected. We used a fluorescence-dissecting microscope to identify the location of the progeny of the injected bone-marrow cells that expressed activated M-Ras and GFP. All mice exhibited grossly enlarged spleens and livers that fluoresced brightly as a result of dense infiltration by GFP-positive cells. The latter were abundant in blood and also in the bone marrow, lungs and kidneys. In the gut, GFP-positive cells had infiltrated the microvilli of jejunum, rendering them brightly fluorescent. Staining of histological sections with toluidine blue confirmed that the liver, spleen, lung, gut and kidney had been infiltrated with large numbers of cells that exhibited the typical metachromatic granules of mast cells (Figure 7a). Flow cytometric analysis of cells from the spleen or bone marrow confirmed that the vast majority of the GFP-positive cells expressed c-kit and FcɛRI and thus had the phenotype of mast cells (Figure 7b). Thus, mice transplanted with bone-marrow cells expressing constitutively active M-Ras developed a systemic mastocytosis that affected multiple organs and, in the late stage of the disease, was associated with a mast-cell leukemia. We also transplanted cells from the enlarged spleens of primary recipients of bone-marrow cells expressing active M-Ras to sublethally irradiated mice. These secondary recipients developed a similar mastocytosis and leukemia of GFP-positive cells, demonstrating the presence in the donors of oncogenic cells with a capacity for extensive self-renewal.
Quite different results were obtained in mice that were injected intravenously with bone-marrow cells transduced with G12V H-Ras. These mice developed a much more acute disease and became moribund only 2 weeks after injection. These mice exhibited enlarged spleens and livers that were infiltrated with large numbers of GFP-positive cells and fluoresced brightly. Histological examination of spleens and livers showed infiltration by mononuclear cells that lacked cytoplasmic granules (Figure 7a). Flow cytometric analysis of cells from spleen of these mice (Figure 7b) showed that the major population of GFP-positive cells expressed Mac-1, with the majority of these also expressing CD8. GFP-positive cells were also present in the blood. When cells from the spleens of these mice were cultured in the absence of growth factors, they gave rise to GFP-positive macrophages as well as nonadherent GFP-positive cells with extensive cytoplasmic processes that resembled dendritic cells. In summary, while mice injected with bone-marrow cells expressing activated M-Ras developed a mastocytosis, mice injected with bone-marrow cells transduced with activated H-Ras developed a malignant histiocytosis and monocytic leukemia.
Our results demonstrate that expression of activated M-Ras in hemopoietic stem/progenitor cells was sufficient to permit their survival, growth and differentiation in the absence of exogenous growth factors. In this respect activated M-Ras shared properties with activated H-Ras, although there were clear and intriguing differences in the types of differentiated cells that were generated. Likewise, while in both cases expression of the activated Ras protein appeared to be sufficient for immortalization so that, upon transplantation to mice, bone-marrow cells expressing activated mutants of either M-Ras or H-Ras gave rise to neoplastic diseases, there were striking differences in the cell lineages involved, presumably reflecting the differences in the signals downstream of M-Ras and H-Ras.
The cells generated in short-term cultures and colony assays are likely to be the progeny of transduced committed progenitors. In contrast, those cells generated from the continuous cell lines or leukemias must have been the progeny of either transduced stem cells, or, transduced progenitor cells that had acquired an abnormal capacity for self-renewal and had been immortalized.
The observation that the colonies supported by signals from activated M-Ras contained both macrophages and neutrophils whereas those supported by signals from activated H-Ras contained only macrophages, is consistent with evidence that expression of activated H-Ras in bipotential granulocyte macrophage-progenitors inhibited neutrophil differentiation (Darley and Burnett, 1999). Other evidence supports a role for p21 Ras proteins in the differentiation and maturation of monocytes (Skorski et al., 1992; Hibi et al., 1993; Maher et al., 1994; Jin et al., 1995).
In short-term liquid cultures, activated M-Ras resulted in the generation of a variety of cell types, suggesting that it supported the survival and proliferation of multiple types of progenitors and did not inhibit the generation of any particular lineage. In contrast, our observation that expression of activated H-Ras supported the generation of mainly macrophages and dendritic cells suggests that it inhibited the generation of other types of cells. Such an inhibitory effect may account for the failure of activated H-Ras to support the generation of mast cells in the absence of exogenous IL-3. Thus, although in the presence of exogenous IL-3, cells transduced with activated H-Ras did generate mast cells, there was clearly selection against mast cells that expressed levels of activated H-Ras sufficient to support viability and growth without exogenous IL-3. Thus, those mast cells expressing activated H-Ras that were generated in the presence of exogenous IL-3 died when IL-3 was removed. The levels of activated H-Ras that were compatible with the generation of mast-cells in the presence of IL-3 were 30–50% of those of endogenous p21 Ras and were ∼5–10 times lower than the levels of activated H-Ras seen in dendritic cells generated in the absence of exogenous IL-3 (Figure 5d).
