Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Correspondence to: L Sachs, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: leo.sachs@weizmann.ac.il

The establishment of a system for in vitro clonal development of hematopoietic cells made it possible to discover the cytokines that regulate hematopoiesis. These cytokines include colony stimulating factors and others, which interact in a network, and there is a cytokine cascade which couples growth and differentiation. A network allows considerable flexibility and a ready amplification of response to a particular stimulus. A network may also be necessary to stabilize the whole system. Cells called hematopoietic stem cells (HSC) can repopulate all hematopoietic lineages in lethally irradiated hosts, and under appropriate conditions give rise to neuronal, muscle, and epithelial cells. Granulocyte colony stimulating factor induces migration of both HSC and in vitro colony forming cells from the bone marrow to peripheral blood. Granulocyte colony stimulating factor is also used clinically to repair irradiation and chemotherapy associated suppression of normal hematopoiesis in cancer patients, and to stimulate normal granulocyte develpment in patients with infantile congenital agranulocytosis. It is suggested that there may also be appropriate conditions under which in vitro colony forming cells have a wider differentiation potential similar to that shown by HSC. An essential part of the developmental program is cytokine suppression of apoptosis by changing the balance in expression of apoptosis inducing and suppressing genes. Decreasing the level of cytokines that suppress therapeutic induction of apoptosis in malignant cells can improve cancer therapy. Cytokines and some other compounds can reprogram abnormal developmental programs in leukemia, so that the leukemic cells differentiate to mature non dividing cells, and this can also be used for therapy. There is considerable plasticity in the developmental programs of normal and malignant cells.

Oncogene (2002) 21, 3284-3294 DOI: 10.1038/sj/onc/1205319

hematopoiesis; in vitro colony forming cells; cytokine network; stem cells; reprograming leukemic cells; apoptosis

Introduction

Normal embryonal development requires survival of essential cells, their multiplication and differentiation to mature cells of different lineages, and elimination of unnecessary, damaged or potentially harmful cells by programed cell death (apoptosis). These processes are also essential for the proper functioning of the body throughout adult life. An excellent model system to study these processes is the hematopoietic system. The formation of different types of blood cells, hematopoiesis, is essential for the development and survival of a normal individual. New blood cells belonging to different cell lineages are formed from stem cells during embryogenesis and throughout the lifetime of the adult to replace cells that have completed their life span. Abnormalities in the normal developmental program for blood cell formation result in hematological diseases including leukemia. Understanding the cellular and molecular controls of normal blood cell development makes it possible to answer questions about the origin and treatment of these diseases. This became a reality about four decades ago with the development of the first cell culture system in which normal blood cells could be cloned and made to develop to different cell lineages (Ginsburg and Sachs, 1963). This system allowed the identification, purification and gene cloning of the molecules (cytokines) that regulate hematopoiesis. Developmental programs include control of apoptosis, growth and differentiation potentials. We will discuss the role of the cytokine network in controlling the developmental programs in normal hematopoiesis and leukemia.

Clonal development of normal hematopoietic cells in culture

From the very early days of hematopoiesis research by using cells in culture, it was found that normal hematopoietic cells did not survive in culture unless certain feeder cells such as fibroblasts were added to the cultures (Ginsburg and Sachs, 1963). When feeder cells were added, the hematopoietic cells survived, multiplied and gave rise to colonies of mast cells (Figure 1) or granulocytes in various stages of differentiation. The cultures also showed differentiation to macrophages. The concluding sentence of the first study describing this system for the culture of normal mouse hematopoietic cells in liquid medium therefore stated 'The described cultures thus seem to offer a useful system for a quantitative kinetic approach to hematopoietic cell formation and for experimental studies on the mechanism and regulation of hematopoietic cell differentiation' (Ginsburg and Sachs, 1963). To make it simpler to distinguish and isolate separate colonies, the system using feeder layers was then applied to the development of hematopoietic cell colonies in semi-solid medium containing agar (Pluznik and Sachs, 1965) or methylcellulose (Ichikawa et al., 1966). The colonies obtained in agar or methylcellulose using these feeder layers contained macrophages, granulocytes (Figure 1), or both macrophages and granulocytes, in various stages of differentiation to mature cells. It was also shown that the hematopoietic cell colonies in culture can originate from single cells (Ginsburg and Sachs, 1963; Pluznik and Sachs, 1966; Paran and Sachs, 1969) and are therefore clones which develop from precursor cells that survived, multiplied and differentiated to mature cells in culture. The procedure for clonal growth of mouse macrophages and granulocytes in agar (Pluznik and Sachs, 1965) was then also used by others (Bradley and Metcalf, 1966), and was applied to the clonal development of normal human macrophages and granulocytes (Paran et al., 1970; Pike and Robinson, 1970) and other blood cell lineages (Stephenson et al., 1971; Metcalf et al., 1975; Gerassi and Sachs, 1976).

Discovery of colony stimulating factors

When hematopoietic cells were cloned in agar, another more solid agar layer was placed between cells of the feeder layer and the hematopoietic cells seeded for cloning. This showed that the inducer(s) required for the formation of macrophage and granulocyte clones were secreted by the feeder layer cells and can diffuse through agar (Pluznik and Sachs, 1965). This finding led to the discovery that the inducers required for the formation of macrophage and granulocyte clones are present in conditioned medium produced by the feeder cells (Ichikawa et al., 1966; Pluznik and Sachs, 1966). Such conditioned media were then used to purify these inducers and a similar approach was later used to identify the protein inducers for cloning of T and B lymphocytes (reviewed in Sachs, 1995, 1996).