One candidate for the signaling pathway that accounted for this difference between H-Ras and M-Ras was the Erk pathway. We observed greater activation of Erk downstream of activated H-Ras than of M-Ras in hemopoietic cells (Figure 2b), agreeing with results in nonhemopoietic cells (Kimmelman et al., 1997; Matsumoto et al., 1997; Ehrhardt et al., 1999). Moreover, differences in levels of activation of Erk clearly dictated the obvious morphological difference between the macrophages generated from bone-marrow cells expressing activated M- or H-Ras (Figure 3). Thus, treatment with the MEK1 inhibitor, PD98059, converted the activated phenotype of macrophages expressing activated H-Ras to the spindle-shaped phenotype typical of macrophages expressing activated M-Ras.
That expression of constitutively active M-Ras in bone-marrow cells gave rise to lines of mast cells and systemic mastocytosis in vivo, is consistent with observations that expression of v-ErbB in bone-marrow cells led to a lethal mastocytosis (von Ruden et al., 1992), as we have recently shown that EGF induces strong activation of M-Ras (and relatively weak activation of H-Ras) (Ehrhardt et al., 2004). Likewise, consistent is the report of transformation of immature mast cells by the v-abl oncogene (Gurish et al., 1995). Expression of v-abl in hemopoietic cells is known to activate PI3K (Oki et al., 2002) and probably M-Ras, given that it activated p21 Ras (Danial et al., 1998). Moreover, the degree of activation of endogenous p21 Ras appears to have been modest, and thus was likely to be well below the levels of activated p21 Ras (equivalent to 30–50% of endogenous p21 Ras) that were compatible with mast cell differentiation in the presence of IL-3 (Figure 5c). The increased numbers of peritoneal and cutaneous mast cells in neurofibromin-1 (Nf1) haploinsufficient mice (Ingram et al., 2000, 2001) may also be consistent with our findings, as Nf1 is a Ras GAP that inactivates M-Ras (Ohba et al., 2000).
In others' hands, expression of activated H-Ras in primary bone-marrow cells usually resulted in transformation of lymphoid cells (Pierce and Aaronson, 1982; Dunbar et al., 1991; Hawley et al., 1995), although there are reports of effects on myeloid cells (Pierce and Aaronson, 1985; Maher et al., 1994; Hawley et al., 1995). The rapidity of the development (within 2 weeks) of the fatal histiocytosis/monocytic leukemias that we observed after transplantation of primary bone-marrow cells expressing activated H-Ras could account for our failure to observe the lymphomas seen in other studies (Pierce and Aaronson, 1982; Dunbar et al., 1991; Hawley et al., 1995), which took 2–3 months to arise.
It is not clear how expression of activated M-Ras protein or activated H-Ras in normal bone-marrow stem or progenitor cells reproducibly promoted the emergence of immortal, oncogenic cells. In other types of primary cells, expression of activated p21Ras alone is not oncogenic, at least in part because it induces senescence. Our results raise the possibility that, at least in a stem cell or committed progenitor cell, expression of activated M-Ras or H-Ras may be sufficient to enhance the probability of self-renewal and result in immortalization. Signalling pathways downstream of p21Ras have been reported to upregulate the levels and activity of Notch-1 (Weijzen et al., 2002), and expression of activated Notch-1 results in increased self-renewal of hemopoietic stem/progenitor cells (Varnum-Finney et al., 2000). Expression of either activated Ras protein may have enhanced DNA methylation and suppression of genes promoting senescence (MacLeod et al., 1995).
This is the first report on the effects of expression of activated M-Ras in primary cells. It will be of interest to see whether expression of activated M-Ras in primary cells of other types is sufficient to induce immortality. Given the relatively low ability of activated M-Ras to activate the Erk pathway that is known to be important in inducing senescence (Lin et al., 1998; Zhu et al., 1998), it is possible that activated M-Ras will be relatively poor at inducing senescence. In the course of preparation of this manuscript it has been reported that expression of activated p21 Ras in fibroblasts at levels equivalent to those of the endogenous allele was sufficient to induce limited transformation of primary fibroblasts (Tuveson et al., 2004). This phenotype included a lack of induction of senescence and the loss of contact inhibition, although, in contrast with our results in hemopietic cells, it failed to induce tumorigenesis or anchorage-independent growth. In our experiments, the levels of activated H-Ras that were sufficient to fully transform hemopoietic cells were higher, being ∼5–10-fold higher than the levels of endogenous p21 Ras. The levels of activated M-Ras that were sufficient for transformation of hemopoietic cells were approximately the same as those needed for transformation by activated H-Ras although, lacking antibodies specific for M-Ras, we were not able to compare these with those of endogenous M-Ras.
Our data provide some clues to the signals downstream of activated M-Ras that promoted the survival and growth of hemopoietic progenitor cells. PI3 kinase activity was essential for the growth and survival of primary hematopoietic cells expressing either activated H-Ras or M-Ras (Figure 2a), consistent with results in a myeloid cell line (Matsuguchi and Kraft, 1998), and in nonhemopoietic cells (Rodriguez-Viciana et al., 1997; Sheng et al., 2001). Our finding that p38MAPK activity was needed for optimal growth of cells expressing activated H-Ras contrasts with other observations suggesting that p38MAPK activity blocked transformation by activated p21 Ras (Chen et al., 2000; Pruitt et al., 2002). However, it is possible that p38MAPK activity was required for optimal secretion of IL-3 downstream of activated H-Ras (Nair et al., 1992), as p38MAPK has been implicated in the regulation of mRNA stability and translational efficiency (Chen et al., 1998; Ming et al., 1998, 2001; Winzen et al., 1999). Our finding that SB 203580 enhanced the proliferation of hematopoietic cells expressing activated M-Ras is consistent with the report that SB 203580 increased the proliferation of NIH3T3 cells transformed by activated M-Ras (Quilliam et al., 1999). Thus, it is plausible that expression of activated M-Ras or H-Ras is mimicking the action of growth factors in activating M-Ras and p21Ras (Satoh et al., 1991; Duronio et al., 1992; Louahed et al., 1999; Ehrhardt et al., 2004), MAP kinases (Welham et al., 1992; Okuda et al., 1994; Terada et al., 1995, 1997; Foltz et al., 1997; Foltz and Schrader, 1997; Rausch and Marshall, 1997, 1999; Kimmelman et al., 2000; Grill and Schrader, 2002) and PI3K (Gold et al., 1994; Kimmelman et al., 2000; Grill and Schrader, 2002).
Intriguingly, bone-marrow cells expressing activated N-Ras did not survive or proliferate in the absence of exogenous growth factors, and failed to give rise to leukemias when transplanted into mice (unpublished observations). This is consistent with the results of MacKenzie et al. (1999), who concluded that expression of activated N-Ras in murine bone-marrow cells had primarily an antiproliferative, proapoptotic effect, and observed that only some mice injected with bone-marrow cells transduced with activated N-Ras developed myeloproliferative diseases, and that only after long latent periods. It is also consistent with evidence that, expression of activated N-Ras in a hemopoietic cell line was insufficient to support growth and viability (Levings et al., 1999), and that, in human myeloid leukemias, mutations in N-Ras are a relatively late event in disease progression (Bashey et al., 1992). However, it is unclear why mutations in N-Ras are seen more commonly in human myeloid leukemias and preleukemias than are mutations in other p21 Ras family members (Bos, 1989). Certainly, given the evidence of differences in the mechanisms of p21 Ras-mediated oncogenesis in mice and humans, it will be important to repeat these studies in human cells and determine the frequency of mutations of M-Ras in human leukemias.
Materials and methods
Complementary DNA encoding mutated Q71L M-Ras or G12V H-Ras was cloned into a retroviral vector pMXpie that contains an IRES (internal ribosomal entry site)-GFP expression cassette and a puromycin-resistant gene (Ehrhardt et al., 1999). Recombinant mouse interleukin-6 (rmIL-6) was from Intergene (Purchase, NY, USA), recombinant mouse SLF (rmSLF) from R&D systems (London, Ontario, Canada), MethoCult M3134 from StemCell Technologies (Vancouver, BC, Canada), PD98059 and Ly294002 from Upstate (Lake Placid, NY, USA), and SB 203580 from Calbiochem (San Diego, CA, USA). The anti-β-actin antibody AC-15, 5-FU, and mouse thrombin were from Sigma (Louis, MO, USA). Citrated bovine plasma was from Pel Freeze (Arkansas). Antibodies specific for phospho-ERK were from New England Biolabs (Missisauga, Ontario, Canada) and for the myc-tag (9E10) or IL-3 were gifts from Dr H Ziltener (The Biomedical Research Centre, UBC, Vancouver, Canada).