In cells belonging to the myeloid cell lineages, four different proteins that induce cell viability and multiplication and can thus induce the formation of clones (colony-inducing proteins) have been identified (reviewed in Sachs, 1986, 1987a,b, 1990, 1995; Metcalf, 1985). The same proteins have been given different names. After they were first discovered in cell culture supernatant fluids (Ichikawa et al., 1966; Pluznik and Sachs, 1966), the first inducer identified was called mashran gm from the Hebrew word meaning to send forth with the initials for granulocytes and macrophages (Ichikawa et al., 1967). This and other colony-inducing proteins were then renamed macrophage and granulocyte inducers (MGI) (Landau and Sachs, 1971) and MGI-type 1(MGI-1). They are now called colony-stimulating factors (CSF) (reviewed in Metcalf, 1985; Sachs, 1987b) and one protein is called interleukin-3 (IL-3) (Ihle et al., 1982). Of these four CSFs, one (M-CSF), induces the development of clones with macrophages, another (G-CSF), clones with granulocytes, the third (GM-CSF), clones with granulocytes, macrophages, or both macrophages and granulocytes, and the fourth (IL-3), clones with macrophages, granulocytes, eosinophils, mast cells, erythroid cells, or megakaryocytes. The CSFs induce cell viability and cell multiplication (Lotem et al., 1991a; and reviewed in Sachs, 1987b, 1990; Sachs and Lotem, 1993) and enhance the functional activity of mature cells (reviewed in Metcalf, 1985). Cloning of genes from mice and humans for IL-3, GM-CSF, M-CSF and G-CSF have shown that these proteins are coded by different genes (reviewed in Clark and Kamen, 1987). Since the discovery of these CSFs, other cytokines that act on cells of the myeloid lineage have been found, including stem cell factor (SCF) (Witte, 1990), Flt3/Flk2 ligand (FL) (Lyman et al., 1993) and thrombopoietin (TPO) (Kaushansky, 1995), some of which can synergize with different CSFs. Granulocyte CSF (G-CSF) is now used clinically to repair irradiation and chemotherapy associated suppression of normal hematopoiesis in cancer patients, to stimulate normal granulocyte development in patients with infantile congenital agranulocytosis, and to induce migration of hematopoietic stem cells from the bone marrow to peripheral blood for stem cell transplantation (reviewed in Sachs, 1995, 1996)

The different cytokines carry out similar and sometimes overlapping functions that are mediated by a family of cytokine receptors. M-CSF, SCF and FL receptors have a related intracellular tyrosine kinase domain, whereas GM-CSF G-CSF, IL-3 and TPO receptors do not have a tyrosine kinase domain and recruit src-related cytoplasmic protein tyrosine kinases and JAK protein tyrosine kinases to transmit their signals intracellularly (reviewed in Kishimoto et al., 1994; Ihle, 1995; Taniguchi, 1995). Activated JAKs phosphorylate and activate cytoplasmic signal transducers and transcription activators (STATs) that translocate into the nucleus and activate gene expression (reviewed in Kishimoto et al., 1994; Ihle, 1995; Taniguchi, 1995). The ability of the different cytokines to carry out similar and sometimes overlapping functions, endows the hematopoietic system with sufficient flexibility to prevent complete hematopoietic failure when one or more of the cytokine genes or their signaling receptors are genetically targeted (reviewed in Lotem and Sachs, 1999). This flexibility also applies to the control of development and function of T lymphocytes and neuronal cells by different cytokines (reviewed in Lotem and Sachs, 1999) and presumably also to other cell types.

Differentiation inducing cytokines and a cytokine network

The development of clones with terminally differentiated, nondividing, mature cells such as granulocytes and macrophages from single precursor cells requires induction of cell viability, cell multiplication, and differentiation associated with the arrest of cell multiplication. How can one explain the ability of a cytokine to transmit signals that activate these distinct and apparently contradicting processes in the same cell? One possible explanation is that the cytoplasmic region of CSF receptors may have distinct domains, that recruit different signal-transducing molecules which induce the different processes by activating different transcription factors. Some support for this possibility was obtained in transfection experiments with certain factor dependent cell lines using G-CSF receptor constructs with different mutations in their cytoplasmic region. The results indicated that the N-terminal or C-terminal parts of the G-CSF receptor cytoplasmic region were sufficient to confer cell multiplication or cell differentiation, respectively, when transfected into these factor-dependent hematopoietic cell lines (Dong et al., 1993; Fukunaga et al., 1993). However, the finding that a growth signal provided to the same cells by GM-CSF or IL-3 did not lead to cell differentiation and even inhibited the G-CSF induced differentiation (Fukunaga et al., 1993), indicates that this system does not really represent the situation in normal hematopoietic cells.

We have taken a different approach and postulated the existence of a cytokine cascade in the signaling pathways for multiplication and differentiation induced by the CSFs. We looked for a cytokine that acts as a myeloid cell differentiation inducer but does not have CSF activity, and found such a protein which was called macrophage and granulocyte inducer-type 2 (MGI-2) (reviewed in Sachs, 1987b, 1990). Studies on amino acid sequence of purified MGI-2 and myeloid cell differentiation inducing activity of recombinant interleukin 6 (IL-6) showed that MGI-2 is IL-6 (Shabo et al., 1988), and it was suggested that there are presumably other hematopoietic cell differentiation inducers. Studies on myeloid leukemic cells have identified other differentiation inducing proteins with no CSF activity, including leukemia inhibitory factor (LIF) (reviewed in Sachs, 1995, 1996). Another cytokine, interleukin 1 (IL-1), also induced differentiation in some clones of myeloid leukemic cells, and this is mediated by induction of IL-6 (Lotem and Sachs, 1989a). IL-6 and IL-1 induce viability and differentiation without apparently inducing cell multiplication in normal myeloid precursors (Lotem and Sachs, 1989b, 1990). Oncostatin M (OSM) and interleukin 11 (IL-11), which use the same cell surface signal transducing protein, gp130, that is used by IL-6 and LIF (reviewed in Kishimoto et al., 1994; Taga and Kishimoto, 1997), also induce myeloid differentiation (Taga and Kishimoto, 1997) without CSF activity.

The existence of CSFs and distinct differentiation inducing cytokines led to the prediction, that CSFs which induce growth and non-CSF cytokines which induce differentiation act in a cytokine cascade that ensures effective coupling of growth and differentiation. This was confirmed by showing that all four CSFs, GM-CSF, G-CSF, M-CSF and IL-3, can induce in normal hematopoietic precursors the production of IL-6 which does not induce the formation of colonies but can induce myeloid precursor cells to differentiate (Lotem and Sachs, 1982, 1983 and reviewed in Sachs, 1987b, 1990, 1995, 1996). The ability of IL-3 to induce production of IL-6 in normal hematopoietic precursor cells was then confirmed by others (Schneider et al., 1991). The use of various factor dependent hematopoietic cell lines has also shown the ability of IL-3 and GM-CSF to induce IL-6 (Hültner et al., 1989). This cytokine cascade is part of a network that includes other cytokines. Production of specific cell types has to be induced when new cells are required and has to stop when sufficient cells have been produced. This requires an appropriate balance between inducers and inhibitors of development. The network of interactions between hematopoietic cytokines (Figure 2) (reviewed in Sachs, 1995, 1996) thus includes cytokines that can function as inhibitors of hematopoietic cell multiplication such as tumor necrosis factor (TNF). Another inhibitory cytokine, transforming growth factor 1 (TGF1), which is part of this network (Lotem et al., 1991b), selectively inhibits the activity and the production of some CSFs and ILs (Lotem and Sachs, 1990). Other cytokines which may also participate in the network include interferons / and , interleukins 4, 10 and 13 (IL-4, IL-10, and IL-13) which exert negative effects on the responsiveness of hematopoietic myeloid cells to certain CSFs and on the production of these cytokines (reviewed in Sachs, 1996). This network has also to be taken into account in the clinical use of cytokines. What can be therapeutically useful may be due to the direct action of an injected cytokine, or to an indirect effect due to other cytokines that are switched on in vivo.

Experiments with myeloid leukemic cells have shown that a cytokine network is maintained in these leukemic cells including the ability of IL-6 and GM-CSF to positively autoregulate their own expression (Shabo et al., 1989a) and induce expression of other cytokines including M-CSF, GM-CSF, IL-6, IL-1, IL-1, TNF and TGF1 (Shabo et al., 1989a,b; Lotem et al., 1991b). However, unlike in normal precursors, the CSFs did not induce IL-6 in leukemic cells that can be induced to differentiate by adding IL-6 (Lotem and Sachs, 1982, 1989b). The hematopoietic system is thus controlled by a cytokine network that allows amplification of signals and indirect activity of cytokines by inducing production of other cytokines, and parts of this network are maintained in leukemic cells.

The network of interactions between cytokines that regulate the hematopoietic system has yet another level of control involving cytokine receptors. For example, induction of differentiation with IL-6 induced expression of surface receptors for GM-CSF, M-CSF and IL-3 which were not expressed or expressed at low levels before differentiation (Lotem and Sachs, 1986, 1989b). TGF-1 inhibited cytokine production (Lotem and Sachs, 1990) and expression of cytokine receptors (Jacobsen et al., 1991), thus ensuring the feedback inhibition of hematopoiesis at the level of both cytokines and receptors. Expression of cytokine and cytokine receptor genes can also be separately regulated. This is shown in IL-6 treated leukemic cells that develop IL-3 and M-CSF receptors during differentiation but do not express IL-3 or M-CSF, and before differentiation have G-CSF receptors and do not express G-CSF (Lotem et al., 1991b). Other leukemic cells which can be differentiated with GM-CSF are induced to express IL-6 and M-CSF (Lotem et al., 1991b), but do not express receptors for IL-6 (Lotem and Sachs, 1987) or M-CSF (c-fms) (Lotem et al., 1991b).

A network of cytokine interactions allows considerable flexibility. It also allows a ready amplification of response to a particular stimulus such as bacterial lipopolysaccharide (reviewed in Sachs, 1990). A separate regulation of cytokine genes and cytokine receptor genes provides cells with an additional control system to prevent uncontrolled autocrine growth. In addition to the flexibility of this network both for the response to present-day infections and to different types of infections that may develop in the future, a network may also be necessary to stabilize the whole system.