Cell lines and cell cultures
Cells were grown at 37°C in humidified incubators gassed with 5% CO2. Cells of Bosc23, a human retroviral packaging cell line, were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen), supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μ M 2-mercaptoethanol, 100 μ/ml of penicillin, 50 μg/ml of streptomycin (StemCell Technologies, Vancouver, BC, Canada) and 10% heat-inactivated fetal calf serum (FCS). BaF/3 cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 2% of 10-fold concentrated WEHI-3B-conditioned medium as a source of IL-3.
Transfection and retroviral infection of bone-marrow cells
Bone-marrow cells were harvested from the femurs and tibias of Balb/c mice treated 3 days before with 5-FU (150 mg 5-FU/kg body weight). Red blood cells were lysed with ammonium chloride. For retroviral infection of bone-marrow cells, cells were precultured for 24 h in a cytokine cocktail (Guo et al., 2003) containing IL-3, SLF and IL-6, after which they were cocultured for 48 h in the presence of 6 μg/ml of polybrene with retrovirus-producing, γ-irradiated Bosc23 cells, generated as described before (Guo et al., 2003). Nonadherent bone-marrow cells were collected and cultured for another 48 h in fresh medium containing the same cocktail of cytokines with the addition of puromycin (Sigma) at 2 μg/ml. Retrovirally transduced bone-marrow cells were then cultured in the presence or absence of growth factors in liquid cultures or in colony assays.
Bone-marrow cells (5 × 103) expressing either activated M-Ras, H-Ras or GFP alone were seeded in triplicate in 3-cm dishes in 1.5 ml of 1% methylcellulose containing DMEM, 15% FCS, in the presence or absence of cytokines. After 7 days, the colonies were counted with a dissecting microscope. For the plasma-clot assay, 2 × 103 cells were suspended in DMEM supplemented with 14% citrated bovine plasma and 15 μg/ml thrombin, and were plated into 24-well plates. After 7 days, the clots were loosened from the wells and slipped onto glass slides. Filter papers were placed over the clots to absorb water, and the slides were air-dried and stained with Hema 3 Stain (Fisher Scientific, Nepean, Ontario, Canada).
The transduced bone-marrow cells were starved of serum and growth factors by incubation in DMEM at 37°C for 3 h, and were lysed in lysis buffer (Ehrhardt et al., 2001). The concentrations of proteins in the lysates were determined by BCA protein assay (Pierce). Samples of lysates containing equal amount of proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted with the indicated antibodies.
Flow cytometric analysis
2 × 105 cells were washed and then held for 15 min on ice in PBS with 1% BSA containing the indicated primary antibodies. Cells were washed twice in the same solution and held on ice with appropriate secondary reagents for another 15 min. After washing, cells were analysed using the FACScalibur instrument and Cellquest software (BD Biosciences).
Syngeneic Balb/c mice were γ-irradiated with 800 rads, and transplanted intravenously within 3–6 h with bone-marrow cells expressing either GFP alone, or GFP plus activated M-Ras or activated H-Ras. The recipient mice were monitored, and, when showing signs of ill-health, were killed and dissected. The mice and organs were examined for distribution of GFP-positive cells derived from the injected cells using a fluorescence-dissecting microscope. Organs were fixed in 4% paraformaldehyde for histological staining. Blood or cells dissociated from the spleen were analysed by flow cytometry or by staining of smears or Cytospin preparations.
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We thank Dr H Ziltener for providing the anti-IL-3 and 9E10 antibodies and Mrs H Merkens for technical assistance. This project was supported by a grant from the Canadian Institutes of Health Research (CIHR).
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Guo, X., Schrader, K., Xu, Y. et al. Expression of a constitutively active mutant of M-Ras in normal bone marrow is sufficient for induction of a malignant mastocytosis/mast cell leukemia, distinct from the histiocytosis/monocytic leukemia induced by expression of activated H-Ras. Oncogene 24, 2330–2342 (2005). https://doi.org/10.1038/sj.onc.1208441
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