Differentiation potential of hematopoietic cells

The discovery of CSFs and the other cytokines in the network have identified the regulators of differentiation of hematopoietic precursor cells to mature cells of different hematopoietic cell lineages. The bone marrow which contains hematopoietic precursor cells in the adult that can be assayed by in vitro colony formation, also contains cells which have been called hematopoietic stem cells (HSC) that can repopulate all hematopoietic lineages in lethally irradiated hosts (reviewed in Dzierzak et al., 1998; Weissman, 2000). This ability to repopulate lethally irradiated hosts is the basis for bone marrow transplantation. The finding that G-CSF and GM-CSF can induce the migration of HSCs from the bone marrow to the peripheral blood (reviewed in Gazitt, 2001), has simplified the procedure for stem cell harvesting for transplantation. This shows that like the hematopoietic in vitro colony forming cells, HSCs also respond to CSFs and both types of cells are induced to migrate to the peripheral blood by G-CSF and GM-CSF (Horsfall et al., 2000; Gazitt, 2001).

All cells in the body are derived from a single fertilized egg. Embryonic stem (ES) cells derived from the inner cell mass of the blastocyst have a very broad differentiation potential and can also give rise to hematopoietic cells (Kennedy et al., 1997). The ability of HSCs to repopulate all hematopoietic lineages in lethally irradiated hosts raises the question whether their differentiation potential is restricted to hematopoietic cells, or can extend even further to other non-hematopoietic cell types in a manner similar to ES cells. Experiments using bone marrow cells enriched for HSCs have shown that under appropriate conditions they can differentiate in vivo to glial cells in the brain (Eglitis and Mezey, 1997), skeletal muscle (Ferrari et al., 1998), epithelial cells such as liver hepatocytes during liver regeneration (Petersen et al., 1999; Alison et al., 2000; Lagasse et al., 2000; Theise et al., 2000) and to myocytes, endothelial cells and smooth muscle cells in regenerating infarcted myocardium (Orlic et al., 2001). Even a single injected HSC showed a multi-lineage differentiation potential to epithelial cells in liver, lung, gastrointestinal tract and skin (Krause et al., 2001). It has also been shown that neuronal stem cells (Bjornson et al., 1999) and muscle satellite cells (Jackson et al., 1999) can differentiate to myeloid and lymphoid blood cells in irradiated hosts. Various stem cells therefore show considerable plasticity and can reprogram their gene expression in response to signals produced at sites such as regenerating tissues, which presumably have the right combination of different cytokines, and can thus display a multipotential differentiation capacity (Figure 3). It has been proposed that in vitro colony forming cells have a more limited differentiation potential than HSC (reviewed in Weissman, 2000). It can however be suggested that in vitro hematopoietic colony forming cells might also behave like HSCs under appropriate conditions. The ability of some types of apparently differentiated somatic cells to develop into a complete adult when placed into an enucleated unfertilized egg (reviewed in Gurdon and Colman, 1999), indicates that cells with an apparently restricted differentiation potential can reprogram gene expression under the right conditions and show totipotent differentiation. It will be interesting to determine whether hematopoietic precursor cells including HSCs can show such a totipotent differentiation.

Reprograming of gene expression in malignant cells: control of leukemia by cytokines and other compounds

Leukemic cells have abnormal developmental programs. This raises the question whether leukemic cells can be reprogramed to regain normal behavior. We found that there are myeloid leukemic cells that can be induced to differentiate to non dividing mature granulocytes and macrophages by different cytokines (Paran et al., 1970; Fibach et al., 1972; Lotem and Sachs, 1977; Shabo et al., 1988; and reviewed in Sachs, 1978, 1986, 1987a,b, 1995, 1996). This showed that these leukemic cells can be reprogramed to behave like non malignant cells. It was also found that different clones of myeloid leukemic cells have different blocks in the ability to be induced to undergo differentiation by cytokines (Fibach et al., 1973; Lotem and Sachs, 1974). Analysis of the pattern of protein expression by two dimensional gel electrophoresis in normal myeloid precursors and clones of myeloid leukemic cells that can be induced to differentiate has shown, that certain cytokine induced protein changes in the normal cells were constitutively expressed in the leukemic cells (Liebermann et al., 1980; Hoffman-Liebermann et al., 1981). It was suggested that the change from inducible to constitutive expression of these proteins leads to the loss of cytokine dependence for cell viability and growth in these leukemic cells (Liebermann et al., 1980; Sachs, 1980; Hoffman-Liebermann et al., 1981). Comparison of the pattern of protein expression in differentiation competent and differentiation defective clones of myeloid leukemic cells showed, that constitutive expression of other proteins was associated with blocks in differentiation (Liebermann et al., 1980; Sachs, 1980). The protein changes during differentiation are induced as a series of parallel multiple pathways of gene expression. Normal differentiation requires synchronous initiation and progression of these pathways. The presence of constitutive rather than induced gene expression of some pathways produces asynchrony in the coordination required for development, resulting in blocks in differentiation (Liebermann et al., 1980; Sachs, 1980).

The cytokines that control the developmental program regulate various transcription factors. Deficiencies in these transcription factors lead to severe hematopoietic failure during embryonic development (reviewed in Tenen et al., 1997; Bonifer et al., 1998; Sieweke and Graf, 1998). The conclusion on the role of constitutive gene expression in producing blocks in differentiation is supported by studies on constitutive expression of transcription factors. Expression of some transcription factors such as c-myc, c-myb and E2F1 is reduced when myeloid cells differentiate (Gonda and Metcalf, 1984; Blatt et al., 1992; Melamed et al., 1993). Constitutive expression of these genes (Hoffman-Liebermann and Liebermann, 1991; Selvakumaran et al., 1992; Amanullah et al., 2000) as well as of transcription factor genes such as the homeobox gene Hox B8 (Hox 2.4) (Blatt et al., 1992) or GATA-1 (Tanaka et al., 2000), disrupted the ability of cells to undergo cytokine induced differentiation. Constitutive expression of proteins that are normally up regulated during myeloid differentiation, including the cytosolic form of the protein tyrosine phosphatase PTPC (Tanuma et al., 2000) and the JAK2 kinase inhibitor SSI-1/JAB/SOCS-1 (Naka et al., 1997; Starr et al., 1997), also suppressed the ability of the cells to undergo cytokine induced differentiation. The study of these genes has thus also shown that constitutive expression of certain pathways results in blocks in differentiation. Reversion from the constitutive to the induced state of these pathways (Symonds and Sachs, 1983), showed that these cells can then again differentiate.

Studies in vivo have shown that normal differentiation of myeloid leukemic cells can be induced not only in culture but also in vivo (Lotem and Sachs, 1978, 1988) (Figure 4). After injection of mouse myeloid leukemic cells into fetuses, leukemic cells were shown to participate in normal hematopoietic cell differentiation to mature granulocytes and macrophages in apparently healthy adult animals (Gootwine et al., 1982; Webb et al., 1984). It has also been shown that mature granulocytes in human myeloid leukemia patients were derived from leukemic cells (Fearon et al., 1986). The development of leukemia was inhibited in mice inoculated with leukemic cells by increasing the amount of differentiation inducing cytokine, either by injecting it or by injecting a compound that increased its production by cells in the body (reviewed in Sachs, 1987a,b, 1990, 1995, 1996). Induction of differentiation in vivo, like in vitro, can occur directly, or by an indirect mechanism that involves induction of the appropriate differentiation inducing cytokine by other cells in the body (Lotem and Sachs, 1981, 1984, 1988, 1992a).

The finding that myeloid leukemic cells can be induced to differentiate to mature cells in vitro and in vivo by differentiation inducing cytokines and in doing so have regained normal growth control and lost their leukemogenicity in vivo, created the basis for differentiation therapy of leukemia (reviewed in Sachs, 1978, 1996). The ability of the leukemic cells to be induced to differentiate by the cytokines was not due to correction of their chromosomal abnormalities (reviewed in Sachs, 1987a). The results indicate that an abnormal developmental program in leukemic cells can be reprogramed epigenetically by appropriate differentiation inducing cytokines (reviewed in Lotem and Sachs, 2002).

Studies with a variety of compounds, other than normal hematopoietic cytokines, have shown that many compounds can induce differentiation in clones of myeloid leukemic cells. These include glucocorticoid hormones, compounds that are used today in cancer chemotherapy, such as cytosine arabinoside, methotrexate and others, and irradiation. At high doses, irradiation and the compounds used in cancer chemotherapy kill cells by inducing apoptosis, whereas at low doses they can induce differentiation. Not all these compounds are equally active on the same leukemic clone (reviewed in Sachs, 1978, 1982). Analysis of the pattern of protein expression in glucocorticoid sensitive or resistant myeloid leukemic clones has shown, that resistance to glucocorticoid hormones was also associated with constitutive expression of certain pathways (Cohen and Sachs, 1981). A variety of compounds can also induce differentiation in clones that are not induced to differentiate by a hematopoietic cytokine, and in some of these clones induction of differentiation requires combined treatment with different compounds (reviewed in Sachs, 1982). In addition to certain steroids, chemotherapeutic compounds and radiation, other compounds that can induce differentiation in myeloid leukemic cells include insulin, bacterial lipopolysaccharide, tumor promoting phorbol esters (reviewed in Sachs, 1978, 1982) and retinoic acid (reviewed in Degos, 1992). This effect of retinoic acid on differentiation of promyelocytic leukemia cells is now used clinically in the therapy of these leukemias (Degos, 1992) showing the successful application of the concept of differentiation therapy in the clinic. It is possible that all myeloid leukemic cells which are no longer susceptible to the normal hematopoietic cytokines by themselves can be epigenetically induced to differentiate by the appropriate combination of compounds. The experiments with myeloid leukemic cells have shown that there are different pathways of gene expression for inducing differentiation, and that genetic changes which suppress induction of differentiation by one compound need not affect differentiation by another compound using alternative pathways (Cohen and Sachs, 1981 and reviewed in Sachs, 1982). These results show that leukemic cells can be reprogramed and that there is considerable plasticity in the myeloid differentiation program. This presumably also applies to other cell types.

Cytokines as suppressors of apoptosis

Normal hematopoietic cells, like other normal cell types, die by the process of apoptosis when deprived of viability inducing cytokines that include CSFs and various other cytokines. Normal hematopoietic cells require these cytokines to suppress apoptosis throughout cell differentiation, and although some of these cytokines can also induce cell multiplication, suppression of apoptosis and cell multiplication can be separately regulated (reviewed in Sachs and Lotem, 1993; Sachs, 1995, 1996; Lotem and Sachs, 1999). Genetic changes that result in increased expression of apoptosis suppressing genes or decreased expression of apoptosis inducing genes make cells less dependent on apoptosis suppressing cytokines. A change in the balance of expression of the apoptosis inducing gene bax and the apoptosis suppressing genes bcl-2 or bcl-x_L (reviewed in Lotem and Sachs, 1999; Masuda et al., 2001) can explain the ability of various cytokines to suppress apoptosis (Table 1). In addition, cytokines can also change this balance by up regulation of other bcl-2-related genes such as the apoptosis suppressing mcl-1 (Chao et al., 1998; Puthier et al., 1999) and A1 (Lin et al., 1993) and by down regulation of apoptosis inducing genes hrk (Sanz et al., 2000, 2001), bim (Dijkers et al., 2000; Shinjyo et al., 2001) and bak (Welniak et al., 2001) (Table 1). Cytokines also transmit a viability inducing effect by changing the activity of apoptosis regulating proteins rather than by changing their expression levels. One such mechanism involves activation of phosphatidyl inositol-3-kinase (PI3K) which phosphorylates and activates the Akt/PKB kinase which in turn phosphorylates the apoptosis suppressor protein Bad. This causes dissociation of Bad from Bcl-x_L and uncovers the apoptosis suppressing function of Bcl-x_L (reviewed in Lotem and Sachs, 1999 and Table 1). In addition to Bad phosphorylation, Akt/PKB can also phosphorylate and inactivate other intracellular components of the apoptotic machinery including procaspase 9 (reviewed in Datta et al., 1999). Another mechanism of cytokine mediated apoptosis suppression involves prevention of mitochondrial translocation of Bax (Gross et al., 1998; Khaled et al., 1999) (Table 1). These cytokine induced changes in the balance of gene expression of apoptosis suppressing versus apoptosis inducing genes prevent disruption of normal mitochondrial physiology and release of molecules such as cytochrome c or Smac/Diablo that initiate caspase activation and apoptosis (Vander Heiden et al., 1999, 2000, 2001; Chauhan et al., 2001). Cytokines also activate expression of other apoptosis suppressing genes such as Survivin (Carter et al., 2001), XIAP and cIAP2 (Digicylioglu and Lipton, 2001) that are caspase inhibitors (reviewed in Deveraux and Reed, 1999), and FLIP, that may disrupt the ability of cell surface molecules such as Fas to activate apoptosis (Kovalovich et al., 2001) (Table 1).

Like normal myeloid cells, myeloid leukemia cells from most patients still require hematopoietic cytokines for viability (Griffin and Löwenberg, 1986) and in the absence of cytokines die by apoptosis. An increased ability of cells to survive can be an important step in tumor development (Korsmeyer, 1992). There are myeloid leukemic cells from some patients that are autonomous and do not require an exogenous source of cytokines for viability (Griffin and Löwenberg, 1986), and myeloid leukemic cell lines have been established in culture that are independent of apoptosis suppressing cytokines for viability (reviewed in Sachs, 1996). The viability of cells that are independent of apoptosis suppressing cytokines can be due to overexpression of the apoptosis suppressing gene bcl-2 (reviewed in Korsmeyer, 1992) or other apoptosis suppressing genes. Cytokine-independent viability gives these cancer cells an advantage over cytokine-dependent cells in vivo when there is a limited supply of such cytokines. We found that when certain cytokine independent myeloid leukemic cells are induced to differentiate with IL-6, they regain a cytokine dependent state for viability before terminal differentiation and undergo apoptosis following withdrawal of IL-6 (Fibach and Sachs, 1976; Lotem et al., 1991a; Lotem and Sachs, 1989b, 1995a). These IL-6 primed cells were rescued from apoptosis by re-adding IL-6, or by adding IL-3, M-CSF, G-CSF, SCF, IFN- or IL-1 (Fibach and Sachs, 1976; Lotem et al., 1991a; Lotem and Sachs, 1989b, 1995a). The cytokine dependent IL-6 primed mouse myeloid leukemic cells were less leukemogenic in vivo in syngeneic mice than cytokine independent leukemic cells (Fibach and Sachs, 1976), presumably because of a limited supply of the required cytokines in vivo. It may thus be possible to suppress leukemia not only by cytotoxic agents or by induction of terminal differentiation, but also by decreasing the in vivo supply of apoptosis suppressing cytokines or the response of leukemic cells to these cytokines (reviewed in Sachs, 1996). This suggestion has been supported by experiments showing in vivo inhibition of multiple myeloma by injection of neutralizing antibody to IL-6 (Bataille et al., 1995). The IL-6 receptor shares the gp130 signaling chain with the LIF, IL-11, OSM, ciliary nerotrophic factor (CNTF) and corticotrophin 1 (CT-1) receptors (reviewed in Kishimoto et al., 1994; Taga and Kishimoto, 1997) and some of these cytokines also support the viability of multiple myeloma cells (Zhang et al., 1994; Taga and Kishimoto, 1997). The development of an antagonist to gp130 may be an even better way to suppress multiple myeloma in vivo (Renné et al., 1998).

Apoptosis can be induced not only by cytokine withdrawal from cytokine dependent normal and cancer cells, but also by various DNA damaging and other cytotoxic agents (reviewed in Sachs and Lotem, 1993; Sachs, 1995, 1996; Thompson, 1995). We found that expression of the tumor suppressor wild-type p53 is an important physiological signal for activation of apoptosis (Yonish-Rouach et al., 1991). Overexpression of wild-type p53 is sufficient to induce apoptosis in myeloid leukemic cells (Yonish-Rouach et al., 1991; Lotem and Sachs, 1995a, 1996a, 1998; Lotem et al., 1996) and other cell types (Shaw et al., 1992; Abrahamson et al., 1995). The use of p53 deficient mice has shown that wild-type p53 is required for induction of apoptosis in normal in vitro colony forming cells by -irradiation (Lotem and Sachs, 1993a) and in thymocytes by -irradiation and certain DNA-damaging compounds (Lotem and Sachs, 1993a; Clarke et al., 1993; Lowe et al., 1993). Wild-type p53 also induces apoptosis following cytokine deprivation in normal in vitro colony forming cells (Lotem and Sachs, 1993a). Not all apoptosis pathways are mediated by p53 as shown by the equal sensitivity in normal and p53 deficient mice to dexamethasone induced apoptosis in thymocytes (Lotem and Sachs, 1993a; Clarke et al., 1993; Lowe et al., 1993) and to -irradiation in activated T-cells (Strasser et al., 1994). The ability of wild-type p53 to induce apoptosis appears to require its physical or functional interaction with other gene products including c-Abl, E2F-1, ATM or the DNA helicass XPB and XPD (reviewed in Lotem and Sachs, 1999). Expression of other cellular genes including mdm-2 and Rb can negatively regulate the ability of wild-type p53 to induce apoptosis (reviewed in Lotem and Sachs, 1999). Accumulation of wild-type p53 protein following DNA damage is mainly due to post-translational events that decrease interaction of p53 with the mdm-2 protein, which is an E3 ubiquitin ligase that targets p53 to proteasomal degradation (Haupt et al., 1997; Kubbutat et al., 1997). We have shown that p53 protein stability is also regulated by the enzyme NADH quinone oxidoreductase (NQO1), and NQO1 inhibition caused p53 degradation and thus protected cells against p53-mediated apoptosis (Asher et al., 2001). Signaling of apoptosis by wild-type p53 is thus subject to positive and negative regulation by these other gene products.

In view of the role of wild-type p53 in apoptosis pathways, its inactivation endows cells with a viability advantage under conditions of limited cytokine concentrations or after DNA damage. Mutations in p53 occur in more than 50% of human cancers (reviewed in Levine, 1997; Vogelstein et al., 2000), suggesting that suppression of apoptosis is an important step in formation and progression of cancer. We found that a p53 mutation can suppress induction of apoptosis in cells with deregulated myc that lack wild-type p53, thus showing a gain of function property of mutant p53 (Lotem and Sachs, 1993b, 1995b). These c-myc and mutant p53 co-expressing cells showed increased leukemogenicity in vivo compared to cells that only express deregulated c-myc (Lotem and Sachs, 1995b). In addition to increasing the amount of wild-type p53, decreasing the amount of mutant p53 in cancer cells could thus enhance their sensitivity to apoptosis inducing cytotoxic agents used in therapy. We showed that the NQO1 inhibitor dicoumarol can also cause degradation of mutant p53 protein in leukemic cells, raising the possibility of combining dicoumarol with cytotoxic agents in therapy against cancer cells that express high levels of mutant p53 (Asher et al., 2001).

Wild-type p53 transcriptionally activates some genes and transcriptionally represses other genes (reviewed in Oren, 1992; Ko and Prives, 1996). Several of the p53 inducible genes encode proteins that have been implicated in having a potential role in apoptosis. These include the bcl-2 related proapoptotic genes bax (reviewed in Lotem and Sachs, 1999) and Noxa (Oda et al., 2000a), death domain containing genes DR5, FAS (reviewed in Lotem and Sachs, 1999) and Pidd (Lin et al., 2000), the mitochondrial genes p53AIP1 (Oda et al., 2000b) and PUMA (Nakano and Vousden, 2001; Yu et al., 2001), the ced-4 homolog Apaf-1 (Kannan et al., 2001; Moroni et al., 2001) and the genes PERP (Attardi et al., 2000) and p53DINP1 (Okamura et al., 2001). Some of the p53 repressible genes are apoptosis suppressors including bcl-2 (Lotem and Sachs, 1995a), the IGF-1 receptor (Prisco et al., 1997), heat shock protein 70 (HSP70) (Agoff et al., 1993) and p202 (D'Souza et al., 2001). Wild-type p53 appears to change the balance in expression of apoptosis-inducing versus apoptosis-suppressing genes in favor of the former and thus induce apoptosis in a manner similar to apoptosis induced by a deficiency of viability inducing cytokines. The contribution of each of these p53-regulated genes to induction of apoptosis can vary in different cell types as shown for the essential role of bax in p53 mediated apoptosis in neuronal cells but not in thymocytes (reviewed in Lotem and Sachs, 1999). However, when both bax and bak are knocked out, p53 mediated apoptosis in thymocytes is also impaired (Lindsten et al., 2000; Wei et al., 2001). Such bax and bak defective mice are also defective in apoptosis pathways not driven by p53 (Lindsten et al., 2000; Wei et al., 2001).

Induction of apoptosis by -irradiation or by different compounds used in cancer chemotherapy such as vincristine, doxorubicin, methotrexate and cytosine arabinoside was suppressed by IL-6, IL-3, G-CSF, GM-CSF and IFN- even in myeloid leukemic cells that are cytokine independent for viability (Lotem et al., 1991a; Lotem and Sachs, 1992b, 1995a; and reviewed in Sachs, 1995, 1996; Sachs and Lotem, 1993). Cytokines also suppressed induction of apoptosis in cytokine independent myeloid leukemic cells by transforming growth factor 1 (Lotem and Sachs, 1992b), and by overexpression of wild-type p53 in myeloid leukemic (Yonish-Rouach et al., 1991; Lotem and Sachs, 1995a, 1996a,b, 1997, 1998; Lotem et al., 1996) and erythroleukemic cells (Abrahamson et al., 1995). IL-3 and GM-CSF suppressed induction of apoptosis by the chemotherapeutic compound doxorubicin in primary cultures of human myeloid leukemia cells (Kaplinsky et al., 1996) and IL-3 suppressed -irradiation induced apoptosis in normal myeloid hematopoietic cells and IL-3 dependent cell lines (Collins et al., 1992; Canman et al., 1995). Studies on normal and malignant lymphoid cells have shown that IL-1, IL-2, IL-4, IL-6, IL-7, and IL-9 suppress induction of apoptosis by dexamethasone (reviewed in Lotem and Sachs, 1999). IL-6 was also shown to suppress FAS-mediated apoptosis in normal hepatocytes (Kovalovich et al., 2001) and multiple myeloma cells (Chauhan et al., 1997) and IL-15 suppressed FAS-mediated apoptosis in normal mast cells and in mast cell lines (Masuda et al., 2001). Similar studies on neuronal cells have shown that induction of apoptosis by neurotoxic agents is suppressed by IL-6, NGF, BDNF, CNTF, and GDNF (reviewed in Lotem and Sachs, 1999). Cytokines can thus protect against induction of apoptosis by different cytotoxic agents in normal and cancer cells. They decrease cytotoxic effects in normal cells and also decrease the effectiveness of cancer radiotherapy and chemotherapy. This decreased effectiveness can explain the poorer remission rates and survival following chemotherapy in acute myeloid leukemia patients whose cells show a higher responsiveness to hematopoietic cytokines in vitro (Griffin and Löwenberg, 1986). We have suggested that reducing the level of viability inducing cytokines in vivo or the responsiveness of cancer cells to these cytokines may be clinically useful in cancer therapy (Lotem and Sachs, 1992b, 1996b; Sachs and Lotem, 1993; Kaplinsky et al., 1996; Sachs, 1996). This would decrease viability of cytokine dependent cancer cells and increase their susceptibility to the anticancer effects of irradiation or chemotherapy.

Induction of apoptosis is not only suppressed by cytokines, and we have shown that apoptosis induction by overexpression of wild-type p53 can also be suppressed by antioxidants, Ca²⁺-mobilizing compounds through calcineurin activation, and protease inhibitors (reviewed in Lotem and Sachs, 1999). This showed that apoptosis induction by wild-type p53 involves steps that can be regulated by the degree of cellular oxidative stress, calcium concentration and protease activation. The cytokines as well as these other compounds suppressed apoptosis upstream to caspase activation (Lotem and Sachs, 1997, 1998). Apoptosis suppression by the cytokines did not depend on calcium activated calcineurin (Lotem and Sachs, 1998) or a decrease in oxidative stress (Lotem et al., 1996). The different pattern of the ability of cytokines, antioxidants, Ca²⁺-mobilizing compounds and protease inhibitors to suppress induction of apoptosis in the same cells by cycloheximide, doxorubicin, vincristine or cytokine withdrawal (reviewed in Lotem and Sachs, 1999), indicated that there are multiple pathways leading to apoptosis that can be selectively suppressed by different anti-apoptotic agents and that cytokines can suppress pathways of apoptosis that are not suppressed by these other agents. The ability of cytokines to suppress apoptosis under different conditions is an essential part of the developmental program that regulates cell viability, growth and differentiation.

Acknowledgements

This work was supported by a research grant from the Dolfi and Lola Ebner Center for Biomedical Research.

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Figure 1 Cell culture system for cloning and clonal differentiation of normal hematopoietic cells. (a) Culture of mouse mast cells that have multiplied and differentiated on a feeder layer of mouse embryo cells (Ginsburg and Sachs, 1963). (b-d) Clones of macrophages and granulocytes in cultures of normal hematopoietic cell precursors incubated with the appropriate inducer in semisolid medium containing agar. (b) Petri dish with clones (Pluznik and Sachs, 1965), (c) granulocyte clone, and (d) macrophage clone (Ichikawa et al., 1966)

Figure 2 Network of hematopoietic cytokine interactions (references in the text)

Figure 3 Differentiation of stem cells to various cell types (references in the text)

Figure 4 In vivo differentiation of myeloid leukemic cells. Leukemic blast cells (a) and cells in various stages of differentiation (b) (Lotem and Sachs, 1978)

Table 1 Cytokine regulation of genes of the apoptotic machinery